15th Congress of the INTERNATIONAL SOCIETY FOR ANIMAL ...

15th Congress of the

INTERNATIONAL SOCIETY FOR ANIMAL CLINICAL

PATHOLOGY

14th Conference of the

EUROPEAN SOCIETY OF VETERINARY CLINICAL

PATHOLOGY

3rd – 7th July, 2012

University of Ljubljana, Veterinary Faculty

Ljubljana, SLOVENIA

15th

Congress of the

International Society for Animal Clinical Pathology

14th

Conference of the

European Society of Veterinary Clinical Pathology

3rd

– 7th July, 2012, Ljubljana, SLOVENIA

Edited by

Martina Klinkon

Jožica Ježek

Jože Starič

2

Published by: UNIVERSITY OF LJUBLJANA, VETERINARY FACULTY

Editors: Martina KLINKON, Jožica JEŽEK, Jože STARIČ

Printed by: Birografika BORI d.o.o., Linhartova cesta 1, 1001 LJUBLJANA

Number of copies: 250

CIP - Kataložni zapis o publikaciji

Narodna in univerzitetna knjižnica, Ljubljana

636.09(082)

CONGRESS of the International Society for Animal Clinical Pathology (15 ; 2012 ;

Ljubljana)

15th Congress of the International Society for Animal Clinical Pathology [also] 14th

Conference of the European Society of Veterinary Clinical Pathology, Ljubljana, Slovenia,

3rd-7th July, 2012 / edited by Martina Klinkon, Jožica Ježek, Jože Starič. - Ljubljana :

Veterinary Faculty, 2012

ISBN 978-961-6199-58-2

1. Klinkon, Martina 2. Conference of the European Society of Veterinary Clinical Pathology

(14 ; 2012 ; Ljubljana)

262333952

3

ISACP Executive Board (2010-2012):

President: Morrie CRAIG

([email protected]) Oregon State University, USA

Vice president (2012

Congress Chair):

Martina KLINKON-OGRINEC

([email protected]) University of Ljubljana, Slovenia

Immediate

Past-President:

Josep PASTOR

([email protected]) University of Barcelona, Spain

Executive Director: Mary CHRISTOPHER

([email protected]) University of California-Davis, USA

Secretary: Guillaume COUNOTTE

([email protected]) Animal Health Services, Deventer, the

Netherlands

Treasurer: Carolina RIOS

([email protected]) Universidad Santo Tomás, Santiago,

Chile

Account Manager: John HARVEY

([email protected]) University of Florida, USA

ESVCP: Current Excecutive Board

President: Stefano COMAZZI Milan, Italy

Past President: Jose CERON Murcia, Spain

Vice president: Peter O'BRIEN Dublin, Ireland

Secretary: Peter GRAHAM Poulton-le-Fylde, UK

Treasurer: Mark PINCHES Surrey, UK

Councillors: Josep PASTOR

Stefan NEUMANN

Barcelona, Spain

Göttingen, Germany

International Scientific Committee

Mary CHRISTOPHER, USA

Peter O’BRIEN, Ireland

Kendal HARR, USA

Leslie SHARKEY, USA

Jose CERON, Spain

Jožica JEŽEK, Slovenia

Jože STARIČ, Slovenia

Local Organizing Committee:

Martina KLINKON

Matjaž OCEPEK

Jože STARIČ

Jožica JEŽEK

Marija NEMEC

Petra ZRIMŠEK

Nina ČEBULJ KADUNC

Tomaž ZADNIK

Nataša TOZON

Silvestra KOBAL

mailto:[email protected]







4

2012 ISACP –ESVCP Sponzors & exhibitors

We are very grateful for support from the following sponsors and exhibitors

Platinum sponsor

Silver sponsor

Sponsor or exhibitor

Demeditec

Randox Laboratories & Sanolabor

Iris

Meditrade

Dipros

Tridelta Development

COST Action: Farm animals proteomics (FA1002)

The 15th

ISACP & 14th

ESVCP was supported by Slovenian Research Agency,

grant number E4-4532-0406-12.

Others:

Novartis

Pfizer

Merck

Kefo ŠOVF

5

ISACP AWARD

HEINER SOMMER AWARD

Heiner Sommer was one of four founding members of the

ISACB (now the ISACP). Each biannual congress an active or

retired member of the Society is selected by the Board to receive

the Heiner Sommer Prize in recognition of their lifetime

contributions in the field of animal clinical biochemistry. The

recipient is invited to present a keynote Heiner Sommer Lecture

at the Congress. The award includes travel, lodging, and

registration for the Congress.

Past Heiner Sommer

Award Winners:

2000 J. Jerry Kaneko , University of California-Davis, USA

2002 Eitan Bogin, Kimron Veterinary Institute, Israel

2004 John H. Lumsden, University of Guelph, Canada

2006 Jean-Pierre Braun, University of Toulouse, France

2008 P. David Eckersall, Glasgow University, UK

2010 John W. Harvey, University of Florida, Gainesville,

USA

2012 Heiner Sommer

Award Winner:

Fernando G Wittwer

Fernando G Wittwer, graduated as Veterinarian at the University

of Chile in 1968, Master in Veterinary Science University of

Liverpool, UK in 1977. With post-degree training on Veterinary

Clinical Pathology, mostly in Clinical Biochemistry and

Hematology of farm animals in Great Britain: Universities of

Liverpool and Edinburgh, Germany: Free University Berlin and

Tiho Hannover, Israel: Kimrom Institute and New Zealand:

University of Lincoln.

Professor at the Universidad Austral de Chile, Faculty of

Veterinary Sciences, as responsable for teaching activities on

Animal Clinical Patholgy for veterinary and graduated students.

Administrative activities as Dean and vice-Dean of the

Veterinary Faculty, head of the Department of Veterinary

Clinical Sciences, international consultant on diagnostic and

control of metabolic disturbances on farm animals and member

of evaluation panel for accreditation systems of veterinary

schools in Latinoamerica.

Research activities on metabolic unbalances in farm animals,

with proyects associated to mineral deficiencies in dairy cows

and the use of metabolic profiles in herd medicine. Author or co-

author of 112 papers in ISI journal, 38 reviews and many papers

presented in scientific meetings. Editor of 12 proceedings and

referee of scientific journals and research agencies.

Member of the International Society of Animal Clinical

Pathology, Chilean Society of Animal Production, Chilean

Society of Animal Clinical Pathology and the Chilean Buiatric

Society.

6

2012 ISACP Travel Grant Awards

Afshin Davasaztabrizi, Department of Clinical Sciences, Tabriz Branch, Islamic Azad

University, Tabriz, Iran. [email protected]

Alireza Rocky, Khuzestan, Shahid Chamran University of Ahvaz, Ahvaz, Iran.

[email protected]

Monika Kerestes, Faculty of Veterinary Science, Budapest, Hungary.

[email protected]

Jani Mavromati, Veterinary Medicine Faculty, Tirana, Albania. [email protected]

Antonio Melendez Lazo, Barcelona, Spain. [email protected]

Ana Cristina Silvestre Ferreira, University Tras-os-Montes e Alto Douro, Vila Real, Portugal.

[email protected]

2012 ISACP Young Investigator Prizes

J.J.Kaneko Prize

Presented for the best oral presentation by a young investigator

Eitan Bogin Prize

Presented for the best poster presentation by a young investigator

2012 ESVCP Best Free Communication Award

Presented for the best platform presentation

2012 ESVCP Best Poster Award

Presented for the best poster presentation

2012 ESVCP Best Case Presentation Award

Presented for the best case presentation






7

Invited Speakers

Ilias Alexandrakis Ilias Alexandrakis. Research and Development Manager Mr Alexandrakis took his first degree in Medical Laboratories studies at the University of Thessaloniki, Greece, acquiring a wide spectrum of knowledge in the medical area with a six month placement in Hippocrates hospital of Thessaloniki specialising in biochemistry, haematology, histopathology and endocrinology techniques. After graduation, he spent four years working in a private surgery, responsible for carrying out tests and maintaining the laboratory. In 2008, he took a full time MSc in Applied Bio-molecular Technology in the University of Nottingham. The most up-to-date techniques were introduced in microbiology, biotechnology, structural biology and drug delivery giving the opportunity to be competitive in the job market. He joined PetScreen in 2009 and has since been involved in establishing core technologies for the company including cell-based chemosensivity assays, ELISA development, obtaining and analysing SELDI-TOF MS data, chemiluminescence assays and health and safety management. The combination of an extensive knowledge of pertinent laboratory techniques and clinical experience make Mr Alexandrakis ideally qualified to lead the emerging PLS research team.

Andre Almeida

André de Almeida (born Lisbon, 1974) is a young researcher at the Tropical Research Institute in Lisbon, Portugal and an invited researcher at the Mass Spectrometry laboratory of the Chemical and Biological Technologies Institute of the New University of Lisbon. André de Almeida has earned a Degree in Animal Science by the University of Évora (1998), an MSc in Tropical Animal and Veterinary Sciences by the Faculty of Veterinary Medicine of Lisbon (2000) and a PhD in Biology by the New University of Lisbon (2005). André de Almeida has conducted field and laboratory works in several countries: South Africa, Spain, Australia and Guinea-Bissau. André de Almeida research is dedicated to the application of Proteomics and Mass Spectrometry to Animal and Veterinary Sciences, with diverse areas of interest, particularly at the level of animal production and physiology and more recently tick-borne diseases. Research is done in collaboration with Australian, French, Spanish, Slovakian, German and British institutions, among others. André de Almeida is the Vice-Chair of the COST action FA1002 – Proteomics in Farm Animals, Associated Editor of Tropical Animal Health and Production (Springer) and invited Editor of the Journal of Proteomics (Elsevier). André de Almeida has published over 35 papers in international peer-reviewed journals and has participated in several national and international research projects in animal and veterinary sciences.

Anna Bassols Anna Bassols graduated in Pharmacy in 1980 at the Universitat de Barcelona and defended her PhD Thesis in 1986 dealing with regulation of glucose metabolism in rats. She is also Specialist in Clinical Biochemistry (1987). After her PhD, she took a postdoctoral position in the Department of Biochemistry at the University of

8

Massachusetts Medical School (1986-1987), where she was involved in the study of growth factors and cell signaling. Back in Spain, she was appointed in 1988 as a professor at the Faculty of Veterinary Medicine at the Universitat Autònoma de Barcelona, where she is now Full Professor at the Department of Biochemistry and Molecular Biology, and Director of the Veterinary Clinical Biochemistry Service. Her scientific interests are focused in the search for biomarkers useful for animal medicine and animal production, as well as in the role of the innate immune system and the involvement of extracellular matrix molecules in the inflammatory process.

Barbara Von Beust DVM, PhD, Dipl ACVP & ECVCP Switzerland Dr. Barbara von Beust, a board certified veterinary clinical pathologist (Diplomate ACVP & ECVCP) received her PhD from Washington State University, USA, in veterinary cell-mediated immunology research. She is an experienced laboratory manager with a profound understanding of Good Laboratory Practice guidelines. She has extensive experience in planning, directing, evaluating and monitoring preclinical research and safety studies in general and in clinical laboratory data analysis, interpretation and reporting in particular. She has authored and peer reviewed numerous clinical pathology reports and assisted in the audits of clinical pathology laboratories in several leading European CRO’s. Dr. von Beust’s special expertise is in the area of cytology diagnostics and more specifically in hematology and bone marrow data analysis, the interpretation of acute phase response parameters, and the scientific evaluation of cell – mediated specific immune responses (including cytokine profiles). Dr. von Beust works as an international consultant and scientific expert. She is currently the acting chair of the Education Committee of the ECVCP and an active member of the Regulatory Affairs Committee of the American Society of Veterinary Clinical Pathology (ASVCP) and the Career Development Outreach Committee of the Society of Toxicologic Pathology (STP).

Denise I. Bounous

Denise I. Bounous, DVM, PhD Senior Veterinary Pathology Fellow, Clinical Pathology for Discovery Toxicology Bristol Myers Squibb Company Princeton, NJ Dr. Bounous is a Board Certified Veterinary Clinical Pathologist. She is a Senior Veterinary Pathology Fellow in Clinical Pathology for Discovery Toxicology at Bristol Myers Squibb. Her previous pharmaceutical experience includes Astra Zeneca Pharmaceuticals and DuPont Pharmaceuticals. She was professor of Clinical Pathology at the University Of Georgia College Of Veterinary Medicine for 10 years prior to joining the pharmaceutical industry. She obtained her undergraduate degree from the University of the South in Sewanee, Tennessee; MT (ASCP) from Baptist Memorial Hospital in Memphis; DVM from Oklahoma State University;

9

residency in Clinical Pathology and PhD from Louisiana State University. She is a member of American College of Veterinary Pathology, American Society of Veterinary Clinical Pathology, American Association of Clinical Chemists, and Society of Toxicologic Pathology. Her scientific interests and experience include: biomarkers of pharmacologic toxicity; mechanisms of bone marrow toxicity; laboratory animal clinical pathology; and diagnostic clinical pathology. She has served as an active member of cross-company/academic working groups developing novel biomarkers of toxicity: HESI Renal and Cardiac, C-Path Renal and Muscle Toxicity groups. These groups work with FDA and EMEA to bring new biomarkers into acceptance for use in pharmaceutical studies. Dr Bounous has been author/co-author of over 75 scientific publications and abstracts in these areas.

Fabrizio Ceciliani

Fabrizio Ceciliani, DVM, PhD, graduated in Veterinary Medicine at the University of Milano. After military duty as Veterinary Officer in Italian Army, he defended in 1994 a PhD thesis in Biochemistry. From 1995 to 1997 he held a post doc fellowship in Molecular Biology. In 1998 he worked as visiting scientist in the Laboratory of Central Nervous Studies and Strokes – The Prion Unit - of the National Institute of Neurological Disorders and Stroke (NINDS), Bethesda. Since 2001 Fabrizio Ceciliani is a Researcher in Veterinary Pathology at the School of Veterinary Medicine, University of Milano. His scientific interest lies in innate immunity of domestic animals, focusing on acute phase proteins, and how post-translational modifications modify their functions and molecular mechanism of action. The species of interest are basically dairy cows, but also cats, goats and wildlife ruminants are currently investigated. Monocytes and polymorphonulcear cells are mostly studied as biological models. The basic activities investigated so far included chemotaxis, phagocytosis, oxidative burst, killing and gene expression and profiling after challenging the cells with acute phase proteins, as well pro- and anti-inflammatory molecules, such as for example immunomodulatory polyunsaturated fatty acids.

Jose Ceron

Jose Ceron is the head of the Clinical Pathology Laboratory at Murcia Veterinary School. He is an ECVCP Diplomate, has been invited speaker at different congresses organized by ESVIM, NAVC or ESVCP and was the coordinator of a CD-book entitled “Veterinary Clinical Pathology: an undergraduate integrated course”. He was the past president of the European Society of Veterinary Clinical Pathology. His main topics of interest are the study of haematological and biochemical biomarkers of animal health and welfare and education in clinical pathology.

Didier Concordet

Didier Concordet, Professor of statistics at the National Veterinary School of Toulouse

10

Email: [email protected] Scientific profile: statistician, amateur of informatics. Mathematical taste: stochastic modelling and analysis for biology (pharmacology, biostatistics). Main research topics (up to now): -Markov processes/semigroups, their equilibrium -Sobolev type inequalities -Random algorithms, stochastic simulation -Stochastic models and stochastic inverse problems in biology -population pharmacokinetics/pharmacodynamics -epidemiology -statistics for medical decisions -biostatistics

Mary Christopher

Dr. Mary Christopher is a Professor of Clinical Pathology at the University of California-Davis School of Veterinary Medicine and is board-certified by the American College of Veterinary Pathologists and the European College of Veterinary Clinical Pathology. Dr. Christopher received her DVM from Iowa State University in 1980 and completed her PhD and NIH postdoctoral training at the University of Minnesota. She was on the faculty at the University of Florida for 5 years and has been at UC Davis since 1994, where she served as Chief of Clinical Pathology for more than 10 years and as Associate Director of the Teaching Hospital. She is a recipient of the North American Outstanding Teacher Award, the Norden Distinguished Teacher Award, the ASVCP Education Award, and a Fulbright Scholar Award. She was Editor-in-Chief of Veterinary Clinical Pathology from 1997 to 2009, founded the International Association of Veterinary Editors, and is Executive Director of the International Society for Animal Clinical Pathology. Her research interests include erythrocytes and anemia, diagnostic clinical pathology, pathology reporting, and scientific writing.

Guillaume Counotte

Guillaume Counotte, trained as biochemist and toxicologist and now a European Registered Toxicologist, works at the Animal Health Service (AHS) in the Netherlands. The AHS is an independent private organization, dedicated to animal health, expert veterinary knowledge, with a large veterinary diagnostic laboratory and extensive support facilities. Guillaume Counotte is a member of the R&D-department and works on projects in the field of veterinary toxicology (trace metals, residues, plant toxins, drinking water) and biomarkers (bone-metabolism in horse and swine, immune resistance in cows).

Nina Čebulj

Nina Čebulj-Kadunc has graduated in 1985 at Veterinary Faculty, University of Ljubljana. After a period of traineeship at a dairy farm, she was employed as an assistant (Physiology of Domestic Animals) in 1986 and elected as the associate professor of Veterinary Physiology in 2010. In 1988 she has passed a master degree entitled Glutathione peroxidase activity in calves and in 1997 a doctor degree (PhD)

11

Biorhythmical changes of tyroxine, triiodotyronine, cortisol and progesterone concentrations in Jezersko-Solchava eves. Now, dr. Čebulj-Kadunc is actively integrated in the research project of Veterinary Faculty entitled Endocrine, immune and enzyme response in healthy and sick animals. Her main field of research activity is endocrinology of domestic animals with emphasis on biorhythmical fluctuations of hormone profiles and non-invasive methods for hormone determination. Currently, she’s mostly engaged with physiological characteristics of Slovene autochthonous domestic animal breeds and their reintroduction to sustained agriculture. She also participates in studies of antioxidant status in domestic animals.

Manica Černe

Manica Černe graduated in Veterinary medicine in University of Ljubljana 1984. She defended her M.Sc. and Ph.D. Thesis in the same faculty dealing with pathology and patohistology of pig intestinal disease. She also finished M.Sc. study of Toxicology on Medical University of Vienna. She started her professional career on the Institute of Pathology, forensic and administrative veterinary medicine, first as researcher than as assistant and assistant professor. As researcher she took an active part in different projects from prevention and curative veterinary medicine. Later found new challenge in Lek Pharmaceuticals. From 2002 she works there as a researcher expert and manager on preclinical development of active pharmaceutical ingredient and final products. She planned, performed and assesses the results from in-house or outsourced in-vivo pharmacologic and toxicity studies. She has published in international peer-reviewed journals and has participated in several national and international research projects in animal and veterinary sciences as well as pharmaceutical and chemical science. Manica Černe, Lek Pharmaceuticals d.d. Verovškova 57 SI-1526 Ljubljana SLOVENIA [email protected]

Denis DeNicola

Dr. DeNicola earned his DVM in 1978 and PhD in 1981 from Purdue University at which time he began his academic career. For more than 20 years he served as educator of veterinary students in clinical pathology, veterinary pathology residents in clinical pathology and surgical pathology, and veterinarians and technicians in clinical pathology. He served as Director of the Veterinary Clinical Pathology Laboratory and the National Veterinary Cytology Resource Center at Purdue and just recently joined IDEXX as Chief Veterinary Educator. He maintains an adjunct professorship at Purdue. He has responsibilities for developing educational efforts for IDEXX in addition to his pathology service responsibilities. His primary areas of interest beyond education include diagnostic cytology, hematology and surgical pathology.

David Eckersall After graduating from the University of Liverpool with a BSc in

12

Biochemistry and the University of Edinburgh with a PhD while at the Animal Breeding Research Organisation, Prof David Eckersall has been based for over two decades at the Faculty of Veterinary Medicine of the University of Glasgow where he is now the Professor of Veterinary Biochemistry. His main focus of research has been on acute phase proteins in domestic animals and in particular the use of these blood proteins as biomarkers for disease. He has pioneered the use of specific protein assays in veterinary clinical biochemistry inventing the biochemical reagents for an all-species haptoglobin assay as well as developing immunoassays for proteins such as canine C-reactive protein and feline alpha-1 acid glycoprotein. His research covers both companion and production animals and has identified the use of alpha-1 acid glycoprotein in the differential diagnosis of feline infectious peritonitis that the acute phase proteins haptoglobin and serum amyloid A are produced in the mammary gland during mastitis in dairy cows. He is a Fellow of the Royal College of Pathologists and was awarded the Heiner Sommer Prize of the International Society for Animal Clinical Pathology for Lifetime Contribution to Animal Clinical Biochemistry in 2008 and the Siemens Prize of the Division of Animal Clinical Chemistry of the American Association of Clinical Chemistry for Contributions to Animal Clinical Chemistry in 2010. Since 2011 he has been the Chair of the EU COST Action on Farm Animal Proteomics.

Kathleen P. Freeman

Dr. Freeman is a Senior Veterinary Clinical Pathologist working at IDEXX Laboratories, Wetherby, United Kingdom. She received her DVM, MS and PhD from Oklahoma State University, College of Veterinary Medicine. She has been working in the UK since late in 1997 and was a founding diplomate of the ECVCP. She also is a consultant for Veterinary Information Network. She is the Course Director for the Laboratory Quality Management courses offered regularly on VIN. She is past Chair of the ASVCP Qualtiy Assurance and Laboratory Standards Committee and continues to be a working member of this committee. She was the first chair of the ECVCP Laboratory Standards Committee and continues to work on that committee and on the Cytology Subcommittee of the Examination Committee. She has served as Treasurer of ESVCP and ECVCP and as a Councillor for ESVCP. She has special interests in equine clinical pathology and cytology, quality assurance/quality control, customer service and laboratory management. She is delighted to be speaking to you today.

Alessia Giordano

Dr. Giordano is Assistant Professor at the School of Veterinary Medicine of the University of Milan. She is board-certified by the European College of Veterinary Clinical Pathology (ECVCP) and she is the clinical pathologist in chief of the clinical pathology laboratory at the Faculty Large Animal Teaching Hospital. Dr. Giordano has been member of the ECVCP exam committee of which is currently the chair. She received her DVM degree and her PhD in Animal Pathology and Veterinary Hygiene at the University of Milan where she works since 2007 as Researcher. Her main scientific interests include: diagnostic clinical pathology, feline infectious peritonitis, acute phase

13

proteins and dysproteinemias, diagnostic electrophoresis, biomarkers of inflammation, method validation. Dr. Giordano is co-author of several peer-reviewed publications and she has been reviewer for several international scientific journals.

Hugues Guyot

Born in Belgium, 1976. Graduated in Veterinary Medicine (Doctor of Veterinary Medicine) in 2000 (University of Liege, Belgium). Private practice in France (dairy) and Belgium (beef). Assistant in the Clinic for Ruminants in ULg, Liege, Belgium (2001). Activity focused on herd health management. Master of Veterinary Science (Herd Health Management, ULg, 2002). Doctor of Veterinary Sciences (PhD) in 2007. Externships in Bern (Switzerland), Ithaca (Cornell, NY, USA), St-Hyacinthe (Québec, Canada), Lyon (France), Oberschleissheim (Munich, Germany). Diplomate by examination of the European College of Bovine Health Management (2008). Member of the Education & Residency Committee of ECBHM (2009). Creation of the Bovine Ambulatory Clinic in Liege (2010). Activity both in individual medicine and herd health management. Senior lecturer in the clinical department for production animals (ULg) in the area of herd health medicine (2012). Research topics: trace elements deficiencies and supplementation, side-cow diagnosis test, emerging diseases in Belgium (Bluetongue, Ehrlichiosis), lameness.

Kendal Harr

Dr. Kendal Harr has had an interest in wildlife since she worked at the New York State Fisheries Diagnostic Laboratories at Cornell University in the early 1990s. She received her DVM from Cornell University and her Master of Science, thesis: Diagnostic Development of Acute Phase Proteins in Manatees, from the University of Florida. For this work, she won the Excellence in Clinical Research Award from the University of Florida. While completing her residency, she won the CL Davis Award for Scholarship and the ACVP Young Investigator Award (2001) First Place in Toxicology for poster presentation of Loss of hepatic glutathione S-transferaseexpression in large mouth bass (Micropterus salmoides) by estradiol as demonstrated by real-time polymerase chain reaction, among others. Since that time, she has worked at UF as the associate director of Aquatic Animal Healthy, worked for IDEXX, and now owns her own pathology company, URIKA, LLC, with consultation to Abaxis.

John W. Harvey

Dr. John W. Harvey earned his DVM degree from Kansas State University and his PhD degree from the University of California at Davis. He is board certified as a clinical pathologist by the American College of Veterinary Pathologists, and is currently Professor and Executive Associate Dean of the University of Florida College of Veterinary Medicine. Dr. Harvey is a recognized expert in comparative hematology, having published over 160 journal articles and book

14

chapters. He is author of “Veterinary Hematology. A Diagnostic Guide and Color Atlas,” and coeditor of "Clinical Biochemistry of Domestic Animals.” Awards and honors that Dr. Harvey has received include the Alumni Recognition Award from the Kansas State University College of Veterinary Medicine, the Award for Outstanding Contribution to Animal Clinical Chemistry, Division of Animal Clinical Chemistry, American Association for Clinical Chemistry, the Lifetime Achievement Award from the American Society for Veterinary Clinical Pathology, the Heiner Sommer Prize for Lifetime Contributions in the Field of Animal Clinical Pathology from the International Society for Animal Clinical Pathology, and the Mark L. Morris, Sr, Lifetime Achievement Award from Hill’s Pet Nutrition, Inc.

Regina Hofmann-Lehmann

Curriculum Vitae (Excerpt): Since 2011 Head Clinical Laboratory, Head Center for Clinical Studies, University of Zurich. Since 2010 Professor of Clinical Laboratory Medicine, University of Zurich. President of the Swiss Association for Veterinary Laboratory Diagnosticians SVVLD. 2004 – 2010 Professorship Swiss National Science Foundation. 2003 Habilitation in Laboratory and Experimental Medicine, University of Zurich. 1997 – 2001 Research assignment, Harvard Medical School & Viral Pathogenesis, Dana-Farber Cancer Institute, Boston, USA. 1997 Specialization in Laboratory Medicine and Basic Medicine. 1983 – 1992 Studies in Veterinary Medicine & doctoral studies, University of Zurich, Switzerland. Research Funding Information (Summarized): Principle/senior investigator of 26 research grants (~3.7 million CHF). Swiss National Science Foundation grants since 1998. Major Research Interests: Retroviral infections, Haemotropic Mycoplasmas, Vector-borne infections and infections in wild felids, Laboratory medicine: molecular diagnostics, hematology, clinical chemistry Honors, Awards, Fellowships (Excerpt) 2004/08-Swiss National Science Foundation Professorship, 1999-Stratton Fellowship, Boston, USA, 1998-Swiss National Science Foundation Scholarship for advanced researchers, 1997 Scholarship, Canton Zurich, 1992-Best doctoral thesis of the year, VSF, UZH, Zurich, Switzerland. 168 publications in scientific peer-reviewed journals. Miscellaneous: Reviewer for 34 scientific journals, Patent holder.

Danijela Kirovski

Danijela Kirovski is an Associate Professor in the Department of Physiology and Biochemistry and the Faculty of Veterinary Medicine University of Belgrade, Serbia. She graduated as DVM in 1995, Master of Science (in 1999) and PhD (in 2005) at FVM University of Belgrade. Until 2005 her research was mainly focused on endocrinology and growth performance in neonatal calves. Thereafter, her research interest was reorientated mainly to transition cow metabolic problems. She attained the Norman E. Borlaug Fellows

15

grant in year 2005 at the Department of Animal Science at Iowa State University, Iowa, USA where she was involved in research related to fatty liver problems in high-yielding cows. She attended post-doctoral fellowship at the Department of Dairy Science, Virginia Polytechnic and State University, Virginia, USA in 2007. At Virginia Tech prof Kirovski was involved in a Project related to manipulation of milk yield and composition using different nutrients for lactating cows. She enrolled Specialization course on Bovine Health Management at the FVM University of Belgrade and accomplished DVSc in 2010. Currently, her research focuses on the effects of heat stress on metabolic performances of dairy cows. She published over 40 scientific papers and had numerous oral presentations.

Milica Kova čević Filipovi ć

Milica Kovacevic Filipovic, DVM, PhD, graduated from the Faculty of Veterinary Medicine (FVM), University of Belgrade, Serbia, in 1993. Since 1994 she has been employed at FVM and since 2011 she has been holding a position of associate professor at the Department of Pathophysiology. Her PhD thesis is in a domain of experimental hematology - hematopoietic stem cell research using pig and mouse experimental models. She completed the total of 18 months of postdoctoral studies (research on stem cells) in France (Institute for Natural Substances Chemistry, National Centre of Scientific Research, Paris and National French Institute for Blood, Bordeaux). She is also working on developing clinical pathology field at FVM and she published several papers on clinical hematology issues, as well as on acute phase proteins.

Björn Kuhla

Björn Kuhla finished an apprenticeship as skilled worker for cattle breeding in 1990. From 1991 to 1996, he studied Chemistry at the University of Rostock, Germany and obtained his doctoral degree in 1999. His PostDoc carrier started at the agricultural research centre of the Bayer AG in Monheim in 1999. From 2000 to 2005, he continued his PostDoc at the Universities of Rostock and Leipzig by focusing on molecular and metabolic mechanisms underlying neurodegenerative and aging processes in the brain. From 2005 to 2006, he held a PostDoc fellowship at the Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, USA. In 2006 he became a scientist for proteome analysis at the Research Unit „Nutritional Physiology“ of the Leibniz Institute for Farm Animal Biology (FBN), Dummerstorf, Germany. Since 2009, he is the junior research group leader for „Feed Intake Regulation“ at the FBN. His scientific interests lay in the elucidation of metabolites, hormones and cytokines as central signals controlling feed intake during the periparturient period of dairy cows. One of his major research projects focuses on the identification of hepatic and muscular metabolic pathways activated or deactivated at the onset of lactation and thereby triggering autonomous nervous signals controlling feed intake.

Ernst Leidinger

Ernst Leidinger studied veterinary medicine in Vienna, Austria. During his studies he worked as a demonstrator and teaching assistant at the Institute of Anatomy. After completion of the diploma program in 1986 to 1995 he was at the Institute of Medical Chemistry, Veterinary University working as an assistant. During that time, Ernst

16

Leidinger did his doctoral thesis on the production of monoclonal antibodies against synthetic peptide antigens. In 1994 Ernst Leidinger together with his wife, Dr. Judith Leidinger founded the independent veterinary diagnostic laboratory "In Vitro" in Vienna. In 1995 he was appointed as national specialist in veterinary clinical laboratory diagnostics, and is a member of the examination commission for this specialty. End of 2005, Ernst Leidinger received the defacto recognition as "Diplomate of the European College of Veterinary Clinical Pathology" (ECVCP). He is webmaster and chair of the College's website editorial board. Ernst Leidinger is author of several publications and regularly gives national and international lectures. He enjoys travelling and SCUBA diving; preferably both things combined.

Inger Lilliehöök

Dr Lilliehook is a Clinical Pathologist and the Laboratory Director at the Clinical Pathology Laboratory at the University Animal Hospital (UDS). She is Associate Professor of the Faculty of Veterinary Medicine and Animal Science, Swedish University of Agricultural Sciences (SLU), Uppsala, Sweden. She received her PhD in Veterinary Clinical Pathology 1999 at the Swedish University of Agricultural Sciences in Uppsala. Inger Lilliehook became defacto Diplomate of the European College of Veterinary Clinical Pathology (ECVCP) 2005 and is a member of the ECVCP Education committee. The Clinical Pathology Laboratory at SLU is an ESVCP approved training site and has an approved residency program. Inger is mainly in charge of diagnostic clinical pathology services, but additionally is course director for Clinical Pathology and teaches veterinary students, veterinarians, medical technologists and residents. Her research areas are hematological changes of animals in heath and disease and evaluation of hematology instruments.

Gregor Majdi č

Gregor Majdic graduated at the University of Ljubljana Veterinary school in 1992, obtained a Ph.D. at the University of Edinburgh, Scotland in 1996 and has worked as a postdoctoral fellow at University of Texas Southwestern medical center in Dallas, Texas, from 1998 to 2001. Currently he is associate professor for physiology at Veterinary School, University of Ljubljana. He is also the head of the department Center for Animal Genomics. He has secondary appointment as associate professor of physiology at Medical school, University of Maribor. He has published 40 peer reviewed papers, which have been cited more than 1000 times. He has been awarded several national and international grants including two NIH grants in collaboration with colleagues from the US. His research work is mostly in the field of neuroendocrinology and reproductive biology. Recently, his laboratory developed a novel procedure of stem cell treatment in dogs and horses and they established a spin-out company to commercialize this novel method. For this achievement, he and his colleagues were awarded two Slovenian national awards for the best innovations in Slovenia in 2011.

17

Gregor Majdic is also very active in communicating science to general public and has received two national awards for excellence in communicating science to general public.

Carlo Masserdotti

Carlo Masserdotti graduated in Veterinary Medicine in 1990 at the University of Milan. From 1993 his interest was mainly focused on clinical pathology, particularly in diagnostic cytopathology, attending specialistic courses and references institutions in Italy and in foreign countries. He is author of scientific papers concerning cytopathology and he had some lectures at national and international meetings. From 1998 he is teacher and lecturer at course of Cytology, organized by SCIVAC. From 2001 to 2004 he was President of SICIV (Italian Society of Veterinary Cytology). From 2003 to 2006 he was Vice-president of European Society of Veterinary Clinical Pathology. In 2005 he received the de-facto recognition as Diplomate of the European College of Veterinary Clinical Pathology. In 2008 achieves post-graduate Specialization in Clinical Biochemistry, at the University of Brescia. Actually he is consultant in clinical pathology (cytopathology, histopathology) at San Marco Veterinary Laboratory in Padua. His researchs was mainly focused on cytologic features of spontaneous tumors and inflammatory diseases of companion animals; actually the scientific interest is mainly in hepatic cytology and histopathology. His personal hobby is triathlon, preferably on the long distances; he loves whales as greatest expression of grace.

Matjaž Ocepek

Dr. Matjaž Ocepek DVM is a Senior Research Fellow at the Veterinary Faculty, University of Ljubljana, Slovenia. He is the person in charge of the Laboratory for especially dangerous bacterial diseases and the Laboratory for molecular bacteriology at the Institute of Microbiology and Parasitology at the Veterinary Faculty. He is engaged in development and implementation of the methods for diagnostics of zoonoses and of especially dangerous bacterial diseases of animals, and molecular methods for animal bacterial diseases. He is also a leader of several scientific projects and a principal investigator one of the main research groups at the Veterinary Faculty. Besides the research work he also gives lectures to undergraduate (Microbiology) and postgraduate (Microbiological techniques) students. Currently, he is a mentor for six PhD students. He has published more than 60 original scientific papers and he is a member of the editorial board of the Slovenian Veterinary Research, a reviewer for several international scientific journals, and a member of the European Society of Mycobacteriology (chairman of the 31st ESM congress), International Association for Paratuberculosis, International Society for Infectious Diseases and Slovenian Microbiological Society (vice president).

Alessandro Poli

Alessandro Poli completed a DVM degree at the School of Veterinary Medicine of the University of Pisa in 1977. Assistant professor of Veterinary Pathology at the Department of Veterinary Pathology of the University of Pisa since 1981, Associate Professor of Veterinary

18

Pathology since 1987, he is Full Professor of Veterinary Pathology at the Department of Animal Pathology, University of Pisa, since 2000. Charter member of the European College of Veterinary Pathologists he is author of 130 publications and 70 of them have been published on peer-reviewed international Journals. His scientific activity has been focused on immunopathology and oncology, with a special regard to either veterinary or comparative pathology, particularly to animal models for the pathogenesis of human diseases. The main fields of research in immunopathology have been the study of canine and feline immunomediated glomerulonephritis and the FIV-induced feline immunodeficiency syndrome as a model for HIV-1 infection and AIDS syndrome in humans. Particularly, the pathogenesis of FIV-induced lesions has been was investigated by home made immunohistochemical and in-situ PCR methods to reveal viral presence and host response. His research activity in oncology has been focused on three main topics: the classification and the staging of tumours by immunohistochemical detection of cytoskeleton, membrane and cytoplasmic antigens; the development of morphological criteria for the staging and classification of tumours; and the study of markers of malignancy. In this sense, the lipid associated sialic acid (LASA) has been studied in canine neoplasms. Recently, the research has been focused on the study of feline and canine mammary tumours, in order to characterize the pattern of steroid hormones expression in healthy, dysplastic and neoplastic tissues, to evaluate the angiogenic activity of VEGF in neoplastic tissues and, recently, to study the expression of the Cyclooxygenase-2. The purpose of this investigation is to evaluate the role of feline and canine mammary tumours as a useful animal model for the human breast tumour pathogenesis, to identify some reliable prognostic markers, and to develop new therapeutic strategies in the small animal practice.

Ilse Schwendenwein Dr. Ilse Schwendenwein Dipl. ECVCP Head of the Division of Clinical Pathology – Central Laboratory University of Veterinary Medicine Vienna, Austria The Central Laboratory is the central diagnostic unit for haematology, clinical chemistry and cytology of the universities teaching hospital, but we also receive samples from outside practitioners and external research units. We see about 17 000 patients per year covering all species including exotic animals. The Central Laboratory has an ISO 9001-2008 certified quality management System in place and we try to approach the ISO 15 189 regulations as far as it is possible for a veterinary setting. We are an ESVCP approved training site and have an approved residency program in place. Our main interest is focussed on quality assessment, analysis of variance components and efficacy testing to provide guidelines for test interpretation.

Leslie Sharkey

Dr. Leslie Sharkey completed her DVM, PhD, and clinical pathology residency training at the Ohio State University. After two years in small animal practice, she was a clinical pathologist at Tufts

19

University for 5 years, before joining the faculty at the University of Minnesota in 2003. At the University of Minnesota, Leslie is the director of the clinical pathology laboratory and the residency training program and is research faculty at the Comprehensive Cancer Center. She teaches a core clinical pathology course in the second year and co-teaches a Problems in Small Animal Medicine course with Dr. Jane Armstrong in the third year. Leslie's research interests include evaluation of the diagnostic performance of laboratory testing, complications of cancer therapy, and the scholarship of teaching and learning. Recent work involves studies of the diagnostic performance of cytology of the canine liver and pancreas, evaluation of radiation induced damage and repair of bone marrow in a murine model, and outcomes assessment of a case-based approach to teaching veterinary clinical pathology.

Lindsay Tomlinson

Lindsay Tomlinson, DVM, DVSc, graduated from the Ontario Veterinary College in Guelph, Ontario, Canada in 1995 and 1999, for her respective degrees. Lindsay researched and defended her thesis on chlamydial disease in sheep in the DVSc program. She spent two years as a Visiting Instructor at North Carolina State University and obtained board certification with the American College of Veterinary Pathologists (ACVP) in Anatomic Pathology during that time. Lindsay worked as a Toxicologic Pathologist in the Anatomic and Clinical Pathology Departments and as a Project Representative for Drug Safety at Bristol-Myers Squibb (BMS) in New Brunswick, NJ, for just under 10 years. She earned board certification with the ACVP in Clinical Pathology and with the American Board of Toxicologists (ABT) in Toxicology during her time at BMS. Her current position is Associate Research Fellow at Pfizer Inc. in Cambridge, MA. Lindsay has a leadership role in the pathology of discovery and developmental drug safety. She is the Project Representative for Drug Safety on several projects and maintains strong ties to the clinical pathology community through the Regulatory Affairs Committee of the American Society for Veterinary Clinical Pathologists (ASVCP) as current chair.

Nataša Tozon

Prof. Nataša Tozon graduated at Veterinary faculty, University of Ljubljana at 1991. 1995 she took a muster degree and 1998 she concluded the Ph.D. study with final thesis »The occurrence and progression of renal pathological changes in feline immunodeficiency virus-infected cats« From 2009 she is an Associate Professor in Veterinary Internal medicine for small animals at Veterinary Faculty in Ljubljana. From 1997 she is care a lecture cours on Infection diseases and Oncology and from 2007 is a chair of Clinical Oncologyin Veterinary Medicine (elective subject) at the same faculty. She was a supervisor for three student research thesis and two PhD final theses. At the moment she supervises two student researches and four PhD final theses. She is a mamber of many national and international association: European College of Veterinary Internal Medicine – ECVIM, European Society of Veterinary Oncology – ESVONC, International Society for Companion Animal Infectious Diseases - ISCAID (since

20

2010), Federation of European Companion Animal Veterinary Associations - FECAVA and World Small Animal Veterinary asssociation – WSAVA. Since 2006 she is representativ for Slovenia in WSAVA and President of Slovene Small Animal Veterinary Association - SZVMZ (3rd election, the last from 2010) She was an invited reviewer for Slovenian Veterinary Resarch, Journal of Small Animal Practis, Current medical chemitry, Veterinary Arhiv Zagreb. She is author of more than 25 publications on referred journals and her activities account for a total of more than 180 published works. As associated professor and as clinicion in Veterinary internal Medicine of Small Animals, she has focused on Infection disease and clinical oncology. She introduce and adaapted the alternativ treatment option of neoplastic disease in veterinary medicine, electrochemotherapy and electrogene terapy. Her main resarech area regards infection diseases is artropode trasmited diseases as well as clnical immunology in infection disease in general. The main topic of the artorpode trasmited disease research project is pathogensis and host immune respons in dogs infected with A. phagocytophilum.

Catherine Trumel

Professor Catherine Trumel graduated from Toulouse Veterinary School (ENVT) in 1992 and obtained her PhD on platelet signalling transduction at Toulouse University in 1999. She was first assistant-professor in Small Animal and Horse Internal Medicine at ENVT and participated in the creation of the Laboratoire Central de Biologie Médicale (LCBM) of the ENVT. She passed the Board examination of the European College of Veterinary Clinical Pathology in 2006 and was recruited as Professor of Animal and Comparative Clinical Pathology by ENVT in 2006. Her teaching activities cover undergraduate, post-graduate, continuing education (as responsible of the Certificate of Hématologie et Biochimie Clinique Animales), and resident tutoring for ECVCP. She is also the person in charge of the LCBM (accredited by ECVCP in 20112) which is operated by A Geffré. Her research interests (now within an INSERM associated mixed-service unit currently under construction) are mainly in hematology and cytology and cover pre-analytical and analytical evaluation of methods, reference intervals and physiological factors of variation.

Romana Turk

Faculty of Veterinary Medicine University of Zagreb Heinzelova 55, 10000 Zagreb, Croatia Phone: +385 1 2390 180 Fax: +385 1 2390 184 E-mail: [email protected] Romana Turk (PhD) is a Researcher at the Faculty of Veterinary Medicine University of Zagreb, Croatia. She obtained her B.Sc. (1993), M.Sc. (2002) and PhD (2005) in Medical Biochemistry at the Faculty of Pharmacy and Biochemistry University of Zagreb, Croatia. Her M.SC and PhD theses were devoted to the role of anti-oxidative enzymes in reproduction and fertility of dairy cows. From 1993-1994

21

she worked at Clinical Laboratory for Hematology and Biochemistry, Hospital for Pulmonary Diseases and Tuberculosis and at Biochemical Laboratory, General Hospital Varaždin, Croatia. In 1994, she moved to the Faculty of Veterinary Medicine University of Zagreb and started with research at the Department of Pathophsyiology and teaching in pathophysiology of domestic animals, veterinary laboratory diagnostics and specific hormonal and metabolic disorders in domestic animals. She is also a supervisor for master and PhD students. Her main research interest is focused on biomarkers of oxidative stress and inflammation in reproductive and productive disorders of dairy cows, including periparturient metabolic diseases, subfertility and mastitis pointing at antioxidative enzymes and lipoproteins.

Fernando G Wittwer

Fernando G Wittwer, graduated as Veterinarian at the University of Chile in 1968, Master in Veterinary Science University of Liverpool, UK in 1977. With post-degree training on Veterinary Clinical Pathology, mostly in Clinical Biochemistry and Hematology of farm animals in Great Britain: Universities of Liverpool and Edinburgh, Germany: Free University Berlin and Tiho Hannover, Israel: Kimrom Institute and New Zealand: University of Lincoln. Professor at the Universidad Austral de Chile, Faculty of Veterinary Sciences, as responsable for teaching activities on Animal Clinical Patholgy for veterinary and graduated students. Administrative activities as Dean and vice-Dean of the Veterinary Faculty, head of the Department of Veterinary Clinical Sciences, international consultant on diagnostic and control of metabolic disturbances on farm animals and member of evaluation panel for accreditation systems of veterinary schools in Latinoamerica. Research activities on metabolic unbalances in farm animals, with proyects associated to mineral deficiencies in dairy cows and the use of metabolic profiles in herd medicine. Author or co-author of 112 papers in ISI journal, 38 reviews and many papers presented in scientific meetings. Editor of 12 proceedings and referee of scientific journals and research agencies. Member of the International Society of Animal Clinical Pathology, Chilean Society of Animal Production, Chilean Society of Animal Clinical Pathology and the Chilean Buiatric Society.

Osamu Yamato

The area of my current research interests is focused on a variety of inherited metabolic disorders in domestic and wild animals including lysosomal storage diseases. These include GM1 and GM2 gangliosidosis, neuronal ceroid lipofuscinosis, mucopolysaccharidosis, disorders of purine and pyrimidine metabolism, and other metabolic and neurodegenerative disorders. Research is being performed to clarify molecular defects and pathogenesis as well as to develop diagnostic, therapeutic, and preventive methods relating to the above diseases. Previously, I was studying Allium plant toxicity such as onion-induced hemolytic anemia in domestic animals and was able to identify some of the causative agents, i.e., sodium alk(en)nyl thisulfates. Currently research on the functionalily of these natural compounds is being performed. These compounds have demonstrated a variety of functions

22

including anti-tumor and anti-thrombogenic effects beneficial for human health.

Karen M. Young

Karen M. Young, VMD, PhD received a BA in Russian language and history in 1973, a VMD in 1978, and a PhD in hematopathology in 1985 from the University of Pennsylvania where she was also an Intern in Small Animal Medicine and Surgery from 1978-79 and a Resident in Medical Oncology from 1979-81. Dr. Young has been a faculty member in the Department of Pathobiological Sciences at the University of Wisconsin-Madison since 1985 and is currently Professor of Clinical Pathology and Chief of Staff for Diagnostic Services. A recipient of the Norden Distinguished Teacher Award and Chancellor's Hilldale Award for Excellence in Teaching, she was also the founding chair of the Education Committee of the American Society for Veterinary Clinical Pathology. Dr. Young is currently Editor-in-Chief of Veterinary Clinical Pathology and is a co-author with Dr. Mary Christopher of Writing for Publication in Veterinary Medicine: A Practical Guide for Researchers and Clinicians. Research interests include teaching problem-solving effectively, assessment-as-learning, hematopoietic neoplasia, and ocular cytology.

23

INDEX

INVITED LECTURES

1. STRENGTHENING GLOBAL ALLIANCES IN VETERINARY CLINICAL PATHOLOGY. M.

M. Christopher ................................................................................................................................. 33

2. CONTRIBUTION OF CLINICAL BIOCHEMISTRY IN RUMINANT PRODUCTION. F. G.

Wittwer ............................................................................................................................................ 33

3. EVALUATION OF METABOLIC AND ENDOCRINE STATUS IN TRANSITION DAIRY

COWS IN PREDICTION AND DIAGNOSIS OF FATTY LIVER SYNDROME. D. Kirovski ... 40

4. OXIDATIVE STRESS AND INFLAMMATORY RESPONSE IN BOVINE REPRODUCTIVE

AND PRODUCTIVE DISORDERS: INTERACTION OF HDL-ASSOCIATED PROTEINS. R.

Turk, P. Roncada ............................................................................................................................. 43

5. TRACE MINERALS DEFICIENCY DIAGNOSIS IN RUMINANTS. H. Guyot ......................... 45

6. ONCOLOGIC CYTOLOGY OF THE CANINE LIVER AND PANCREAS. L. Sharkey ............. 50

7. ADIPOSE TISSUE DERIVED STEM CELLS FOR TREATMENT OF OSTEOARTHRITIS

AND OTHER JOINT PROBLEMS IN DOGS. K.Čeh, G. Majdič ................................................. 52

8. THE ROLE OF THE COX-2/PGE2 PATHWAY IN PROMOTING CANCEROGENESIS OF

CANINE AND FELINE TUMOURS. A. Poli ................................................................................ 54

9. CYTOLOGIC DIAGNOSIS OF MESENCHYMAL TUMORS: NOT JUST “SARCOMA”. C.

Masserdotti ...................................................................................................................................... 55

10. NON HEPATOCYTE CELLS: DESCRIPTION OF NORMAL AND PATHOLOGIC

CYTOMORPHOLOGY OF THE “OTHERS” LIVER CELLS. C. Masserdotti ............................ 57

11. LABORATORY TESTS FOR METABOLIC INBORN ERRORS IN DOMESTIC ANIMALS:

CLUES TO DIAGNOSIS. O. Yamato ............................................................................................ 59

12. LABORATORY TESTS FOR THE DIAGNOSIS OF ACUTE PANCREATITIS IN DOGS AND

CATS – SERUM LIPASE ACTIVITY REVISITED. I. Schwendenwein, E. Hooijberg, B.

Ruetgen, I. Flickinger, F. Zeugswetter, K. Hittmair ........................................................................ 59

13. COMPARATIVE ASPECTS OF THE ACUTE PHASE PROTEIN RESPONSE. P.D. Eckersall 62

14. SERUM AMYLOID A AS BIOMARKER OF SUBCLINICAL INFLAMMATION IN CATTLE.

M. Kovačević Filipović ................................................................................................................... 63

15. THE USE OF CART ALGORITHMS TO COMBINE SERUM ACUTE PHASE PROTEIN

LEVELS AS A DIAGNOSTIC AID IN CANINE LYMPHOMA. I. Alexandrakis ....................... 65

16. HAEMOTROPIC MYCOPLASMA INFECTION IN CATS AND DOGS. R. Hofmann-Lehmann,

M. Novacco, J. Baumann, B. Willi, F. S. Boretti ............................................................................ 66

17. TICK-BORNE DISEASES IN DOGS IN SLOVENIA. N. Tozon .................................................. 68

18. MOLECULAR TECHNIQUES USED IN DIAGNOSIS AND CONTROL OF VETERINARY

INFECTIOUS DISEASES. M. Ocepek, I. Toplak, D. Kušar .......................................................... 70

19. PROTEOMICS, ELECTROPHORESIS AND MASS SPECTROMETRY: A TOOL IN

BIOMARKER DETECTION AND DIAGNOSIS? A. M. Almeida ............................................... 74

20. CLINICAL PATHOLOGY AND STATISTICS. D.Concordet ...................................................... 76

21. ASVCP FINALIZED REFERENCE INTERVAL GUIDELINES. K. E. Harr ............................... 76

22. ADIPOKINES IN COMPANION ANIMALS: AN UPDATE. J. J. Cerón, A. Tvarijonaviciute .... 79

23. LEPTIN LEVELS AND SEASONAL REPRODUCTIVE ACTIVITY IN LIPIZZAN FILLIES. N.

Čebulj-Kadunc ................................................................................................................................. 80

24. VARIANCE COMPONENTS AND CRITICAL DIFFERENCES IN DOGS AND CATS. I.

Schwendenwein, E. Hooijberg, B. Hanus, F. Frommlet , M. Pagitz ............................................... 84

25. URINE CHEMISTRY - AN EVIDENCE-BASED APPROACH. E. Leidinger, J. Leidinger ........ 87

26. ERYTHROCYTE ENZYME DEFICIENCIES IN DOMESTIC ANIMALS. J. W. Harvey .......... 94

27. MAMMALIAN HEMATOPOIESIS. J. W. Harvey ........................................................................ 94

28. PLATELET COUNTING IN THE CAT. C. Trumel., F. Granat , N. Bourgès Abella, A. Geffré ... 97

24

29. BONE MARROW EXAMINATION IN DOGS AND CATS. J. W. Harvey ................................. 98

30. RETICULOCYTE COUNTS – WHAT IS TRUTH AND WHAT DO WE NEED TO KNOW FOR

INTERPRETING RETICULOCYTOSES? D. DeNicola ............................................................. 101

31. TOTAL ALLOWABLE ERROR: A UNIFYING CONCEPT IN THE VETERINARY CLINICAL

PATHOLOGY LABORATORY. K. P. Freeman .......................................................................... 101

32. ALLOWABLE TOTAL ERROR AND ITS USE IN VETERINARY PRACTICE. K. E. Harr ... 101

33. QC VALIDATION – AN INTRODUCTION. E. Leidinger ......................................................... 106

34. GLP PRINCIPLE IN PRECLINICAL TESTING. M. Černe, E. Lovšin Barle ............................. 110

35. VETERINARY TOXICOLOGY: AN INTEGRATED APPROACH. G. Counotte ..................... 111

36. THE GULF OIL SPILL: PETROLEUM INTOXICATION. K. E. Harr ....................................... 114

37. SERUM IRON ANALYTES IN HEALTHY AND ILL FLORIDA MANATEES. J. W. Harvey 117

38. HAEMATOLOGICAL CHANGES IN HORSES WITH ENDOTOXAEMIA. I. Lilliehöök, H. W.

Tvedten, J. Brojer, A. Edner, K. Nostell ........................................................................................ 117

WORKSHOPS

TOXICOLOGY

1. OVERVIEW OF ROLES AND RESPONSIBILITIES, LABORATORY PERSONNEL AND THE

EXPANDING ROLES OF THE CLINICAL PATHOLOGIST IN DRUG DEVELOPMENT. L.

Tomlinson ...................................................................................................................................... 118

2. DATA ANALYSIS / INTERPRETATION OF DATA. B. Von Beust ......................................... 118

3. BEST PRACTICES IN ROUTINE LABORATORY INVESTIGATIONS: HEMATOLOGY,

COAGULATION, CLINICAL BIOCHEMISTRY, AND URINALYSIS. L. Tomlinson ............ 119

4. NON ROUTINE LABORATORY INVESTIGATIONS: BONE MARROW, CYTOLOGY,

ENDOCRINOLOGY AND OTHERS. D. Bounous...................................................................... 119

5. INSTRUMENT AND METHOD VALIDATION. B. Von Beust ................................................. 119

6. POINTS TO CONSIDER FOR NEW BIOMARKER QUALIFICATION. D. Bounous .............. 120

HOW TO PUBLISH

1. WRITING FOR PUBLICATION IN VETERINARY MEDICINE:

KEY TO SUCCESS. M. Christopher, K. Young .......................................................................... 120

PROTEOMICS AND ANIMAL HEALTH (COST- FA 1002 Workshop)

1. PROTEOMICS IN VETERINARY SCIENCE: AN OVERVIEW. P. D. Eckersall ..................... 120

2. PROTEOMICS IN VETERINARY SCIENCES: A CASE STUDY IN TICK-BORNE DISEASES.

A. M. Almeida, M. Ventosa, D. Meyer, D. Martinez, T. Lefrançois, N. Vachiéry, A.V. Coelho, I.

Marcelino ....................................................................................................................................... 122

3. INCREASED OXIDATIVE STRESS AND PEROXISOMAL FATTY ACID DEGRADATION

IN EARLY LACTATING HOLSTEIN COWS DEVELOPING FATTY LIVER SYNDROM -

LESSIONS FROM PROTEOMIC ANALYSIS. B. Kuhla, C. Schäff, D. Albrecht, M. Röntgen, C.

C. Metges, H. M. Hammon............................................................................................................ 125

4. POST TRANSLATIONAL MODIFICATIONS OF ACUTE PHASE PROTEINS IN

VETERINARY MEDICNE: A PROTEOMIC APPROACH. F.Ceciliani .................................... 127

5. SEPARATION TECHNIQUES AND THE USE OF DIGE FOR PROTEOMIC ANALYSIS OF

VETERINARY SAMPLES. A. Marco-Ramell, A. Bassols .......................................................... 129

25

ORAL PRESENTATIONS

RUMINANT CLINICAL PATHOLOGY

1. EFFECT OF TIME RELATED TO CALVING AND RUMEN PROTECTED CHOLINE (RPC)

SUPPLEMENTATION ON BLOOD ANALYTES AND ON LIVER LIPID AND GLYCOGEN.

T. Gaal, P. Elek, F. Husveth .......................................................................................................... 132

2. ASSOCIATION BETWEEN HEMATOLOGICAL AND ELECTROPHORETIC PROFILES

AND PREVALENCE OF POST-PARTUM DISEASES IN DAIRY COWS. S. Paltrinieri, P.

Moretti, M. Venturini, N. Morandi, A. Giordano .......................................................................... 132

3. HEMATOLOGICAL CHANGES ASSOCIATED WITH RETAINED PLACENTA IN CATTLE.

P. Moretti, A. Giordano, M. Venturini, N. Morandi, S. Paltrinieri ................................................ 133

4. SURVEY OF VITAMIN A, BETA-CAROTENE, CALCIUM AND PHOSPHORUS

CONCENTRATIONS IN SERUM OF EWES WITH RETAINED PLACENTA. A.

Davasaztabrizi ............................................................................................................................... 133

NOVEL BIOMARKERS

5. URINE PROTEOME IN DOGS AFFECTED BY LEISHMANIASIS. A. Buono, F. Dondi, E.

Ferlizza, G. Isani, J. Duque, P. Tapia, C. Zaragoza, R. Barrera .................................................... 139

6. EVALUATION OF THE REVISED QUANTITATIVE INSULIN SENSITIVITY CHECK

INDEX IN DAIRY COWS SHOWING VARIOUS FORMS OF KETONE PATTERN AND

PUERPEAL METRITIS M. Kerestes, V. Faigl, M. Kulcsár, O. Balogh, J. Földi, H. Fébel, Y.

Chilliard, G. Huszenicza ................................................................................................................ 140

7. DIAGNOSTIC VALUE OF ACUTE PHASE INDEX OF MILK IN DETECTION OF

SUBCLINICAL MASTITIS IN DAIRY COWS.S. H. Shirazi-Beheshtiha, S. Safi, V. Rabbani,

M.B. Bolourchi .............................................................................................................................. 140

CASE PRESENTATIONS

8. A CASE OF CANINE SURRA IMPORTED INTO GERMANY. M. Defontis, J. Richartz, N.

Engelmann, C. Bauer, V.M. Schwierk, P. Büscher, A. Moritz...................................................... 141

9. CHRONIC LYMPHOCYTIC LEUKEMIA/SMALL CELL LYMPHOMA IN A HORSE. F. Cian,

G. Tyner, V. Martini, S. Comazzi, J. Archer ................................................................................. 141

10. CASE REPORT: CUTANEOUS T-CELL LYMPHOMA. B. Rütgen, I. Flickinger, B.

Wolfesberger, B. Litschauer, A. Fuchs-Baumgartinger, S.E. Essler, A. Saalmüller, I.

Schwendenwein ............................................................................................................................. 141

ACUTE PHASE PROTEINS

11. CHANGES IN THE CONCENTRATIONS OF SERUM AMYLOID A (SAA) FOLLOWING

SURGERY, TRAUMA OR SNAKE ENVENOMATION IN DOGS: A STUDY OF THE

KINETICS OF SAA. M. Christensen, E. Moldal, R. Langhorn, A. Goddard, A. Tvarijonaviciute,

M. Kjelgaard-Hansen ..................................................................................................................... 142

12. EVALUATION OF A CANINE SPECIFIC QUANTITATIVE POINT- OF- CARE (POC)

IMMUNOASSAY FOR MEASURING C-REACTIVE PROTEIN IN DOGS. M. Costa, K.

Papasouliotis .................................................................................................................................. 143

IMMUNOLOGY AND INFECTIOUS DISEASES

13. PCR-BASED DETECTION OF A. PHAGOCYTOPHILUM DNA IN PARAFFIN EMBEDDED

SKIN BIOPSIES FROM DOGS SEROPOSITIVE AGAINST A. PHAGOCYTOPHILUM. I.

Berzina, N. Müller, C. Krudewig, I. Matise, R. Ranka, M. Welle................................................. 145

26

NOVEL DIAGNOSTIC TECHNIQUES

14. PRELIMINARY REFERENCE INTERVALS FOR FLUOROGENIC MEASUREMENT OF

THROMBIN GENERATION IN THE BEAGLE DOG. M. Defontis, S. Côté, M. Stirn, D. Ledieu147

15. FLOW CYTOMETRIC EVALUATION OF DRUG RESISTANCE IN CANINE LYMPHOMA

SHORT TERM CELL CULTURES P. Vajdovich ........................................................................ 148

16. MEASUREMENT OF SOLUBLE MESOTHELIN RELATED PEPTIDES (SMRP) IN CANINE

BODY CAVITY EFFUSIONS. M. Costa, K. Murphy, D. Wilson, K. Papasouliotis ................... 148

CLINICAL PATHOLOGY AND STATISTICS

17. PREDICTIVE VALUE ADVISOR: A SOFTWARE FOR THE EVALUATION OF THE

IMPRECISION OF PREDICTIVE VALUES OF TEST RESULTS ACCORDING TO

IMPRECISION OF SENSITIVITY, SPECIFICITY AND PRE-TEST PROBABILITY. N.

Bourgès-Abella, P. Bourdaud'hui, A. Geffré, D. Concordet, J.P. Braun, O. Dossin, C. Trumel ... 150

18. REFERENCE VALUE ADVISOR (V.2) NOW PERFORMS REGRESSION-BASED

REFERENCE INTERVALS WITH CONTINUOUS COVARIABLES. N. Bourgès-Abella, A.

Geffré, D. Concordet, J.P. Braun, C. Trumel ................................................................................ 150

BIOCHEMISTRY

19. ACTIVATION OF LIPID METABOLISM IN CANINE BABESIOSIS CAUSED BY BABESIA

CANIS N. Kučer, V.Mrljak, R. Rafaj Barić, J.Kuleš, I.Šmit, J. Selanec, M. Crnogaj .................. 151

20. LACTATE DEHYDROGENASE (LDH) ISOENZYMES IN DOGS AFFECTED BY

LYMPHOMA. A. Giordano, D. Stefanello, M. Pastore, R. Ferrari, L. Giori, P. Moretti, S.

Paltrinieri ....................................................................................................................................... 151

21. EFFECTS OF PLATELET-ACTIVATING FACTOR RECEPTOR ANTAGONIST (PAFRA) ON

SELECTED INFLAMMATORY AND BIOCHEMICAL PARAMETERS IN A RAT

ENDOTOXEMIA MODEL. R. Col, E. Keskin............................................................................. 151

22. URINE PROTEOME FROM HEALTHY DOGS, CATS, HORSES AND COWS. E. Ferlizza, E.

Armuzzi, A. Buono, E. Carpenè, G. Andreani, F. Dondi, G. Isani ................................................ 152

23. EVALUATION OF THREE URINE DIPSTICKS FOR DOGS, CATS, AND COWS. S. Klenner,

M. Defontis, N. Bauer, K. Failing, A. Moritz ................................................................................ 152

24. CLINICAL RELEVANCE OF TGF-ß1 IN DOGS. S. Neumann, S. Gaedcke ............................. 153

25. LDH IN BODY CAVITY FLUIDS: IS IT DIAGNOSTICALLY USEFUL? C. Smuts, J. Mills, T.

Gaál ................................................................................................................................................ 153

26. CANINE PANCREATIC LIPASE (SPEC CPL) AND C-REACTIVE PROTEIN (CRP) IN DOGS

TREATED WITH ANTICONVULSANTS (PHENOBARBITAL AND POTASSIUM

BROMIDE). J. Pastor, V. Albarracin, A. Meléndez-Lazo, J. Rodón ............................................ 153

27. IMMULITE 1000 MEASUREMENTS IN VETERINARY SAMPLES. R. Pato, R. Pena, Y. Saco,

A. Bassols ...................................................................................................................................... 154

28. USE OF BIOMARKERS IN ANIMAL MODELING OF HEART FAILURE AND

CARDIORENAL DYSFUNCTION. C. McClay, J. Urban, R. Gerhart, N. Papenfuss ................. 154

HEMATOLOGY

29. KINETICS AND CHARACTERISTICS OF FELINE PLATELET AGGREGATION IN VITRO.

B. Riond, A.K. Waßmuth, S. Hartnack, R. Hofmann-Lehmann, H. Lutz ..................................... 161

27

30. EFFECTIVE PREVENTION OF PSEUDOTHROMBOCYTOPENIA IN FELINE EDTA BLOOD

SAMPLES WITH THE PROSTAGLANDIN I2-ANALOGUE ILOPROST. B. Riond, A.K.

Waßmuth, S. Hartnack, R. Hofmann-Lehmann, H. Lutz .............................................................. 162

31. EXPLORATIVE REFERENCE INTERVALS FOR BIOCHEMISTRY AND HAEMATOLOGY

DATA IN HEALTHY PIGLETS DURING THE FIRST 27 DAYS OF LIFE. B. Rütgen, E.

Hooijberg, L. Schwarz, H.L. Worliczek, I. Schwendenwein ......................................................... 162

32. THE “QUATREFOIL” ERYTHROCYTES: UN-EXPECTED PATTERN IN CANINE RED

BLOOD CELL MORPHOLOGY. A. Gavazza, G. Lubas, M. Ricci, B. Gugliucci, A. Pasquini . 162

33. PLATELET INDICES IN VIRULENT CANINE BABESIOSIS AND THEIR ASSOCIATION

WITH OUTCOME. A. Goddard, A. Kristensen, A. Leisewitz, P. Thompson, J. Schoeman ........ 163

34. D-DIMER MEASUREMENT BY LATEX AGGLUTINATION;COMPARISON OF A

SEMIQUANTITATIVE AND QUANTITATIVE METHOD AND EVALUATION OF THE

CLINICAL VALUE OF THE TEST IN DOGS. J. Archer, A. Pastorello, P. Monti, P. Kuczybska163

35. DIAGNOSTIC PERFORMANCES OF FLOW CYTOMETRIC DETECTION OF BLOOD AND

BONE MARROW INFILTRATION IN DOGS WITH LARGE B-CELL LYMPHOMA. V.

Martini, M.E. Gelain, S. Comazzi ................................................................................................. 164

36. DOUBLE ERYTHROCYTE POPULATIONS : RETROSPECTIVE STUDY ON CASES

OBSERVED WITH SYSMEX XT-2000iV (2008-2012). N. Bourgès-Abella, B. Cuq, F. Granat, A.

Geffré, C. Hanot, C. Trumel .......................................................................................................... 164

37. LEEK DIET MAY CAUSE HEMOLYTIC ANEMIA : A CASE REPORT IN A CAT. N.

Bourgès-Abella, F. Granat, A. Geffré, C. Trumel ......................................................................... 164

38. GASTRIC DILATATION AND VOLVULUS SYNDROME – ARE THERE ANY CLUES IN

HEMATOLOGIC EXAMINATION? I. Uhrikova, L. Rauserova-Lexmaulova, K. Rehakova, I.

Hajek, J. Doubek ........................................................................................................................... 165

39. EFFECT OF STORAGE CONDITIONS ON KAOLIN/ NONKAOLIN-ACTIVATED

THROMBOELASTOGRAPHY AND HEMOSTATIC PARAMETERS IN CATS. R. Col, C.M.

Iazbik, G. Couto ............................................................................................................................. 165

TOXICOLOGY & FREE TOPICS

40. PRELIMINARY REPORT OF HEMOSTATIC PARAMETERS IN THE EXPERIMENTAL

GÖTTINGEN MINIPIG. M. Defontis, S. Côté, M. Stirn, D. Ledieu ............................................ 174

41. AFLATOXINS IN SLOVENE MILK AND FEED SAMPLES. B. Jakovac-Strajn, G. Tavčar-

Kalcher, I. Ujčič-Vrhovnik, K. Pavšič-Vrtač, K. Fon-Tacer, A. Vengušt ..................................... 174

42. IS ALKALINE PHOSPHATASE ACTIVITY IN BOVINE NASAL SECRETION A RESULT OF

LOCAL SYNTHESIS? M.F. Ghazali, N.N. Jonsson, P.D. Eckersall .......................................... 175

WILDLIFE CLINICAL PATHOLOGY

43. SERUM CALCIUM AND INORGANIC PHOSPHATE EVALUATION IN HERMANN'S

TORTOISES (Testudo hermanni). M. Stvarnik, M. Klinkon, A. Dovč ........................................ 180

44. HEMATOLOGY REFERENCE INTERVALS IN CYNOMOLGUS MONKEYS. N. Bourgès-

Abella, A. Geffré, E. Moureaux, M. Vincenti, J.P. Braun, C. Trumel .......................................... 180

EQUINE CLINICAL PATHOLOGY

45. EVALUATION OF SERUM AMYLOID A, HAPTOGLOBIN, NUCLEATED CELL COUNT,

TOTAL PROTEIN AND HEMOLYSIS IN PERITONEAL FLUID FOR DIFFERENTIATION

OF MEDICAL AND SURGICAL COLIC IN HORSES. E. Scheepers, T. Holberg Pihl, M. Sanz,

P. Page, A. Goddard, S. Jacobsen .................................................................................................. 181

28

46. MEASUREMENT OF SERUM ALBUMIN BY PROTEIN ELECTROPHORESIS AND

BROMOCRESOL GREEN METHODS IN CANINE AND EQUINE PATIENTS E. Ramery, F.

Bureau ........................................................................................................................................... 182

POSTER PRESENTATIONS


1. THE EFFECT OF PARITY ON BONE FORMATION DURING LATE PREGNANCY AND

EARLY LACTATION IN SAANEN GOATS M. Belić, V. Kušec, M. Robić, J. Grizelj, A.

Svetina, Z. Vrbanac, R. Turk ......................................................................................................... 133

2. AGREEMENT BETWEEN ELECTROCHEMILUMINESCENCE AND

CHEMILUMINESCENCE IMMUNOASSAY METHODS FOR DETERMINATION OF

SERUM FREE TETRAIODOTHYRONINE VALUES IN HOLSTEIN COWS. B. Eshratkhah,

A.R. Mirzaei, M. Mostafaei Bigham ............................................................................................. 134

3. SUBCLINICAL MASTITIS IN SHEEP: NOVEL DIAGNOSTIC FINDINGS. A. Miglio, M.T.

Antognoni, L. Moscati, C. Maresca, A. Valiani, E. Scoccia, V. Mangili, G. Fruganti .................. 134

4. OVINE MALIGNANT THEILERIOSIS: THE STATUS OF ANTIOXIDANT VITAMINS,

SERUM LIPID PROFILE, LIPID PEROXIDATION AND ERYTHROCYTE ANTIOXIDANT

DEFENSE. R. Sadoughifar, S. Nazifi, Razavi .............................................................................. 135

5. MEASURING GLUCOSE CONCENTRATION IN BLOOD OF COWS WITH THE OPTIUM

XCEED TM METER AND THE BIOCHEMICAL ANALYSER – COMPARISON OF

METHODS. J. Ježek, J. Starič, M. Klinkon, M. Nemec ............................................................... 135

6. CHEEK TOOTH ABNORMALITIES IN SHEEP FLOCKS. V. Erjavec .................................... 135

7. GLUTAVAC® TEST SET (GLUTARALDEHYDE BLOOD TEST) – A DIAGNOSTIC

SUPPORT FOR FOREGIN BODY DISEASE. T. Zadnik, S. Rakulič Zelov ............................... 136

8. MONITORING SUBCLINICAL KETOSIS IN TRANSITION COWS PREGNANT WITH A

SINGLE CALF OR TWINS. T. Zadnik, R. Lombar .................................................................... 136

9. LOGISTIC REGRESSION MODELING FOR THE PREDICTION OF LEFT DISPLACED

ABOMASUM IN DAIRY COWS. S. Safi, N. Basiri, A. Rahimi foroushani ............................... 137

CYTOLOGY AND ONCOLOGY

10. CANINE CUTANEOUS HISTIOCYTOMA: A RETROSPECTIVE STUDY OF 55 CASES

(2002-2011). C. Pressanti, F. Granat, A. Geffre, M.C. Cadiergues ............................................... 137

11. CANINE NEOPLASTIC EFFUSION: MORPHOMETRIC ANALISYS OF CYTOLOGICAL

SAMPLES. G. Ghisleni, V. Pizzatti Sertorelli, A. Forlani, V. Bronzo, M. Caniatti ..................... 137

12. EFFECT OF NEEDLE GAUGE ON THE QUALITY OF CANINE SPLEEN FINE NEEDLE

ASPIRATION SPECIMENS – A PRELIMINARY STUDY. N. Bourgès-Abella, C. Layssol, A.

Geffré, C. Trumel .......................................................................................................................... 138

13. DETERMINATION OF PROLIFERATIVE ACTIVITY IN CANINE LEUKEMIAS BY FLOW

CYTOMETRY.A. Poggi, B. Miniscalco, L. Aresu, M.E. Gelain, S. Comazzi, V. Martini, E.

Morello, F. Gattino, F. Riondato ................................................................................................... 138

14. PROGNOSTIC VALUE OF KI67 IN CANINE LYMPHOMA DETERMINED BY FLOW

CYTOMETRY. A. Poggi, B. Miniscalco, L. Aresu, M.E. Gelain, S. Comazzi, V. Martini, E.

Morello, F. Gattino, F. Riondato ................................................................................................... 139

29

CASE PRESENTATIONS

15. »WATER DIABETES« IN A GREYHOUND ASSOCIATED WITH CORTICOSTEROIDS. L.

Blythe ........................................................................................................................................... 142


16. ACUTE PHASE PROTEIN RESPONSE IN PIGS AFTER EXPERIMENTAL INFECTION

WITH DIFFERENT STRAINS OF PORCINE REPRODUCTIVE AND RESPIRATORY

SYNDROME VIRUS. Y. Saco, M. Cortey, J. Segalés, R. Pato, R. Pena, F. Martínez-Lobo, C.

Prieto, A. Bassols ........................................................................................................................... 143

17. DIAGNOSTIC VALUE OF MILK ACUTE PHASE INDEX IN DETECTION OF BOVINE

SUBCLINICAL MASTITIS. S. Safi, S.H. Shirazi-Beheshtiha, V. Rabbani, M. Bolourchi ......... 144

18. SERUM AMILOID A AS A MARKER OF INFLAMMATION IN CANINE JOINT. J. Francuski,

N. Andrić, M. Lazarević Macanović, D. Marković, A. Radovanović, M. Kovačević Filipović ... 144


19. PRO-INFLAMATORY CYTOKINES QUANTIFICATION IN PIGS EXPERIMENTALLY

INOCULATED WITH ACTINOBACILLUS PLEUROPNEUMONIAE. M. Quezada, A. Islas, A.

Ruiz, T. Casanova, C. Lecoq, D. Muñoz ...................................................................................... 145

20. EVALUATION OF LUNG LESIONS OF PIGS INOCULATED WITH A LOW PATHOGENIC

SEROTYPE 6 OF ACTINOBACILLUS PLEUROPNEUMONIAE. A. Islas, M. Quezada, A.

Ruiz, D. Muñoz, C. Lecocq ........................................................................................................... 146

21. CRAYFISH PLAGUE IN SLOVENIA: FIRST REPORT OF THE PLAGUE AGENT

APHANOMYCES ASTACI. D. Kušar, A. Vrezec, M. Ocepek, V. Jenčič................................... 146

22. PROTEINURIA IN DOGS NATURALLY INFECTED WITH THE BACTERIUM

ANAPLASMA PHAGOCYTOPHILUM. U. Ravnik, B. Premrov Bajuk, K. Babnik, N. Tozon . 146

23. EPIDEMIOLOGICAL SITUATION OF IBR/IPV, MB/BVD AND SOME RESPIRATORY

VIRUSES IN SLOVENIAN AUTOCHTHONOUS BREEDS M. Nemec, P. Hostnik, Z. Klinkon,

T. Zadnik, M. Klinkon ................................................................................................................... 147


24. FEASIBILITY OF DETECTION OF TESTOSTERONE AND ESTRADIOL IN HAIR USING

COMMERCIAL ELISA KITS. T. Snoj, A. Nemec Svete, N. Cebulj-Kadunc, S. Kobal ............. 148

25. ELECTRON MICROSCOPIC EXAMINATION OF BUFFY COAT: SIMPLE METHOD USING

PLASTIC MEMATOCRIT TUBE AND APPLICATION FOR DIAGNOSIS OF LYSOSOMAL

STORAGE DISEASES IN DOGS AND CATS. A. Yabuki, S. Yoneshige, K. Mizukami, M.M.

Rahman, M.M. Uddin, O. Yamato ................................................................................................ 149

26. LYMPHOCYTE ANTIGENS: STABILITY OF EXPRESSION AMONG HEALTHY DOGS

AND UPON STORAGE. F. Riondato, A. Poggi, A. Rota, B. Miniscalco .................................... 149

BIOCHEMISTRY

27. DIAGNOSTIC MEASUREMENTS OF CANINE SERUM AMYLOID A USING AUTOMATED

LATEX AGGLUTINATION TURBIDIMETRY: AN INTERLABORATORIAL COMPARISON.

M. Christensen, J. Ceron, A. Tvarijonaviciute, M. Kjelgaard-Hansen .......................................... 155

28. ELEVATED PLASMA ADIPONECTIN LEVEL AND PERIPHERAL BLOOD LEUKOCYTE

ADIPONECTIN RECEPTOR EXPRESSION IN INSULIN DEFICIENT DOGS. N. Mori ........ 155

29. COMPARING THE USE OF BODY FAT PERCENTAGE VERSUS 5-POINT BODY

CONDITION SCORE FOR ASSESSING OVERWEIGHT STATUS IN DOGS BY SCREENING

PLASMA METABOLITE PROFILES. G. Li, P. Lee, N. Mori, I. Yamamoto, K. Koh, T. Arai .. 155

30

30. cDNA CLONING AND mRNA EXPRESSION OF DOG CDKAL1. I. Yamamoto, A. Mori, H.

Takemitsu, N. Mori, G. Li, K. Kawasumi, T. Arai ........................................................................ 156

31. DETECTION OF PROTEINURIA IN DOGS AND CATS – AGREEMENT BETWEEN AN IN

HOUSE DRY CHEMISTRY SYSTEM (AUTION MICRO ™) AND CONVENTIONAL WET

CHEMISTRY B. Rütgen, I. Schwendenwein ................................................................................ 156

32. HEME-MEDIATED BINDING OF ALPHA-CASEIN TO FERRITIN: EVIDENCE OF ALPHA-

CASEIN BINDING TO FERROUS IRON. K. Orino, Y. Yoshikawa, K. Watanabe ................... 156

33. C-REACTIVE PROTEIN AND SOME BIOCHEMICAL PARAMETERS IN FEMALE DOGS

AFTER OVARIOHYSTERECTOMY. N. Hadzimusic ................................................................ 157

34. COMPARATIVE STUDY OF PLASMA LIPIDS IN HEALTHY DOGS AND DOGS WITH

SKIN DISEASES. R. Barrera, P. Ruiz, C. Zaragoza, F.J. Duque, A. Buono ................................ 157

35. RELIABILITY OF A SINGLE COLLECTION OF URINE SAMPLE IN DETERMINATION OF

URINE PROTEIN TO CREATININE RATIO. G. Rossi, A. Zatelli, S. Paltrinieri ...................... 158

36. MEASUREMENT OF SERUM PROGESTERONE IN MARES: A METHOD COMPARISON

STUDY. G. Lubas, A. Gavazza, A. Rota, D. Panzani, S. Demi .................................................... 158

37. ISCHEMIA MODIFIED ALBUMIN IN HORSES EXERCISED AT DIFFERENT INTENSITIES

ON A TREADMILL. L.A. Yonezawa, T.S. Barbosa, M.J. Watanabe, J.L. Knaut, C.L. Marinho,

L.E.S. Michima, A. Kohayagawa .................................................................................................. 158

38. HIGH MOBILITY GROUP BOX 1 AS A PREDICTOR OF GASTRIC NECROSIS. I. Uhrikova,

L. Rauserova-Lexmaulova, K. Rehakova, I. Hajek, J. Doubek ..................................................... 159

39. COMPARISON OF THE SCHMIDT HAENSCH AND ATAGO PAL-USG

REFRACTOMETERS FOR DETERMINATION OF URINE SPECIFIC GRAVITY. H. Tvedten,

Å. Noren ........................................................................................................................................ 159

40. EFFECT OF PHENOBARBITAL THERAPY ON SELECTED LABORATORY PARAMETERS:

A RETROSPECTIVE STUDY (2007-2012). I. Hajek, P. Schanilec, I. Uhrikova ........................ 160

41. PROTEOMICS APPLICATION IN SERA: DIFFERENCES BETWEEN VACCINATED AND

NONVACCINATED SHEEP. J. Mavromati, G. Mazzuchelli ...................................................... 160

42. COMPARISON OF URINE PROTEIN / CREATININE RATIO IN URINE SAMPLES

COLLECTED BY CYSTOCENTESIS AND MANUAL COMPRESSION IN CATS –

PRELIMINARY RESULTS. H. Vilhena, R. Santos, T. Sargo, T. Lima, S. Dias, F.L. Queiroga,

A.C. Silvestre-Ferreira ................................................................................................................... 160

43. EVALUATION OF SOME SERUM PARAMETERS AFTER OVARIECTOMY IN THE RAT.

R. Mirzaei, B. Amoughli Tabrizi, A. Balila .................................................................................. 161

HEMATOLOGY

44. CHARACTERIZATION OF CANINE BONE MARROW MESENCHYMAL STEM CELLS. C.

Tejero, L.K. Ostronoff, M.L. Fermín, C. Fragio, L.G. León, E. Kremmer ................................... 166

45. FIBRINOGEN CONCENTRATION MEASUREMENT IN DOGS: A COMPARISON

BETWEEN THE QBC-VET AUTOREADER AND MC1PLUS SEMIAUTOMATIC

COAGULOMETER. L.V. Athanasiou, T.A. Petanides, M.K. Chatzis, M.N. Saridomichelakis .. 166

46. REFERENCE INTERVALS FOR HEMATOLOGIC PARAMETERS OF ARABIAN HORSES.

A. Rocky, S. Gooraninejad, M. Razi Jalali, M. Pourmahdi, F. Naghashpour ............................... 167

47. A NOVEL POINT MUTATION IN THE BETA1-TUBULIN GENE IN ASYMPTOMATIC

MACROTHROMBOCYTOPENIC NORFOLK AND CAIRN TERRIERS. Gelain, W. Bertazzolo,

G. Tutino, E. Pogliani, F. Cian, M.K. Boudreaux ......................................................................... 167

48. DETERMINATION OF COMPLETE BLOOD COUNT REFERENCE INTERVALS IN

PIRENEAN MOUNTAIN DOGS. A. Pasquini, I. Annibali, G. Biagi, B. Gugliucci, G. Lubas, A.

Gavazza ......................................................................................................................................... 167

49. CANINE BONE MARROW CYTOLOGICAL EXAMINATION: A RETROSPECTIVE STUDY

OF 295 CASES. V. Turinelli, A. Gavazza, G. Lubas .................................................................... 168

31

50. A RETROSPECTIVE STUDY OF INCIDENCE AND CLASSIFICATION IN FELINE BONE

MARROW CYTOLOGICAL EXAMINATION. A. Gavazza, V. Turinelli, G. Lubas ................ 168

51. USE OF THE MCDh* STAINING FOR HAEMATOLOGY OF THE DOG, CAT, HORSE AND

CATTLE : PRELIMINARY RESULTS. N. Bourgès-Abella, C. Hanot, C. Marche, J.P. Braun, C.

Trumel ........................................................................................................................................... 169

52. INCREASED ERYTHROCYTE OSMOTIC RESISTANCE IN DOGS WITH SINGLE

EXTRAHEPATIC PORTOSYSTHEMIC SHUNT. K. Rehakova, I. Uhrikova, L. Rauserova–

Lexmaulova, J. Lorenzova, L. Stehlik, O. Skor, J. Doubek .......................................................... 169

53. COMPARISON OF THROMBOTIC MICROSCOPIC PLATELET COUNTING METHOD TO

THE LEUKOPLATE MICROSCOPIC METHOD, ADVIA 2120 AND IDEXX

VETAUTOREAD™ PLATELET METHODS. H. Tvedten, J. Ljusner, I. Lilliehöök ................. 169

54. FACTOR VIII LEVELS IN A GROUP OF BELGIAN SHEPHERD MALINOIS DOGS BREED

IN ITALY. G. Lubas, A. Gavazza, M. Caldin ............................................................................... 170

55. PROTHROMBINE TIME MEASUREMENTS BY A NOVEL POINT OF CARE DEVICE IN

CANINE WHOLE BLOOD. P. Vajdovich, J. Antal ..................................................................... 170

56. RETICULOCYTE PARAMETERS IN DOGS WITH LEISHMANIASIS. A PRELIMINAR

STUDY. J. Pastor, A. Meléndez-Lazo, C. Miret, M. Planellas, M. Mesalles................................ 171

57. BLOOD PARAMETERS OF CIKA CALVES AND BULLS. M. Klinkon, G. Gorjanc, Z. Klinkon,

J. Ježek, M. Simčič ........................................................................................................................ 171

58. COMPARISON OF THE ABC VET AND BC 2800 VET VETERINARY HAEMATOLOGICAL

ANALYZERS. M. Nemec, M. Klinkon, J. Ježek .......................................................................... 171

59. CANINE CBC ANALYSIS WITH THE LASERCYTE AND PROCYTE DX AUTOMATED

HEMATOLOGY ANALYZERS. D. DeNicola, M. Haynes, C. Lee, J. Hammond, M. Wellman, G.

Couto ............................................................................................................................................. 172

60. CANINE RETICULOCYTE ANALYSIS WITH THE LASERCYTE AND PROCYTE DX

AUTOMATED HEMATOLOGY ANALYZERS. D. DeNicola, C. Lee, M. Haynes, J. Hammond,

J. Christian, J. Knoll, M. Scott, H. Wamsley ................................................................................. 172

61. PORCINE HEMATOLOGY EVALUATION ON THE IDEXX PROCYTE DX HEMATOLOGY

ANALYZER D. DeNicola, J. Chase, J. Hammond, J. Russell ...................................................... 173

62. AGE-ASSOCIATED ALTERATION IN PERIPHERAL LEUKOCYTE SUBPOPULATION

AND ITS CYTOKINE SYNTHESIS RESPONSIBILITY TO INSULIN IN BEAGLE DOGS. M.

Fujiwara, H. Otsuka, T. Yonezawa, I. Yamamoto, T. Arai ........................................................... 173

63. HEME-MEDIATED BINDING OF ALPHA-CASEIN TO FERRITIN: EVIDENCE OF ALPHA-

CASEIN BINDING TO FERROUS IRON. K. Orino, Y. Yoshikawa, K. Watanabe ................... 173


64. AMELIORATIVE EFFECTS OF DIETARY ALUMINOSILICATES ON BIOCHEMICAL

CHANGES IN BROILER CHICKENS DURING AFLATOXICOSIS. D. Prvulovic, D. Kojic, M.

Popovic, G. Grubor-Lajsic ............................................................................................................. 175

65. DETERMINATION OF ATRAZINE IN SERUM AND URINE OF CATTLE BY HPLC, USING

MOLECULAR IMPRINTED POLYMERS (MIP). S.Z. Peighambarzadeh, S. Safi, S.J. Shahtaheri,

M. Javanbakht, A.Rahimi Forushani ............................................................................................. 175

66. CHARACTERIZATION OF STEM CELL MARKERS ON BONE MARROW AND ADIPOSE

TISSUE-DERIVED CANINE MESENCHYMAL STEM CELLS. Y. Takemitsu, D. Zhao, I.

Yamamoto, T. Arai ....................................................................................................................... 176

67. RARELY USED METHODS FOR INVESTIGATING PORTO-SYSTEMIC SHUNTS IN DOGS.

C. Smuts, M. Bennett, M. Sharman, J. Mills, T. Gaál ................................................................... 176

68. RELATIONSHIP BETWEEN COLLIE EYE ANOMALY- AND IVERMECTIN TOXICOSIS-

ASSOCIATED MUTATIONS: ANALYSIS ACROSS MULTIPLE DOG BREEDS. K. Mizukami,

H-S. Chang, D. Endoh, A. Yabuki, O. Yamato ............................................................................. 177

32

69. ABERRANT ANTIGEN EXPRESSION IN CANINE LYMPHOMA: A RETROSPECTIVE

STUDY. V. Martini, A. Pasqua, M.E. Gelain, S. Comazzi ........................................................... 177

70. CLINICAL PRESENTATION CURRICULUM IN VETERINARY EDUCATION: A

COGNITIVE PERSPECTIVE S.Safi, P. Hemmati, S.H. Shirazi-Beheshtiha ............................... 177

71. STATISTICAL STUDY ON PREVALENCE OF TUMORS IN INSURED DOGS IN JAPAN A.

Shimamura, T. Arai ....................................................................................................................... 178

72. N-ACYLHOMOSERINE LACTONES OF PSEUDOMONAS AERUGINOSA IN CLINICAL

SAMPLES FROM CANINE OTITIS EXTERNA: VALIDATION OF AN ANALYTICAL

METHOD USING LIQUID CHROMATOGRAPHY WITH TANDEM MASS SPECTROSCOPY

(LC-MS/MS). K. Srimpf, J. Hosseini, T. Kotnik, D. Kušar, M. Vengušt, G. Tavčar-Kalcher ...... 178

73. LEG WEAKNESS PATHOLOGY IN BROILER CHICKENS. I. Dinev..................................... 179

74. PATHOMORPHOLOGICAL INVESTIGATIONS ON THE INCIDENCE OF AXIAL

SKELETON PATHOLOGY ASSOCIATED WITH POSTERIOR PARALYSIS IN

COMMERCIAL BROILER CHICKENS. I Dinev ....................................................................... 179

75. EFFECTS OF ANTIPARASITIC AND IMMUNOSTIMULATIVE DOSES OF LEVAMISOLE

ON HEMATOLOGIC PARAMETERS IN DOGS. S.H. Shirazi-Beheshtiha, A. Shariat, N. Taefi-

Nasrabadi, A. Golmohammadi, M.R. Taghdiri ............................................................................. 179


76. HISTOPATHOLOGICAL FINDINGS IN ABOMASA OF FALLOW DEER FROM BRIJUNI

REGION IN CROATIA. M. Robić, B. Artuković, A. Beck, R. Turk, M. Belić, Ž. Grabarević ... 181


77. COMPARISON OF TOTAL LEUKOCYTES RESPONSE OF HORSES SUBJECTED TO

EXERCISE ON HIGH SPEED TREADMILL – PRELIMINARY RESULTS. T. Barbosa, L.

Yonezawa, C. Marinho, M.J. Watanabe, A. Kohayagawa ............................................................ 182

78. EFFECT OF EXERCISE ON HIGH-SPEED TREADMILL ON DNA DAMAGE IN

PERIPHERAL LEUKOCYTES OF HORSES. T. Barbosa, L. Yonezawa, C. Marinho, J. Knaut,

M.J. Watanabe, D. Salvadori, D. Almeida, A. Kohayagawa ......................................................... 182

79. REFERENCE VALUES FOR HAEMATOLOGICAL PARAMETERS IN LUSITANO HORSE.

A.C. Silvestre-Ferreira, M. Cotovio, M. Maia, F. Queiroga, M.J. Pires, A. Colaço ..................... 183

80. MONONUCLEAR CELLS CONCENTRATION IN FRACTIONED SAMPLES OF BONE

MARROW ASPIRATE OF HORSE’S STERNUM. F. Correa, J. Galecio, H. Bustamante, F.

Wittwer, A. Ramirez, B. Menarim................................................................................................. 183

33

INVITED LECTURES

ISACP PLENARY LECTURE

STRENGTHENING GLOBAL ALLIANCES IN VETERINARY CLINICAL PATHOLOGY

M. M. Christopher. Department of Pathology, Microbiology, and Immunology, School of Veterinary Medicine,

University of California–Davis, Davis, CA, USA

Correspondence: [email protected]

Veterinary clinical pathologyhas come a long way over the past 50 years, expanding with trends in medical

pathology and veterinary medicine to fill the growing demand for diagnostic medicine and to improve the health

of animals and humans worldwide. Growth in laboratory medicine has been facilitated byimpetus (eg,

specialization), technological capability, and people with the training necessary to develop, use, and advance

laboratory methods to meet clinical needs. Clinical pathology in many countriesremains underdeveloped, where

clinical medicine and specialization are less advanced and diagnostic priorities may differ.The shrinking world in

which we livecreates opportunities for alliances—individual, institutional, and organizational—that buildfuture

capacity,advancetechnology,research, and education, and address important issues in clinical pathology

worldwide.

Residencyprogramsand board certificationprepare highly qualifiedclinicalpathologistsbut residency training is

notwidely recognizedoutside Europe and North America where a PhD remains the gold standard, even for

individuals primarily involved in diagnostics, teaching, and clinical research. A PhD isprimarily a research

degree thatcomplements but does not replace residency training. This divergence in research and diagnostic

training can create a dilemma for individuals from many countries who seek advanced training in clinical

pathology but are expected by their university or sponsor to obtain a PhD. How can we create flexible training

programs and options that link diagnostics and research that meet international expectations for research while

also filling important clinical-diagnostic and educational needs? How can we promote the value of board

certified specialistsinternationally?

Growth in companion animal medicine together with consolidation of the food animal industry has led to a

decline in food animalcaseloads and the de facto specialization of many clinical pathology

laboratoriesincompanion animal diagnostics. This affects training opportunitiesandthreatens tomarginalize our

ability to contribute meaningfully tolaboratory diagnostics and researchof food animals, a priority in most

countries of the world. How can we use international alliances to ensure clinical pathologists are more fully

engaged inthe laboratory testing of food animals and in government laboratories?Global alliances between the

ASVCP, ESVCP, and ISACPand key international organizationssuch as the OIE (World Organisation for

Animal Health), FAO (Food and Agriculture Organization), and WAVLD (World Association of Veterinary

Laboratory Diagnosticians)are essential toensure the expertise and leadership of clinical pathologists are used in

establishing internationalguidelineson laboratory accreditation, personnel, standards, and best practices in

diagnostic testing.

HEINER SOMMER LECTURE

CONTRIBUTION OF CLINICAL BIOCHEMISTRY IN RUMINANT PRODUCTION

F. G. Wittwer. Faculty of Veterinary Sciences, Universidad Austral de Chile, Valdivia, Chile.


Introduction

Animal clinical biochemistry is the discipline of the veterinary sciences dealing with the examination of diseases

that affect tissues, fluids, or other materials from a living or death patient. Blood chemistry has been used for the

diagnosis of clinical and nutritional problems in production animals. The advances in the scientific knowledge

and technical facilities in this discipline have coincided with the increasing intensification in farm livestock


34

management, when greater efforts were made by farmers to achieve higher production at minimum cost. Under

this context, ruminant biochemistry appears as a tool in research and applied medicine to help understand and

solve the biological factors that limit the achievement of the goals in high producing animals. The aim of this

paper is to show the support of animal clinical biochemistry on ruminant medicine and accordingly in the

development of the milk and meat industry.

The ISACP and ruminant medicine

The International Society of Animal Clinical Biochemistry (ISACP) formerly ISACB, had it first meeting in

1984, in Bonn, Germany, organized by H Sommer. Since then the Society has organized every two years 14th

meetings. A mean of 88 papers (28 conferences and 60 abstracts) has been presented in each 11 of these

meetings, most of them related to clinical biochemistry (Figure 1).

Figure 1. Number of papers presented at ISACP (ex ISACB) meetings. (1986, 1992 and 1998 are not included)

In each congress a mean of 26 papers related to ruminants have been presented, a larger number than other

species, 19 related to small animals, 16 to general clinical pathology and 8 for horses, but a tendency to move to

small animals is observed since ISACB move to ISACP (Figure 2).

Figure 2. Distribution of the papers presented at ISACP (ex ISACB) Congress according to species

35

Figure 3. Analysis of the papers presented at ISACP (ex ISACB) meetings: A. Distribution by specie of

production animals (n=393) and B. Distribution by topic of the papers related to ruminants (n=292)

Considering only production animals (cattle, sheep, goats, camels, pigs, poultry and fish) most of the papers

(63%) have dealt with cattle medicine. The most common topics of these papers were metabolic disturbances

(39%) and diseases studies (37%) (Figures 3A and B).

Studies of mineral metabolism, especially in the diagnosis and prevention of mineral (Ca, Mg, Cu and Se) and

vitamins (A, E) deficiencies, as well as of energy (NEB, ketosis, fatty liver) in groups of animals are the most

frequent. A large percentage is related to dairy cows during the transition period. Also a large number of papers

make reference to the use of metabolic profiles in herd medicine (Figure 4A). Papers on ruminant diseases are

related to inflammation markers (APP), digestive disturbances (mostly on liver damage) mastitis (diagnostic test

and milk quality), infectious and parasitic diseases and oxidative stress (associated to Se or Vit E deficiency)

(Figure 4B).

Figure 4. Distribution by topic of the papers presented at ISACP (ex ISACB) meetings and related to A:

metabolic disturbances and B: ruminant’s diseases.

Clinical biochemistry in the diagnosis and control of metabolic disturbances

Metabolic problems in ruminants are mostly related to nutritional unbalances, including single nutritional

deficiencies (inappropriate mixing of the diet, inadequate feeding facilities) or more complex problems due to

the interactions between nutrition, environment and management. It presentation is more frequent in animals

reared under grazing conditions, as used in Latin-American, because of the large variation in the disposition and

contents of nutrient in the grasses used as forages, mostly associated to soil conditions and grass growth and

maturity according to season. This unbalances affect a complete group of animals within a herd, usually those

metabolically more susceptible, where a group will be presenting a chronic and subclinical disease. Under this

36

condition higher difficulty in the diagnoses are observed essentially before the production of the animals is

affected.

The conventional approach of sampling individual animals with clinical signs of disease is a useful way to

confirm or differentiate veterinary diagnoses. It is the first step in applying clinical chemistry to a new problem

and may confirm or suggest the treatment required to restore the animal normality. However, the basic aetiology

of the disease, needed to be determined in order to to eradicate the cause and prevent it spread in the herd, may

not be so apparent. In this situation a blood biochemical profile on a random sample of animals at a particular

stage of production may prove useful in demonstrating abnormalities before clinical signs of the disease have

developed (Manston et.al., 1981).

Clinical biochemistry measures are commonly used in veterinary practices as cow side test (dry chemistry for

ketosis, milk fever; wet chemistry for inmunoglobulins in calves or refractometry for serum protein); or

laboratory test as an aid in the diagnosis of individual or herd health problems. Nowadays individual medicine in

production animals has limited use. In contrast herd diagnostic procedures have been advocated as a useful tool

to monitor and diagnose metabolic diseases as well other health problems (Bertoni, 1999; Herdt, 2000; Wittwer,

2000; Oetzel, 2004; Van Saun, 2008).

Biochemical markers are body tissues or fluids that define the degree of homeostasis in a biochemical process in

one animal or a group of them. Following the use of the “Metabolic Profiles” (MP) reported by Payne et al,

1972, this technique has been adapted to different productive systems, either incorporating or eliminating

analyses according to the local reality and needs, and to the new scientific and technological knowledge. At

present, besides the use of blood samples, the use of milk and urine samples has been encouraged because of the

less invasive and easy to perform sampling process (Cucunubo et al., 2012).

According to the parturition syndrome theory, parturition is a natural but decisive stress affecting the high

producer dairy cow. It induces the homeostatic system to collapse and the animal becomes ill when the control

capacity is exhausted by abiotic (housing, milking) and trophologic (diet not according with needs and

performance) risks (Sommer, 1985).

A MP is defined as a series of specific analytical tests run in combination and used as a herd-based diagnostic

aid (Van Saun, 2008). The use of a MP is the result of technologic improvements in analytical instrumentation,

which can complete multiple analyses in a short time period. The Compton Metabolic Profile, first introduced in

the early 1970’s, has traditionally been used in this approach. The original intent was to monitor the metabolic

health of the herd, help diagnose metabolic problems and production diseases, and identify metabolically

superior animals. Although similar samples and analytical methods are used in assessing disease diagnosis or

metabolic profiling, their approaches to sampling and interpretation have been adapted over time (Herdt et al.,

2001). Application of MP has undergone a number of transformations as more information related mainly to

periparturient disease risks has been elucidated (Duffield, 2007). Two perspectives on the application of

metabolic profiles have been promoted.

Risk assessment. The targeted diagnostic approach utilizes well defined diagnostic analytes (non esterified fatty

acids (NEFA) and ßOHbutirate (BHB) for negative energy balance (NEB) (Duffield, 2009; Ospina et al., 2010),

(or cholesterol and AST for the parturition syndrome) (Sommer, 1985) to determine herd risk for specific

periparturient diseases. Under this approach, a specific analyte concentration is determined and compared to

defined threshold criteria (Table 2). Percent of individuals above or below the threshold value are used to

interpret herd disease risk. To assess disease risk, sampling strategies should be focused on time periods prior to

disease diagnosis. This places the greatest burden on prepartum samples or those immediately postpartum

(Duffield, 2007; Van Saun, 2010).

37

Table 2. Associations between pre and postpartum plasma ßOHbutyrate concentrations and postpartum disease

risk.

Cut point

mmol/L

Time

Days

Disease Risk factor Reference

>1.2 1 to 7 LDA. 8.0 LeBlanc et al., 2005

>1.2 1 to 7 Metritis 3.3 Duffield et al., 2009

>1.0 3 to 14 Ketosis 2.8 Ospina et al., 2010

>1.1 3 to 21 Any disease 3.5 Van Saun, 2006

LDA= left dysplacement abomasum (Adapted from Van Saun 2010)

Diagnostic screening. The screening tool approach is consistent with traditional MP where multiple analytes are

determined within selected group or groups of cows. A screening tool approach can be used as a broad-based

diagnostic evaluation of herd nutritive status, assessment of disease risk factors, or indicator of potential factors

responsible for disease conditions. This approach allows for more flexibility for sampling time periods (up to 21

days prior to and following calving). Limitations to the screening tool approach are high testing costs. The

approach to this application is to evaluate metabolic balance as well as changes that occur over the transition

period in cows or other specific critical periods in groups of animals. Testing is broad based and includes

analytes reflecting energy balance: NEFA and BHB, protein status: blood or milk urea (BU or MU) and albumin,

macro and micro minerals: Ca, P, Mg, Cu, Zn, Se. The test panel can be reduced or expanded according to

circumstances. Analytes for general health condition as liver function as been used, including enzymes (AST,

GMD, GGT) and cholesterol (Wittwer, 2000). One approach that has been described is to measure analytes in

pooled samples of individuals and evaluate pooled samples over a series of predefined cow groupings (Van

Saun, 2010).

An association between nutrition mistakes and reproductive and lactation performance as well as the occurrence

of deficiencies and health problems has been documented (Ferguson, 1991; Duffield et al., 2009). Energy

balance can be assesses through the use of body condition score (chronic marker) or by measurement of plasma

NEFA (prepartum) and BHB (pospartum) as acute markers of lipid mobilization. Prolonged NEB around calving

lead to ketosis, fatty liver, lower fertility and increased disease susceptibility during the transition period of cows

(Herdt, 2000). Protein nutritional status is difficult to evaluate; low plasma albumin concentration has a low

specificity and moderate sensibility as a chronic marker of protein deficiency. Low BU or MU are sensitive and

specific markers to evaluate a low protein intake. By the opposite an increase of this metabolite reflects a waste

of nitrogen associated with an excess of dietary protein leading to an energy inadequacy, which is caused by

rumen asynchrony of degradable protein with fermentable energy (Whitaker et al., 1995). This is a common

situation in grazing cows in temperate climates produced by the consumption of forages with more than 20% of

dietary protein and a limited intake of energy. A change in the blood concentration of minerals and vitamins will

be observed when the body reserves are exhausted and the homeostatic regulation mechanism has been

exhausted. Calcium is a insensible marker because of the homeostatic mechanism. Phosphorus is a sensitive but

unspecific marker. Magnesium is a sensitive and specific marker for hyopmagnesemia, a frequent problem

associated to sudden death and tetany in dairy and beef cows in southern Chile and New Zealand. Cooper and

zinc have low sensitivity but decrease when the reserves are exhausted. Selenium, usually determined by the

blood activity of GPx, is a sensitive and specific marker for the detection of Se deficiency, frequent to diagnose

in ruminants fed with low selenium content forages and has been associated to higher predisposition to oxidative

stress and mastitis (Wittwer et al., 2002).

The control of metabolic problems due to nutritional deficiencies are based in the correct an opportune

diagnosis and the supplementation with the deficient nutrient. The use of clinical biochemistry test to

evaluate response after supplementation trials is a valuable diagnostic tool, especially for mineral

deficiencies. Its use is harmless for the animals since it is treated with a product normally eat by the

animal. To keep a control group and to define a response marker or measurable parameter are absolutely

necessary. The type of supplement and the lag time to produce a change must be considered. For example,

38

the fast response (1 day) in the Mg concentrations in blood or urine following supplementation with MgO

(Table 3), or the slow response (months) in blood GPx activity determined when cattle is supplemented

with Se (Figure 2).

Table 3. Plasma (Mg-p) and urinary magnesium (Mg-u and clearance CUM) (x ± SE) in grazing cattle (n=120)

supplemented with MgO (50 g/d).

PERIOD Mg-p (mmol/l) Mg-u (mmol/l) CUM (mmol/l)

Pre-supplementation 0.64±0.05a 0.54±0.07

a 0.15±0.02

a

Supplementation 0.78±0.04b 2.53±0.82

b 0.37±0.08

b

Post-supplementation 0.64±0.04 a 1.80±0.39

a 0.15±0.02

a

a-b = P < 0.05

Wittwer et al., 1997

Figure 2 Effect of a single selenium supplementation over 14 months on blood GPx activity (means ± SD) in

grazing cows and calves. (adapted from Oblitas et al 2000 and Wittwer et al 2002)

This antecedents show the support given by the animal clinical biochemistry in the development of the animal

production systems throughout research and clinical activities using the dairy cattle industry as an example. The

ISACP has been involved in this target being ruminant medicine one of the subjects with more papers presented

at its Congress, mostly related to the diagnosis and control of herd health and preventive medicine programs.

References

Bertoni G. Guida alla interpretazione dei profili metabolici. Univ Perugia, 1999:1-135.

Cucunubo LG, Strieder-Barboza C, Wittwer F, Noro M. Diagnóstico de cetosis subclínica y balance energético

negativo en vacas lecheras en condiciones de pastoreo mediante el uso de muestras de orina y leche. Rev Cient.

FCV-LUZ. 2112 in press.

Duffield TF. Peripartum metabolic profiling. Proc Am Assoc Bov Pract. 2007;40:213-218.

Duffield TD, Lissemore KD, McBride BW, Leslie KE. Impact of hyperketonemia in early lactation dairy cows

on health and production. J Dairy Sci. 2009;92:571-580.

Ferguson J D. Nutrition and reproduction in dairy cows. Vet. Clin. N Am Food A. 1991;7:483-507

* = cows

**= calves

39

Herdt TH. Ruminant adaptation to negative energy balance. Influences on the etiology of ketosis and fatty liver.

Vet. Clin. N Am-Food A. 2000;16:215-230.

Herdt TH, Dart B, Neuder L. Will large dairy herds lead to the revival of metabolic profile testing? Proc Am

Assoc Bov Pract. 2001:27-34.

LeBlanc SJ, Leslie KE, Duffield TF. Metabolic predictors of displaced abomasum in dairy cattle. J Dairy Sci.

2005;88:159-170.

Manston BR, Allen WM. Modern diagnostic methods in practice. Br vet J. 1981;137:241-247.

Oblitas, F, Contreras PA, Böhmwald H, Wittwer F. Efecto de la suplementación con selenio en la actividad de

glutatión peroxidasa (GSH-Px) y ganancia de peso en bovinos selenio deficientes a pastoreo. Arch Med Vet.

2000;32:55–62.

Ospina PA, Nydam DV, Stokol T, Overton TR. Evaluation of nonesterified fatty acids and ß-hydroxybutyrate in

transition dairy cattle in the northeastern United States: Critical thresholds for prediction of clinical diseases. J

Dairy Sci. 2010;93:546-554.

Oetzel GR. Monitoring and testing dairy herds for metabolic disease. Vet Clin NA: Food Anim Pract.

2004;20:651-674.

Payne JM. The future of presyntomatic diagnosis. Proc.Roy.Soc.Med. 1972;65:181-183.

Sommer H. Control of health and nutritional status in dairy cows. Vet. Med. Rev. 1985;13:135-155.

Van Saun RJ, Wustenberg M. Metabolic profiling to evaluate nutritional and disease status. Bov Pract.

1997;31:37-42.

Van Saun RJ. Metabolic profiles for evaluation of the transition period. Proc Am Assoc Bov Pract.2006;39:130-

138.

Van Saun RJ. Metabolic Profiling. In: Anderson DE, Rings M, eds. Current Veterinary Therapy, Food Animal

Practice, 5th vol., Saunders Elsevier. 2008;153-162.

Van Saun RJ. Indicators of dairy cow transition risks: metabolic profiling revisted. In: Wittwer F, Chihuailaf R,

Contreras H, Gallo C, Kruze J, Lanuza F, Letelier C, Monti G, Noro M. eds. Updates on ruminant production

and medicine. Andros; 2010:65-77

Whitaker D, Kelly JM. Use and interpretation of metabolic profiles in dairy cows . Dept Vet Clin Studies, Univ

Edimburgh. 1995:1-13.

Wittwer F, Contreras PA, Silva N, Böhmwald H. Efecto de la suplementación con magnesio en alimento y agua

sobre el control de tetania hipomagnesémica en rebaños Hereford. Arch Med Vet. 1997;29:25-34.

Wittwer F. Marcadores bioquímicos no controle de problemas metabólicos nutricionais em gado de leite. In:

González FHD, Barcellos JO, Ospina H, Ribeiro LAO, eds. Perfil metabólico em ruminantes: seu uso em

nutrição e doenças nutricionais. UFRGS, Brasil, 2000:53-62.

Wittwer F, Araya O, Contreras P. 2002. Strategic procedure for important herd problems of heifers. In: Kaske

M., Scholz, H., Höltershinken, eds. Recent Developments and Perspectives in Bovine Medicine. Keynote

Lectures, XXII World Buiatric Congress. Hannover 2002:396–409.

40


EVALUATION OF METABOLIC AND ENDOCRINE STATUS IN TRANSITION DAIRY COWS IN

PREDICTION AND DIAGNOSIS OF FATTY LIVER SYNDROME

D. Kirovski. Department of Physiology and Biochemistry, Faculty of Veterinary Medicine University of

Belgrade, Belgrade, Serbia


Introduction

Fatty liver syndrome is a major health problem in high yielding dairy cows in early lactation. Fatty liver

usually develops after calving with peak incidence at about 10 days in milk (Mulligan and Doherty, 2008).

Nutritional factors such as obesity are crucial in fatty liver etiology (Šamanc et al., 2010). Obese cows have a

greater decrease in feed intake during the peripartal period and, therefore, have a more severe negative energy

balance during early lactation. That leads to increased lipolysis of adipose tissue. Nonesterified fatty acids

(NEFA) that are released from the stored triacylglycerol (TAG) in the adipose tissue are readily taken up by liver

where they provide energy for liver function. If more NEFA arrive at the liver that needed for energy purposes,

the excess may be oxidized incompletely and generate ketone bodies or be converted to TAG for deposition.

Under normal conditions, TAG is secreted from the liver as very-low-density lipoproteins (VLDL). Inadequate

secretion of VLDL may contributes to development of fatty liver. However, obesity in dry cows is not always

associated with fatty liver indicating that there are risk factors other than obesity that predispose the cow to

development of fatty liver. Inadequate endocrine adjustments during initiation of lactation may play a role in

fatty liver pathogenesis. Genetic factors may increase probability of fatty liver due to mutation that affect lipid

metabolism in adipose tissue, or lipid metabolism and secretion in the liver (Bobe et al., 2004). Finally, fatty

liver may develop secondary to other diseases and disorders, meaning that factors triggered by trauma or disease

may contribute to development of fatty liver. These compounds (cytokines-mainly cachectin and interleukin-1)

are released from white blood cells in response to injury and infection, and may increase hepatic conversion of

fatty acids to TAG (Ametaj et al., 2005).

Categories of fatty liver

Fatty liver may be evaluated either by chemical or histological analysis of liver samples for liver TAG

or total lipid (Collins et al., 1985). Fatty liver can be categorized into mild (liver TAG 1-5% wet weight; total

lipid 5-20% of volumen), moderate (liver TAG 5-10% wet weight; total lipid 20-40 % of volumen) and severe

fatty liver (liver TAG > 10% wet weight; total lipid > 40% of volume). Liver samples for histological

measurement of liver fat content are obtained by liver biopsy.

Histological pathology of fatty liver

Histological findings in cows with fatty liver include (a) fatty cysts in liver parenchyma; (b) increased

volume of individual hepatocytes; (c) mitochondrial damage; (d) compression and decreased volumen of nuclei,

rough endoplasmatic reticulum, sinusoids, and other organelles; and (e) decreased number of organelles

(Johannsen et al., 1993). Accumulation of TAG in liver with fatty liver is limited to the centrilobular section of

the liver near the hepatic vein, but the accumulation extends to the midzonal section and than spreads to the

periportal sections in cows with moderate and severe fatty liver. Increased concentrations of liver TAG are

accompanied with decreased concentrations of structural lipids (free holesterol, cholesteryl ester, and

phospholipids), energy precursors (citrate), and energy storage molecules (glycogen). Decreased concentrations

of glycogen increase risk for metabolic disorders that are associated with fatty liver. Therefore, it is

recommended using rather ratio of liver TAG to liver glycogen, rather than liver TAG alone, as a diagnostic

indicator for fatty liver.

Accumulation of liver TAG in liver reverses slowly (Veenhuizen et al., 1991) and probably has only

minor long term negative association with the liver itself because liver cells can regenerate within days. The

detrimental associations of fatty liver with other diseases, however, are probably longer and less reversible.

Metabolic changes associated with fatty liver

The pathological changes associated with fatty liver affect concentrations of metabolites in plasma.

Concentrations of total protein (TP), albumin, urea N, α-amino N, glucose, TAG and cholesterol are usually

decreased, while concentrations of ammonia, NEFA, BHBA, and acetoacetate are usually elevated. Decreased

concentrations of metabolites such as α -amino N and glucose can decrease function of some organs (Rehange et


41

al., 2001). Elevated concentrations of ammonia, NEFA and BHBA can decrease physiological function of organs

because of their toxicity at high concentrations and because of their metabolic effect. We have done investigation

related to metabolic changes in blood of the cows affected by fatty liver (Šamanc et al., 2011). Forty dry cows

from a commercial dairy herd were involved in the study. Four blood samples were taken from each animal: the

first 5 to 7 days before expected calving (dry period), the second 12 days after calving (puerperal period), the

third 30 days after calving and fourth 60 days after calving. Blood glucose, BHBA, TP, albumin, urea N, total

bilirubin, Ca and P concentrations were measured. On day 12 after calving liver percutaneous biopsies were

obtained and total lipid content in the hepatocytes was determined. Cows were divided into two groups based on

the degree of lipid accumulation in the liver: healthy cows (<20% fat, n = 20) and cows with fatty liver

syndrome (>20% fat, n = 20). Results showed that BHBA concentrations were significantly higher and glucose

concentrations were significantly lower in diseased cows compare with healthy cows at dry and puerperal

periods. Additionally, at day 12 after calving total bilirubin concentrations were significantly higher in diseased

cows. At day 30 after calving concentrations of TP, albumin, glucose, BHBA and Ca were significantly lower in

diseased compared to healthy cows indicating on functional disruption of hepatocytes. Trend of changes in

metabolic parameters that characterized cows at day 30 of lactation were mainly, still present at day 60 of

lactation, although differences in blood parameters between groups were not significant.

Hormonal changes associated with fatty liver

There is strong relation between endocrine status during the peripartal period (3 weeks before to 3

weeks after calving), when the homeostatic mechanisms are under the greatest challenges, and postpartal

metabolic disorders in dairy cows. It is generally observed that insulin, insulin like growth factor-I (IGF-I) and

thyroid hormone (TH) concentrations decrease immediately after calving due to the adaptation of organism to

negative energy balance and initiation of lactation (Stojić et al, 2001). If reductions are pronounced or not

synchronized with other metabolic and endocrine adjustments, the decrease may cause a metabolic disturbance

that leads to TAG accumulation in parenchymal tissue, especially the liver. Namely, decreased levels of blood

insulin combined with decrease glucose concentrations suppress fat mobilization from adipose tissue and blood

NEFA reuptake and reesterification in adipose tissue, leading to increase in blood NEFA concentrations.

Decreased levels of TH in blood are associated with reduced mitochondrial capacity to oxidize fatty acids which

lead to diffuse lipid infiltration of hepatocytes and pronounce hepatic function impairment. When fatty liver

develops, concentrations of those hormones decrease more rapidly. Anyway, the hormonal status of cows in the

far-off and mid dry period and its relationship to postpartal metabolic disorders may be of great importance for

prediction of fatty liver. Namely, identification of risk factors that are associated with homeorhetic mechanisms

leading to metabolic disorders would provide a tool that could be used to better manage cows to prevent fatty

liver or provide early warning signs that could be used to segregate cows and mark them for intensive

interventional protocols after calving. This strategy requires an understanding of the point in time when

adjustments of regulatory mechanisms break through physiological limits thus predisposing the cow to metabolic

problems. Our results related to TH concentrations in cows affected with fatty liver showed that cows, that were

affected by severe fatty liver during early lactation, were in a hypothyroid state from day 30 before calving (mid

dry period) until day 12 after calving (puerperium), which means prior to development of the condition. We

concluded that the hypothyroid state during the mid dry period may both be a risk factor and an early indicator of

fatty liver, while, during early lactation, it is important etiopathogenetic factor of fatty liver (Šamanc et al.,

2010). In the same study, insulin concentrations decreased more pronounced in cows with fatty liver than in

healthy cows but only after calving (unpublished data). The same trend was established for IGF-I, since its

concentrations decreased more rapidly in cows affected by fatty liver, but only after calving (Kirovski et al.,

2012).

It should be emphasizes that fatty liver is also associated with altered hormonal sensitivity of adipose

tissue and the pancreas. Lipogenesis is less promoted by insulin. Additionally, clearence rates of insulin in

hepatocytes and the uptake of glucose by peripheral tissues are decreased (Ohtsuka et al., 2001).

Diagnostic indicators of fatty liver

Liver biopsy is the only reliable method for the detection of fatty liver, especially for the determination

of its severity through the estimation of total lipids and triglycerides. Since some metabolic and endocrine

parameters are significantly correlated with fatty liver degree, they may be used as diagnostic indicators of fatty

liver. Namely, concentrations of puerperal NEFA, BHBA, and total bilirubin are highly positively correlated

with fatty liver degree, while glucose, insulin, IGF-I, and thyroid hormone concentrations are highly negatively

correlated with fatty liver degree (Šamanc et al., 2011). Accordingly, puerperal BHBA, NEFA and glucose

concentrations may be good diagnostic indicators of fatty liver. Insulin, IGF-I and thyroid hormone

concentrations may also be used as diagnostic indicators of fatty liver.

42

The indicators for predicting fatty liver

Fatty liver may be predicted by identifying those factors that initiate changes in metabolism that lead to

fatty liver. According to our results and literature data, elevation of NEFA and BHBA concentrations

simultaneously with a decrease of glucose concentrations in the blood 5 to 7 days before calving may be

indicators of development of postpartum fatty liver. Hypothyroid condition during the dry period in dairy cows

may predispose them to fatty liver and therefore may be used as an indicator for fatty liver prediction.

References

1. Ametaj BN, Bradford BJ, Bobe G, Nafikov RA, Lu Y, Young JW, Beitz DC. Strong relationships

between mediators of the acute phase response and fatty liver in dairy cows. Can J Anim Sci. 2005;

85:165-175.

2. Bobe G, Young JW, Beitz DC. Invited review: pathology, etiology, prevention, and treatment of fatty

liver in dairy cows. Journal of Dairy Science. 2004;87:3105–3124.

3. Collins RA, Reid IM, Williams LA, Dennis PF. Histological measurement of fat content of liver of

dairy cows. J Comp Pathol. 1985; 95:437-441.

4. Johansen U, Menger S, Staufenbiel R, Rossow N. Investigation on morphology and function of the liver

of high-yielding cows two weeks post partum. Dtsch. Tieraerztl. Wochenschr. 1993; 100:177-181.

5. Kirovski D, Šamanc H, Vujanac I, Stojić V, Sladojević Ž, Lazarević M, Nedić O. Effect of peripartum

dietary energy supplementation on insulin-like growth factor-I, its binding proteins and thyroid

hormones in early lactation dairy cows. Acta Veterinaria (Beograd). 2010; in press.

6. Mulligan FJ, Doherty ML. Production diseases of the transition cow. Veterinary Journal. 2008;176:3–9.

7. Ohtsuka H, Koiwa M, Hatsugaya A, Kudo K, Hoshi F, Itoh N, Yokota H, Okada H, Kawamura S.

Rrelationship between serum TNF activity and insulin resistance in dairy cows affected with naturally

occuring fatty liver. J Vet Med Sci. 2001; 63:1021-1025.

8. Rehange J, Meier C, Kaske M, Scholz H, 2001, Changes in the amino acid ratio and ammonia

concentration in plasma and cerebrospinal fluid of dairy cows suffering from hepatosteatosis and liver

failure, J Dairy Sci, 84 (Suppl 1), 152 (Abstract).

9. Šamanc H, Kirovski D, Jovanović M, Vujanac I, Bojković-Kovačević S, Jakić-Dimić D, Prodanović R,

Stajković S. New insights into body condition score and its association with fatty liver in Holstein dairy

cows. Acta Veterinaria (Beograd). 2010; 60:525-540.

10. Šamanc H, Kirovski D, Stojić V, Stojnović D, Vujanac I, Prodanović R, Bojković-Kovačević S, 2011,

Application of the metabolic profile test in the prediction and diagnosis of fatty liver in Holstein cows,

Acta Veterinaria (Beograd), 61, 5-6, 543-553.

11. Šamanc H, Stojić V, Kirovski D, Jovanović M, Cernescu H, Vujanac I, 2010, Thyroid hormones

concentrations during the mid-dry period: an early indicator of fatty liver in Holstein – Friesian dairy

cows. Journal of Thyroid Research. 2010: ID 897602.

12. Stojić V, Gvozdić D, Kirovski D, Nikolić JA, Huszenicza G, Šamanc H, Ivanov I. Serum thyroxine and

triiodothyronine concentrations prior to and after delivery in primiparous Holstein cows. Acta

Veterinaria. 2001;51:3–8.

13. Veenhuizen JJ, Drackley JK, Richard MJ, Sanderson TP, Miller LD, Young JW. Metabolic changes in

blood and liver during development and early treatment of experimenta fatty liver and ketosis in cows. J

Dairy Sci. 1991; 74: 4238-4253.

43

Acknowledgements

This work was supported by Ministry of science and technology, Republic of Serbia, Project Grant No

46002.

OXIDATIVE STRESS AND INFLAMMATORY RESPONSE IN BOVINE REPRODUCTIVE AND

PRODUCTIVE DISORDERS: INTERACTION OF HDL-ASSOCIATED PROTEINS

R. Turk1, P. Roncada

2. 1Department of Pathophysiology, Faculty of Veterinary Medicine, University of Zagreb,

Zagreb, Croatia; 2Istituto Sperimentale Italiano L. Spallanzani, Milano, Sezione di Proteomica c/o Facoltà di

Medicina Veterinaria, Università degli Studi di Milano, Milano, Italy.


In recent years, there has been growing knowledge on the importance of the cell redox metabolism and

the recognition of oxidative stress as the process implicated in many pathophysiological conditions of dairy cows

including reproductive disorders and productive diseases such as mastitis. Increased oxidative stress might be an

important mechanism by which periparturient disorders increase the incidence of reproduction and production

diseases in advanced lactation.

Increasing evidence indicates that oxidative stress lead to cell and tissue damage. However, free

radicals that are generated during oxidative stress are also produced during normal metabolism and consequently

are involved in both animal health and disease. Thus, there is a delicate balance between oxidants and

antioxidants in health and disease. In some diseases, free radicals are secondary to the disease process, but in

some cases a causality is identified by free radicals. The radical-radical reaction is of great importance and is

among the most rapid non-enzymatic reaction yielding even more potent non-radical oxidant. Hydrogen

peroxide is one of the most crucial redox signalling molecules being involved in activation of transcription

factors, such as nuclear factor-kappaB (NFkB), that are involved in a large number of normal cellular processes,

such as immune and inflammatory responses, stress response, cellular growth and apoptosis (Nordberg and

Arnér, 2001). The reducing environment inside the cell helps to prevent oxidative damage. Such an environment

is maintained by a number of anti-oxidant molecules, most of them are enzymes and other proteins. Of great

importance is the role that micronutrients play in regulation of redox cell signalling which establish the strong

link between diet and health and disease.

Oxidative stress is more and more considered as a notable component in the signalling processes

involved in inflammatory responses including stimulation of cell adhesion molecules and production of chemo-

attractant. During inflammatory conditions, phagocytes produce reactive oxygen species (ROS) that are needed

for killing bacteria (Thannickal and Fanburg, 2000). However, raised amount of ROS can overcome the

antioxidant system and compromise the immune function of cows. Directly, ROS can oxidize macromolecules

such as lipids, proteins and DNA and cause oxidative cell injury. Indirectly, ROS can damage cellular

components and membranes and thus modify metabolic pathways. Oxidative stress can enhance the adhesion of

activated neutrophils to mammary endothelial cells and intensify inflammation during mastitis in cows (Maddox

et al., 1999). Several studies have shown that supplementing dairy cows with an adequate level of antioxidants

increases immune function of cows, i.e. phagocytosis, bacterial killing and oxidative metabolism of neutrophils

(Grasso et al., 1990). Furthermore, oxidatively fragmented lipids which are generated during oxidative stress are

pro-inflammatory and provoke the acute phase response, APR (Steinberg, 1997) suggesting the strong linkage

between oxidative stress and inflammation.

Pregnancy and early lactation are physiological states accompanied by high metabolic and energy

demands and elevated requirements for tissue oxygen which provoke oxidative stress (Turk et al., 2011). A

major role in the development of oxidative stress in cows is a negative energy balance (NEB) which often occurs

in late pregnancy and early lactation. Periparturient period has a crucial role for subsequent health and fertility of

cows. Most of the metabolic diseases such as milk fever, ketosis, retained placenta, and displacement of

abomasum occur within the first two week of lactation. In addition, some of infectious diseases, in particular

mastitis, become clinically apparent during the first two weeks of lactation (Goff and Horst, 1997). Intensified

processes of NEFA oxidation preceded in the liver during periparturient period, result in the increased

production of ROS and oxidative stress development (Bionaz et al., 2007). Particularly, peroxidation of

steroidogenic enzymes by ROS can inactivate their function and directly impair reproduction. Physiological

level of ROS play an important role in the regulation of reproductive processes such as folliculogenesis, oocyte

maturation, corpus luteum, uterine function, embryogenesis, embryonic implantation and feto-placental

development (Agarwal et al., 2008). However, exceeded amount of ROS can modify cell functions and endanger

cell survival leading to an increased interval to first oestrus, poor oocyte quality, weak oestrus, poor embryonic


44

development and even early embryo death. Thus, ROS must be inactivated keeping only a small amount of ROS

necessary to maintain normal cell function.

Mammalian cells possess natural anti-oxidative system, which is, in part, due to activities of various

enzymes and proteins involving in the removal of reactive oxygen molecules and repair of oxidative damage

(Wiese et al., 1995). Among a number of enzymes and proteins being involved in oxidative stress and

inflammatory response, several of them are associated with lipoproteins. High-density lipoprotein (HDL) plays

an important role as an anti-oxidative/anti-inflammatory particle providing a non-specific defence of a host. This

action of HDL is mediated by several structural proteins and enzymes carried on the particle, including

paraoxonase-1 (PON1), platelet-activating factor acetylhydrolase (PAF-AH) and apolipoprotein AI (apAI)

(Turk, 2009). High density lipoprotein is a particle consisting of about 50% proteins and 50% lipids. The major

proteins of HDL are apolipoproteins (apo) AI and AII. There are also a number of less abundant proteins

including apoCI, apoCII, apoCIII, apoE, apoJ, apoI, lecitin: cholesterol acyltransferase (LCAT), serum

paraoxonase (PON1) and platelet-activating factor acetylhydrolase (PAF-AH) (Van Lenten et al., 2001).

PON1 and PAF-AH are both anti-oxidative/anti-inflammatory enzymes which hydrolyze lipid

hydroperoxides and oxidatively fragmented phospholipids produced during oxidative stress (Turk et al.,

2008a,b). ApoAI is a negative acute-phase protein whose level is decreased during the acute phase response. It

might play the role of an anti-inflammatory factor by the inhibition of both the neutrophil activation and

inflammatory cytokines production from monocytes and macrophages (Yan et al., 2006). Serum amyloid A

(SAA) and haptoglobin (Hp) are the major APPs in ruminants whose concentrations rise dramatically during the

acute phase response (APR) (Eckersall and Bell, 2010). During APR, SAA associates with HDL particles

leading to an enrichment of HDL with SAA (Van Lenten et al., 1995). Concomitantly with these changes,

cytokines which mediate APR lead to the depletion of anti-inflammatory/anti-oxidative proteins apoAI, PON1,

PAF-AH and lipids from SAA-enriched HDL (Cabana et al., 1996). Since PON1 decreases during APR, it could

be considered as a negative acute phase protein. Apart from an enrichment of HDL with SAA, there is an

increased abundance of haptoglobin and complement C3 on HDL (Watanabe et al., 2009) all suggesting the

remodelling of HDL particles during inflammatory and oxidative stress conditions. Once remodelled, HDL

particle becomes functionally defective and turns from anti-oxidative/anti-inflammatory to inflammatory profile

which alters anti-oxidative and anti-inflammatory properties of HDL which in general compromise immune

function of the body and impair anti-oxidative system.

Better understanding of pathophysiology of HDL-associated proteins and their interaction in bovine

reproductive and productive disorders could lead to further advantages in the diagnosis and the therapy of

diseases in cows.

References:

Agarwal A, Gupta S, Sekhon I, Shah R. Redox considerations in female reproductive function and assisted

reproduction: from molecular mechanisms to health implications. Antioxid Redox Signal. 2008; 10: 1375-1403.

Bionaz M, Trevisi E, Calamari L, Librandi F, Ferrai A, Bertoni G. Plasma paraoxonase, health, inflammatory

conditions, and liver function in transition dairy cows. J Dairy Sci. 2007; 90: 1740- 1750.

Cabana V, Lukens JR, Rice KS, Hawkins TJ, Getz GS. HDL content and composition in acute phase response in

three species: triglyceride enrichment of HDL a factor in its decrease. Journal Lipid Res. 1996; 37: 2662-2674.

Eckersall P, Bell R. Acute phase proteins: Biomarkers of infection and inflammation in veterinary medicine. Vet

J. 2010; 185: 23-27.

Goff JP, Horst RL. Physiological changes at parturition and their relationship to metabolic disorders. J Dairy Sci.

1997; 80: 1260-1268.

Grasso PJ, Scholz RW, Erskine RJ, Erberhart RJ. Phagocytosis, bactericidal activity, and oxidative metabolism

of milk neutrophils from dairy cows fed selenium-supplemented and selenium-deficient diets. Am J Vet Res.

1990; 51: 269-274.

Maddox JF, Aherne KM, Reddy CC, Sordillo LM. Increased neutrophil adherence and adhesion molecule

mRNA expression in endothelial cells during selenium deficiency. J Leukoc Biol. 1999; 65: 658-664.

Nordberg J, Arnér ESJ. Reactive oxygen species, antioxidants, and the mammalian thioredoxin system. Free

Radic Biol Med. 2001; 31: 1287-1312.

45

Steinberg D. Low density lipoprotein oxidation and its pathobiological significance. J Biol Chem. 1997; 272:

20963-20966.

Thannickal VJ, Fanburg BL. Reactive oxygen species in cell signaling. Am J Physiol-Lung C. 2000; 279:1005-

1028.

Turk R. The role of HDL-associated enzymes in bovine reproduction. In: Pagano SI, Strait BN, eds. HDL and

LDL cholesterol: Physiology and clinical significance. NY, USA: Nova Science Publishers; 2009: 129-145.

Turk R, Juretić D, Gereš D, Bačić G, Milešević M, Flegar-Meštrić Z, Turk N, Svetina A. Bovine platelet-

activating factor acetylhydrolase (PAF-AH) activity related to fertility. Anim Reprod Sci. 2008a; 105: 344-353.

Turk R, Juretić D, Gereš D, Svetina A, Turk N, Flegar-Meštrić, Z. Influence of oxidative stress and metabolic

adaptation on PON1 activity and MDA level in transition dairy cows. Anim Reprod Sci. 2008b; 108: 98-106.

Turk R, Samardžija M, Bačić, G. Oxidative stress and reproductive disorders in dairy cows. In: Marek ER,

ed.Dairy cows: Nutrition, fertility and milk production. NY, USA: Nova Science Publishers; 2011: 59-98.

Van Lenten, B.J.; Navab, M.; Shih, D.; Fogelman & Lusis, A.J. (2001). The role of high-density lipoproteins in

oxidation and inflammation. Trends in Cardiovascular Medicine, 11, 155-161.

Watanabe J, Grijalva V, Hama S, et al. Hemoglobin and its scavenger protein haptoglobin associate with apoA-

1-containing particles and influence the inflammatory properties and function of high density lipoprotein. J Biol

Chem., 2009;284:18292-18301.

Wiese AG, Pacifici RE, Davies KJA. Transient adaptation to oxidative stress in mammalian cells. Arch Biochem

Biophys. 1995; 318: 231-240.

Yan, YL, Li Y, Lou B, Wu, MP. Beneficial effects of ApoA-I on LPS-induced acute lung injury and

endotoxemia in mice. Life Sci. 2006; 79: 210-215.

TRACE MINERALS DEFICIENCY DIAGNOSIS IN RUMINANTS

H. Guyot1 1

University of Liege, Faculty of Veterinary Medicine, Clinical Department of Production Animals,

Bovine Ambulatory Clinic, Liège, Belgium.


Introduction

Trace elements are essential in human and animal’s nutrition. They play a major role in health, immunity and

reproduction. The production of milk or meat as well as the housing and management conditions of high-

performing animals can generate oxidative stress and health troubles in cattle. Some trace elements and vitamins

have an anti-oxidative action and increase immunity. The most important trace elements in cattle are selenium

(Se), copper (Cu), zinc (Zn), iodine (I), cobalt (Co), molybdene (Mo), manganese (Mn) and iron (Fe). Co is

associated with vitamin B12 synthesis in the rumen, so the assessment of Co deficiency can also be done by

measuring vitamin B12 blood concentration. The Mo is particularly known for his antagonist effect on Cu

absorption. Regarding Mn and Fe, these two elements are generally not deficient in cattle but often in excess in

the feed.

Are trace elements deficiencies important in cattle practice?

During the last 20 years, a discrepancy has been observed between the animals’ need and the amount of trace

elements given by the ration. The progresses in genetics and cattle performance, as well as the impoverishment

of soils have accentuated this disequilibrium. This has consequences on animal’s health, reproduction and

productivity. Numerous papers have described the negative effects of trace elements deficiencies on cattle’s

health (Graham, 1991). In Europe, trace elements deficiencies are nowadays widely spread in cattle. A recent

study (Guyot et al., 2009) presented the Belgian epidemiology of Se, Zn, Cu and I deficiencies in herds with

health problems, compared to healthy herds. As expected, lower blood trace elements concentrations were found

in unhealthy herds. These concentrations in Belgian herds were compared to those in Ireland and France with


46

similar values (Mee and Rogers, 1994; Enjalbert et al., 2006). These results can be extrapolated to other

European countries, as the depletion in the soils and the nutrition of cattle is similar.

Why to diagnose?

There is a wide range of symptoms associated with trace element deficiencies and thus the clinical signs are not

pathognomonic. The laboratory is needed to confirm the diagnosis. Moreover, most of the time, the deficiencies

are multiple (Guyot et al., 2009) and it is important to know the elements that are deficient in order to adequately

correct and avoid competition between minerals. Finally, some trace elements are synergetic, thus the situation

might be exacerbated if only one of the 2 minerals is given to face the deficiency (Guyot et al., 2011).

How to diagnose?

*Samples

As previously mentioned, the clinic signs are not sufficient to make a diagnosis. Laboratory analyses are needed

to confirm the diagnosis of deficiency. Moreover, samples must be taken directly from the animals rather than

from feedstuffs because the availability and absorption of minerals as well as amounts fed to cattle can widely

vary.

Different type of samples can be taken from animals: blood, milk, urine, hair or tissues (principally liver). Blood

is the most frequently taken and analysed. Blood samples are easily taken and the modern techniques of assay

are accurate, precise, sensitive and fast enough to make a good diagnosis. The inductively coupled plasma –

mass spectroscopy (ICP-MS) has recently proven its efficacy at this point of view. Most of the time, blood is

centrifuged and plasma or serum are used for ICP-MS assays. Except for Cu (plasma > serum), plasma and

serum values are similar. The blood is generally an early indicator of the deficiency phase as serum/plasma

represents the transport pool of the mineral. Milk can give an idea of the trace mineral status of a lactating herd.

It is very easy to sample but limited to certain elements (Se, I). Urine samples are of low interest, mainly due to

the disparity of urine density and thus, concentration within a day. Furthermore, reference ranges are missing.

Hair is may be the easiest sample to collect however the accuracy of trace elements determination in this sample

is poor and subject to external contaminations. Finally, for some elements (Cu, Co), liver biopsies can be helpful

in precisely determining trace elements status of animals. Nevertheless, the liver biopsy is often considered as

complicated and risked for patients by numerous European practitioners. Farmers are also generally reluctant to

this procedure.

*Sampling

It is not the goal to remind the blood sampling procedure or liver biopsy, but to focus on the misinterpretation

that can be made from wrong samples. The major risk is of course the haemolysis that can falsely increase Se,

Cu and Zn plasma/serum concentrations. The delay between sampling and analysis may also lead to false results.

A long delay will increase the risk of haemolysis but also the diffusion of Se from erythrocytes (contained in

glutathione-peroxidase) to serum/plasma. The activity of enzymes (e.g. glutathione-peroxidase or superoxide

dismutase) may also decrease with the time. Finally, some tubes with rubber plugs may contain Zn, leading to a

false increase of plasma/serum Zn concentration. Specific plastic tubes, without rubber and free of trace of

minerals should be used.

Diagnosis of trace elements deficiencies in cattle should be done at a herd level because this is clearly a herd

disease. The diagnosis consists in evaluating the nutritional status of the herd. For determining a disease, sick

animals should be tested. Thus, for assessing nutritional status, healthy animals should be taken. At a herd level,

several healthy animals must be chosen for sampling. The number depends on the deficiency prevalence and the

intra-herd variation of trace elements concentration. According to different authors, this number fluctuates from

7 to 15 (Herdt, 2000; Herdt et al., 2000; Guyot and Rollin, 2007). Animals to be sampled must be selected to

form a homogeneous group in the target population. Analysis can be made individually or by pool. The pool has

the advantage to be less expensive. It is accurate if the number of selected animals is sufficient (about 15

animals) and if the group of sampled animals is homogenous. One pool per target population is necessary.

Nevertheless, pool is effective on plasmatic Se, Zn, Cu, I, but is useless for determining activity of glutathione-

peroxidase in erythrocytes (GPX), superoxide-dismutase (SOD) or thyroid hormones. While using pool,

attention must be paid in the interpretation of results if outliers (heterogeneous group) are present in the sampled

population. In general, if the group is homogeneous, an “all-or-nothing” rule is applied as the majority of

sampled animals (>70%) are either deficient or healthy (Guyot et al., 2009).

*Source of variation

47

Haemolysis is a huge source of variation as it artificially increases plasmatic Cu, Zn and Se concentrations. GPX

and SOD in the red blood cells contain various amounts of these trace elements. Inflammation and diseases, as

the Cu-containing ceruloplasmine, an inflammatory protein, will secondarily increase plasmatic Cu

concentration, acting like a positive acute-phase protein. Concomitantly, a decreased plasmatic Zn concentration

will occur while inflammation is present. Inflammation has also an impact on Se metabolism and thyroid

hormones (e.g. Euthyroid Sick Syndrome). Other factors, such as gestation, range of lactation, age (calves versus

dams) may influence blood trace elements levels. Teat-dipping with I-containing products of I interfere with the

diagnosis of I deficiency (in milk or plasma) by trans-cutaneous absorption of I. The administration of some

deworming drugs (closantel, nitroxinil) in cattle will also increase, for a quite long period, the plasmatic I. The

concentration of Se in milk can vary depending on the form of Se given in the ration with more Se transferred to

colostrum or milk while cows fed selenomethionine compared to Na-Selenite. Moreover, at equal dosage of Se

in the feed, plasmatic Se and, in a lesser extend, the activity of GPX, reaches higher concentrations if

selenomethionine is given in the ration, compared to Na-Selenite. The correlation between GPX and plasmatic

Se is well described in the literature; however this correlation may change according to the form of Se as well. In

any form of Se, following an oral supplementation, plasmatic Se will increases gradually to reach a steady state

between 30-60 days, while GPX will continue to progressively increase at least up to 120 days (Guyot et al.,

2007c).

*Analyses and interpretation

Evaluating trace mineral status on blood or animal’s tissues is subject to some limitations and variations. Two

different ways are often present: determining nutritional status (e.g. plasmatic or hepatic concentration of the

trace element) or functional status (e.g. thyroid hormones, GPX, SOD). Even the thresholds are often discussed.

The table 1 proposes different thresholds for assessing nutritional and functional status of the major trace

elements Se, Zn, Cu, I and Co. According to the Fig.1, they have been chosen considering the concentration of

trace elements needed in order to optimize health, welfare, food quality and productivity and not just avoiding

clinical diseases or barely reaching N.R.C. requirements (NRC, 2000 and 2001). Beside this, the blood or tissue

(e.g. liver) represents different pools of trace elements in the body. When dietary intakes are not sufficient, the

pools of storage and transport will decrease, respectively, but they must be depleted before seeing dysfunction

and disease (Fig.2). Ideally for the diagnosis, the storage pool must be sampled but in practice, it is not always

accessible.

Fig.1: Threshold for determination of correct Fig.2: Representation of the kinetic of the

trace element status in cattle (adapted from pools of trace elements, according to the

Chung, 2003). mineral level (from Underwood & Suttle,

1999).

48

Table 1: Threshold for plasma zinc (Zn), plasma copper (Cu), activity of glutathione peroxidase in erythrocytes

(GPX), plasma selenium (Se), serum cobalt (Co), plasma thyroxine (T4), plasmatic inorganic iodine (PII) in

blood and iodine (I) in milk for adult cattle (Herdt et al., 2000; Guyot et al., 2007c; Guyot and Rollin, 2007;

Guyot et al., 2009; Guyot et al., 2011).

Herd status Zn

(µmol/L)

Cu

(µmol/L)

GPX

(IU/gHb)

Se

(µg/L)

Co

(µg/L)

PII

(µg/L)

T4

(nmol/L)

Milk I

(µg/L)

Adequate 14-21 13-18 220-600 80-140 0.17-2 45-650 30-120 80

Marginal 8-14 8-13 75-220 65-80 - 15-45 20-30 30-80

Deficient <8 <8 <75 <65 - <15 <20 <30

*Specific diagnosis for Se, Cu, Zn, I and Co

° Selenium

Serum/plasma Se is good indicator of dietary intake. Plasma Se is a nutritional marker that quickly increases

(within 2-6 days) after oral supplementation (Ellis et al., 1997). For functional marker, or long-term marker, the

assay of GPX in red blood cells is often used. Because the Se-containing GPX is formed at the time of

erythrocyte’s development, the GPX activity allows determining Se status of the 100-120 previous days (mean

of half-life of red blood cells). As Se is well transferred from dam to calf, the cow in late gestation will show

lower Se plasma concentration. After calving, Se levels will progressively increase in the dam. In calves, a

higher proportion of Se is in the GPX rather than the plasma, so the GPX activities are higher and plasma Se

lower, compared to their dams (Guyot et al., 2011). I supplementation has an impact on Se metabolism with low

Se status while animals fed supplementary I, compared with I-deficient diets (Pavlata et al., 2005; Guyot et al.,

2011). Liver biopsy is also a good means to assess Se status, if realisable. Finally, milk Se can also be assessed

but is widely influenced by the form of Se given in the ration (Guyot et al., 2007c).

° Copper

For Cu, the storage pool is the liver. It reflects the long-term availability of dietary Cu. A decreased liver Cu

concentration is an early marker of insufficient Cu dietary intake. The most sensitive way to assess Cu

deficiency is therefore the liver biopsy, even if in Europe, practitioners and farmers are unenthusiastic to this

method of sampling. Instead, plasma/serum Cu is more often used. The transport pool is constituted by the blood

and, in a certain extent, also the functional pool. In fact, the ceruloplasmin, a protein of inflammation produced

by the liver, contains about 80% of the Cu in the blood. Plasma Cu assessment seems to be better rather than

serum because a variable amount of Cu may be captive in the clot. There is a curvilinear relationship between

liver and blood Cu concentration. However, hepatic Cu concentration must fall drastically before a decreased

blood Cu concentration can be seen (Underwood and Suttle, 1999). Plasma Cu is thus a late (long-term) marker

of Cu deficiency but remains useful to be performed at a herd level, with at least 10-15 animals sampled per herd

(Herdt, 2000). The SOD is a Cu-containing enzyme sometimes used in determining Cu status but it is very

delicate to interpret. Milk Cu does not present interest as Cu milk concentration is usually poor.

° Zinc

The storage pool for Zn is not well-defined, making the evaluation of Zn status tricky. Even if hepatic Zn

concentration decreases after a long inadequate Zn dietary intake, the liver Zn is not a good means for assessing

Zn status in cattle. Thus, the plasma/serum Zn concentration is preferred because it is reduced in Zn deficiency.

However, there might be dysfunctions (e.g. reduced feed efficiency) before observing a decreased serum Zn

concentration. A low serum Zn concentration is indicative of a deficiency while a normal serum Zn does not

necessarily plead for a deficiency (Underwood and Suttle, 1999).

° Iodine

I deficiency can also be assessed by nutritional or functional markers. For the nutritional ones, the most used is

the plasmatic inorganic iodine (PII). The PII reflects the dietary I intake and its concentration fluctuates very

quickly according to the I fed in the ration. For functional markers, the hypothalamus-pituitary-thyroid axis is

49

investigated with thyroid stimulating hormone (TSH) and total thyroid hormones (thyroxine T4 or tri-iodo-

thyronine T3) (Guyot et al., 2011). Huge variations can physiologically exist (pregnancy, circadian rhythm,

birth, age) and are important to be included in the interpretation of the status. Clinical pathology laboratories can

easily assay thyroid hormones while there is no laboratory able to measure bovine TSH at the moment. TSH, T4

and T3 are usually taken for diagnosing hypothyroidism, originates, in cattle, mainly from I deficiency (Guyot et

al., 2007a,b). Total blood I is constituted with PII and I from thyroid hormones, setting the interpretation of a

short-term and long-term marker not obvious. Milk I can be used for assessing herd I status. It also vary quickly

with dietary I, but in a lesser extent than PII. On the field, weighing thyroid gland in (stillborn or goitrous) calves

is useful to appreciate I status. The maximal weight of the gland has been determined as follows (Hernandez et

al., 1972): Y (weight of thyroid gland, in grams) = 0.348 x Z0.944

(Z is the body weight, in kg).

° Cobalt

Co status in ruminants can be determined by Co or vitamin B12 blood concentration. Vitamin B12 is constituted

with 4% of Co. The blood concentration of these two elements is very low, necessitating highly-performing

techniques of dosage (ICP-MS, radioimmunoassay, chemiluminescence, high-performance liquid

chromatography). As liver contains the largest concentration of Co, it is the sample of choice and represents the

storage pool of Co. Nevertheless, there is a variable proportion of Co that is not associated with vitamin B12,

inducing misinterpretation of Co status either in liver or in blood. Correlation between serum Co and serum

vitamin B12 is low. According to these elements, the assessment of Co status in ruminants, either in liver or

blood, is complicated and poorly reliable with routine laboratory assays (Underwood and Suttle, 1999: Herdt et

al., 2000).

Conclusions

Trace elements deficiencies are frequent in Europe in cattle. As there are no pathognomic signs, clinical

pathology diagnosis is necessary. Sampling at a herd-level increases the reliability of the diagnosis, if samples

are taken from a homogeneous group of healthy animals. Blood is most often used because of its higher

feasibility on the field. But the storage pool, that is the most effective one for assessing mineral status, is not

always represented by the blood. Specific herd thresholds for determining trace elements status in cattle must

include health, welfare, food quality and productivity.

References

Chung TK. Make the most of vitamin supplementation. Poultry Int. 2003;1:28-32.

Ellis RG, Herdt TH, Stowe HD. Physical, hematologic, biochemical, and immunologic effects of

supranutritional supplementation with dietary selenium in Holstein cows. Am J Vet Res. 1997;58:760-764.

Enjalbert F, Lebreton P, Salat O. Effects of copper, zinc and selenium status on performance and health

in commercial dairy and beef herds: Retrospective study. J Anim Physiol Anim Nutr. 2006;90:459-466.

Graham TW. Trace elements deficiencies in cattle. Vet Clin North Am Food Anim Pract 1991;7:153-

215.

Guyot H, Sulon J, Beckers J-F, Closset J, Lebreton P, Alves de Oliveira L, Rollin F. Development and

validation of a radioimmunoassay for thyrotropin in cattle. J Vet Diagn Invest. 2007a;19:643-651.

Guyot H, Lebreton P, Alves de Oliveira L, Sulon J, Beckers J-F, Rollin F. Thyrotropin in newborn

calves as a tool for diagnosing hypothyroidism. Cattle Pract. 2007b;15:271-275.

Guyot H, Spring P, Andrieu S, Rollin F. Comparative responses to sodium selenite and

organic

selenium supplements in Belgian Blue cows and calves. Livestock Sci. 2007c;111:259-263.

Guyot H, Rollin F. Le diagnostic des carences en Iode et Sélénium chez les bovins. Ann Med Vet.

2007;151:166-191.

Guyot H, Saegerman C, Lebreton P, Sandersen C, Rollin F. Epidemiology of trace elements

deficiencies in Belgian beef and dairy cattle herds. J Trace Elem Med Biol. 2009;23:116-123.

Guyot H, Alves de Oliveira L, Ramery E, Beckers J-F, Rollin F. Effect of a combined iodine and

selenium supplementation on I and Se status of cows and their calves. J Trace Elem Med Biol. 2011;25:118-124.

50

Herdt HT. Variability characteristics and test selection in herd-level nutritional and metabolic profile

testing. Vet Clin North Am Food Anim Pract. 2000;16:387-383.

Herdt HT, Rumbeiha W., Braselton WE. The use of blood analyses to evaluate mineral status in

livestock. Vet Clin North Am Food Anim Pract. 2000;16:423-444.

Hernandez MV, Etta KM, Reineke EP, Oxender WD, Hafs HD. Thyroid function in the prenatal and

neonatal bovine. J Anim Sci. 1972;34:780-785.

Mee JF, Rogers PAM. Base-line survey of blood trace element status of 50 dairy herds in the south of

Ireland in the spring and autumn of 1991. Irish Vet J. 1994;47:115-122.

National Research Council (NRC). Nutrient requirements of beef cattle. 7th

ed. Washington DC:

National Academy Press; 2000: 248p.

National Research Council (NRC). Nutrient requirements of dairy cattle. 7th

ed. Washington DC:

National Academy Press; 2001:408p.

Pavlata L, Slosarkova S, Fleischer P, Pechova A. Effects of increased iodine supply on the selenium

status of kids. Vet Med.-Czech. 2005; 50:186-194.

Underwood EJ, Suttle NF. The Mineral Nutrition of Livestock. 3rd

ed. Oxon: CABI Publishing; 1999.


ONCOLOGIC CYTOLOGY OF THE CANINE LIVER AND PANCREAS

L. Sharkey. University of Minnesota, Veterinary clinical sciences, USA.


Introduction

Examination of cytologic samples from the liver and pancreas can be an important component in the diagnostic

evaluation of patients with suspected disease in these organs. The frequency of samples from the liver and

pancreas has increased with the more routine availability of ultrasound for evaluation of patients with abdominal

disease, which reveals lesions that are not appreciable with abdominal palpation or survey radiographs. Routine

evaluation of the liver for metastatic disease in patients with neoplasia is another common indication for

cytology. In contrast, cytologic evaluation of the pancreas has been slower to gain acceptance, partially due to

concerns about the safety of collecting samples. Optimal application of cytology for hepatic and pancreatic

disease relies on an understanding of the diagnostic performance for specific conditions. We will focus this

discussion on the diagnosis of neoplastic disease in these organs.

Diagnostic performance of cytology for the presence of hepatic neoplasia

Indications for hepatic cytology in general include hepatomegaly, the detection of mass lesions, altered

echogenicity, and clinical suspicion of malignancies that have the potential to metastasize to the liver. It is

important to note that many changes may be observed in the liver secondary to the presence of neoplasia,

including necrosis, inflammation, extramedullary hematopoiesis, vacuolar change, and reactive hyperplasia (both

hepatocellular and biliary). These may be observed whether or not malignant cells are present in the samples.

Several studies have examined cytology:histopathology correlation for the diagnosis of hepatic neoplasia and

found relatively low sensitivity but high specificity. Wang et al found that only 22% of histologically identified

liver tumors were detected by cytology, however the false positive rate was only 2% (Wang et al. 2004). One

study of 34 ultrasonographically detected focal liver lesions found neoplasia in some but not all lesions sampled,

and identified neoplastic cells spreading beyond the focal lesion in some cases (Faverazani 2006). The authors

note that the pattern of echogenecity was not consistently related to any particular tumor type. A more recent

larger retrospective cytology:histopathology correlation study of 140 ultrasound detected focal liver lesions in

dogs found a sensitivity of 52% and a positive predictive value of 87% for neoplasia (Bahr et al, in press).

Within those general parameters of diagnostic performance, there was variation in the ability of cytology to

detect tumor subtypes. For example, cytology was most sensitive for round cell tumors (60%) and non-

hepatocellular carcinomas (55%), but was lower for hepatocellular carcinomas (35%) and sarcomas (17%),

51

although the number of sarcomas in that study was very small. The positive predictive value of cytology for

hepatocellular carcinoma was excellent (100%) and also high for non-hepatocellular carcinoma (86%) and round

cell neoplasia (75%). All of these studies were retrospective, which raises the concern that selection bias may

influence the diagnostic performance of cytology that was reported. Bahr et al used the STARD criteria to

characterize the bias by evaluating patients that met all study criteria compared with those meeting all study

criteria except no histology was performed. The data suggest that patients with a cytologic diagnosis of

inflammation and necrosis of the liver were more likely to undergo biopsy than patients with other cytologic

diagnoses (neoplasia, hyperplasia, extramedullary hematopoiesis, cholestasis, vacuolar change, normal). The use

of cytology for the diagnosis of hepatic metastasis in canine cutaneous mast cell tumors has been studied in more

detail.

Independent investigators have shown that cytologic involvement of the liver is a negative prognostic indicator

for survival (Stefanello et al 2009, Book et al 2011). Suggested cytologic criteria to distinguish neoplastic cell

involvement from non-neoplastic mastocytosis included 1: clustering of well differentiated mast cells, 2: large

numbers of well differentiated mast cells, and 3: mast cells with atypical morphology, including pleomorphic

and poorly granulated cells. Of critical importance is that both studies identified patients with metastatic mast

cell involvement of the liver in the absence of ultrasonographic abnormalities, so a normal abdominal ultrasound

study does not exclude hepatic metastases associated with cutaneous mast cell tumors in dogs.

Primary hepatic malignancies include hepatocellular carcinoma, cholangiocellular neoplasia, and rarely, hepatic

carcinoid. Hepatocellular carcinomas can be reliably identified when there are marked criteria of malignancy,

however well differentiated tumors may be impossible to distinguish from benign hepatomas or reactive

hepatocellular hyperplasia without histopathology. Cholangiocellular tumors and hepatic carcinoids are often

difficult to distinguish from metastatic lesions (see below).

Metastatic neoplasms include hematopoetic origin tumors (lymphoma, histiocytic malignancies, mast cell tumor,

infiltration by leukemic cells), carcinomas (often pancreatic or intestinal), and less frequently sarcomas, with

hemangiosarcoma being prominent. Round cell tumors can often be specifically identified based on cytology

provided there are sufficient numbers of cells. In some low cellularity samples, histopathology or sampling of

other involved organs may be required to support the diagnosis, and rarely special stains or markers may be

required to define the cell line of origin. Occasionally, extramedullary hematopoiesis can imitate round cell

neoplasia. It is often not possible to definitively determine the cell of origin of non-hepatocellular origin

carcinomas, so imaging is important to characterize the scope of involvement and in some cases may help with

identification of a primary tissue. Likewise, sarcomas often lack specific identifying features for

subclassification.

Pancreatic cytology

Very little information is available on the cytology of the canine pancreas due to lack of robust cytology:

histopathology correlation studies. Concerns are expressed about the safety of aspiration cytology and biopsy

procedures involving the pancreas in dogs and cats that limit the use of cytology and biopsy, however studies are

underway at the University of Minnesota, where pancreatic cytology samples are collected fairly routinely and

anecdotal evidence suggests few complications. A prospective study at our institution demonstrated that

ultrasound guided aspiration and clam shell biopsy of the pancreas in healthy dogs failed to increase the serum

pancreatic lipase activity, however sampling did result in slightly increased trypsin like immunoreactivity levels

in serum and mild peracute necrosis, inflammation, hemorrhage, and fibrin deposition at the biopsy sites

(Cordner et al 2010). In this study of normal dogs, no cytologic samples collected by ultrasound guidance were

of diagnostic cellularity, however 65-75% of intraoperative aspirates or touch impressions from biopsy samples

were of diagnostic quality. Our clinical experience suggests that diseased tissue is more exfoliative than the

normal pancreatic tissue, which is encased in a fibrovascular stroma. Preliminary unpublished data from a

retrospective study of canine pancreatic cytology at the University of Minnesota suggests that in sick dogs, only

approximately 25% of ultrasonographically collected pancreatic cytology samples were nondiagnostic, with the

remainder being of adequate to excellent quality (samples from neoplastic lesions were not analyzed separately).

At the University of Minnesota, indications for pancreatic cytology are similar to those for the liver and include

ultrasonographic changes including altered echogenicity and the identification of mass lesions. Histology studies

of the pancreas suggest that like the liver, nodular hyperplasia is a common nonspecific change that will have to

be distinguished from well differentiated acinar tumors. Like hepatocellular tumors, our experience is that

pancreatic acinar tumors exhibiting marked cytologic pleomorphism are relatively easy to identify, but moderate

to well-differentiated acinar tumors will likely require histopathology for confirmation of the diagnosis. The

veterinary medical literature describing the cytology of pancreatic neoplasia in the dog is largely limited to case

52

reports and small case series, and more comprehensive studies are needed. Preliminary unpublished data from a

series of sick dogs that had pancreatic cytology performed at the University of Minnesota identified neoplasia in

approximately 15% of the samples evaluated, with a predominance of carcinomas, occasional neuroendocrine

neoplasia, and a single case of lymphoma involving the pancreas. In a very limited number of pancreatic

cytology cases, histology was eventually performed by surgical biopsy or at necropsy; for all cases in which a

cytologic diagnosis was available, it matched the ultimate histologic diagnosis.

In summary, preliminary studies and anecdotal evidence suggest that pancreatic cytology has the potential to be

a safe and useful diagnostic procedure. More information will become available as current descriptive

retrospective studies are completed. There is still a significant need for more comprehensive prospective

cytology:histopathology correlation studies that will better define optimal techniques and safety precautions, the

diagnostic performance of pancreatic cytology for specific disease processes, and more refined criteria for the

cytologic diagnosis of pancreatic neoplasia.

References:

Bahr K, Sharkey LC, Murakami T, Feeney D. Accuracy of ultrasound guided fine needle aspiration of focal liver

lesions in dogs: 140 cases (2005-2008). Journal of the American Animal Hospital Association. In press.

Book AP, Fidel J, Wills T, Bryan J, Sellon R, Mattoon J. Correlation of ultrasound findings, liver and spleen

cytology, and prognosis in the clinical staging of high metastatic risk canine mast cell tumors. Vet Rad Ultrasound.

2011;52:548-554.

Cordner AP, Armstrong PJ, Newman SJ, Novo R, Sharkey LC, Jessen C. Effect of pancreatic tissue sampling on

serum pancreatic enzyme levels in clinically healthy dogs. J Vet Diagn Invest 2010;22:702-707.

Faverzani S, Chinosi S, Valenti P, Caniatti M. Comparison between ultrasonography and cytology of liver focal

lesions and parenchyma in the dog and cat. Vet Res Comm. 2006;30(S):293-296.

Stefanello D, Valenti P, Faverzani S, Bronzo V, et al. Ultrasound guided cytology of spleen and liver: a

prognostic tool in canine cutaneous mast cell tumor. J Vet Intern Med. 2009;23:1051-1057.

Wang KY, Panciera DL, Al-Rukibat RK et al. Accuracy of ultrasound-guided fine needle aspiration of the liver

and cytologic findings in dogs and cats: 97 cases. J Am Vet Med Assoc. 2004;224:75-78.

ADIPOSE TISSUE DERIVED STEM CELLS FOR TREATMENT OF OSTEOARTHRITIS AND

OTHER JOINT PROBLEMS IN DOGS

K.Čeh1, G. Majdič

1,2 . 1Center for Animal Genomics, Veterinary Faculty, University of Ljubljana, Gerbiceva 60,

1000 Ljubljana, Slovenia; and 2Institute of physiology, Medical faculty, University of Maribor, Maribor,

Slovenia


Although stem cells have become very fashionable both in research and public media during the last 10 years,

they have been around for much longer. The first stem cell transplantations were performed in the fifties by

transplantation of bone marrow(Mathe et al., 1959) and are today routinely used in treatments of different

leukaemias. These treatments are very successful in human medicine. However, interestingly, even though

many experiments leading to successful procedure in humans were done in dogs, bone marrow transplantation

has not yet become widely used treatment in veterinary medicine.

Stem cells started to attract wide interest in the late nineties with the first isolation of human embryonic stem

cells (Thomson et al., 1998), which are, by some, still seen as the holy grail of regenerative medicine. However,

since the isolation of human embryonic stem cells requires destruction of a human embryo, production of these

cells is accompanied by ethical dilemmas(Vogel and Holden, 2008). Therefore, scientist were looking for

different ways to overcome these ethical problems, and soon after the first isolation of embryonic stem cells, it

has been reported that stem cells could be found in many different tissues also in adult humans, not just in bone

marrow, which is rich in hematopoetic stem cells, but also in tissues like muscles, brain and adipose tissue(van

der Kooy and Weiss, 2000). Stem cells from adipose tissue attracted a lot of attention since adipose tissue is easy

to obtain and could therefore represent an easy source of stem cells for auto transplantation. Although adipose

tissue derived stem cells do not have the same differentiating potential as embryonic stem cells, they still have

../../../../Documents%20and%20Settings/staricjo/Local%20Settings/Temporary%20Internet%20Files/Content.Outlook/48IFC8T5/SKUPAJ-predavanja-vabljena.doc#_ENREF_10





53

the capacity to develop into different types of differentiated cells, mostly from the same lineage as adipose

tissue, although full transdifferentiation potential of these cells is not yet known(Bailey et al., 2010; Ding et al.,

2011; Gimble and Guilak, 2003).

In addition to adipose stem cells from humans and laboratory rodents, adipose derived stem cells have been

shown to exist also in different domestic animals such as dog, swine and horses. Although we still do not have a

general marker for characterizing stem cells, International society for Cellular therapy has recently launched a

guideline, suggesting that mesenchymal stem cells need to have the following characteristics: they must express

CD73, CD90, and CD105; they should not express CD45, CD34, CD14 [CD11b], CD79 [CD19] and they should

be able to show ability for transdifferentiation into bone, adipose and cartilage cells in in vitro

conditions(Dominici et al., 2006). Although these guidelines were issued for work with human mesenchymal

stem cells, it is probably opportune to adopt the same guidelines in the veterinary medicine.

In parallel with the popularity of stem cell research in human medicine, it has been soon recognized that stem

cells have potential applications also in the veterinary medicine.The first attempts using adult stem cell in horses

were performed in 2003 for treatment of suspensory ligament desmitis, using bone marrow mesenchymal stem

cells (Herthel, 2002). Since this first report, stem cell treatments have been used increasingly in the equine

medicine, primarily for tendonitis and osteoarthritis/cartilage injury. Although several hundreds of horses have

been reportedly treated with stem cells, either bone marrow or adipose tissue derived (reviewed in (Fortier and

Travis, 2011), there are no clinical trials that would undisputable demonstrate effectiveness of such treatments.

Nevertheless, experimental evidence suggest that treatments do have some benefits, and a pilot study with

adipose derived stem cells for treatment of tendonitis in horses have shown significant improvement in

histological score (Nixon et al., 2008). On the other hand, one study in horses also didn’t detect any

improvement in articular health after osteoarthritis treatment with adipose derived stem cells and even reported

increase in proinflamatory TNF-alpha concentration in the synovial fluid, although this had no clinical

effect(Frisbie et al., 2009). The first treatments of dogs with stem cells were reported several years later than in

horses. In dogs, adipose derived stem cells are mostly used, and to date, two small clinical trials reported

improvement of osteoarthritis in dogs (Black et al., 2008; Black et al., 2007).

In our laboratory, we started experimental treatments with autologous adipose derived stem cells in 2010, mainly

in dog patients with osteoarthritis. Instead of using nucleated cell fraction, freshly obtained from a dog patient

adipose tissue, we are culturing cells in vitro to obtain large enough numbers of cells. We believe this procedure

has two important benefits over using freshly prepared cells. Firstly, the amount of starting material (adipose

tissue taken from the patient) is much smaller and, secondly, the cells are enriched since many nucleated cells

presented in the adipose stromal fraction would not divide and expand in cell culture. This procedure also allow

as to characterize the cells obtained from the adipose tissue and our experiments do show that cells growing in

culture are rich with stem cells,as determined by the expression of stemness markers CD 44 and CD 73, and the

ability to differentiate into bone and cartilage cells. Interestingly, in direct comparison with human adipose

tissue derived stem cells and studied at Educell company in Ljubljana, our preliminary experiments suggest that

dog adipose tissue is much richer with stem cells in comparisonto human tissue, what might represent an

advantage in future developments of regenerative therapies in veterinary medicine. In our procedure, cells

obtained from the adipose tissue are expanded in the laboratory for a week, until enough cells are obtained for

harvesting and treatment. Usually, 3 – 4 millions of cells are injected directly into diseased joints, and all

patients are evaluated regularly by orthopaedic surgeon after the treatment. So far, 44 dogs have been treated

with this procedure, mostly with diagnosis of knee osteoarthritis, although some cases with elbow osteoarthritis,

rupture of ligamentacruciata and hip dysplasia were also treated experimentally. Analysis of subjective

estimation by dogs’ owners and clinical examination of dogs by orthopaedic surgeon(s) suggest moderate to

significant improvements in lameness, pain and general activity of treated dogs. Furthermore, radiography of

knee in several patients with osteoarthritis revealed marked improvement in the status of knee cartilage,

suggesting beneficial effect of stem cells treatment.

Despite accumulating experimental data about benefit of stem cell treatments for joint problems in dogs, no large

double blinded clinical trials have been performed. Therefore, our next step will be to perform proper clinical

trial to establish whether stem cell treatments really represent a viable option for orthopaedic problems in dogs

and horses. Although stem cells offer promising novel treatments, potentially for many different diseases, also

in the veterinary medicine, such developments are still in their early phases. Besides proper clinical trials to

undoubtedly show the benefit of stem cell treatments, many other questions will have to be answered. What are

the optimal numbers of cells used for treatments? Which stem cells are the best for treatments, and perhaps

different stem cells will prove to be differentially useful for different problems? What is the best delivery method

for stem cells? And last, but certainly very important question, how do stem cells work – by trans-differentiation

into different tissues or perhaps by secreting some unknown growth factors/immunomodulatory factors, in which













54

case live stem cells might become obsolete once we will learn what factors from stem cells are involved in

healing processes.

References

Bailey, A.M., Kapur, S., Katz, A.J., 2010. Characterization of adipose-derived stem cells: an update. Curr Stem

Cell Res Ther 5, 95-102.

Black, L.L., Gaynor, J., Adams, C., Dhupa, S., Sams, A.E., Taylor, R., Harman, S., Gingerich, D.A., Harman, R.,

2008. Effect of intraarticular injection of autologous adipose-derived mesenchymal stem and regenerative cells

on clinical signs of chronic osteoarthritis of the elbow joint in dogs. Vet Ther 9, 192-200.

Black, L.L., Gaynor, J., Gahring, D., Adams, C., Aron, D., Harman, S., Gingerich, D.A., Harman, R., 2007.

Effect of adipose-derived mesenchymal stem and regenerative cells on lameness in dogs with chronic

osteoarthritis of the coxofemoral joints: a randomized, double-blinded, multicenter, controlled trial. Vet Ther 8,

272-284.

Ding, D.C., Shyu, W.C., Lin, S.Z., 2011. Mesenchymal stem cells. Cell Transplant 20, 5-14.

Dominici, M., Le Blanc, K., Mueller, I., Slaper-Cortenbach, I., Marini, F., Krause, D., Deans, R., Keating, A.,

Prockop, D., Horwitz, E., 2006. Minimal criteria for defining multipotent mesenchymal stromal cells. The

International Society for Cellular Therapy position statement. Cytotherapy 8, 315-317.

Fortier, L.A., Travis, A.J., 2011. Stem cells in veterinary medicine. Stem Cell Res Ther 2, 9.

Frisbie, D.D., Kisiday, J.D., Kawcak, C.E., Werpy, N.M., McIlwraith, C.W., 2009. Evaluation of adipose-

derived stromal vascular fraction or bone marrow-derived mesenchymal stem cells for treatment of

osteoarthritis. J Orthop Res 27, 1675-1680.

Gimble, J., Guilak, F., 2003. Adipose-derived adult stem cells: isolation, characterization, and differentiation

potential. Cytotherapy 5, 362-369.

Herthel, D.J., 2002. Suspensory desmitis therapies, Proceedings of 12th ACVS symposium, pp. 165 - 167.

Mathe, G., Jammet, H., Pendic, B., Schwarzenberg, L., Duplan, J.F., Maupin, B., Latarjet, R., Larrieu, M.J.,

Kalic, D., Djukic, Z., 1959. [Transfusions and grafts of homologous bone marrow in humans after accidental

high dosage irradiation]. Rev Fr Etud Clin Biol 4, 226-238.

Nixon, A.J., Dahlgren, L.A., Haupt, J.L., Yeager, A.E., Ward, D.L., 2008. Effect of adipose-derived nucleated

cell fractions on tendon repair in horses with collagenase-induced tendinitis. Am J Vet Res 69, 928-937.

Thomson, J.A., Itskovitz-Eldor, J., Shapiro, S.S., Waknitz, M.A., Swiergiel, J.J., Marshall, V.S., Jones, J.M.,

1998. Embryonic stem cell lines derived from human blastocysts. Science 282, 1145-1147.

van der Kooy, D., Weiss, S., 2000. Why stem cells? Science 287, 1439-1441.

Vogel, G., Holden, C., 2008. Stem cells. Ethics questions add to concerns about NIH lines. Science 321, 756-

757.

THE ROLE OF THE COX-2/PGE2 PATHWAY IN PROMOTING CANCEROGENESIS OF CANINE

AND FELINE TUMOURS

A. Poli, Department of Animal Pathology, Prophylaxis and Food Hygiene, Veterinary Faculty, University of

Pisa, Viale delle Piagge 2, 56124 Pisa, Italy.


Abstract

Since 1992 it has been recognised that non-steroidal anti-inflammatory drugs (NSAIDs) inhibit

prostaglandin synthesis and tumor growth in a wide variety of experimental animal models: aspirin,

indomethacin, piroxicam, and sulindac inhibit chemically induced tumours of the colon, tongue, esophagus,

pancreas, bladder, breast, liver, and various sarcomas, as well as numerous transplantable tumours.

Afterwards, a large body of evidence from population based studies, case control studies, and clinical

trials indicate that the regular use of NSAIDs over a 10-15 year period reduces the relative risk of developing

colorectal cancer in humans. Furthermore, use of NSAIDs leads to regression of pre-exiting adenomas in

patients with familiar adenomatous polyposis. As many other human cancers are reported to have elevated

levels of COX-2 and overproduced PGs, there is a great interest in evaluating the role of NSAIDs for prevention

and treatment strategies for other cancer such as breast, stomach, pancreas, urinary tract, lung and prostate.

Prostaglandin production, particularly PGE2 and its receptors play a predominant role in promoting

cancer progression, through enhancing cellular proliferation, promoting angiogenesis, inhibiting of apoptosis,

stimulating of invasion/mobility, and suppression of immune response. The only other COX-2 derived

prostaglandin implicated in oncogenesis is TXA2, which has been reported to promote angiogenesis.

Three kinds of enzymes, phospholipase A2 (PLA2), cyclooxygenase (COX) and terminal PGE synthase

(PGES) are involved in the biosynthesis of PGE2, and each of the three enzymatic steps involves multiple

enzymes that can act in different phases of cell activation. Arachidonic acid released from membrane


55

glycerophospholipids by PLA2 enzymes is then supplied to either of the two COX isoenzymes, COX-1 or COX-

2. The constitutive COX-1 contributes mainly to immediate PG generation and is responsible for homeostasis,

while the inducible COX-2 mediates delayed PG generation and is induced by various inflammatory stimuli and

neoplasia. The COX metabolite PG H2 (PGH2) is then isomerized to PGE2 by terminal PGES enzymes. To

date, three PGES enzymes, microsomal PGES-1 (mPGES-1) and -2 (mPGES-2) and cytosolic PGES (cPGES),

have been identified. PGE2 exerts its biological action through binding to specific receptors, known as EP

receptors. Four subtypes of EP receptor have been identified: EP1, EP2, EP3 and EP4. These receptors differ

in structure, binding profiles and use different intracellular signalling pathways.

In veterinary medicine COX-2 overexpression has been identified in several canine and feline epithelial

and mesenchymal tumours. In the dog, histopathological studies have identified COX-2 expression in

neoplastic cells of transitional cell carcinomas, prostatic carcinomas, mammary benign and malignant tumours,

oral and cutaneous squamous cell carcinomas, intestinal adenoma and carcinomas, nasal carcinomas, renal cell

carcinomas, prostatic carcinomas, melanoma, osteosarcoma and recently lymphoma. In the feline species COX-

2 expression was detected by immunohistochemistry in transitional cell, squamous cell, mammary, pulmonary,

intestinal carcinomas and in lymphoma and vaccine associated sarcoma.

In the last years the use of selective anti COX-2 NSAIDs has reduced the onset of adverse side effects

deriving from COX-1 inhibition. However, a long term use of specific COX-2 inhibitors often leads to the onset

of adverse cardiovascular side effects, due to the decreased synthesis of both PGe2 and PGI2, these latter being

important for normal vascular integrity. Since mPGES-1 is not involved in PGI2 biosynthesis, therefore its

inhibition might be a useful strategy to reduce PGE2 levels with a decreased risk of side effects. The basis of

this therapy it is supported by the induced expression of mPGES-1 in cancers of the stomach, colon,

endometrium, lung and skin in humans. Furthermore, studies based on genetic deletion and ⁄or pharmacological

manipulation of EP receptors with selective agonists ⁄antagonists have shown that EP1, EP2 and EP4 are all

implicated in intestinal tumourigenesis. In particular, EP2 receptor expression is associated with enhanced

development of several tumours and is associated with an unfavourable prognosis in squamous cell carcinoma of

the lung. EP2 receptor expression is also increased in pre-malignant lesions.

On this basis it is relevant to investigate COX-2, mPGES-1 and EP2 receptor expression in normal,

reactive and neoplastic canine and feline tissues and to determine whether the expression of these three

molecules is correlated with tumorogenesis in companion animals.

Preliminary investigations have been performed on canine non neoplastic and neoplastic bone tissues

and in canine and feline non neoplastic and neoplastic mammary tissues.

No immunoreactivity to mPGES-1 or EP2 receptor as well as COX-2 was observed in normal bone,

while 50% of reactive bone samples expressed COX-2 and 57% expressed mPGES-1 and EP2 receptor, although

with weak labelling intensity. Ninety-three percent of osteosarcomas expressed COX-2, while mPGES-1 was

expressed by 85% and EP2 receptor by 89% of the tumours.

Also in canine and feline mammary tissues mPGES-1 and EP2 receptor expression was significantly

higher than that detected in non neoplastic lesions, with percentages similar to those described in human breast

cancers.

These preliminary results provide a rationale for further investigations to study the role of PGE2 in the

tumorogenesis of companion animals may have important implications for the prevention and treatment of

selective canine and feline tumours by antogonising COX-2/PGE2 signaling, inhibiting the EP2 pathway or

targeting mPGES-1.

CYTOLOGIC DIAGNOSIS OF MESENCHYMAL TUMORS: NOT JUST “SARCOMA”

C. Masserdotti. San Marco Veterinary Laboratory, Padua, Italy

Correspondence:[email protected]

Introduction: The aim of this lecture is to discuss the cytological presentation of mesenchymal tumors, and to

describe the specific features of the different neoplastic conditions on the base of published data and personal

experience. The recognition of some cytologic peculiarities may allow the identification of specific types of

mesenchymal tumors that otherwise are only recognized by histopathologic examination of tissue sections. Why

should we try to recognize the different types of sarcomas? In cytologic preparations, mesenchymal tumors are

characterized by variable numbers of spindle, tailed or stellate cells that are scattered individually or are found in

small accumulations. In many cases the generic diagnosis of “sarcoma” is the only one possible because a low

number of cells is present, or because specific features are lacking. In many circumstances spending considerable

amount of time on the smear may be rewording, because the finding specific cytologic features can lead to the

identification of a specific neoplastic lineage. Although some soft tissue sarcomas are approached similarly (that

is: care for wide excision, monitoring mostly for local recurrence rather than metastasis) in many others a correct

56

identification before surgery and tumor staging may lead to the choice of significantly different surgical

procedures and the option of chemioterapeutic treatments.

Cytological features: The key for the recognition of different types of sarcoma is the identification of those

features that allow the determination of lineage of the neoplastic cells. In the following notes, I will try to

describe the cytological features that can help in the refinement of the general diagnosis of “mesenchymal

tumor”.

Neuronal sarcoma. This type of sarcoma arises preferably in the subcutaneous tissues of the extremities.

Cellularity is generally moderate to high, depending on the degrees of differentiation. Cytologic features are few

and represented by palisading arrangements of the neoplastic cells.

Perivascular tumors. They originate in the wall of blood vessels and include myopericytoma and

haemangiopericytoma; these two perivascular tumors cannot be distinguished on cytologic samples, and only the

histopathologic examination with immunohistochemistry can help in their differentiation. Cytologic features are

represented by typically numeorus plump spindle cells that sometimes are arranged in perivascular pattern;

occasional whorls may be present; cytoplasm often has ill-defined borders and a few small clear vacuoles may be

noted. In some cases, binucleated cells, with a less prominent fusiform morphology, have oval to kidney-shaped

protruding nuclei which give the cell the typical appearance of an insect head with composite eyes. Others cells

are multinucleated, with peripheral localization of the nuclei just beneath the cytoplasmic membrane.

Vascular tumors. These neoplasms arise from endothelial cells everywhere in the body; haemangioma is a

benign tumor, but cytologically the sample appears bloody and a final diagnosis cannot be made. In selected

cases, a very few number of elongated spindle cells are present in a bloody background. Haemangiosarcoma is

the malignant counterpart and can be recognized by cytological examination but again many aspirates may be

just bloody. Depending on the degree of differentiation of the tumor, aspirates may contain a variable number of

spindle, stellate, epithelioid mesenchymal cells that have basophilic, sometimes vacuolated cytoplasm, round to

ovoid nuclei, irregularly clumped chromatin, and variably visible nucleoli. Cells are dispersed in variable

amount of blood and anisocytosis and anisokaryosis can be moderate to marked. A few binucleated or

multinucleated cells can be present. In some cases, pseudoacinar distribution of the cells, with or without

erythrocytes or leukocytes into the lumen, can be observed and suggests the neoplastic cells are attempting to

form a vessel. Erythrophagocytic macrophages, extramedullar hematopoiesis and cell apoptosis can be

distinctive, although not pathognomonic features. The diagnosis can be confirmed by immunocytochemistry.

Smooth muscle tumors. Tumors arising from smooth muscle have been subdivided into benign and malignant

tumors that include leiomyomas and leiomyosarcomas, respectively. Smooth muscle tumors are frequent in the

gastrointestinal or genitourinary systems, but can arise also from arrector pili muscles in the skin, or from

smooth muscle within other organs, such as in the hepatobiliary system. Leiomyoma is composed from spindle,

elongated cells, arranged in densely packed fascicles that mimic normal smooth muscle tissue. Cytological

features are generally not detectable by fine needle aspiration (FNA) or fine needle capillary suction (FNCS)

because cells do not exfoliate easily. By vigorously aspirating the tissue, in some circumstances it is possible to

observe rare mensenchymal cells, with fusiform cytoplasm and elongate, cigar-shaped nuclei that are scattered

singly or arranged in tridimensional cohesive accumulations. Leiomyosarcoma can be composed of densely

packed fascicles of fusiform cells or by pleomorphic cells with variable arrangement; cytological features do not

differ from other sarcomas, with the exception of well differentiated tumors, where the nuclei retain the classic

cigar-like shape.

When features of malignancy are observed in a mesenchymal tumor that exfoliated from an intestinal mass, the

presence of gastrointestinal stromal tumor (GIST) must be considered in as a differential. This type of sarcoma

arises from Cajal cells and not from smooth muscle, but a cytological differentiation between these two sarcomas

is not possible.

Striated muscle tumors. Benign and malignant tumors originating from skeletal striated muscle are quite

infrequent and include rhabdomyoma and rhabdomyosarcoma, respectively. There are very few descriptions of

striated muscle sarcomas. Although in people the presence of cells with striated cytoplasm has been described,

this feature is observed very uncommonly in veterinary cytologic specimens. The cytologic diagnosis of

rhabdomyosarcoma can be very challenging: the most common feature consists in plump, round to polygonal

cells with round macronucleolated nuclei, although the round shape of many poorly differentiated cells can

resemble large lymphocytes. Sometimes, the neoplastic cells can show a ribbon-like, elongated shape, with

multiple nuclei arranged in row (strap cells). Immunocytochemistry is necessary in most of the cases to

differentiate this neoplasm from a lymphoid or histiocytic proliferative disorder.

Granular cell tumor of the tongue/larynx. This tumor most often involve the the tongue or larynx region. The

origin of this tumor is still controversial and can be differentiated from rhabdomyoma and oncocytoma only on

the basis of immunohistochemistry and ultrastructural analysis. Morphologically these cells show similar

cytological features with round cells that have finely granular eosinophilic cytoplasm and round nuclei.

Liposarcoma. Liposarcoma is a rare neoplasia arising from adipose tissue. Cells that exfoliate from this tumor

are plump, spindle cell, with round to ovoid nuclei that are often distorted by variable accumulations of

57

intracytoplasmic refractile clear globules that represent lipid. Perivascular arrangements are observed frequently.

Stains specific for lipid, like the Oil Red-O stain or Sudan Black, help in the diagnosis of liposarcoma in

cytologic samples.

Myxosarcoma. This sarcoma produces mucin that appears as abundant eosinophilic amorphous material

dispersed in the background. The cells are immersed in this material and show spindle cytoplasm and nuclear

pleomorphism. Care must be taken in order to differentiate this myxoid material with others substances, like

chondroid matter or the presence of mucinous degeneration.

Osteosarcoma. This neoplasm arises from bone tissue, although sometimes can originate in soft tissues as result

of metaplastic transformation. Specific features are the presence of roundish, mostly single cells, whose nuclei

localize eccentrically, so that the profile of the nuclear membrane seems to overlap with the cytoplasmic outline.

Nuclei show moderate to marked anisokaryosis, granular or clumped chromatin and sometimes prominent

nucleoli. Among neoplastic cells is generally present a little amount of dense eosinophilic fibrillar material,

presumptively osteoid matrix. The presence of giant multinucleated neoplastic cells is an inconstistent finding.

Chondrosarcoma. This neoplasm arises from cartilage. Specific features are represented by ovoid, plump or

spindle cells, with round to ovoid nuclei. The cells are distributed singly or in small groups, and often are

embedded in a large amount of dense eosinophilic material, most consistent with chondroid matrix. In the well

differentiated form of chondrosarcoma, some single cells can lie in an envelope of dense material.

Suggested readings

- R. Höinghaus, M. Hewicker-Trautwein and R. Mischke Immunocytochemical differentiation of canine

mesenchymal tumors in cytologic imprint preparations Vet Clin Path Volume 37, Issue 1, March 2008,

Pages: 104–111

- Akerman M. Fine-needle aspiration cytology of soft tissue sarcoma: benefits and limitations. Sarcoma.

1998;2(3-4):155-61.

- Fleshman R, Mayerson J, Wakely PE Jr. Fine-needle aspiration biopsy of high-grade sarcoma: a report

of 107 cases. Cancer. 2007 Dec 25;111(6):491-8.

NON HEPATOCYTE CELLS: DESCRIPTION OF NORMAL AND PATHOLOGIC

CYTOMORPHOLOGY OF THE “OTHERS” LIVER CELLS.

C. Masserdotti. San Marco Veterinary Laboratory, Padua, Italy


Introduction: The main target of hepatocyte cytopathology is the evaluation of the hepatocyte morphology and

arrangement in oder to identify pathologic and neoplastic states. Nevertheless, the liver is composed also by cell

lines different from hepatocytes, many of which play an integrated and crucial role in many biological functions

of the liver. The aim of this lecture is to describe non-hepatocyte liver cells, and to discuss their roles and

cytomorphologic features in normal and pathologic conditions.

In the traditional hepatic lobule there is an efferent venule at its centre and portal tracts at its periphery; from the

portal tract to the central vein, hepatocytes are arranged in rows separated by sinusoidal spaces. Sinusois are

lined by specialised fenestrated endothelial cells, which form a porous barrier that allows the exchange of

substances transported in the blood stream. Kupffer cells are specialised hepatic macrophages that lie within the

sinusoidal lumen. The space of Disse lies between the sinusoidal endothelium and the hepatocytes and contains a

little amount of extracellular matrix, the hepatic stellate cells, also called Ito cells and a small number of mast

cells. The rows of hepatocytes line the bile canaliculi, where bile drains into the canal of Hering near the margins

of the small portal tracts, and finally into the bile duct.

In the following notes I will describe the cytological features of Kupffer cells, cholangiocytes, hepatic stellate

cells and hepatic mast cells, both in normal and pathological conditions.

Kupffer cells. This specialised hepatic macrophages live attached to the luminal surface of the sinusoidal

endothelial cells; this position is strategic for engulfing bacteria, virus and toxins arriving through the portal

blood. Kupffer cells produce many chemical substances, such as heicosanoid, free radicals, cytokines, interferon

and lysosomal enzymes, that can act as defense mechanisms or they can stimulate specific pathways, like in

fibrosis processes.

Cytology: When not stimulated, Kupffer cells have features identical to macrophages in other tissues and are

round cells with a little amount of cytoplasm and small hyperchromatic nuclei; Kupffer cells generally are not

recognizable in normal conditions, but sometimes they can be observed as small cells wedged between the

cytoplasm of hepatocytes. When Kupffer cells become activated, they increase in size and have irregularly

http://onlinelibrary.wiley.com/doi/10.1111/j.1939-165X.2008.00017.x/abstract

http://onlinelibrary.wiley.com/doi/10.1111/j.1939-165X.2008.00017.x/abstract

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Akerman%20M%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/pubmed/17941014


58

vacuolated cytoplasm. Kupffer cells can have phagocytic activity against many pathogens, but they can also

remove lipid, necrotic debris, lipofuscin and bile, especially during cholestasis.

Cholangiocytes. These cells line the bile ducts up to the choledocus. The interlobular bile ducts have a small

diameter and are lined by cholangiocytes with small cuboidal shape, whereas larger intrahepatic bile ducts are

lined by tall columnar cholangiocytes. Cholangiocytes have secretory activity.

Cytology: Cholangiocytes are very rare in cytologic smears of normal liver; sometimes these cells are dispersed

among hepatocytes and can be recognized because of their cuboidal shape, the small amount of cytoplasm, the

hyperchromatic nuclei and their arrangement in cohesive sheets; if a large bile duct is sampled, cholangiocytes

can be columnar with basal hypercromatic nuclei and arrangement in palisades.

Cholangiocytes can increase in number (hyperplasia), when the liver is affected by a chronic cholangitis or a

chronic cholestasis. Cholangiocytes can show a variable degree of cytoplasmic vacuolation, secondary to many

conditions. This changes is possibly non-specific and of unknown significance.

Cholangioma is a rare benign tumor that is mostly described in cats and is characterized by by replication of

variably dilated biliry ducts lined by a single layer of cholangiocytes and supported by thin fibrous septa.

Aspirates from cholangiomas yields a very low number of cholangiocytes that often are arranged in small sheets.

The cholangiocarcinoma is the malignant counterpart of this tumor and it occurs more frequently than the

cholangioma, especially in dogs. The cells that exfoliate from this tumor are found in variable-sized clusters ad

sheets and can have acinar or palisade arrangement. The cells show moderate to marked features of atypia such

as deeply basophilic cytoplasm that occasionally is vacuolated, and roundish, macronucleolated nuclei with

clumped chromatin. Anisokaryosis is generally moderate to marked. Atypical mytoses can be observed.

Although very impressive, the features of cholangiocarcinoma do not differ substantially from other extrahepatic

carcinomas and must to be differentiated from tumors that metastatize to the liver such as pancreatic, intestinal,

renal or prostatic carcinomas.

Hepatic stellate cells. Also called Ito cells, lipocytes or fat-storing cells, hepatic stellate cells are not easily

identified in normal conditions, although sometimes they can be recognized by their cytoplasm dilated by lipid

material. The principal function of hepatic stellate cells is to store vitamin A in their cytoplasm. These cells

probably are involved in contractile activity, which can contribute to control the blood pressure through the

sinusoids. Hepatic stellate cells produce apolipoprotein E, growth factors and many cytokines and control the

normal homeostasis and production of extracellular matrix in the space of Disse. The hepatic stellate cells are

highly sensitive to the action of chemical insult that can results from acute hepatic injury. If the stimulation

becomes chronic, the hepatic stellate cells transform into myofibroblasts and produce extracellular matrix

components. Therefore, stellate cells are the most important cells leading to the development of hepatic fibrosis.

Cytology: Hepatic stellate cells have round shape and cytoplasm that is distended by round lipid vacuoles that

displaced the hyperchromatic nuclei at the periphery of the cell. Hepatic stellate cells can resemble lipid-laden

hepatocytes but the latter typically have large and granular nuclei, whereas hepatic stellate cells have smaller and

hyperchromatic nuclei. Also, the number of stellate cells generally is very low, whereas in fatty liver, lipid-

containing hepatocytes are very numerous. When a pathologic insult induces the transformation of hepatic

stellate cells into myofibroblasts, the cells lose their round shape and the cytoplasmic vacuoles, become spindle

and are often arranged in bundles that can cross the hepatocyte clusters.

Mast cells. In healthy dogs, the presence of a small number of resident mast cells in the space of Disse space is

well documented. Some authors observed mast cells in the proximity of hepatic stellate cells, supporting the

occurrence of some interaction between these two type of cells. Mast cells appear to be more numerous near the

central vein. The role of mast cells as inflammatory cells is well documented in many pathological conditions,

such as in acute and chronic hepatopathy, as well as in immunomediated and biliary diseases. Mast cells play a

key role in fibrogenesis, in the deposition of extracellular matrix and in sinusoidal capillarization, a pathological

phenomenon that leads to altered exchanges between blood and hepatocytes. An increased number of mast cells

have been observed in hepatic fibrosis. Mast cells promote transformation and proliferation of hepatic stellate

cells into myofibroblasts, the cells responsible for the production of extracellular matrix. Mast cells induce the

proliferation of myofibroblasts by releasing the enzyme chymase from their cytoplasmic granules.

Mast cells also are able to inhibit the action of proteolytic enzymes in the fibrous septa and are therefore

responsible for the progression and worsening of the sclerosing lesions in advanced hepatic fibrosis.

Cytology: Mast cells that exfoliate from liver aspirates are scattered individually. They show round shape the

presence of abundant red to purple cytoplasmic granules and a round nucleus. Since granules are sometimes

inconsistent, mast cells may be identified easily with the use of special stains, such as Toluidine Blue. When

features of fibrosis are present, it is not infrequent to observe mast cells in the proximity of spindle cells that

interconnect the clusters of hepatocytes. It is important to understand the role of mast cells in the liver in both

normal and pathological conditions in order to avoid the misdiagnosis of an inflammatory/fibrotic process with

metastatic mast cell tumor.

Suggested readings

59

- Meyer DJ. The Liver, in Raskin RE, Meyer DJ Atlas of canine and feline Cytology, Saunders 2001:

226-248

- Stockhaus C ,Teske E ,Van Den Ingh T., Rothuizen J .The influence of age on the cytology of the liver

in healthy dogs .Veterinary Pathology 2002;39 :154-158

- Brandão DF, Ramalho LN, Ramalho FS, Zucoloto S, Martinelli Ade L, Silva Ode C. Liver cirrhosis and

hepatic stellate cells. Acta Cir Bras. 2006;21 Suppl 1:54-7. Review.

- Strazzabosco M, Fabris L. Functional anatomy of normal bile ducts. Anat Rec (Hoboken). 2008

Jun;291(6):653-60. Review.

- Masserdotti C, Bertazzolo W. Cytologic features of hepatic fibrosis in dogs: description of 11 cases.

Veterinaria 2008;22(3):9-14

- Yamamoto K. Electron microscopy of mast cells in the venous wall of canine liver. J Vet Med Sci,

2000, 62(11):1183-1188

NOVEL BIOMARKERS

LABORATORY TESTS FOR METABOLIC INBORN ERRORS IN DOMESTIC ANIMALS: CLUES

TO DIAGNOSIS

O. Yamato. Kagoshima University, Veterinary Medicine, 1-21-24 Kohrimoto, Kagoshima 890-0065, Japan.


LABORATORY TESTS FOR THE DIAGNOSIS OF ACUTE PANCREATITIS IN DOGS AND CATS –

SERUM LIPASE ACTIVITY REVISITED

I. Schwendenwein1, E. Hooijberg

1, B. Ruetgen

1, I. Flickinger

2, F. Zeugswetter

2, K. Hittmair

3. University of

Veterinary Medicine, Vienna Austria, 1Central Laboratory, Department of Pathobiology,

2 Clinic for Internal

Medicine and Infectious Diseases, Department of Clinical Sciences, 3 Division for diagnostic Imaging,

Department of Clinical Sciences.


Acute pancreatitis is a commonly sterile inflammation of the pancreas of acute onset and characterized by

necrosis and edema (Van den Bossche 2010). It is completely reversible as pancreatic architecture remains

undisrupted in acute cases (Watson 2004; Watson, Roulois et al. 2007). In chronic pancreatitis tissue architecture

is destroyed and thus the condition becomes irreversible. The true prevalence of acute pancreatitis is difficult to

estimate because of the difficult ante mortem diagnosis (Watson, Roulois et al. 2007). In a series of 200 canine

post mortems acute pancreatitis was seen in 2 % and chronic pancreatitis in 26% (Newman, Steiner et al. 2004).

In cats histopathologic signs of acute pancreatitis were found at a prevalence of 67% with 45% detected in

apparently healthy cats (De Cock, Forman et al. 2007). This seems to be extremely high, but serial cuts were

performed so that even mild cases were detected. In another study (cats with pancreatitis n=21, healthy cats n=8)

where computed tomography was validated against histopathology, fPL and f-TLI (Trypsin like

immunoreactivity) only 8/18 (44%) of cats suffered from a combination of acute and chronic pancreatitis

(Forman, Marks et al. 2004). It has to be kept in mind, that single foci of acute pancreatitis might be missed

when biopsies are taken in living animals (Forman, Marks et al. 2004). Thus even histopathology on surgical

biopsies has its limitations as a gold standard in diagnosing acute pancreatits.

Pathomechnisms leading to acute pancreatitis are not yet completely understood – duct ligation and duct

perfusion with sodium tauro-cholate produce mild to moderate necrotizing acute pancreatitis in cats and dogs

(Banerjee, Galloway et al. 1994). Patients suffering from blunt abdominal trauma, such as cats with high rise

syndrome (Liehmann, Dörner et al. 2012). or dogs hit by cars (de Arespacochaga, Hittmair et al. 2006) might

develop acute pancreatitis as a complication. Early activation of digestive enzymes secreted as zymogens within

the organ lead to autodigestion thus inciting necrosis and neutrophilic inflammation. Autohydrolysis of trypsin

and temporary binding of trypsin to panceratic secretory trypsin inhibitor (PSTI) are autoprotective mechanisms

which seem to be continuously activated in the healthy pancreas (Watson 2004) and are overwhelmed in the case

of acute pancreatitis. As a complication, a severe systemic inflammatory response syndrome may occur. The

inciting causes in naturally occurring disease usually remain unknown. The final common pathway is initiated by

the already mentioned premature activation of trypsin within the pancreas leading to acinar necrosis, circulating

proteolytic enzymes which are mopped up by alph1- antitrypsin and alpha 2-macroglobulin, and resorption of

endotoxins from the gut. Peripancreatic fat necrosis with or without generalized peritonitis might develop. Local

and systemic cytokine release might induce a severe systemic inflammatory response syndrome leading to DIC






60

(Watson 2004). In severe cases other organs such as kidneys and lungs may be affected leading to azotemia and

acute respiratory distress syndrome.

Clinical signs and course of the disease show a high variability, from mild episodes to severe, fatal disease.

Single episodes might be encountered as well as recurrent bouts (Watson 2004).

Depending on the severity acute pancreatitis might become a life threatening disease and early diagnosis is

desirable, despite the fact that specific therapeutic agents are lacking. On the other hand, as no potentially

detrimental therapy will be initiated, a low specificity of diagnostic tests is less problematic as a non specific

therapy will not be harmful to the patient.

Clinical signs, such as lethargy, anorexia, vomiting and diarrhea and pain in the epigastric region are non

specific, thus laboratory tests combined with diagnostic imaging and/or biopsy are needed to establish a

diagnosis. Histopathology of a tissue sample is regarded as gold standard, despite its limitations as focal disease

might be missed by the biopsy.

In veterinary medicine the determination of pancreatic enzyme activity in serum or plasma such as lipase and

amylase are considered insensitive and non specific.

As a result a quest for the ultimate tests for the diagnosis of acute pancreatitis was going on over the last few

years. Initially increases of canine and feline trypsin like immunoreactivity (c-TLI and f-TLI) in peripheral blood

were considered to be good indicators of acute pancreatitis in these species (dogs: (Ruaux 2003) cats:(Gerhardt,

Steiner et al. 2001)). However they were soon replaced by an assay which determines the concentration of feline

and canine pancreas specific lipase in serum. Currently two in-house ELISA-assays for quantification of canine

and feline pancreatic lipase are considered state of the art diagnostics for diagnosing acute pancreatitis (Steiner

JM 2009; Huth, Relford et al. 2010; Jaensch 2010)

In human medicine determination of serum amylase and lipase activities is still in use for the diagnosis of acute

pancreatitis. Lipases are defined as enzymes that hydrolyze glycerol esters of long-chain fatty acids (Panteghini

2006). For appropriate function co-lipases and bile salts are necessary. Most of the serum lipase activity is

considered to be of pancreatic origin, but other tissues like gastric, pulmonary and intestinal mucosa also secrete

lipases. The lipase molecule is small enough to be filtered by the kidney but it is completely reabsorbed by the

tubulus (Panteghini 2006).In humans the diagnostic sensitivity of lipase activity is 0.8 to 1 depending on the

selected diagnostic cutoff. Increases are seen within 4 to 8 hours and decreases within 8 to 14 days. Elevations

between 2 and 50 times above the reference interval have been reported. The height of the increase is not

necessarily proportional to the severity of the attack (Panteghini 2006). Many methods to determine lipase

activity have been described. Triglyceride and non-triglyceride substrates, titrimetric, turbinimetric,

spectorphotmetric, fluorometric and immunological techniques have been used. In veterinary medicine

turbidimetric methods measuring the hydrolization of triolein containing substrates were used. Data on reference

intervals for cats and dogs are scarce and nothing is known about variance components or diagnostic reference

changes in these species. A couple of years ago a new synthetic substrate 1,2-O-dilauryl-rac-glycerso-3 glutaric

acid –(4-methyl.resorufin) ester was introduced in automated assays. This is a bluish chromophore with peak

absorption at 580 nm. The stability of the substrate in contrast to fatty emulsions which tend to segregate again is

the main advantage of this new method. Colipase and bile acids are added in abundance to ensure appropriate

enzyme activity. This assay has been validated for the dog (Graca, Messick et al. 2005) where a much lower and

more narrow reference interval as with conventional assays was established for dogs (n= 30; 20-120 U/L) with a

diagnostic sensitivity of 0.93 and a specificity of 0.53. To the authors best knowledge there is no published

evidence of further studies investigating the recommended reference intervals in dogs or cats. In a study on how

hyperglycemia affects pancreatic enzyme activity in cats the authors refer to a reference interval from 8-26 U/L

(Zini, Osto et al. 2010).

Shortcomings in the diagnostic inefficacy of an assay can be caused by a variety of factors such as analytical

problems, lack of adequate diagnostic cutoffs or reference intervals, non specific changes in the

activity/concentration of the analyte caused by other diseases. Thus revisiting the conventional lipase assay in

terms of diagnostic sensitivity and specificity at various cutoffs combined and compared with ultrasound

imaging and species specific lipase concentrations seemed appropriate.

The Spec cPL™has been in use in the authors lab since December 2010 and the Spec fPL™ since July 2011.

These respective tests were requested in 235 dogs and 84 cats showing clinical signs typical of acute pancreatitis.

Simultaneously a “conventional” serum lipase assay based on 1,2-O-dilauryl-rac-glycerso-3 glutaric acid –(4-

methyl.resorufin) ester was performed. Ultrasound results were available in 186 dogs and 58 cats. Agreement

was assessed by the kappa test between the species specific pancreatic lipase assays and ultrasound results, as

well as between the new tests and different cut-off levels for the conventional lipase. Furthermore the state of the

art assays were compared to combinations of ultrasound findings consistent with acute pancreatitis combined

with results of conventional lipase assays at different cutoff levels. Kappa values below 0.2 are classified as

poor, between 0.2 and 0.4 as fair, between 0.4 and 0.6 as moderate, between 0.6 and 0.8 as good, and very good

above 0.8. Diagnostic performance was assessed by a Bayesian diagram in two ways. First the specific lipase

assays were evaluated against ultrasound alone as well as the conventional lipase assay results at different cut-off

61

levels alone or in combination with ultrasound results. Secondly the specific lipase assays served as a gold

standard and the diagnostic performance of the conventional tests and test combinations were assessed.

The kappa values in dogs when the biochemical assays were compared with ultrasound results were fair and

varied between 0.31 for the cPL and 0.33 for lipase at a cutoff at 125U/L. Agreement between cPL and

conventional lipase activity and cPL and in combination of positive Ultrasound and conventional lipase activity

was good ranging between 0.63 up to 0.77.

When ultrasound results were selected as the gold standard the conventional lipase assay showed a sensitivits or

0.51 and a specificity of 0.8 compared with a sensitivity of 0.45 and a specificity of 0.84 for cPL.

When cPL was chosen as the gold standard, ultrasound alone correctly classified 67.2%, the conventional lipase

assay at a cutoff of 125U/L 90.2%, at 250 U/L again 90.2% of cases; The combination of ultrasound and lipase

activities at 125 U/L correctly classified 86% of cases, and the combination at 250U/L 87.1% of cases.

The kappa vaules in cats when the biochemical assays were compared with ultrasound results were poor in the

case of fPL at the threshold to fair (0.21). The kappa for fPL and conventional lipase at a cutoff of 30U/L and a

cutoff of 50/L was good, and for a cutoff of 100U/L only fair (0.29). The kappa for fPL versus positive

ultrasound and positive lipase above 30U/L was good, for the combination at a cutoff of 50U/L only moderate

(0.53) and poor when the cutoff for lipase was set at 100 U/L.

When ultrasound changes were selected as the gold standard the sensitivity and specificity of fPL was 0.375 and

0.864 respectively and for conventional lipase at a cutoff of 30U/L 0.344 and 0.731 respectively.

When fPL was chosen as the gold standard ultrasound alone correctly classified 58.6%, the conventional lipase

assay at a cutoff of 30U/L 85.7%, at 50U/L again 85.7% and at a cutoff of 100 U/L only 78.6%; A combination

of ultrasound and lipase at 30 U/L correctly classified 86.2%, at 50U/L 84.5% and at 100 U/L 75.9% of cases.

According to these findings the inclusion of a conventional lipase assay in first opinion consultations to screen

for acute pancreatitis might be a useful cost effective tool.

References

Banerjee, A. K., S. W. Galloway, et al. (1994). "Experimental models of acute pancreatitis." British Journal of

Surgery 81(8): 1096-1103.

de Arespacochaga, A. G., K. M. Hittmair, et al. (2006). "Comparison of Lipase Activity in Peritoneal Fluid of

Dogs with Different Pathologies – A Complementary Diagnostic Tool in Acute Pancreatitis?" Journal

of Veterinary Medicine Series A 53(3): 119-122.

De Cock, H. E. V., M. A. Forman, et al. (2007). "Prevalence and Histopathologic Characteristics of Pancreatitis

in Cats." Veterinary Pathology Online 44(1): 39-49.

Forman, M. A., S. L. Marks, et al. (2004). "Evaluation of Serum Feline Pancreatic Lipase Immunoreactivity and

Helical Computed Tomography versus Conventional Testing for the Diagnosis of Feline Pancreatitis."

Journal of Veterinary Internal Medicine 18(6): 807-815.

Gerhardt, A., J. M. Steiner, et al. (2001). "Comparison of the Sensitivity of Different Diagnostic Tests for

Pancreatitis in Cats." Journal of Veterinary Internal Medicine 15(4): 329-333.

Graca, R., J. Messick, et al. (2005). "Validation and diagnostic efficacy of a lipase assay using the substrate 1,2-

o-dilauryl-rac-glycero glutaric acid-(6′methyl resorufin)-ester for the diagnosis of acute pancreatitis in

dogs." Veterinary Clinical Pathology 34(1): 39-43.

Huth, S. P., R. Relford, et al. (2010). "Analytical validation of an ELISA for measurement of canine pancreas-

specific lipase." Veterinary Clinical Pathology 39(3): 346-353.

Jaensch, S. (2010). "Associations between serum amylase, lipase and pancreatic specific lipase in dogs."

Comparative Clinical Pathology: 1-4.

Liehmann, L. M., J. Dörner, et al. (2012). "Pancreatic rupture in four cats with high-rise syndrome." Journal of

Feline Medicine and Surgery 14(2): 131-137.

Newman, S., J. Steiner, et al. (2004). "Localization of Pancreatic Inflammation and Necrosis in Dogs." Journal of

Veterinary Internal Medicine 18(4): 488-493.

Panthegini, M., Renze Bais, Wouter W. .can Solinge in Enzymes in Tietz Textbook of Clinical Chemistry and

Molceular Diagnostics 4th

Edition ed. by Car. A. Burtis, Edward R. Ashwood, David E.Bruns, Elsevier,

Saunders Inc. St. Lous, Missouri 2066; p 621-22

Ruaux, C. G. (2003). "Diagnostic approaches to acute pancreatitis." Clinical Techniques in Small Animal

Practice 18(4): 245-249.

Steiner JM, T. S., Lees GE, Willard MD, Williams DA, Ruaux CG (2009). "Stability of canine pancreatic lipase

immunoreactivity concentration in serum samples and effects of long-term administration of prednisone

to dogs on serum canine pancreatic lipase immunoreactivity concentrations." Am J Vet Res 70(8):

1001-1005.

TVan den Bossche, D. P., S. Daminet (2010). "Acute pancreatitis in dogs and cats: pathogenesis, clinical signs

and clinicopathologic findings." Vlaams Diergeneeskundig Tijdschrift 79(1): 09.

62

Watson, P. (2004). "Pancreatitis in the dog:. dealing with a spectrum of disease." In Practice 26(2): 64-77.

Watson, P. J., A. J. A. Roulois, et al. (2007). "Prevalence and breed distribution of chronic pancreatitis at post-

mortem examination in first-opinion dogs." Journal of Small Animal Practice 48(11): 609-618.

Zini, E., M. Osto, et al. (2010). "Hyperglycaemia but not hyperlipidaemia decreases serum amylase and

increases neutrophils in the exocrine pancreas of cats." Research in Veterinary Science 89(1): 20-26.


COMPARATIVE ASPECTS OF THE ACUTE PHASE PROTEIN RESPONSE

P.D. Eckersall. Institute of Infection, Immunity & Inflammation and the School of Veterinary Medicine,

University of Glasgow, Bearsden Rd., Glasgow G61 1QH, UK


The acute phase protein (APP) response has been studied in humans for over 50 years. Though the study of these

proteins in domestic animals has been less intensive, they have been the object of research and diagnostic use for

nearly 20 years. Although a lot of progress has been achieved in the development of assays for their

measurement and in application to clinical pathology, there has been limited investigation into one of the

fundamental features of the comparative biology of the acute phase protein reaction.

Since the initial discovery of the acute phase nature of C-reactive protein (CRP) in man it has been evident that

there are notable between species variations in the biochemistry and pathophysiology among the APP. Thus,

although it is well established that APP can be split into major (increasing ~100 fold) moderate (increasing up to

10 fold), minor (increasing 2 fold) and negative (decreasing) APP, there are clear differences between species in

the identity of APP in each group.

It is known that in cattle and other ruminants the major APP are haptoglobin (Hp) and serum amyloid A (SAA)

with moderate APP being α1-acid glycoprotein (AGP), lipid binding protein (LBP) and ceruloplasmin (Cp). In

pigs, CRP and SAA are major APP, with pigMAP (inter α-trypsin inhibitor H) being a major/moderate reacting

protein. In pigs AGP has given equivocal results as an APP, possibly as the serum concentration of this protein

is high at birth in piglets and takes some time to reduce to the resting level of a healthy animal.

In dogs the major APP, as in humans, is CRP with Hp, AGP and Cp being a moderate reactants. A particularly

noticeable species difference in the dog is that Hp is not only an APP but an increase in serum concentration is

also stimulated by cortisosteroids, whether by endogenous as in Cushing's syndrome or exogenous material as in

steroid therapy. SAA is a major APP and AGP is a moderate APP in dogs. Cats are another species where CRP

does not act as a major APP while AGP, Hp and SAA have been shown to give an APP response to infection and

inflammation. Horses have a particularly strong SAA response with Hp concentration in serum also increasing

during an acute phase response.

In species that have been less well studied APP are also evident. In chickens ovotransferrin, Cp and a Hp-like

protein called PIT54 show APP activity with SAA also being reported as a major APP. In fish such as the

Atlantic salmon, Cp and Hp give APP responses while there are also reports that CRP is present.

Given the differences between species of the APP responses, there are two, connected major questions which

have not been satisfactorily answered. Why should there be such a diversity between species in the APP

response and why would a protein be a major APP in one species but a moderate or minor APP in another

species of animal?

These can be illustrated by consideration of the pathophysiology of three of the APP with significant species

differences. C-reactive protein is a major APP in man, dog and pig but is not an APP or is a moderate reacting

protein in species such as cat, horse and cow. Haptoglobin is a moderate APP in many species but is a major

reactant in ruminants. Serum amyloid A is a major APP in all species with the only exception being the rat

which does not have this serum protein.

In the case of Hp in ruminants, the protein structure includes an extra sequence on the α-haptoglobin chain which

has a free cysteine group allowing the formation of cys-cys disulphide bonds between Hp tetramers and the

formation of the large polymeric form (>106 Da) of Hp found in cattle and sheep. Potentially there is a

disadvantage to the circulation of such large protein complexes in a healthy animal which would direct toward

the evolution of an APP with a major response during disease but minimal production in the absence of disease.

For SAA, it is understandable why this protein would be a major APP as it is known to be the precursor of

amyloid A deposits found in alpha amyloidosis. Therefore a continuous production of the protein could lead to

an evolutionary unfavourable pathological or fatal condition. If a similar evolutionary driver is the cause of the

acute phase response of CRP it is necessary to identify why a continuous high level of this protein in serum

would be detrimental to health in relevant species such that it only being produced during disease. In humans

there are associations between cardiovascular disease and moderately elevated serum CRP, but during an

evolutionary timescale the over nutrition needed to develop atherosclerotic diseases has not been prevalent in the


63

human population. It is more likely that the evolutionary stimulus has derived from the functions and activities

of the protein. C-reactive protein has a high affinity for phosphorylcholine, the phospholipid present in plasma

membranes. This phospholipid is at a much lower abundance in erythrocyte membranes from ruminants than in

membranes from other species and it is suggested that the response of the erythrocyte to bacterial toxins may

depend on the membrane phospholipid composition. Though this may not be the key factor in species variation

of the APP it does open up avenues for investigation and leads to the hypothesis that:

C-reactive protein has a pathological impact in species such as dog and pig where it exhibits a major APP

response which is not present in the cow or horse where it is at best a minor APP.

SERUM AMYLOID A AS BIOMARKER OF SUBCLINICAL INFLAMMATION IN CATTLE

M. Kovačević Filipović. Department of pathophysiology,Faculty of Veterinary Medicine, University of

Belgrade, Belgrade, Serbia


Subclinical inflammation in cattle: Subclinical inflammation of different etiology elicit an neuroendocrine-

metabolic response including activation of sympathetic neuronal pathways, sympathetic-adrenomedullary and

hypothalamic-pituitary-adrenal axis, progressing in acute phase response that further induces endocrine,

metabolic and immune system disturbances. In its chronic subclinical form these disturbances cause direct and

indirect losts in farm animal production systems. In dairy industry, subclinical mastitis is one of leading causes

of chronic inflammation. It is characterised with local reaction evident through changes in milk composition, but

subclinical systemic response is also present (Eckersall et al., 2001). Production diseases such as subacute

ruminal acidosis (SARA) and fatty liver could also be a reason for persistent or temporary induction of low level

subclinical inflammation (Gerloff et al, 1986, Zebeli et al., 2010). We propose that in experimental setting,

serum amyloid A (SAA) concentration and isoform pattern in combination with other analysis could be helpfull

in monitoring specific subclinical/clinical disease progression, remission or recovery.

Acute phase proteins (APPs): Only few hours after tissue injury, hepatocytes stimulated with glucocorticoids

and proinflamatory cytokines, enhance the synthesis of more than several dozens of APPs (mainly globulins),

even in expense of albumin synthesis (Eckersall and Conner, 1988). The fast synthesis of large amount of

different APPs demonstrate their importance in body defense including positive and negative regulation of

inflammatory response as well as tissue protection from excessive or inappropriate inflammatory effects. Major

APPs in bovine are haptoglobin (Hp), serum amyloid A, lipopolisaharid binding protein and α 1 acid

glicoprotein. Multiple roles for every APP have been demonstrated. Recent literature is abundant with

description on variety of different putative SAA functions. Yet, the main recognised SAA function is regulation

of cholesterol reutilisation during inflammation, when large amount of cells die and injured tissues have to be

remodeled (rev. Kisilevsky et Manley, 2012). On the other side, it is proposed that large amount of SAA 3,

isoform synthesized by mammary gland and secreted in colostrum, have a role in protection of mucosal barrier

in new born (Mack et al, 2003) and antibacterial role in infected mammary gland (Molenaar et al., 2009). In last

decade it became clear that a spectrum of different cells could be stimulated to synthesize specific isoforms of

SAA. This could be the mechanism that promptly provide proteins of interest at the site of tissue injury.

SAA as a marker of inflammation: During severe acute inflammation (example: severe clinical mastitis) 2,5%

of total protein production in liver is SAA (rev. Kisilevsky et Manley, 2012), increasing SAA concentration up

to 1000 times from baseline values (1-3µg/mL to 2500 µg/mL). Except in few recent publications, it is

considered that raise in SAA is fast, but non specific to etiology of tissue injury. In bovines, SAA raise has dose

dependent manner when different amounts of lipopolysaccharide are injected intravenously (Jacobsen et al.,

2004). In cases of subclinical mastitis, plasma SAA is higher when two quarters were infected comparing to one

quarter infection, reflecting magnitude of inflamed or injured mammary gland tissue (Kovačević Filipović et al.,

2010). In Table 1, serum and milk SAA concentrations found in cattles affected with various diseases (mostly

with different forms of mastitis), are summarized. It could be noticed that in average, subclinical inflammation is

characterized with 10 times, while experimental as well as spontaneous clinically manifested diseases are

characterized with few hundred times higher plasma SAA concentrations than baseline. Yet, high individual

variation after challenge with same dose of LPS are found (Jacobsen et al., 2004).

Table 1.

SAA isoforms: SAAs are very small proteins (12-14kDa), encoded by multiple genes. In human and mice, clear

classification and nomenclature for isoforms and its allelic variations have been done (Sipe et al., 1999). In

humans SAA1 and SAA2 genes code for acute phase proteins SAA1 and SAA2 synthesized by liver (rev.

Kisilevsky et Manley, 2012) and SAA1 seems to be predominant constitutive and inducible form (Xu et al,

2006). It is interesting that very recent publication suggests that elevated SAA2 is a biomarker for lung

adenocarcinoma in humans (Sung et al., 2012), demonstrating a possibility that SAA isoforms could be used as

64

specific biomarkers of disease. Constitutive and inducible bovine plasma SAAs are produces in hepatocytes.

They are heterogeneous in their amino acid sequences, molecular weight and isoelectric points - pI values

(Westermark et al., 1986; Horadagoda et al., 1993; Alsemgeest et al., 1995). Inducible bovine SAA isoforms

synthesized extrahepatically in mammary epithelial cells (Kho et al., 2000), abomasal epithelial cells (Dilda et al,

2011) and adipocytes (Mukesh et al., 2001) are designated SAA3. Hepatic isoform isolated by Rossevatin et al.,

(1992) and mammary isoform isolated by Kho et al., (2000) have 60% of amino acid homology. Our group and

others detected in healthy cattle plasma/serum, isoforms with pI 7.4 and pI 6.8 (Jacobsen et al., 2005; Orro et al.,

2008, Kovačević Filipović et al., 2012). Analysis of literature data reveal that acute and chronic inflammation

augment the number of isoforms that appear in plasma (Jacobsen et al., 2005, Takahashi et al., 2009, Kovačević

Filipović et al., 2012). Also, it seems that chronic inflammation (example Staphy. aureus subclinical or clinical

mastitis) induce appearance of more acidic isoforms in plasma (Kovačević Filipović et al., 2012). If this

observation is going to be confirmed, using SAA concentration and isoform pattern, it will be possible to make

discrimination between acute and chronic inflammatory process, and to monitor acute phase reaction to specific

disease.

References

1. Alsemgeest SP, Haradagoda A, Hulskamp-Koch CK, Tooten PC, Kim DH, Niewold TA, Gruys E. First

evidence for the existence of multiple isoforms of bovine amyloid-A (apoSAA). Scand. J. Immunol.

1995; 41: 407-413.

2. Dilda F, Pisani LF, Rahman MM, Modina S, Tessaro I, Sartorelli P, Ceciliani F, Lecchi C. Distribution

of acute phase proteins in the bovine forestomachs and abomasum. Vet. J. 2011; 192: 101-105

3. Eckersall PD, Conner JG. Bovine and canine acute phase proteins. Vet Res Commun. 1988; 12: 169-78.

4. Eckersall PD, Young FJ, McComb C, Hogarth CJM, Safi S, Weber A, McDonald T, Nolan AM,

Fitzpatrick JL. Acute phase proteins in serum and milk from dairy cows with clinical mastitis. Vet. Rec.

2001; 148: 35-42.

5. Eckersall PD, Young FJ, Nolan AM, Knight CH, McComb C, Waterston MM, Hogarth CJ, Scott EM,

Fitzpatrick JL. Acute phase proteins in bovine milk in an experimental model of Staphylococcus aureus

subclinical mastitis. J.Dairy Sci. 2006; 89: 1488-1501.

6. Gerardi G, Bernardini D, Azzurra EC, Ferrari V, Iob L, Segato S. Use of serum amyloid A and milk

amyloid A in the diagnosis of subclinical mastitis in dairy cows. J Dairy Res. 2009; 76: 411-417.

7. Gerloff BJ, Herdt TH, Emery RS.Relationship of hepatic lipidosis to health and performance in dairy

cattle. J Am Vet Med Assoc. 1986; 188: 845-50.

8. Horadagoda A, Eckersall PD., Alsemgeest SP, Gibbs HA. Purification and quantitative measurement of

bovine serum amyloid-A. Res. Vet. Sci. 1993; 55: 317-325.

9. Jacobsen S, Andersen PH, Toelboell T, Heegaard PM. Dose dependency and individual variability of

the lipopolysaccharide-induced bovine acute phase protein response. J. Dairy Sci. 2004; 87: 3330-3339.

10. Jacobsen S, Niewold TA, Kornalinalijnslijper E, Toussaint MJM, Gruys E. Kinetics of local and

systemic isoforms of serum amyloid A in bovine mastitis milk. Vet. Immunol. Immunopathol. 2005;

104, 21-31.

11. Kho YJ, Cho KK, Kim SC, Kim SH, Chung MI, Baik MG, Choi YJ. Rapid communication: cloning of

bovine serum amyloid A3 cDNA. J Anim Sci. 2000; 78: 2756-2757.

12. Kisilevsky R, Manley PN. Acute-phase serum amyloid A: perspectives on its physiological and

pathological roles. Amyloid. 2012; 19:5-14.

13. Kovačević Filipović M, Stevanović J, Stevanov Pavlović M, Debeljak Martačić J, Knežević M,

Mijačević Z, Božić T. Acute phase protein response in cows with Staphylococcus aureus subclinical

mastitis. Acta Vet. Beograd; 2010; 60: 205-216.

14. Kovačević-Filipović M, Ilić V, Vujčić Z, Dojnov B, Stevanov-Pavlović M, Mijačević Z, Božić T.

Serum amyloid A isoforms in serum and milk from cows with Staphylococcus aureus subclinical

mastitis. Vet Immunol Immunopathol. 2012; 145: 120-8.

15. Larsen T, Røntved CM, Ingvartsen KL, Vels L, Bjerring M. Enzyme activity and acute phase proteins

in milk utilized as indicators of acute clinical E. coli LPS-induced mastitis. Animal. 2010;4: 1672-9.

16. Mack DR, McDonald TL, Larson MA, Wei S, Weber A. The conserved TFLK motif of mammary-

associated serum amyloid A3 is responsible for up-regulation of intestinal MUC3 mucin expression in

vitro. Pediatr. Res. 2003; 53: 137-142.

17. Molenaar AJ, Harris DP, Rajan GH, Pearson ML, Callaghan MR., Sommer L, Farr, VC, Oden KE,

Miles MC., Petrova RS, Good LL, Singh K, McLaren RD, Prosser CG, Kim KS, Wieliczko RJ, Dines

MH, Johannessen KM, Grigor MR, Davis SR, Stelwagen K. The acute-phase protein serum amyloid A3

is expressed in the bovine mammary gland and plays a role in host defence. Biomarkers. 2009; 14: 26-

37.

65

18. Mukesh M, Bionaz M, Graugnard DE, Drackley JK, Loor JJ. Adipose tissue depots of Holstein cows

are immune responsive: inflammatory gene expression in vitro. Domest. Anim. Endocrinol. 2010; 38:

168-178.

19. Müller-Doblies D, Arquint A, Schaller P, Heegaard PM, Hilbe M, Albini S, Abril C, Tobler K,

Ehrensperger F, Peterhans E, Ackermann M, Metzler A. Innate immune responses of calves during

transient infection with a noncytopathic strain of bovine viral diarrhea virus. Clin Diagn Lab Immunol.

2004;11: 302-12.

20. Nielsen BH, Jacobsen S, Andersen PH, Niewold TA, Heegaard PM. Acute phase protein concentrations

in serum and milk from healthy cows, cows with clinical mastitis and cows with extramammary

inflammatory conditions. Vet Rec. 2004;154 :361-5.

21. Orro T, Jacobsen S, LePage JP, Niewold T, Alasuutari S, Soveri T. Temporal changes in serum

concentrations of acute phase proteins in newborn dairy calves. Vet J. 2008; 176: 82-87.

22. Pyörälä S, Hovinen M, Simojoki H, Fitzpatrick J, Eckersall PD, Orro T. Acute phase proteins in milk in

naturally acquired bovine mastitis caused by different pathogens.Vet. Rec. 2011; 168: 535-542.

23. Simojoki H, Orro T, Taponen S, Pyörälä S. Host response in bovine mastitis experimentally induced

with Staphy. chromogenes. Vet Microbiol. 2009; 16: 95-99.

24. Sipe J. Revised nomenclature for serum amyloid A (SAA). Nomenclature Committee of the

International Society of Amyloidosis. Part 2. Amyloid. 1999; 6 :67-70.

25. Sung HJ, Jeon SA, Ahn JM, Seul KJ, Kim JY, Lee JY, Yoo JS, Lee SY, Kim H, Cho JY. Large-scale

isotype-specific quantification of Serum amyloid A 1/2 by multiple reaction monitoring in crude sera. J

Proteomics. 2012; 75: 2170-80.

26. Suojala L, Orro T, Järvinen H, Saatsi J, Pyörälä S.Acute phase response in two consecutive

experimentally induced E. coli intramammary infections in dairy cows. Acta Vet Scand. 2008; 50: 18-

28.

27. Takahashi E, Kuwayama H, Kawamoto K, Matsui T, Inokuma H. Detection of serum amyloid A

isoforms in cattle. J. Vet. Diagn. Invest. 2009; 21: 874-877.

28. Westermark P, Johnson KH, Westermark GT, Sletten K. and Hayden DW. Bovine amyloid protein AA:

isolation and amino acid sequence analysis Comp. Biochem. Physiol. B. 1986; 85: 609-614.

29. Xu Y, Yamada T, Satoh T, Okuda Y. Measurement of serum amyloid A1 (SAA1), a major isotype of

acute phase SAA. Clin Chem Lab Med. 2006; 44 :59-63.

30. Zebeli Q, Dunn SM, Ametaj BN. Strong associations among rumen endotoxin and acute phase proteins

with plasma minerals in lactating cows fed graded amounts of concentrate. J Anim Sci. 2010; 88: 1545-

53.

THE USE OF CART ALGORITHMS TO COMBINE SERUM ACUTE PHASE PROTEIN LEVELS AS

A DIAGNOSTIC AID IN CANINE LYMPHOMA

I. Alexandrakis. PetScreen Ltd. Biocity Nottingham, Pennyfoot Street, Biocity, NG1 1GF, Nottingham, United

Kingdom.


Background. Identification of some of the biomarkers used in the original Lymphoma Blood test confirmed a

role for acute phase proteins (APPs) in the detection of lymphoma in dogs. However, it has long been recognised

that measurement of signal alone does not give sufficient test performance due to poor specificity. However,

measuring multiple APPs and combining the results in an analytical algorithm could dramatically improve

performance.

Objectives. To determine the efficacy of algorithms using Classification and Regression Tree (CART) analysis

which combine the measurement of two APPs, Haptoglobin (HAPT) and C-Reactive Protein (CRP), for the

detection of lymphoma in dogs.

Dogs. A sample test set of 194 dogs comprising 64 diagnosed with lymphoma, 51 healthy and 79 other

conditions which commonly present with symptoms similar to lymphoma.

Methods. Serum samples were collected from dogs undergoing differential diagnosis for lymphoma. Lymphoma

positive samples were confirmed by either FNA or excisional biopsy. Non-lymphoma dogs were confirmed to be

free of the disease at a minimum of six months after providing the serum sample.

Results. By combining serum HAPT and CRP levels using a specifically designed algorithm, it was possible to

differentiate between dogs with lymphoma and other diseases with great precision giving specificity of 93% and

sensitivity of 85%. By comparing lymphoma and healthy dogs only, the test showed 100% specificity.

Conclusions. Combining canine serum APP levels using CART analysis enables differentiation between dogs

diagnosed with lymphoma and other commonly encountered conditions in differential diagnosis with high levels

of diagnostic significance.

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Nielsen%20BH%22%5BAuthor%5D

66


HAEMOTROPIC MYCOPLASMA INFECTION IN CATS AND DOGS

R. Hofmann-Lehmann1, M. Novacco

1, J. Baumann

1, B. Willi

1,2, F. S. Boretti

2

1 Clinical Laboratory, Vetsuisse Faculty, University of Zurich, Switzerland.

2 Clinic for Small Animal Medicine,

Vetsuisse Faculty, University of Zurich, Switzerland.


In recent years, there has been a growing interest in haemotropic mycoplasmas, also known as haemoplasmas.

Haemotropic mycoplasmas are small, cell wall-less, discoid-shaped bacteria, closely attached to red blood cells

(RBC) of infected animals. They were formerly known as Haemobartonella and Eperythrozoon species and are

the causative agents of infectious anaemia in a variety of mammalian species. Infections with haemoplasmas

can induce acute haemolysis, associated with anorexia, lethargy, dehydration, weight loss and sudden death of

infected animals. The inability to culture these agents in vitro has limited the possibilities to investigate

haemotropic mycoplasmas. However in recent years, species-specific conventional and real-time TaqMan PCR

assays have been developed for sensitive detection of feline and canine haemoplasmas. In addition, for

experimental studies, a serological assay for the detection of anti-haemoplasma antibodies was developed. The

application of these assays has been allowing investigating the pathogenesis and epidemiology of haemoplasma

infections in cats and dogs in more detail.

Haemotropic mycoplasmas in cats

Early studies described two distinct feline haemoplasma species: the Ohio and the California isolate of

Haemobartonella felis. Along with the reclassification of these bacteria within the genus Mycoplasma, these

isolates were renamed Mycoplasma haemofelis (Mhf) and ‘Candidatus Mycoplasma haemominutum’

(CMhm). In 2002, a third haemotropic Mycoplasma species was identified in a privately owned Swiss cat that

presented with haemolytic anaemia; this third species was designated ‘Candidatus Mycoplasma turicensis’

(CMt). By applying PCR-based methods, haemoplasma infections have been diagnosed in domestic cats and

wild felids worldwide. Co-infections with several feline haemoplasmas were reported.

The pathogenic potential of the different feline haemoplasma species varies and co-factors, such as

immunosuppression or pre-existing retroviral infections, may increase the disease severity. In general, Mhf was

found more pathogenic than CMhm. In naturally infected cats, risk factors for feline haemoplasma infections,

such as male gender, old age, cat bite abscesses, retroviral infection, non-pedigree lineage and outdoor access

have been identified. The clinical presentation may depend on the stage of infection. Overall it is not well

understood why some cats show life-threatening anaemia, while others stay asymptomatic despite high blood

loads.

Doxycycline, enrofloxacin and marbofloxacin treatment have been shown to reduce feline haemoplasma blood

loads and clinical signs in infected cats. To date, it is assumed unlikely that antibiotic treatments completely

eliminate haemoplasma infections and infected cats may become asymptomatic carriers for months or years.

However, reactivation of clinical disease seems rare

Haemotropic mycoplasmas in dogs

Haemobartonella canis infections associated with anaemia have been sporadically reported in dogs.

Subsequently, H. canis was reclassified as Mycoplasma haemocanis (Mhc), and a second canine haemoplasma,

‘Candidatus Mycoplasma haematoparvum’ (CMhp), has been described in an anaemic splenectomised dog

undergoing chemotherapy. Both agents seem to exhibit worldwide distribution. In some populations young

animals and male dogs seemed more susceptible to canine haemoplasma infections than adult and female dogs,

respectively. Severe haemolytic anaemia has only occasionally been described in haemoplasma-infected dogs,

mainly in immune-compromised or splenectomized animals. Most haemoplasmas infected dogs present with

chronic, asymptomatic infections; these animals seem unable to clear the infection. As described for other

haemoplasma infections, antibiotic treatment may be unable to eliminate canine haemoplasma infections

completely, but was found to reduce clinical signs of infection.

Transmission

The natural mode of transmission of feline and canine haemoplasmas has not been definitely elucidated. Blood

transfusions have been reported as a source of Mhf and CMhm infections. Furthermore, blood-sucking

arthropods may be involved in the transmission of feline and canine haemoplasmas. The brown dog tick,

Rhipicephalus sanguineus, is likely to play a role as a vector and reservoir for canine haemoplasmas. In Europe,

the brown dog tick is commonly encountered in areas with Mediterranean and sub-Mediterranean climate and

the high prevalence of canine haemoplasma infections found in these countries supports the hypothesis of it


67

being a possible tick vector for the transmission of the infection. CMt, CMhm and Mhf were detected by PCR in

saliva of infected cats indicating that direct transmission of haemoplasmas via saliva may be important.

However, a recent in vivo study that modelled CMt transmission via social contact among cats was unable to

infect cats by subcutaneous or oral inoculation of CMt PCR-positive saliva. In contrast, in further studies

transmission by subcutaneous inoculation of as little as 10 µl of PCR-positive blood or 1’000 copies of CMt or

Mhf was successful in transmitting the infection; this indicates that aggressive interaction but not social contact

is sufficient to transmit CMt and Mhf between cats. Therefore, to prevent haemoplasma transmission, strict flea

and tick control and avoidance of aggressive interactions is recommended. Moreover, blood donors should be

tested by for haemoplasma infections prior to blood transfusion.

Diagnosis

Specific conventional and quantitative real-time TaqMan PCR assays have been introduced and are now

considered the gold standard for the detection and differentiation of feline and canine haemoplasma species. As

important differences exist in the pathogenic potential of the three feline haemoplasma species, a species

differentiation by molecular methods is pivotal. Of note, molecular methods are unable to distinguish between

acute and chronic infections. PCR assays are also applied to investigate the pathogenesis and haemoplasma

tissue loads in experimental studies. No in vitro culture system has been established to date to propagate feline

and canine haemoplasmas. The light microscopic investigation of Giemsa-stained blood smears from infected

animals was shown unreliable to diagnose haemoplasma infections. A diagnostic sensitivity of less than 20% has

been reported for this method, and the diagnostic specificity is often hampered by confusing organisms with

stain precipitates or Howell-Jolly bodies. In particular, light microscopy is unfit to diagnose CMt infection

because of the usually low CMt blood loads. Even at peak CMt bacteraemia only one CMt copy per 103

to 104

RBC may be expected, a number that is undetectable by routine light microscopy. Most recently, we were able

for the first time to demonstrate CMt using electron microscopy. The discoid-shaped CMt organisms measure

about 0.3 μm in diameter and are closely attached to RBC of infected cats. Serological assays are not yet

routinely available but have been used in experimental studies.

Prevalence and importance in Europe

Feline haemoplasma infections were found quite frequently in all populations and countries investigated by

PCR in Europe. In contrast, canine haemoplasma infections are more prevalent in countries with a

Mediterranean climate than in Switzerland. Infected dogs in Switzerland had either been imported from or

visited regions where R. sanguineus is indigenous. This observation supports the hypothesis that canine

haemoplasmas may be indirectly transmitted by blood-sucking arthropods, in particular those that rely on a

warm climate for their survival. In a recent study on the situation in Europe, we identified several risk factors for

canine haemoplasma infections, e.g. living in kennels, young age, and non-pedigree lineage. A higher prevalence

of canine haemoplasma infections in kennel-kept dogs may be due to the fact that dogs in kennels are group

housed, which could increase the risk of direct haemoplasma transmission among dogs and their risk of exposure

to fleas and ticks.

The close relationship between CMt and rodent haemoplasmas as well as among certain feline and canine

haemoplasmas suggests a potential interspecies transmission of these agents. While CMt has not yet been

detected in rodents, CMhm infection has been reported in a dog in China. Moreover, Mhf has recently been

detected by PCR in the blood of a human AIDS patient, and PCR-positive results for porcine haemoplasma-like

organisms have been obtained from blood of Chinese farm workers and swine veterinarians. This substantiates

the risk of an interspecies transmission of haemoplasmas. Nevertheless, so far the zoonotic potential of

haemoplasma infections has been largely neglected. In addition, recently, the first human hemoplasma species

(‘Candidatus M. haemohominis’) has been described. The isolate showed high similarity with the feline CMt.

The infection with ‘Candidatus M. haemohominis’ was associated with clinical signs of hemoplasmosis. Further

studies are required to characterize the epidemiology and clinical significance of this infection.

New data from experimental studies

Until recently, little was known about the humoral immune response in haemoplasma infected cats. Enzyme-

linked immunoassays (ELISA) were developed based on a recombinant protein to monitor the antibodies to

hemoplasmas. Using this ELISA, seroconversion was demonstrated in all cats experimentally infected with CMt,

Mhf and CMt. In some cats, the ELISA was more sensitive and better able to demonstrate exposure to CMt than

PCR. The peak antibody level occurred after the peak of the bacterial blood loads. Antibiotic treatment,

particularly with doxycycline, resulted in a decrease in antibody levels. Following the CMt-infected cats over

time, remarkably, it was found that the antibodies remained detectable for many months, while the PCR from

blood was negative. The persistence of anti-feline hemoplasma antibodies in the absence of detectable

bacteraemia was associated with low levels of CMt detectable by PCR in the tested tissues of the chronically

68

infected cats (kidney, liver, bone marrow and salivary glands). No reactivation of the bacteraemia took place in

these cats, when they underwent immune suppression. Moreover, the seropositive cats were protected from

reinfection as measured by absence of detectable bacteraemia after a repeated CMt exposure. But no cross-

protection against the more pathogenic Mhf was found. In contrast, cats that had recovered from a CMt

infection became faster Mhf-positive than naïve cats, although they stayed CMt-negative. This observation may

contribute to explain the diverse outcomes of Mhf infections observed in individual domestic cats. Further

studies investigating the pathogenesis and immune response in more depth (clinical chemistry, cytokine

response, blood cell subsets) are underway.

In parts adapted from:

Willi B, Novacco M, Meli M, Wolf-Jäckel G, Boretti F, Wengi N, Lutz H, Hofmann-Lehmann R. Haemotropic

mycoplasmas of cats and dogs: transmission, diagnosis, prevalence and importance in Europe. SAT

2010;152:237-244.

TICK-BORNE DISEASES IN DOGS IN SLOVENIA

N. Tozon. Small Animal Clinic, Veterinary Faculty, University of Ljubljana


More than a half (58.5%) of the territory of Slovenia is covered by forest, which places it in the third place in

European Union, after Sweden and Finland. Slovenia also has very high landscape as well as the biological and

climate diversity, varying from the continental through sub-Mediterranean to the Alpine climate. These facts

make the living conditions for the ticks highly favourable and the country an endemic region for a number of tick

transmitted diseases. The most important and wide-spread tick is Ixodes ricinus (I. ricinus). Besides, about 16

different species of ticks were identified in Slovenia less frequently. The brown dog tick Ripiciphalus

sanguineus (R. sanguineus) could be found in very limited areas in the southern part of Slovenian border with

Croatia (Trilar T, 2004).

Lyme borreliosis is recognized as one of the most important tick-borne diseases in humans worldwide. Its

prevalence in human population in Slovenia is with 245 cases/100.000 one of the highest in the world.

According to the same epidemiological studies, dogs are sensitive indicators of the Lyme borreliosis prevalence

because of a very high tick exposure (Stone et al., 2005). Although about 50% of dogs in the endemic areas

could be infected, only 5-10% of them develop clinical signs of borreliosis (Fritz et al, 2009). So far, no

epidemiological or clinical studies in dogs have been performed in Slovenia, but the results from the Institute of

microbiology of Veterinary Faculty demonstrate expected high seroprevalence in dogs in our country and the

percentage is still growing. Between 2007 and 2011, 1557 dog sera were examined for the presence of B.

burgdorferi specific IgG antibodies using IFA and 558 (35.8%) sera were found positive (cut-off ≥ 1:100). In

2011, the percentage of seropositive dogs has raised to 59.3% of dogs with suspected borreliosis (Gruntar I,

2012). In the study published in 2002, 599 human isolates from blood and other different patient’s tissue samples

were established to be B. burgdorferi sensu latu: 85% of them were B. afzelii, 14% B. garinii and 1% B.

burgdorferi sensu stricto,. In addition, the B. afzelii strains were unique, while heterogenity was found within B.

garinii and B. burgdorferi sensu stricto which may play a significant role in the virulence and pathogenesis of

the infection (Ružić-Sabljić E et al, 2002). There is no report of molecular or bacterial culture diagnosis of

borreliosis in dogs in Slovenia and we consider clinical diagnostic criteria in correlation with level of IgG titre

using IFA.

Anaplasmosis, caused by Anaplasma phagocytophilum (A. phagocytophilum) is the second most important

zoonosis and probably the most important tick-borne disease in dogs in Slovenia. Prevalence of bacterial agent in

I. ricinus ticks is significantly different between countries, ranged from zero to more then 30%. Majority of

human clinical cases came from Central Europe (Slovenia) and Scandinavia (Sweden) (Sterle, 2004). In the

years 2005 and 2006 the prevalence of ticks I. ricinus from 8 different locations in Slovenia, infected with A.

phagocytophilum, was 0.31% and 0.63%, respectively, without significant differences between locations

(Smrdel, 2010). In dogs from Slovenia, two variants of A. phagocytophilum were identified by PCR and

sequence analysis of a part of the groESL operon (Strašek et al., 2009).

The seroprevalence of A. phagocytophilum and B. burgdorferi in dogs in Slovenia according to the results of

Institute of microbiology of Veterinary faculty is presented in the Table 1. Among 396 examined dog sera, 196

(49.5 %) samples were positive for A. phagocytophilum and 558/1557 dog sera were positive for B. burgdorferi

(35.8%) using IFA (Gruntar, 2012).


69

Table 1. Seroprevalence of A. phagocytophilum and B. burgdorferi in dogs in Slovenia (Gruntar, 2012;

Ravnik,2011 ).

year A. phagocytophilum B. burgdorferi

N of tested % positive N of tested % positive

2007 101 42.57 398 24.37

2008 125 52.80 477 30.40

2009 92 56.52 302 44.37

2010 40 45.00 198 37.37

2011 38 44.74 182 59.34

2007-2011 396 49.5 1557 35.8

1999 - 2011 754 73.8 113 30.1

In our retrospective study, we demonstrated 73.8 % (553/754) seroprevalence of A. phagocytophilum infection in

dogs in Slovenia. 5.3 % of the dogs were PCR positive. Co-infection with B. burgdorferi s.l. was confirmed in

30.1 % of the dogs (unpublished data).

From the statistical significance point of view, the infection with the H genetic variant, which infects both dogs

and humans, more frequently resulted in acute signs of the disease.

Increased incidence of human cases in Slovenia in 2007 (188.4/million), in 2008 (251.7/million) and 2009

(308.7/million) and decline of cases in 2010 coincide with the increasing and decreasing percentage of

seropositive dogs (Kraigher 2010).

Seroprevalence for anaplasmosis in dogs, tested at the Veterinary Faculty between 2007 and 2011 is comparable

with that in Germany (43-50%), Austria (56.5%) and Portugal (55%) (Gray JS et al, 2011). Seroprevalence in

dogs tested between 1999 and 2011 is much higher. The reasons for this distinction could be the much longer

period and probably different clinical and laboratory criteria, used as a basis to test the suspected patients for

specific antibody titre.

Tick-borne encephalitis (TBE) is endemic in Slovenia too and the incidence rate is one of the highest in the

European Union. According to the epidemiological study in human medicine, the northern and the central areas

of Europe had the highest risk of TBEV infection. I. ricinus is the principal vector of TBE in Slovenia. In the 2-

year study (2005-2006) the 0.47% prevalence of TBE infection was found in Slovenian ticks in six of eight

locations. In the same study they confirmed that the TBE in ticks is genetically related to the TBE in Slovenian

human patients (Durmisi E at al., 2011). Between 1999 and 2009, 2779 cases of TBE were notified in Slovenia

(Blasko-Markic M, 2012).There is still no evidence of clinical cases of TBE in dogs in Slovenia.

There is only one published study about prevalence of babesiosis in dogs in Slovenia. Based on the clinical,

microscopic and molecular investigation of 238 dogs from years 2000 to 2002, babesial parasites in blood were

determined in 14 dogs (5.9 %). Using PCR and subsequent sequence analysis of portions of nns rRNA gene, B.

canis parasite was confirmed. In addition, two subspecies were determined, namely B. canis canis in 4.6% and

B. canis vogeli in 1.3% (Duh D, 2004). Clinical cases of babesiosis in dogs examined at the Small Animal Clinic

of Veterinary Faculty in Ljubljana between 2000 and 2004 were studied retrospectively. The prevalence of

infection in this study, using the same diagnostic tools, was higher: 25 of 260 dogs (9.6%) were infected.

Described clinical signs and haematological profile were comparable with that described in the literature. In 10

of 19 dogs with complete clinical records the specific antibody for A. phagocytophilum were found (>1:128),

however, no A. phagocytophilum DNA could be detected in the selected patients. The results of this study

demonstrated that infection with B. canis canis alone or coinfection with A. phagocytophilum can result in mild

anaemia but severe thrombocytopenia, which could persist also for a longer period after treatment (2 of 5 dogs

remained anaemic and 4 of 5 thrombocytopenic). For this reason haematological alterations should be carefully

monitored during and after completion of treatment, especially in the endemic regions, like Slovenia (Tozon N,

unpublished data 2004).

It is important to follow epidemiological studies in the wide region, which suggest mostly increased prevalence

of already present pathogens and establish some new pathogen species for different host species, including

humans and dogs as well. One of such important studies is the study of prevalence of different pathogens in the

most important vector tick I. ricinus, because of the region selected, north-eastern region of Italy, lying closed to

the Slovenian border. They detected 11 pathogens in I. ricinus adults, collected from 2006 to 2008 in 31 sites;

furthermore 22% of ticks were co-infected with more than one pathogen. The most frequently detected

microorganism was Borelia bugrdorferi s.l. (17.6%), Rickettsia helvetica (13.1%), and “Ca. N. mikurensis”

(10.5%). Within the B. burgdorferi complex, four genotypes (i.e., B. valaisiana, B. garinii, B. afzelii, and B.

burgdorferi sensu stricto) were found. Less prevalent pathogens included R. monacensis (3.7%), TBE virus

(2.1%), A. phagocytophilum (1.5%), Bartonella spp. (1%), and Babesia EU1 (0.5%) (Capelli G et al, 2012).

70

References

Blasko-Markic M, Socan M. Tick-Borne Encephalitis in Slovenia: Data from a Questionnaire Survey.Vector

Borne Zoonotic Dis. 2012; 26.

Capelli G, Ravagnan S, Montarsi F, Ciocchetta S, Cazzin S, Porcellato E, Babiker AM, Cassini R, Salviato A,

Cattoli G, Otranto D. Occurrence and identification of risk areas of Ixodes ricinus-borne pathogens: a cost-

effectiveness analysis in north-eastern Italy. Parasit Vectors. 2012; 27(5):61.

Duh D, Tozon N, Petrovec M et al. Canine babesiosis in Slovenia : molecular evidence of Babesia canis and

Babesia canis vogeli. Vet res (Paris) 2004;3(35):363-368.

Durmisi E, Knap N, Saksida A, Trilar T, Duh D, Avšič-Županc T. Prevalence and molecular characterization of

tick-borne encephalitis virus in Ixodes ricinus ticks collected in Slovenia.Vector Borne Zoonotic Dis. 2011

Jun;11(6):659-64.

Fritz CL. Emerging tick-borne diseases. Vet Clin North Am Small Anim Pract. 2009; 39(2):265-78.

Gray JS, Dautel H, Estrada-Peña A, Kahl O, Lindgren E. Effects of climate change on ticks and tick-borne

diseases in Europe. Interdiscip Perspect Infect Dis 2009;593232.

Gruntar I. Serological diagnostics of lyme borreliosis and granulocytic anaplasmosis in dogs in slovenia: Results

and trends for a period 2007-2011. In: Zbornik referatov 25. simpozija o aktulanikh boleznih malih živali,

Portorož, 30. - 31. marec 2012: Ljubljana: SZVMŽ; 2012:54-56.

Kraigher A, Sočan M, Klavs I et al. Epidemiološko spremljanje nalezljivih boezni v Sloveniji v letu 2010.

Ljubljana: Inštitut za varovanje zdravja, 2011: 61.

Ružić-Sabljić E Maraspin V, Lotric-Furlan S, Jurca T, Logar M, Pikelj-Pecnik A, Strle F., Wien Klin

Wochenschr. Characterization of Borrelia burgdorferi sensu lato strains isolated from human material in

Slovenia. 2002; 3:544-50.

Smrdel KS, Serdt M, Duh D, Knap N, Zupanc TA. Anaplasma phagocytophilum in ticks in Slovenia. Parasit

Vectors. 2010; 4(3):102.

Stone EG, Lacombe EH, Rand PW. Antibody testing and Lyme disease risk. Emerg Infect Dis. 2005;11(5):722-

4.

Strle F. Human granulocytic ehrlichiosis in Europe.Int J Med Microbiol. 2004;293(Suppl 37):27-35.

Strašek K, Tozon N, Duh D, et al. Diversity of groESL sequences of Anaplasma phagocytophilum among dogs

in Slovenia. Clin microbiol and infect 2009;15(suppl.2):79-80;

Ravnik U, Tozon N, Strašek K et al. Anaplasmosis in dogs: the relation of haematological, biochemical and

clinical alterations to antibody titre and PCR confirmed infection. Vet microbiol 2011; 149 (1/2):172-176.

Tozon N, Duh D, Petrovec M et al. Babezioza pri psih - okuženost psov v Sloveniji z Babesio canis canis in

Babesio canis vogeli. In : Zbornik referatov III. strokovnega srečanja sekcije zasebnih veterinarjev praktikov

Veterinarske zbornice, Podčetrtek, Terme Olimia, 5. in 6. marec 2004

Trilar T. Ticks (Acarina: Ixodidae) on birds in Slovenia Ticks (Acarina: Ixodidae) on birds in Slovenia.

Acrocephalus 2004; 25 (123): 213 – 216.

MOLECULAR TECHNIQUES USED IN DIAGNOSIS AND CONTROL OF VETERINARY

INFECTIOUS DISEASES

M. Ocepek, I. Toplak, D. Kušar. Institute of Microbiology and Parasitology, Veterinary Faculty, University of

Ljubljana, Ljubljana, Slovenia


Introduction

Laboratory diagnostics of infectious diseases in the past was based on,e.g., immunological methods, isolation of

causative agents and determination of their morphology, phenotypic and biochemical characteristics,

determination of cytopathic effects and fungal conidiogenesis. The rapid development of molecular methods in

recent decades has brought almost revolutionary changes to the diagnostics and monitoring of infectious

diseases, since detection based on microbial nucleic acids (NA) bypasses many problems of classical techniques,

e.g., incompatibility with the demand for fast results, irreproducibility of results in relation to certain phenotypic

traits or inadequacy of the limit of detection. Molecular techniques, consequently, are nowadays an almost

indispensable tool for state-of-the-art diagnostics in routine clinical microbiological laboratories.

In comparison to traditional methods, molecular assays have several advantages, such as speed,

increased sensitivity and specificity, better discriminatory power of isolate typing techniques, direct detection

and quantification of viruses or uncultivable bacteria, and possible integration of these assays into automated

http://www.ncbi.nlm.nih.gov/pubmed?term=Capelli%20G%5BAuthor%5D&cauthor=true&cauthor_uid=22452970

http://www.ncbi.nlm.nih.gov/pubmed?term=Fritz%20CL%5BAuthor%5D&cauthor=true&cauthor_uid=19185193

http://www.ncbi.nlm.nih.gov/pubmed?term=Stone%20EG%5BAuthor%5D&cauthor=true&cauthor_uid=15890128


71

liquid-handling platforms. Some disadvantages are also recognized, such as cost, lack of discrimination between

live and dead microorganisms, inaccessibility or instability of microbial NA, and inability to discriminate

between expressed and non-active genes in diagnostic molecular studies. In order to achieve satisfactory results,

both the laboratory expert and the clinician should know the strengths and weaknesses of the various diagnostic

methods that can be used in clinical cases.With improvements in efficiency through automation, the role of

molecular diagnostic techniques will further increase. In general, molecular diagnostic methods are essential

when traditional methods generate poor results, i.e., false positives/negatives, or are of low sensitivity, when

intracellular pathogens such as viruses or mycoplasmas and slow-growing microbes such as mycobacteriaare

concerned, and when sub-typing is mandatory. However, special attention needs to be devoted to diagnostics

concerning toxins or resistance. Although employing molecular techniques, a pre-preparation step of cultivation

to pure cultures is essential, especially for resistance assays, since the presence of resistance- or sometimes also

toxin-codinggenes in clinical sample is not per sesufficient evidence for a conclusive diagnosis.

Molecular techniques in veterinary laboratories

When clinical material arrives at a diagnostic molecular laboratory, there are two possible routes, following, of

course, the procedure for extraction of NA – DNA or RNA: (i) detection of target microorganisms in clinical

material without prior processing or, in collaboration with a bacteriological laboratory, after overnight

enrichment in selective/non-selective nutrient broths or (ii) identification or confirmation of isolated

microorganisms. When target microorganisms are detected or, more commonly, identified from the obtained

isolates, their NA can be employed for additional procedures aimed at determination of selected traits, e.g.,

genes coding for toxins, antibiotic resistance and cell surface proteins or unique mutations and genomic

rearrangements, enabling more specific discrimination. In addition to approaching infectious diseases from a

microbial point of view, it is also possible to focus on the host, i.e., study its susceptibility to infectious diseases

by discovering genomic mutations making the host a pre-disposed candidate for the onset of the studied disease.

Molecular diagnostic methods

There are various kinds of molecular techniques for microbial detection or identification. The first group of

methods is represented by hybridization techniques. They are based on specifically labeled DNA or RNA probes,

which bind to complementary regions in microbial targets and therefore enable direct detection of specific

sequences or genes of isolated microorganisms. There are commercial tests available based on hybridization,

e.g., AccuProbe from Gen-Probe, targeting, e.g., mycobacteria, group B streptococci, Listeria monocytogenes,

Staphylococcus aureus, mycoplasmas or various fungal species. In addition to AccuProbe, a commercial test,

INNO LiPA from Innogenetics can be used to provide rapid identification of microbial isolates, even early liquid

cultures of slow-growing mycobacteria, replacing multiple-probe testing by one-strip technology. Another

commonly used method is in situ hybridization (ISH), employing a fluorescent-, radio- or antigen-labeled probe

to localize a specific nucleotide sequence directly in tissue sections. More than one probe can be used

simultaneously if different labels are selected.

Over the past decade, polymerase chain reaction (PCR) techniques (e.g., the commonly employed

singleplex PCR, multiplex PCR targeting more than one nucleotide sequence of a single or of different

microorganisms present in the test sample, and nested PCR performed in two sequential reactions to increase the

sensitivity of detection), real-time PCR (qPCR) and RT-PCR or RT-qPCR (reverse transcription PCR or qPCR

employing RNA template) have become established scientific tools for the detection of NA. In conventional

PCR, target nucleotide sequences are subjected to end-point detection in the plateau phase of amplification by

gel or capillary electrophoresis. In qPCR with a DNA template and RT-qPCR with an RNA template,

fluorescence generated by dsDNA intercalating dyes, e.g., SYBR Green I or, more commonly, by fluorophore-

labeled sequence-specific DNA probes (or primers), is monitored in real time. Product quantification is thus

achieved in addition to detection, and target specificity is increased when using selected DNA probes in addition

to primers initializing amplification. Based on the advantages of qPCR, numerous robust real-time assays have

been developed, e.g. forthe detection of the majority of known RNA and DNA viruses. The development of

multi-color qPCR assays and ready-to-use commercial multiplex real-time kits allows the detection of several

72

infectious agents in a single tube. However, it is important to optimize these assays in order to limit competitive

interactions, which may have an impact on the assay sensitivity.

In contrast to hybridization techniques, PCR assays generate an amplified signal based on the presence

of the gene-of-interest, thus enabling detection of microbial targets of even low or trace amounts in the host

matrix, i.e., clinical samples. In general, PCR also enables rapid detection of microorganisms that were

previously difficult or impossible to detect by traditional microbiological methods, since their growth

requirements were impossible to define, or too complex. In addition, PCR is especially suitable for slowgrowing

microbes and for viable but non-culturable bacteria (VBNC). Although molecular methods are usually unable to

discriminate between live and dead microorganisms, special variants of PCR reaction have overcome the

obstacle, e.g., the viable PCR method combining the use of special reagents expressing high affinity for DNA

with a photo-chemical reaction blocking the subsequent PCR; since reagents cannot pass through undamaged

cell membranes, only DNA from live microorganisms is detected.

The use of an internal (IC) and/or exogenous control is an important aspect of quality control, both of

DNA or RNA extraction and of subsequent PCR assays. ICs are necessary when NA are extracted from

potentially ‘difficult’ biological samples, needing confirmation of adequate efficiency of extraction and the

absence of PCR inhibitors to avoid false negative results. Validation of individual methods within each

laboratory and inter-laboratory testing of methods as employed by different institutions and for different

infectious agents allow comparison of the sensitivity and specificity of routinely used molecular methods, aiming

at standardization of assays. The use of qPCR in clinical diagnostics is growing rapidly and it is becoming one of

the indispensable laboratory tools, perhaps even replacing conventional PCR and other diagnostic methods.

Some methods combine the two abovementioned principles, i.e., hybridization and PCR. Two of them

arein situ PCR (employing a PCR amplification step prior to ISH in order to increase the sensitivity of

hybridization) and the commercially available GenoType assay from Hain Lifescience based on PCR and

subsequent hybridization employing membrane strip technology to identify, e.g., mycobacteria, MRSA,

Clostridium difficile, enterococci and EHEC. Some less frequently employed methods are founded on alternative

principles, e.g., LCR (ligase chain reaction), SDA (strand displacement amplification) and TMA (transcription-

mediated amplification).

The first step in molecular diagnostics of infectious diseases, i.e., sample preparation, is of great

importance, since it enables the generation of reliable results. The aim of preparatory steps is to make NA

available for further processing (i.e., to extract DNA or RNA from clinical material or isolates obtained from the

first-step sample processing) and to prevent enzymatic degradation in the collected samples, to remove inhibitory

substances affecting subsequent detection steps, e.g., PCR, and prepare NA as a solution of adequate

concentration. After initial, if necessary, mechanical or physical disintegration of the sample matrix (e.g.,

homogenization, bead-beating, freezing and thawing, ultra-sound disintegration), microbial cells are subjected to

thermal, enzymatic and/or chemical lysis, followed by a certain number of preparatory steps involving rinsing

and centrifuging to obtain DNA or RNA of satisfactory purity. NA-extraction procedures are of various kinds;

they need to be selected according to the material type. In addition to classical extraction using organic solutions

of phenol/chloroform, there is also magnetic particle-separation available or, most commonly employed, a

variety of commercial extraction kits based on silica-filter columns to remove matrix-specific inhibitors of

subsequent molecular assays. Some sample types, e.g., stools, require more sophisticated processing since they

contain many inhibitors.

Molecular typing methods

The obtained microbial NA can also be employed for a large assortment of molecular techniques that

enabledetermination of nucleotide sequences of different lengths comprising microbial genomes or the

generation of genetic fingerprints to evaluate the genetic relatedness of isolates; genotyping techniques are

commonly used since they enable epidemiological studies, related to strictly defined areas or encompassing

many isolates from a large geographical area.Many genotyping techniques are available in addition to classical

plasmid typing. They can be based on restriction of the total microbial genome, e.g., the well established PFGE

73

(pulsed-field gel electrophoresis) method, considered to be the gold standard in epidemiological studies of

pathogenic microbes, RFLP (restriction fragment length polymorphism),which was the first genotyping

technique and gained widespread application but is now largely obsolete due to the rise of sequencing methods,

and REA (restriction endonuclease analysis),which is based on genome digestion employing restriction

endonucleases, with frequent recognition sites, and is not followed by hybridization with a nucleotide probe like,

commonly, RFLP.

A second group of molecular typing techniques depend on PCR, e.g., the currently well established

MLVA (multiple locus variable number tandem repeat analysis) method, which is based on variation in the

number of repeats in selected, i.e., VNTR loci of the genome; RAPD (randomly amplified polymorphic DNA)

and AP-PCR (arbitrarily primed PCR) employing short arbitrary primers amplifying anonymous stretches of

DNA; AFLP (amplified fragment length polymorphism), based on PCR preceded by digestion with restriction

enzymes and ligation of specific adapters;a toxinotyping method that combines PCR and restriction analysis for

epidemiological studies on C. difficile; PCR-ribotyping, based on the polymorphism of the 16S/23S rRNA

intergenic spacer region; REP-PCR (repetitive extragenic palindromic sequence PCR) and ERIC-PCR

(enterobacterial repetitive intergenic consensus PCR) methods performed by using primers corresponding to the

interspersed repetitive elements of bacterial genome; and PCR-RFLP combining PCR and subsequent restriction

analysis.

Novel and fast-evolving molecular typing techniques are based on sequencing, e.g., the promising

method MLST (multi-locus sequence typing),which selects a certain number of PCR-amplified housekeeping

gene loci for sequencing and later generates allelic numbers combined to specific sequence types, and SLST

(single locus sequence typing, e.g., spa typing based on sequencing the polymorphic region of a specific protein

gene present in S. aureus). There is also SNP genotyping available, which detects single-nucleotide

polymorphisms by using, e.g., restriction analysis, hybridization (e.g., employing molecular beacons), PCR (e.g.,

SSCP, single strand conformation polymorphism analysis and TTGE, temperature gradient gel electrophoresis)

or sequencing (e.g., pyrosequencing).The automated Sanger method has dominated for almost two decadesfor

fast, reliable and inexpensive sequencing of different genomes.The number of pathogens that have their genome

or selected parts sequenced is growing rapidly, thus providing data for phylogenetic analyses and enabling

interpretation of results from epidemiological studies supported by sequences from outbreaks. The Sanger

method is considered first-generation technology and newer methods are referred to as next-generation

sequencing (NGS), generating an enormous amount of data at lower costs and in a shorter time. The possibilityof

sequencingacomplete genome of,e.g., a large number of related organisms, has enabled large-scale comparative

and evolutionary studies to be carried out, which could not have been imagined just a few years ago. The variety

of NGS strategies makes it likely that multiple platforms will coexist, some having clear advantages over others

for use in veterinary laboratories. In addition, NGS technology will replace the currently used microarray assays

based on PCR and hybridization to screen for a large number of selected genes or genome sequences.

Typing techniques need to enable the repeatability of analyses and must exhibit satisfactory resolving

power. Due to internationally based studies, typing must follow standardized protocols encompassing,e.g., size

markers and computer-assisted result processing. In this manner, constructed dendrograms displaying the genetic

relatedness of microbial agents show reproducible tree-topologies,and the training of staff to undertake such

work is simplified. Standardization is improving the interpretation and comparison of results between different

laboratories. Infection sources and transmission routes can thus be defined or confirmed. If a typing technique is

to gain widespread application, it should meet the requirements of relative simplicity, speed, low-budget,

feasibility and flexibility, e.g., simple protocol modification when applied for a different microbial

taxon,allowing use of the same principles, equipment and knowledge.

References

1. Quinn PJ, Markey BK, Leonard FC, FitzPatrick ES, Fanning S, Hartigan PJ. Veterinary microbiology

and microbial disease. Chichester: Willey-Blackwell, 2011

2. Thompson RCA. Molecular epidemiology of infectious diseases. London: Arnold, 2000

74

3. Wilson BA, Salyers AA, Whitt DD, Winkler ME. Bacterial pathogenesis: a molecular approach.

Washington DC: ASM Press, 2011


PROTEOMICS, ELECTROPHORESIS AND MASS SPECTROMETRY: A TOOL IN BIOMARKER

DETECTION AND DIAGNOSIS?

A.M. Almeida. Instituto de Investigação Científica Tropical, Centro de Veterinária e Zootecnia, Lisboa,

Portugal


The control of diseases is heavily dependent on the establishment of adequate diagnosis tools, vaccination

strategies and, although highly costly and controversial, on selective culling of infected animals. The

establishment of efficient diagnosis tools is directly connected to the establishment of a certain biomarker (e.g. a

specific protein) that may be detected in a given tissue or fluid (e.g. muscle or in most cases, serum). Examples

abound in the literature for instance MAP1 (Major Antigenic Protein) a protein related to the infectivity of the

bacteria Ehrlichia ruminantium the agent of the tick borne disease cowdriosis or heartwater (Marcelino et al.,

2012a, 2012b) or the amino acid 3-methyl-Histidine, a know indicator of muscle breakdown in cases of seasonal

weight loss, as we have demonstrated in goats (Almeida et al., 2004, 2008).

Recently the advent of the so called “Omics” approaches: genomics, proteomics, transcriptomics, and

metabolomics has allowed, at least in theory a much deeper knowledge on numerous physiological and infection

processes that have in turn allowed a broader range of putative biomarker candidates of a given disease of

physiological/metabolic condition from which researchers are able to use in the scope of diagnosis tools or

vaccine development.

Being proteomics the study of the proteome i.e. all proteins being expressed in a given organelle, cell, tissue,

organ, organism or body fluid, it may be expectable that, of all the Omics, proteomics will be the one most likely

to strongly contribute to the establishment of adequate biomarkers, of use in diagnostics or vaccine development.

Albeit a strong integration with other Omics, particularly transcriptomics and metabolomics is very important.

Proteomics is heavily reliant on two major components: protein separation and identification. Protein separation

is normally associated to electrophoresis techniques, particularly 2DE (two-dimensional electrophoresis),

whereas protein identification is strongly associated to mass spectrometry techniques (Soares et al., 2012),

particularly MALDI TOF/TOF (Matrix Assisted Laser Desorption Ionization – Time of Flight).

We have been using these approaches in the field of animal and veterinary sciences over the last years. Despite

it’s important limitations related to the fact that only a percentage of the (frequently the same) proteins (Petrak et

al., 2008) in the proteome are able to be analyzed by 2DE and to the fact protein identification using mass

spectrometry is strongly dependent on the level of completion of a genome sequencing of a certain species (in

farm animals and veterinary disease agents and vectors this is seldom a reality), gel electrophoresis and mass

spectrometry is still the power house of proteome analysis providing the bulk of results regarding biomarker

determination.

In this communication, and as illustrated in figure 1, we will briefly address these two techniques (gel

electrophoresis and MALDI), presenting their principles, major characteristics and results that may be obtained.

We will illustrate this last component with three different study cases directly related to our research activities,

1) the use of proteomics in the study of cattle tick-borne diseases, particularly Ehrlichia ruminantium (Marcelino

et al., 2012a, 2012b); 2) the use of proteomics in the establishment of biomarkers of adaptation to seasonal

weight loss (Almeida et al., 2009, 2010) and 3) the use of proteomics in the definition of markers of quality in

meat science and meat processing (Paredi et al., 2012; Almeida and Bendixen, 2012).


75

Figure 1 – The proteomics workflow in the scope of biomarker and diagnosis tool establishment

References

Almeida AM, Bendixen E. Pig Proteomics: a review of a species in the crossroad between biomedical and food

sciences. Journal of Proteomics (2012 in press)

Almeida AM, Campos A, Francisco R, van Harten S, Cardoso LA, Coelho AV 2010. Proteomic investigation of

the effects of weight loss in the gastrocnemius muscle of wild and New Zealand white rabbits via 2D-

electrophoresis and MALDI-TOF MS. Animal Genetics 2010;41: 260-272.

Almeida AM, Campos A, van Harten S, Cardoso LA, Coelho AV. Establishment of a proteomic reference map

for the gastrocnemius muscle in the rabbit (Oryctolagus cuniculus). Research in Veterinary Science 2009;87:

196-199.

Almeida AM, Schwalbach LM, deWaal HO, Greyling JP, Cardoso LA. 3-Methyhistidine in the male goat as an

experimental model: serum concentrations and an indicator of protein breakdown. Scandinavian Journal of

Laboratory Animal Science 2008;35: 259-263.

Almeida AM, Schwalbach LM, deWaal HO, Greyling JP, Cardoso LA.. Serum amino acid and myofibrillar

protein profiles in Boer Goat bucks following undernutrition. Small Ruminant Research 2004; 55: 141-147.

Marcelino I, Almeida AM, Brito C, Meyer DF, Barreto M, Sheikboudou C, Franco CF, Martinez D, LeFrançois

T, Vachiery N, Carrondo MJT, Coelho AV, Alves PM. Proteomic analysis of Ehrlichia ruminantium in host

endothelial cells. Veterinary Microbiology 2012;156: 305-314.

Marcelino I, Almeida AM, Ventosa V, Pruneau L, Martinez D, LeFrançois T, Vachiery N, Coelho AV. Tick-

borne diseases in cattle: applications of Proteomics to develop new generation vaccines. Journal of Proteomics

(2012 in press)

Paredi GL, Raboni S, Bendixen E, Almeida AM, Mozzarelli A. "Muscle to meat" molecular events and

technological transformations: the proteomics insight. Journal of Proteomics (2012 in press)

76

Petrak J, Ivanek R, Toman O, Cmejla R, Cmejlova J, Vyoral D, Zivny J, Vulpe CD. Déjà vu in proteomics. A hit

parade of repeatedly identified differentially expressed proteins. Proteomics. 2008;8: 1744-1749.

Soares R, Franco C, Pires E, Ventosa M, Palhinhas R, Koci K, Almeida Almeida, Coelho AV. Mass

Spectrometry and Animal Science: Protein identification strategies and particularities of farm animal species.

Journal of Proteomics (2012 in press)


D.Concordet. National Veterinary School of Toulouse.


ASVCP FINALIZED REFERENCE INTERVAL GUIDELINES

K. E. Harr. URIKA, LLC, Seattle, WA


COPYRIGHT STATEMENT AND ACKNOWLEDGEMENT

The following abstract is partially excerpted from ASVCP Reference Interval Guidelines finalized 2011

which will be published as a special report in the Veterinary Clinical Pathology Journal in 2012. This guideline

was put together by a core team of laboratorians with consultation from biostatisticians and editorial from the

ASVCP Quality Assurance and Laboratory Standards Committee. The guidelines may be found here:

http://www.asvcp.org/pubs/qas/index.cfm (Accessed May 20, 2012) The author acknowledges the collective

knowledgebase and time commitments of these contributors, especially the subcommittee chair, Dr. Kristen

Friederichs.

INTRODUCTION

When interpreting medical data, there are several factors which must be considered to ensure correct

diagnosis. While many clinicians focus on the physiologic or pathologic mechanism that may be causing the

“abnormal” result(s), there are several“non-animal” factors which may contribute to results outside of reference

intervals including the reference intervals themselves.Within the past couple of years, the ASVCP Quality

Assurance and Laboratory Standards committee has finalized several guidelines to address reference intervals,

preanalytical, analytical, and postanalytical error in both point of care as well as reference laboratories. This

discussion will focus on reference interval generation and maintenance as currently recommended by ASVCP

guidelines.

REFERENCE INTERVALS

History

Reference intervals are a relatively recent concept that was originally proposed in 1969.(Grasbeck,

1969) Remarkably, this cornerstone in medical diagnosis is less than 50 years old. In the 1970s, while the

International Federation of Clinical Chemistry pursued official guidelines and manuscripts, significant

theoretical discussion and publication on reference intervals took place with numerous philosophies and

mathematical approaches suggested and tested. In this soup of ideas, one can find a publication on almost any

approach to the establishment of reference intervals. Although many of these approaches are not currently used,

articles can be cited to justify outdated statistical evaluation, creating significant difficulty in review of

manuscripts submitted for publication and confusion for some authors. Official recommendations have existed in

the human field since the 1980s when the Expert Panel on the Theory of Reference Values (EPTRV) authored a

6-part series on the production of reference values, which was adopted by several professional organizations

including the Clinical and Laboratory Standards Institute (CLSI).(Solberg,1987, PetitClerc, 1987,

Solberg,1988,Solberg,1991,Solberg,1987Dybkaer, 1987) In 2008, the Clinical Laboratory Standards Institute

finalized a 3rd revision of guidelines, promoting transference and easing the strict number of required reference

individuals (previously n = 120) by use of the “robust” method.(Zakowski, 2008) A summary of the guidelines

for human populations has been published.(Geffre, 2009) These guidelines are relevant to veterinary medical

diagnostics, but do not address species differences, the inclusion of low numbers of reference individuals (<80)

that frequently occurs in veterinary medicine, and reference populations that are often afflicted with undiagnosed



http://www.asvcp.org/pubs/qas/index.cfm

77

subclinical disease. Therefore, the ASVCP consensus veterinary guidelines on the establishment of reference

intervals have been developed and are available with free and open access.

Interestingly, both human and veterinary guidelines specifically state that reference intervals should be

population- and method-specific and that reference intervals from the literature should not be used as they may

result in misdiagnosis.Though guidelines specifically state that RIs from the literature should NOT be used, they

are regularly used by veterinary practitioners; therefore, appropriate description of the details of reference

interval generation in a publication has been addressed in the veterinary guidelines. A full description of the

health and population characteristics as well as the preanalytical and analytical methods is mandatory to avoid

misdiagnosis. Until very recently, most articles in the veterinary literature lacked adequate descriptions of

populations and analytical methods to allow appropriate comparison and use. Additionally, in the human field

established consensus reference intervals are partitioned to age and gender and occasionally to race. Partitioned

reference intervals are not typically provided by veterinary diagnostic laboratories, but more publications are

addressing the effect of physiologic variables on histogram dynamics.

Computer support programs have recently been developed,presented upon, and are being updated so

that statistical analysis may be performed according to reference interval guidelines. Scientists at the French

veterinary college in Toulouse have generated freeware that can be downloaded here

http://www.biostat.envt.fr/spip/spip.php?article63 (accessed May 20, 2012) Reference Value Advisor is a set of

macroinstructions for Excel that compute reference intervals using the standard and robust methods with and

without generalized Box-Cox data transformation. Please see the website for further information and citations.

Reference intervals are important in clinical decision making and asking the correct questions can

change understanding and use of the report. Are your reference intervals correct for your patient? Are they

method, species, and population specific? It is the responsibility of veterinary diagnostic laboratories to serve as

many species as possible, but this is frequently logistically not possible and cost prohibitive. Where did they

come from and were they generated according to current ASVCP or other guidelines? Is partitioning required to

improve acuity of diagnosis? One needs to ask the laboratory for this information if it is not apparent in the

medical record. According to current guidelines, if there is any deviation from normal generation in statistical or

population analysis, this must be directly annotated on the report. If RI generation has been in accordance with

guidelines, this should be available to the clinician in a written report (RI study summary document). This

information may include, but is not limited to: Reference population demographics and number of reference

subjects sampled, subject preparation and time or season of collection, if relevant, sample type and handling,

confidence intervals around the reference limits.(Plebani, 2004)

If you are unaware of the specifics of the reference intervals you are currently using, please ask! The

numbers that you use likely on a daily basis for diagnosis are typed in by a technician from data generated by

one or a handful of hopefully qualified quality assurance specialists (a.k.a. human beings who make mistakes).

When was the last time that the reference interval was reviewed and revalidated? Current

recommendations are revision every 3-5 years. Even at reputable institutions this can be problematic. Recently

at UC Davis, it was realized that bovine hematology reference intervals that were used and essentially

disregarded by clinicians as most results were “out of bounds” were last evaluated in the 1950s.(George, 2010)

Amazing what written archives sitting on a slightly older emeritus professor’s shelf found by an exuberant

veterinary student can elucidate; not likely to happen in the digital age.

Table 1.Procedural steps for de novo determination of RI for new methods or new populations.

(Page 27 of ASVCP reference interval guideline, section references are to said guideline.)

1. Perform literature search for information about analytes to be measured (preliminary investigation).

2. Define reference population and establish selection, inclusion and exclusion criteria (Section 1 and Table 1).

3. Develop questionnaire to be completed by examining clinician, owner/caretaker or both in order to determine

if reference individual fits the selection or partitioning criteria (Section 2).

4. Determine number of reference individuals available or the number required to establish reference intervals

with desired level of certainty (as reflected by 90% CI around the reference limits) (Section 3).

5. Select reference individuals, preferably by direct methods (Section 4).

6. Collect and handle reference samples in standardized manner (Section 5).

7. Analyze reference samples using well-controlled methods (Section 6 and 7).

8. Prepare histogram (Section 8).

9. Identify outliers (Section 9). This may require prior transformation to appropriately apply outlier detection

methods and may need to be repeated after initial outliers are eliminated.

10. Determine distribution of reference data (Gaussian or non-Gaussian) (Section 10). If using parametric

methods, transform data if it is not Gaussian and retest distribution. Transformation may improve the

performance of the robust method. Nonparametric methods do not require any particular distribution.

http://www.biostat.envt.fr/spip/spip.php?article63

78

11. Calculate upper and lower reference limits using an appropriate statistical methodbased on distribution of

data and number of samples (Section 11 and Table 2). Calculate confidence intervals for the upper and lower

reference limits.

12. Determine the need for partitioning only if there are sufficient numbers of reference samples or there is

evidence for clinical importance (Section 12).

13. Document all previous steps for a comprehensive reference interval summary report

(Section 13)

REFERENCES 1. Dybkǽr R. Approved recommendation (1987) on the theory of reference values. Part 6. Presentation of

observed valued related to reference values. ClinChimActa 1987;170:S33-S42.

2. Geffré A Friedrichs K, Harr K, Concordet D, Trumel C, Braun JP. Reference values: a review. Vet

ClinPathol 2009;38:288-298.

3. George JW, Snipes J, and Lane VM. Comparison of bovine hematology reference intervals from 1957

to 2006. Vet ClinPathol 2010; 39: 138–148.

4. Grasbeck R, Saris NE. Establishment and use of normal values. Scand J Clin Lab Invest 1969;26(Suppl

110):62-63.

5. PetitClerc C, Solberg HE. Approved recommendation (1987) on the theory of reference values. Part 2.

Selection of individuals for the production of reference values. ClinChimActa. 1987;170:S1-S11.

6. Plebani M. What information on quality specifications should be communicated to clinicians, and how?

ClinChimActa 2004;346(1):25-35.

7. Solberg HE, Stamm DJ.IFCC recommednation: The theory of reference values. Part 4. Control of

analytical variation in the production, transfer and application of reference values. Automat Chem

1991;13:231-234.

8. Solberg HE. Approved recommendation (1987) on the theory of reference values. Part 5. Statistical

treatment of collected reference values. Determination of reference limits. ClinChimActa

1987;170:S13-S32.

9. Solberg HE.Approved recommendation (1986) on the theory of reference values. Part 1. The concept of

reference values. ClinChimActa. 1987;165:111-118.

10. Solberg HE. Approved recommendation (1988) on the theory of reference values. Part 3. Preparation of

indivdiduals and collection of specimens for the production of reference values. ClinChimActa.

1988;177:S1-S12.

11. Zakowski J. Clinical and Laboratory Standards Institute (CLSI). Defining, establishing, and verifying

reference intervals in the clinical laboratory; approved guidelines, 3rd

ed, CLSI document C28-A3.

2008;Vol. 28(3).

79

ENDOCRINOLOGY

ADIPOKINES IN COMPANION ANIMALS: AN UPDATE

J.J. Cerón, A. Tvarijonaviciute. Department of Animal Medicine and Surgery, University of Murcia, Murcia,

Spain


Obesity is a major concern in the companion animal population. The prevalence of obesity in pets is increasing

and in the most recently published studies, 29-34% of dogs and 19-29% of cats were classed as overweight and

around 5-8% in both species were considered obese (Lund et al. 2005, 2006). Overweigh and obese dogs have a

shorter lifespan and an increased risk of associated diseases such as diabetes mellitus, orthopaedic disease,

neoplasia, respiratory and urinary tract diseases (German, 2006).

Adipocytes are recognised as active cells that communicate with the brain and peripheral tissues by secreting

and wide range of proteins called adipokines. These adipokines are involved in the physiopathological

mechanisms implied in obesity and can influence many biological systems including glucose homeostasis,

inflammation and immunity (German et al., 2010). Although some 100 different adipokines have been

characterized in humans and rodents, the adipokines more studied in companion animals in the last years are

adiponectin, leptin and ghrelin. In this presentation we will deal with some aspects regarding adipokines that can

be of interest from the clinical pathological point of view such as:

1.Analytical issues. Although in the past RIA assays have been commonly used for adipokine measurements,

they are being replaced by ELISA due to safety and environmental reasons. In case of adiponectin human assays

with cross-reactivity can be used for dogs and cats, and when possible the use of species-specific standards is

recommended in these cases (Tvarijonaviciute et al., 2010a). Adiponectin circulates in serum in 3 forms: trimers,

hexamers and high molecular weight multimers (HMW), and because they have diverse biological activities it

could be of interest in the future to develop assays that could allow an easy measurement of the different forms.

For leptin, it seems that currently it is more difficult to find human assays with enough cross-reactivity for pets.

Also the quite high limit of quantification of some species-specific assays for canine leptin can limit its use to

quantify this adipokine in lean dogs (Tvarijonaviciute et al., 2011a). In case of ghrelin, the main issue seems to

be the storage conditions for the sample due to instability of the acylated-ghrelin or active form of this adipokine.

2. Inflammation. Inflammation seems to influence the concentrations of adipokines, it increases leptin and

ghrelin (Yilmaz et al., 2011) and decreases adiponectin concentrations.

There is a paradox in the relation between inflammation and obesity. Because although obesity is related with an

increasing risk of inflammatory conditions, no significant increases in acute phase proteins (APPs) have been

found in experimentally induced obesity (Tvarijonaviciute et al, 2011b). It seems that despite adipose cells can

produce APPs (Fain, 2006) its concentrations are not be high enough to produce increase in serum APPs in

obesity, at least that can be detected with the assays available nowadays. On the other hand, obesity is associated

with a decrease in adiponectin concentrations, an anti-inflammatory molecule that could explain the

predisposition of obese animals to inflammatory conditions.

3. New analytes associated with obesity.Plasma concentrations before weight loss of paraoxonase type 1 (PON-

1), an ezyme that reflects the oxidative status of the animal were less in cats that failed to complete a weight loss

programme than cats that were successful. Also serum butyrylcholinesterase (BChE) increased in experimentally

overweight dogs and is correlated with other physical and biochemical markers of obesity (Tvarijonaviciute et al.

2010b, 2012). Possibly in the future new serum analytes related with obesity and associated disorders could be

discovered and serve of help for the understanding of their physiopathological mechanisms as well as markers

for the early detection and monitoring of complications associated with this condition.

References

-Fain JN. Release of interleukins and other inflammatory cytokines by human adipose tissue is enhanced in

obesity and primarily due to the non-fat cells.Vitamines and Hormones. 2006; 74: 443-477.

-German AJ. The growing problem of obesity in dogs and cats. Journal of Nutrition. 2006; 136: 1940-1946.

-German AJ, Ryan VH, German AC, Wood S, Trayhurn P. Obesity, its associated disorders and the role of

inflammatory adipokines in companion animals. The Veterinary Journal. 2010; 185: 4-9.


80

-Lund EM, Armstrong PJ, Kirk CA, Klausner JS. Prevalence and risk factors for obesity in adult cats from

private US veterinary practices. Internacional Journal of Research in Veterinary Medicine. 2005; 3: 88-96.

-Lund EM, Armstrong PJ, Kirk CA, Klausner JS. Prevalence and risk factors for obesity in adult dogs from

private US veterinary practices. Internacional Journal of Research in Veterinary Medicine. 2006; 4: 177-186.

-Tvarijonaviciute A, Martinez S, Ceron JJ.(a) Validation of two commercially available enzyme-linked

immunosorbent assays for adiponectin determination in canine serum samples. Canadian Veterinary Journal.

2010; 10:279-286.

-Tvarijonaviciute A, Tecles F, Ceron JJ. (b) Relatioship between serum butyrylcholinesterase and obesity in

dogs: a preliminary report. The Veterinary Journal. 2010; 186: 197-200.

-Tvarijonaviciute A, Ceron JJ, Martinez S (a). Assessment of five ELISAs for measurements of serum leptin

concentrations in dogs. American Journal of Veterinary Research 2011; 2: 169-173.

-Tvarijonaviciute A, Martinez S, Gutierrez A, Ceron JJ, Tecles F (b). Serum acute phase protein concentrations

in dogs during experimentally short-term induced overweight. A preliminary study. Research in Veterinary

Science 2011: 90: 31-34.

-Tvarijonaviciute A, Ceron JJ, Holden SL, Morris PJ, Biurge V, German AL. Effect of weight loss in obese cats

on biochemical analytes related to inflammation and glucose homeostasis. Domestic Animal Endocrinology

2012; 42: 129-141.

-Ylmaz Z, Ilcol YO, Ulus IH. Endotoxin increases plasma leptin and ghrelin levels in dogs. Critical Care

Medicine 2008; 36:828-833.

LEPTIN LEVELS AND SEASONAL REPRODUCTIVE ACTIVITY IN LIPIZZAN FILLIES

N. Čebulj-Kadunc. Institute of physiology, Pharmacology and Toxicology; University of Ljubljana, Veterinary

faculty, Ljubljana, Slovenia.


INTRODUCTION

Leptin is a protein hormone, synthesized and secreted by white adipose tissue. It is the indicator of nutritional

status, informing the central nervous system about the status of body energy reserves and their balance. It

therefore plays important roles in appetite regulation, energy metabolism and body composition. Leptin is an

effective signal of the energy stores for reproduction by interacting with hypothalamic-pituitary-gonadal axis and

gonads (Houseknecht et al. 1998, Barb et al. 2004, Radin et al. 2009). In horses, leptin concentrations are not

only related to body fat mass but also to age, gender and seasonal influences. Mares are seasonally polyoestrous

breeders, exhibiting ovarian activity during spring and summer as the result of a circannual endogenous rhythm,

regulated by the photoperiod. Several indications exist, confirming the interactions of leptin and photoperiod in

regulation of the onset and duration of seasonal ovarian activity in mares (Houseknecht et al. 1998, Gentry et al.

2002, Buff et al. 2002, Fitzgerald et al. 2002, Ferreira-Dias et al. 2005, Cartmill et al. 2006, Kearns et al. 2006,

Salazar-Ortiz et al. 2011).

The aim of this study was to determine the changes of circulating leptin concentrations in 1- to 4-year old

Lipizzan fillies, exhibiting various patterns of reproductive activity (seasonal, prolonged or whole-year) or

anoestrus.

MATERIAL AND METHODS

The study was performed in Lipica stud farm (Slovenia) in 92 Lipizzan fillies, aged one to four years, in good body

condition (BCS of 7 – 8 during the whole study). They were kept together, free in stable, with outdoor release, and

fed twice a day with hay and oats. Drinking water was given ad libitum. Blood was sampled twice monthly (with a


81

10-day interval) for 12 consecutive months (January to December). Serum was kept frozen (To< –20

oC) until

analysed. Serum leptin concentrations were measured monthly by a commercial RIA (Multi-Species Leptin RIA Kit,

Linco). Ovarian activity was evaluated on the basis of serum progesterone concentrations, measured twice monthly

with a commercial EIA (Ovucheck ® Plasma or Serum Progesterone, Biovet Canada). Statistical calculations were

performed using Statistical Package For Social Sciences for Windows, (SPSS for Windows,Release 12.0) with

subprograms Paired Samples T-Test, Analyses of variance and General Linear Model. Results were evaluated as

statistically significant at the level of P<0.05, and presented as mean standard error of mean ( X s.e.m.).

RESULTS

In yearling fillies, ovarian activity was present from May to October, in 2-year olds from March to December

and in 3- and 4- year olds over the whole year. Ovarian activity was not detected in 7 yearlings. From June to

September, ovarian activity was detected in 100% of fillies of all age groups with exception of yearlings. It was

present over the whole year in 12% of 3-year olds and in 24% of 4-year olds. On this, the fillies were classified

into 4 groups: anoestrous fillies, fillies revealing a seasonal cycle from spring to late summer or early autumn,

fillies with a prolonged cycle which started in spring and was still present in autumn and winter and fillies with

whole-year cyclic activity. The patterns of ovarian activity in various age groups of fillies over the year are

presented in Figure 1.

Figure 1: Proportions of fillies, exhibiting ovarian activity during the year; G-1: yearlings (n=15), G-2: 2-year

olds (n=30), G-3: 3-year olds (n=31), G-4: 4-year olds (n=16)

82

Figure 2: Circulating leptin concentrations in Lipizzan fillies with various patterns of reproductive activity; (R-

1: anoestrus (n=7), R-2: seasonal ovarian activity (n= 40); R-3: prolonged ovarian activity (n=14), R-4: whole-

year ovarian activity (n=30).

Mean serum leptin concentration ranged from 2.06±0.36 ng/ml in yearlings to 3.34±0.78 ng/ml in 4-year old

animals. The values for 3- and 4-years old animals were significantly higher than those in yearlings and in 2 year

old fillies (P<0.01).

The lowest mean whole-year serum leptin concentration (2.04±0.44 ng/ml) was detected in fillies with seasonal

cycle, followed by anoestrous fillies (2.64±0.57 ng/ml), fillies with prolonged cycle (2.78±0.67 ng/ml) and,

finally, in fillies exhibiting whole year cycle (3.42±0.83 ng/ml). Mean serum leptin concentrations in fillies with

different patterns of cyclic activity are plotted as a function of months of the year in Figure 2. Significant

differences were established between average monthly leptin concentrations (P<0.001) in every group of fillies.

The highest of all measured monthly values were those, observed in August for fillies with whole-year and

prolonged reproductive activity (P<0.001). On the other hand, the lowest monthly values were measured in

fillies with seasonal ovarian activity.

DISCUSSION

Average circulating leptin concentrations in examined fillies were at the lower end of normal values for horses

(Radin et al. 2009, Salazar-Ortiz et al. 2011). Age dependent variations reported in horses (Fitzgerald et al. 2002,

Gentry et al. 2002, Ferreira-Dias et al. 2005) were also present in the examined Lipizzan fillies, with the lowest

values in yearlings and the highest in 4-year old animals.

The seasonal reproductive activity of mares is synchronized by photoperiod, which is the primary environmental

factor determining the winter anoestrus (Guillaume et al. 1995). Despite this, mares with higher body fat, and

therefore higher leptin concentrations, can remain cyclic during winter months, but in animals with lower fat

stores, winter anoestrus follows decreased leptin values in autumn (Fitzgerald et al. 2002). A similar situation

was observed in Lipizzan fillies. The highest mean leptin concentrations were measured in fillies with whole-

year reproductive activity, which were, on average, also the oldest group of animals, included in the study. On

the other hand, the lowest leptin values were observed in fillies with seasonal reproductive activity, which were

(with the exception of anoestrous fillies) the youngest age group. The difference can be explained by higher

body fat stores in older (and heavier) fillies, influencing the leptin concentration. Surprisingly, mean levels of

leptin in younger, anoestrous group of Lipizzan fillies were higher than in fillies with seasonal pattern of ovarian

activity. This might reflect a relative resistance to leptin in the youngest group of fillies, which could serve to

maintain high levels of food intake and growth, as well as prevent the premature onset of puberty (Houseknecht

83

et al. 1998).

Irrespectively of reproductive pattern in studied Lipizzan fillies, low leptin levels were observed in the period of

January to June, followed by a gradual leptin increase which reached a peak in August or September and then

decreased from October to December. In mares the summer rise of leptin values may reflect an effect of

increased ambient temperature (Fitzgerald et al. 2002), but could also be explained by optimal food quality in

this season. The peak breeding season, with the majority of fillies exhibiting signs of cyclic ovarian activity

(with exception of yearlings) lasted from May to September. Onset of ovarian activity coincided with the period

of low leptin values which peaked in August (in fillies with prolonged or whole-year ovarian activity) or

September (in fillies with seasonal ovarian activity). The decrease of leptin values started two months before the

end of the peak breeding season, irrespectively of ovulation pattern, confirming that the transition from ovarian

activity to anoestrus is preceded by a decrease in leptin value (Ferreira-Dias et al. 2005). Despite the significant

decrease of values in September and October, some 3-and 4-year olds remained cyclic, indicating that the energy

stores in these fillies were still high enough for sustaining the cyclic ovarian activity.

The results demonstrate the age-dependent variations of circulating leptin values in Lipizzan fillies. Seasonal

leptin fluctuations are also present, which can be attributed to seasonal differences in food quality and influence

of climate, and most probably interfere with photoperiod-induced changes of ovarian activity. The highest leptin

values in fillies with whole-year cycle confirm the possible role of leptin in sustaining the cyclic activity in

winter months.

REFERENCES

1. Barb CR, Kraeling RR. Role of leptin in the regulation of gonadotropin secretion in farm animals. Anim

Reprod Sci. 2004;82-83:155-167.

2. Buff PR, Dodds AC, Morrison CD, Whitley NC, McFadin EL, Daniel JA, Djiane J, Keisler DH. Leptin in

horses: Tissue localization and relationship between peripheral concentrations of leptin and body condition.

J Anim Sci. 2002;80:2942-2948.

3. Cartmill JA, Thompson DL, DelVecchio RP, Storer WA, Crowley JC. Leptin secretion in horses: Effects of

dexamethasone, gender, and testosterone. Dom Anim Endocrinol. 2006; 31:197-210.

4. Ferreira-Dias G, Claudino F, Carvalho H, Agricola R, Alpoim-Moreira J, Robalo Silva J. Seasonal

reproduction in the mare: possible role of plasma leptin, body weight and immune staus. Dom Anim

Endocrinol. 2005;29:203-213.

5. Fitzgerald BP, Reedy SE, Sessions DR, Powell DM, McMannus CJ. Potential signals mediating the

maintenance of reproductive activity during the non-breeding season of the mare. Reprod. 2002;59:115-129.

6. Gentry LR, Thompson DL, Gentry GT, Davis KA, Godke RA. High versus low body condition in mares:

Interactions with responses to somatotropin, GnRH analog and dexamethasone. J Anim Sci. 2002;80:3277-

3285.

7. Guillaume D, Arnaud G, Camillo F, Duchamp G, Palmer E. Effect of melatonin implants on reproductive

staus of mares. Biol Reprod Monogr. 1995;1:435-442.

8. Houseknecht KL, Baile CA, Matteri RL, Spurlock ME. The biology of leptin: A review. J. Anim. Sci. 1998;

76: 1405-1420.

9. Kearns CF, McKeever KH, Malinowski K. Changes in adipopnectin, leptin, and fat mass after clenbuterol

treatment in horses. MedSciSports Exerc. 2006;38:262-267.

10. Radin MJ, Sharkey LC, Holycross BJ. Adipokines: a reviewof biological and analytical principles and an

update in dogs, cats, and horses. Vet Clin Pathol. 2009;38:136-156.

84

11. Salazar-Ortiz J, Camous S, Briant C, Lardic L, Chesneau D, Guillaume D. Effects of nutritional cues on the

duration of the winter anovulatory phase and on associated hormone levels in adult female Welsh pony

horses (Equus caballus). Reprod Biol Endocrinol. 2011;9:130-146.

BIOCHEMISTRY

VARIANCE COMPONENTS AND CRITICAL DIFFERENCES IN DOGS AND CATS

I. Schwendenwein1, E. Hooijberg

1, B. Hanus

2, F. Frommlet

3, M. Pagitz

2.University of Veterinary Medicine,

Vienna, Austria: 1Central Laboratory, Department of Pathobiology,

2 Clinic for Internal Medicine and Infectious

Diseases, Department of Clinical Sciences, University of Veterinary Medicine, Vienna; 3 Institute for Medical

Statistics, Center for Medical Statistics, Informatics and intelligent Systems, Medical University of Vienna.


Laboratory tests are performed for a variety of reasons – establishing a diagnosis, ruling in or out a differential

diagnosis, checking the health status of a patient, and last but not least, for monitoring. Comparison of findings is

a fundamental process when making a diagnosis and monitoring. If the condition of a patient resembles that

which is typical for a specific disease, a positive diagnosis is made. When the condition of a patient resembles a

whole set of diseases, these have to be ruled out one by one. This is a diagnosis by exclusion. The interpretation

of laboratory data is an example of decision-making by comparison. To accomplish these purposes laboratory

results are related to the patient’s history, results of physical examination and reference intervals (RI) or previous

laboratory results. The term reference is derived from the Latin word “referre”, which means to report or to

relate to something. This implies laboratory data must only be compared to reference intervals which fulfill

certain criteria of a relationship to them. This relationship is commonly defined by population characteristics,

preanalytical and analytical conditions and statistical means of data processing. Reference intervals are derived

from a healthy population – this reveals a major fundamental limitation as absolute health does not exist. Health

is necessarily a relative concept, defined by the WHO in 1948 as a” state of complete physical, mental and social

well-being and not merely the absence of disease or infirmity” (Preamble to the Constitution of the World Health

Organization as adopted by the International Health Conference, New York, 19-22 June, 1946; signed on 22 July

1946 by the representatives of 61 States (Official Records of the World Health Organization, no. 2, p. 100) and

entered into force on 7 April 1948). In veterinary patients these conditions can hardly ever be met and even less

easily assessed. A thorough description of the reference population is therefore warranted and the reference

population should mirror the patient population. Establishing appropriate RIs is extremely cumbersome and

expensive for veterinary laboratories. Recruiting an adequate number of reference individuals of which a

minimum of 40 is usually required, might be impossible. Even transfer procedures for updating RIs might be

difficult to accomplish as even for this process 20 healthy individuals are needed (Geffré, Friedrichs et al. 2009).

A result outside the reference interval does not indicate disease, as only 95% of values obtained from a reference

population are included into the calculation for reference intervals, which means a healthy individual might have

values outside the reference interval. For establishing diagnoses, reference intervals are complemented by

clinical decision limits. Clinical decision limits are intended to provide optimum separation between clinical

categories and must not be confused with reference intervals. Population based reference intervals are influenced

by variance components such as individual, analytical and inter-individual or group variance. These variances

influence the reference range (Scope, Schwendenwein et al. 2005; Scope, Schwendenwein et al. 2006). It has

been shown that population based RIs are not suitable for identifying changes of quantities where the

intraindividual variance covers only a small range of the RI (Harris 1974).Calculation of the index of

individuality (IoI) allows one to evaluate the suitability of population based reference intervals. This can be

accomplished relatively easily by so called longitudinal studies. Samples are drawn from a small population of

healthy reference individuals at defined intervals minimizing variances in preanalytical conditions and analyzed

in duplicate to assess analytical variation. Total variation among results of a group of animals is the summary of

intraindividual, preanalytical, analytical and interindividual conditions. Thus intra-individual, inter-individual or

group as well as analytical components of variance can be calculated from data derived by these studies. The

study protocols for assessing variance components might vary. Conditions of their generation have thus to be

documented meticulously. If care is taken to minimize preanalytical and analytical sources of variance as few as

8 individuals sampled over a certain time period can give reasonable data (Fraser and Harris 1989). To minimize

analytical variance, samples could be frozen and analyzed batch wise. On the other hand analysis directly after

sampling might more correctly mirror the clinical situation, where analyses take place on different days. Storage

conditions such as duration as well as freezing and thawing might also introduce variances. However for non-

stable analytes immediate analysis is required. Analytical coefficients of variation can be introduced into the

85

calculations from cumulative quality control data. The intra-individual biological variation is represented by the

mean coefficient of variation for separate values from an individual (CVI). The inter-individual or group

variance is represented by the mean CV for values between individuals CVG , the analytical variance by the mean

CV between duplicate analyses CVA (Walton 2012). These coefficients of variation can also be used to calculate

the index of individuality (IoI), which enables one to decide whether population based reference intervals, can be

used or subject-based references values are more appropriate. Basically the IoI is derived by dividing the

individual variance by the group variance; IoI= (CVI2+CVA

2)

1/2/ CVG (Fraser and Harris 1989). If a quantity has

a high IoI, that is >1.4, population based reference intervals can be used. If the IoI is low, that is <0.6, a

cumulative reporting system based on individual reference values is recommended. A low IoI indicates that the

variance of the analyte in the individual spans only a small range of the group range. This means a change in an

individual whose subjective reference interval is far away from the group reference limits will have to be of a

much larger change to be detected as abnormal as it might still be within the reference limits despite representing

a change in the individual’s homeostatic setpoint. Biological variation measurements show that for most analytes

marked individuality is common (Fraser Callum 2004; Walton 2012). The IoI usually does not influence the test

result when a single result is interpreted, but it has an effect when test results outside the reference interval are

repeated to verify a diagnosis (Hyltoft Petersen, Fraser Callum et al. 1999). In a screening situation in which the

disease has a very low prevalence, testing is aimed at identifying all affected individuals with a low false positive

rate. When the quantity/analyte has a high index of individuality, there is a high chance that the results will fall

within the reference interval again. If the quantity has a low index of individuality the result of the repeat test

will be close to the first confirming it as abnormal regardless of whether it is truly abnormal or false positive.

Here an increase of the positive cutoff might decrease the rate of false positives, but at the cost of sensitivity

(Hyltoft Petersen, Fraser Callum et al. 1999). So in quantities with a low index of individuality increasing the

diagnostic cutoff will decrease the rate of false positives and will be more efficient than repeating the test. In

diagnostic situations when clinical evaluation has preceded or inspired the test request, the prevalence of a

disease is already high. Repeating the test in a quantity with a low IoI, the result will be close to the first result,

regardless of being a false positive one or not.

In those situations the cutoff should be decreased to increase nosographic sensitivity instead of repeated testing.

In a diagnostic high prevalence situation repeated testing is useless and actions should be taken on the first

measurement regardless of the IoI (Hyltoft Petersen, Fraser Callum et al. 1999). Originally a high IoI was

desirable, but means to achieve a high IoI are limited, the only rational way is to reduce the CVG, by

stratification of groups (Harris and Boyd 1990). Improvement of analytical variance and partitioning of groups

might increase the IoI. Data on biological variation can also be used to establish analytical quality specifications

(Stamm 1982). Many laboratory tests are ordered for monitoring purposes, where changes between serial

measurements are evaluated. For assessing the clinical relevance of a change between laboratory results RIs are

not very useful. An interpretative aid for the assessment of the significance of changes in laboratory results is the

critical difference (CD) or reference change value (RCV). The terms are used synonymously. CD and RCV help

to determine whether differences between the results of sequential laboratory tests in a single individual are due

to a change in the patient’s homeostatic set-point or the result of analytical or intra-individual variance

components (Harris and Yasaka 1983). RCV can be easily introduced into laboratory information systems and

thus aid the decision making process. RCV /CD can be established by complex mathematical operations on data

derived by longitudinal studies (Fraser, Cummings et al. 1989). Also in those cases, where a low IoI limits the

usefulness of population derived RIs, evaluation of RCV would help detecting clinically relevant changes. The

RCV is defined as “the difference between 2 consecutive test results in an individual that is statistically

significant in a given proportion of all similar persons”. (Harris and Yasaka 1983). Thus if this criterion is

fulfilled, a positive RCV will indicate a change in the patient despite the value being within the reference interval

(Walton 2012). The concept was introduced early in veterinary medicine but has received little attention. RCV is

calculated by the formula RCV=zp* 21/2

*(CVi2+CVA

2)

1/2(Fraser 2012; Walton 2012). For z usually 1.96 is chosen

to reduce the probability of a type I error (false positive) to 5%.When larger z values are used the probability of

detecting significant change increases because the calculated RCV and the power of the statistical function

increase (Walton 2012). Selecting an appropriate z value might further increase the usefulness of RCV-

calculations. If only a one sided change is of interest one sided z-values should be chosen, which are 1.65 for

p<0.05 or 2.58 for p<0.01 (Fraser 2012). Estimates of CVI are generally stable over geographical regions,

methodology as well in chronic stable disease (Fraser 2012) thus RCV can easily be calculated from published

data when CVA is derived from “local” QC data. In humans it has been shown that CVI remains stable for many

quantities even in chronic disease. Only for a few analytes such as tumor markers in cancer or creatinine in

kidney disease, might specific RCVs decrease the false positive rate as for these analytes RCVs derived from

healthy populations are usually smaller due to a lower CVI in healthy individuals (Ricós, Iglesias et al. 2007).

Garner and coworkers demonstrated by comparing different grading schemes for acute kidney injuries in a

hospital population that simple delta checks introduced by the laboratory system identified 99% of patients

detected by 4 different grading schemes plus additional 14 patients who would not have been detected at all

86

(Garner, Lewington et al. 2012). ISO standard 15 189 requires periodical review of biological reference

intervals, which implies much financial and logistic effort (Ceriotti, Hinzmann et al. 2009), and this requirement

can hardly be fulfilled by veterinary laboratories. Fraser points out, that simply calculating RCV by including the

laboratories CVA might be much more efficient in improving patient care than establishing separate reference

intervals for different situations or partitioning reference intervals (Fraser 2009).

The table below contains data, compiled in the author’s lab from two longitudinal studies over 6 months in 24

dogs (Pagitz, Frommlet et al. 2007) and over 9 months in 9 cats. Dog samples were frozen and analyzed batch

wise, whereas feline samples were analyzed on a daily basis. Assay methods were identical in both species,

whereas the analyzer was changed from a Hitachi 911, which was used in the dogs to a Cobas 511c which was

used in the cat study. Animals from both species were fasted before sampling which was scheduled between 7:30

and 10:00 pm. Both species were fed standardized commercial pet food diets.

quantity CVI dog

%

CVG

dog %

CVA dog

%

RCV

dog %

IoI dog CVI cat

%

CVG cat

%

CVA cat

%

RVC cat

%

IoI cat

ALT 61 34.3 3 169 1.78 16.6 30.5 3.65 47 1.78

AP 24.7 72.5 2.35 68 0.34 14.5 35.2 1.9 40 0.41

T-Ca 4 5.19 3.07 14 0.92 5.37 3.66 2.26 16 1.58

Cl- 2.96 1.75 2.48 10 2.2 0.94 0.92 1.6 5 2.02

Creatinine

Picrate

10.45 13.4 4.64 31 0.85 nd nd nd nd nd

Creatinine

Enzymatic

13.6 19.12 4.4 4 0.75 13.53 14.27 3.02 38 0.97

Cys. C 12.3 14.9 7.2 4 0.95 nd nd nd nd nd

GLDH 53.8 33.1 7.5 150 1.64 37.97 31.18 14.51 112 1.3

K+ 7.26 5.09 2.26 21 1.49 6.09 2.71 1.95 19 2.35

Na+ 2.77 1.35 2.23 10 2.63 0.44 0.41 1.7 5 4.22

PO4- 13.65 12.07 3.37 39 1.16 10.9 4.29 1.95 31 2.6

Total Protein 4.39 8.79 2.83 14 0.59 4.75 3.55 1.67 14 1.41

Urea 17.08 19.01 2.7 48 0.9 12.6 12.53 1.99 35 1.01

Lipase nd nd nd nd nd 15.89 38.47 4.03 45 0.42

T4 nd nd nd nd nd 23.5 4 9.68 70 6.3

Bile acids nd nd nd nd nd 57.1 8.78 0 158 6.5

References

Ceriotti, F., R. Hinzmann, et al. (2009). "Reference intervals: the way forward." Annals of Clinical Biochemistry

46(1): 8-17.

Fraser Callum, G. (2004). Inherent biological variation and reference values. Clinical Chemistry and Laboratory

Medicine. 42: 758.

Fraser, C. G. (2009). "Reference change values: the way forward in monitoring." Annals of Clinical

Biochemistry 46(3): 264-265.

Fraser, C. G. (2012). "Making better use of differences in serial laboratory results." Annals of Clinical

Biochemistry 49(1): 1-3.

87

Fraser, C. G., S. T. Cummings, et al. (1989). "Biological variability of 26 clinical chemistry analytes in elderly

people." Clinical Chemistry 35(5): 783-786.

Fraser, G. G. and E. K. Harris (1989). "Generation and Application of Data on Biological Variation in Clinical

Chemistry." Critical Reviews in Clinical Laboratory Sciences 27(5): 409-437.

Garner, A. E., A. J. P. Lewington, et al. (2012). "Detection of patients with acute kidney injury by the clinical

laboratory using rises in serum creatinine: comparison of proposed definitions and a laboratory delta

check." Annals of Clinical Biochemistry 49(1): 59-62.

Geffré, A., K. Friedrichs, et al. (2009). "Reference values: a review." Veterinary Clinical Pathology 38(3): 288-

298.

Harris, E. K. (1974). "Effects of Intra-and Interindividual Variation on the Appropriate Use of Normal Ranges."

Clinical Chemistry 20(12): 1535-1542.

Harris, E. K. and J. C. Boyd (1990). "On dividing reference data into subgroups to produce separate reference

ranges." Clinical Chemistry 36(2): 265-270.

Harris, E. K. and T. Yasaka (1983). "On the calculation of a "reference change" for comparing two consecutive

measurements." Clinical Chemistry 29(1): 25-30.

Hyltoft Petersen, P., G. Fraser Callum, et al. (1999). The Index of Individuality Is Often a Misinterpreted

Quantity Characteristic. Clinical Chemistry and Laboratory Medicine. 37: 655.

Pagitz, M., F. Frommlet, et al. (2007). "Evaluation of Biological Variance of Cystatin C in Comparison With

Other Endogenous Markers of Glomerular Filtration Rate in Healthy Dogs." Journal of Veterinary

Internal Medicine 21(5): 936-942.

Ricós, C., N. Iglesias, et al. (2007). "Within-subject biological variation in disease: collated data and clinical

consequences." Annals of Clinical Biochemistry 44(4): 343-352.

Scope, A., I. Schwendenwein, et al. (2005). "Influence of outlying values and variations between sampling days

on reference ranges for clinical chemistry in budgerigars (Melopsittacus undulatus)." Veterinary Record

156(10): 310-314.

Scope, A., I. Schwendenwein, et al. (2006). "Biological variation, individuality and critical differences of eight

biochemical blood constituents in budgerigars (Melopsittacus undulatus)." Veterinary Record 159(25):

839-843.

Stamm, D. (1982). A New Concept for Quality Control of Clinical Laboratory Investigations in the Light of

Clinical Requirements and Based on Reference Method Values. Clinical Chemistry and Laboratory

Medicine. 20: 817.

Walton, R. M. (2012). "Subject-based reference values: biological variation, individuality, and reference change

values." Veterinary Clinical Pathology: n/a-n/a.

URINE CHEMISTRY - AN EVIDENCE-BASED APPROACH

E. Leidinger, J. Leidinger. Invitro Diagnostic Lab. Rennweg 95, Vienna, Austria.


Complete urinalysis consisting of physicochemical and microscopic evaluation of the urine is frequently done as

part of the database of animals. In addition to a lecture held previously1 the authors were asked to give an

overview on routine urine chemistry by dipstick as well as by some other routine urinalysis methods.

Applicability and limitations of different urine chemistry test are reviewed for different species and some own

data will be presented. Most studies reviewed for this lecture refer to Multistix® reagent strips (Siemens

Diagnostics, formerly Bayer). The authors use Compur 10 UX® dipsticks (Boehringer, Mannheim) with the

semi-automated reader Urilux®. The data presented may therefore differ from published studies, however some

studies showed comparable results in regards of sensitivity and specificity of dipstick.

Furthermore, best practices and standards are discussed. The lecture does not deal with more sophisticated urine

chemistry methods applied in toxicology and research; those were covered in-depth in past meetings2.

The dipsticks commonly used in veterinary diagnostics were developed for human medicine, only few tests have

been adapted to the special needs of veterinary species.

88

General Remarks

URINALYSIS STANDARDS IN HUMAN AND VETERINARY MEDICINE

ASVCP guidelines3

CLSI guideline4

European guidelines for urinanalysis4

STABILITY OF URINE SAMPLES

According to the new ASVCP QC guidelines urine can be examined within 24 h without loss of clinical

information, most chemical parameters seem to be stable for an even longer period.4

A reduction in leukocyte esterase reactivity by 25% after 24 hrs was noted for refrigerated urine samples from

humans, and also haemoglobin declined. This could cause false negatives. Urine protein values increased.

Precision and stability were excellent for glucose, nitrite, ketones6 and pH

7.

TEST PROCEDURE: the urine dipstick tests are semi-quantitative assays and therefore the recommendations of

the manufacturers concerning dipstick storage and application have to be followed strictly; a too short incubation

or insufficient dipping into urine as well as prolonged dipping or delayed reading of the dipsticks may give

erroneous results in many test pads. Dipsticks require careful storage because those ones exposed to air have a

rapid and cumulative loss of specificity over time for nitrite, and give false positive results for glucose after 7

days and false-negative results for blood after 28 days.8

Samples have to be brought to room temperature (20-22°C) and mixed truly prior to any analysis!

QUALITY ASSESSMENT

Instrument performance logs should be kept for each instrument (e.g., refractometer, dipstick reader) to record

maintenance according to manufacturers’ instructions and calibrations. An instrument validation may be useful

to test for interference, particularly the impact of urine colour on dipstick readability, visually or by automated

methods.3

Specific gravity (USG) While the determination of the urine specific gravity (USG) by a test-pad on many dipsticks is of acceptable

precision with human urine9, there is no evidence that the test pads for specific gravity properly work for any

common veterinary species.10

According to our own data there is no correlation between the specific gravity measured by a Goldberg

refractometer and the Combur 10 dipstick (Roche Diagnostics) for dogs and cats (Fig. 1). USG may correlate

better for the Multistix USG test, but still is not acceptable10,11

. Reasons for this may be a different relevant pH-

range and a different overall-composition of the urine in animals.

The determination of USG by refractometry12

or osmometer is essential for valid results.

89

Scatter Plot with Fit

1

1,01

1,02

1,03

1,04

1,05

1,06

1 1,01 1,02 1,03 1,04 1,05 1,06

SG Refract

SG

Dip

sti

ck Identity

Linear f it

(0.92 + 0.10x)

Fig. 1: Linear fit between USG measured by Goldberg refractometer and

Combur 10 dipstick in cats and dogs (n=259, r=0.27)

Urine pH

The pH indicated by dipsticks is usually in the range of 5.0 to 8.0 units, dipstick reading devices report the pH by

0.5 unity steps; due to limitations of the method accuracy it is questionable if the urine pH should be reported as

decimal figure, anyway. 11

There has been some doubt about the accuracy of the pH obtained from dipsticks and for research and

monitoring purposes a pH-meter may be preferred. Portable pH meters gave results with a similar precision as

bench-top pH-meters. Dipsticks and pH paper tend to overestimate the pH-values. Dipstick and pH-papers had

moderate to poor agreement with the reference method. CV for Multistix was 1.6%, and thus 9 times greater than

for pH-meters.13

Nevertheless, the method seems to be sufficiently accurate for most routine applications.

Leukocyte esterase Frequently the leukocyte esterase reaction is regarded unreliable in dogs and cats.

14

In cats a positive leukocyte esterase reaction is highly unspecific in cats (false positive reactions)15

and should

not be regarded for some dipstick tests (Combur, Multistix16

). We found no fit between leukocytes esterase

activity and leukocytes in sediment (Fig. 2a).

In dogs the leukocyte esterase reaction is not as sensitive as required (sensitivity was 50% for the Compur 10

dipstick), and may further decline with storage of the urine sample6. Therefore, moderate pyuria may be missed

if sediment is not examined (Fig. 2b).

90

a b

Fig. 2: Linear fit between WBC in sediment (WBC) and leukocyte esterase (LE) for cats (a) and dogs (b).

Nitrate Urine-Nitrate is reduced to nitrite by many bacteria, but enough time is needed (>4 hrs)

17 to render a sufficient

nitrite concentration. Nitrite in urine indicates >106 Bacteria/ml. Retention time is usually unmanageable in

veterinary patients; besides, nitrate may be present in lower concentration in urine of carnivores27

.

A false positive reaction can be caused by aged voided urine, and ascorbic acid.18,19

The nitrate pad is very

sensitive towards open storage of dipsticks.8

The test should be disregarded in veterinary species.14,20

Sediment analysis (pyuria, bacteria) a Wright-stained

smear21

, and quantitative culture are required to detect bacteriuria.

Protein The dipstick test is a valuable screening assay for the detection of proteinuria. It is more sensitive to albumin

than to globulins and does not detect Ig light chains (Bence-Jones proteins), Fig. 3.

Fig. 3: Causes of proteinuria detected or not detected, respectively by the

dipstick reaction

Alkaline urine may cause a false positive protein reaction but it does not cause a gross overestimation of urinary

protein measured by dipstick.22

91

The sulfosalicylic acid (SSA) test is frequently used confirm positive underestimates urine protein, but

underestimates protein most of the time in dog and cat urine23

.

The test is useful as first diagnostic step if Bence-Jones proteinuria is suspected from clinical findings, the

dipstick test may be negative in this case.

A newer generation of urine dipsticks is capable to measure protein and creatinine simultaneously; the urine

protein/creatinine ratio can be calcultated manually or by a dipstick reader.24,25

The reliability of the urine protein

results vary with species, creatinine possibly is not very accurate. Microhaematuria does not couse an increase in

urinary protein; the UPC doesn't increase even in grossly visible haematuria26

.

Despite some limitations, the protein measurement by dipstick is reasonable accurate.

Glucose

The dipstick reaction is based upon the glucose-oxidase/peroxidase reaction. The enzymes involved in this

reaction limit the shelf live of dipsticks.10

True positives are found at any state that causes a glucosuria (diabetes mellitus, Fanconi syndrome, stress, fluid

therapy).

False positives may be seen in cats with haematuria and oxidative agents, false negatives may be seen in animals

excreting ascorbic acid in their urine11

, with bilirubinuria, formalin, and low temperatures.11

Most modern test

sticks can compensate for ascorbic acid interference.

Confirmatory tests: the Benedict copper reduction test is still described in textbooks.27

If an analyser method (hexakinase) is used for confirmation it has to be considered that up to 10 mg/dl glucose

are consistent with physiologic glucose excretion and not detected by the test pad reaction.10

There is a very

good agreement between glucose-oxidase and hexakinase methods for blood and urine on autoanalysers.28,29

.

Ketone bodies Ketones include acetone, acetoacetate (AcAc), and ß-hydroxybutyrate (3-OHB), the presence in urine primarily

indicates titrational metabolic acidosis.

Only AcAc and acetone reacts with nitroprusside, except glycine is added, than also acetone is measured. 3-

OHB, the predominant ketone body in diabetic ketoacidosis, is not detected.

Sometimes it is mentioned, that the addition of hydrogen peroxide is useful to convert AcAc into 3-OHB.

Unfortunately, the relative insensitivity, and the potential hazard of the routine use of 30% H2O2 render the

method of limited value.

Positive results almost exclusively indicate diabetic ketoacidosis, ketone bodies are also detectable in

starvation.14

Atypical colour reactions may be caused by brownish urine colour, drugs bearing sulfhydryl groups (ACE

inhibitors), and the phenylketone 4-hydroxyphenylpyruvate (HPPA).31

If the ketone test appears positive without

glucoseuria in reflection photometry (automatic dipstick reader), visually regard the colour of the test-pad, only a

pink to purple colour reaction indicates the presence of ketone bodies.31

False negative results are caused by acidic urine (ascorbic acid) and significant bacteriuria.32

The urine ketone test by dipsticks is useful with some limitations. The measurement of 3-OHO from plasma

(analyser, plasma dipstick, also urine dipsticks) can be used to confirm results.33

Bilirubin

Only conjugated (water soluble) bilirubin can freely be filtrated by the glomeruli and detected in urine.

Bilirubinuria may precede hyperbilirubinuria in animals with a low renal threshold for bilirubin (dogs). In this

species haemoglobin can be converted to bilirubin also in the tubular epithlia and contributes to the excretion of

(unconjugated) bilirubin.34

For bilirubin the timely reading of the dipstick – as indicated by the manufacturer - is essential, because delayed

processing may cause false positive reactions. Atypical colour reactions can also be caused by indican and

metabolites of different drugs. 27

Exposure of bilirubin to ultraviolet light leads to its oxidation to biliverdin which is not detected by the diazo

reaction.

Positive or questionable results can be confirmed by tablet test Ictotest® (Siemens Diagnostics), as well as by

wet chemistry.

Urobilinogen Urobilinogen is a colourless metabolite of bilirubin produced in the intestines and partly reabsorbed and excreted

into the urine.

In human medicine it is a marker for liver disease, haemolysis and intestinal obstruction.

There is little evidence that the parameter is useful in veterinary species.35

Consequently, urobilinogen is omitted in some dipsticks designed for the veterinary market.

92

RBC/Haemoglobin The test for the presence of myoglobin or haemoglobin in the urine may be positive in the presence of

haematuria, haemoglobinuria, or myoglobinuria. Myoglobinuria is seen with muscle disease, haemoglobinuria

may be seen with overwhelming haemolysis, and haematuria is seen with haemorrhage anywhere in the uro-

genital tract.

Haematuria in theory should be recognised on the test pads by spots caused by the reaction of haemoglobin set

free after the lysis of RBC, but this hardly ever can be seen by visual evaluation of the dipstick and never by

automatic analysis. Therefore, haematuria always has to be confirmed by the presence of RBC in the urine

sediment.

Myoglobinuria is seen with muscle disease and may be distinguished from haemoglobin by the associated

clinical signs and serum chemistry (CK). Myoglobin is fast eliminated by the kidney, thus the plasma usually is

colourless.

Some authors mention that urine containing myoglobin tends to be red-brown to brown in colour27

, but this is

also true for severe haemolysis, especially caused by babesia36

.

The ammoniumsulfate precipitation reaction to discriminate between haemoglobinuria and myoglobinuria is still

mentioned in several textbooks and may be useful.37

The most sensitive test to distinguish haematuria and

myoglobinuria is absorbance spectroscopy.

False positive reactions may be caused by oxidising reagents (hypochlorite, hydrogen peroxide), high

concentrations of halogens (bromide, iodine in radiopaque material)27

. False negative reactions by inadequately

mixed urine and failure of RBC to lyse on reagent pad.

In summary, the dipstick reaction for haemoglobin yields useful results with reasonable good sensitivity and

specificity compared to RBC in sediment (Fig. 4).

a b

Fig. 4: Linear fit between RBC in sediment (RBC) and dipstick reaction (Blood) for cats (a) and dogs (b). Cats

have a higher percentage of positive reactions.

Literature 1. Leidinger E, Leidinger J. Urinary sediment - an evidence-based approach, ESVCP Congress, Dublin

2011.

2. Pinches, M. Evaluation of 9 novel renal urinary biomarkers. ESVCP Congress. Thessaloniki, 2009.

3. ASVCP Quality assurance guidelines. Available at http://www.asvcp.org/pubs/qas/index.cfm. Accessed 28

April, 2012.

4. Rabinovitch A, Arzoumanian L, Curcio KM, Dougherty B, Halim A. Urinalysis –approved guideline, 3rd

ed. Clinical and Laboratory Standards Institute document GP16-A3, 2009.

5. Aspervall O, Hallander H, Gant V, Kouri T. European guidelines for urinanalysis: a collaborative document

produced by European clinical microbiologists and clinical chemists under ECLM in collaboration with

ESCMID. European Society of Clin Microbiol Infec Diseas 2001;7:173-178.

6. Froom P, Bieganiec B, Ehrenrich Z, Barak M. Stability of common analytes in urine refrigerated for 24h

before automated analysis by test strips. Clinl Chem 2000;46(9):1384-6.

7. Raskin ER, Murray KA, Levy JK. Comparison of home monitoring methods for feline urine pH

measurement. Veterinary Clinical Pathology 2002;31(2):51-5.

http://www.asvcp.org/pubs/qas/index.cfm

93

8. Gallagher, EJ; Schwartz, E; Weinstein, RS. Performance characteristics of urine dipsticks stored in open

containers. Am J Emerg Med, 1990;8(2):121-3.

9. Chu S, Sparks D. Assessment of a solid-phase reagent for urinary specific gravity determination. Clin

Biochem 1984;17:34-36.

10. Osborne CA, Stevens JB. Urinalysis: a clinical guide to compassionate patient care: Bayer; 1999.

11. Stockham SL, Scott MA. Fundamentals in Clinical Pathology. 2nd ed: Blackwell Publishing; 2008:415-494.

12. George JW. The usefulness and limitations of hand-held refractometers in veterinary laboratory medicine: a

historical and technical review. Veterinary Clinical Pathology 2001;30(4):201-10.

13. Johnson KY, Lulich JP, Osborne CA. Evaluation of the reproducibility and accuracy of pH-determining

devices used to measure urine pH in dogs. Journal of the American Veterinary Medical Association

2007;230(3):364-369.

14. Archer, J. Urine analysis. In: Villiers E, Blackwood L (eds). BSAVA manual of canine and feline clinical

pathology. 2nd

. Ed. Cheltenham: BSAVA, 1998:149-168.

15. Holan KM, Kruger JM, Gibbons SN, Swenson CL. Clinical evaluation of a leukocyte esterase test-strip for

detection of feline pyuria. Vet Clin Path 1997;26:126-131.

16. N. Bauer, S. Rettig, A. Moritz. Evaluation the Clinitek status™ automated dipstick analysis device for

semiquantitative testing of canine urine. Res Vet Sci 2008;85:467–472.

17. Mundt LA, Shanahan K. Graff's Textbook of Urinalysis and Body Fluids. Philadelphia: Lippincott, Wiliams

& Wilkins; 2010.

18. Strasinger SK, Dilorenzo, MS. Chemical examination of urine. In: urinalysis and body fluids. Strasinger SK,

Dilorenzo, MS eds. FA Davis, Philadelphia. 2008;p53-80.

19. Klausner, JS; Osborne, CA; Stevens, JB. Clinical evaluation of commercial reagent strips for detection of

significant bacteriuria in dogs and cats. Am J Vet Res, 1976 vol. 37(6) pp. 719-22.

20. Wamsley H, Alleman R. Complete urinalyis. In: BSAVA manual of canine and feline nephrology and

urinology. 2nd

ed. Elliot J, Grauer GF eds. Gloucester: BSAVA, 2008:87-104.

21. Swenson CL, Boisvert AM, Gibbons SN, Kruger JM. Evaluation of modified Wright-staining of a dried

urinary sediment as a method for accurate detection of bacteriuria in cats. Veterinary Clinical Pathology

2011;40(2):256-264.

22. Reagan L, Lind B, Shearer A, Botts S. Influence of urine pH on accurate urinary protein determination in

Sprague-Dawley rats. Veterinary Clinical Pathology 2007;36:73-8.

23. Moore FM, Brum LS, Brown, L. Urine Protein Determination in Dogs and Cats: Comparison of Dipstick

and Sulfasalicylic Acid Procedures. Vet Clin Path 1991;20:95-97.

24. Welles EG, Whatley EM, Hall AS, Wright JC. Comparison of Multistix PRO dipsticks with other

biochemical assays for determining urine protein (UP), urine creatinine (UC), and UP:UC ratio in dogs and

cats. Veterinary Clinical Pathology 2006;35(1):31-6.

25. Bauer N, Rettig S, Moritz A. Evaluation the Clinitek status™ automated dipstick analysis device for

semiquantitative testing of canine urine. Res. Vet. Sci. 2008;85:467–472.

26. Vaden SL, Pressler BM, Lappin MR, Jensen AL. Effects of urinary tract inflammation and sample blood

contamination on urine albumin and total protein concentrations in canine samples. Veterinary Clinical

Pathology 2004;33(1):14-9.

27. Sink CA, Weinstein NM. Practical veterinary urinalysis. Wiley-Blackwell, Ames, Iowa 2012.

28. Lott JA, Turner K. Evaluation of Trinder's glucose oxidase method for measuring glucose in serum and

urine. Clin Chem 1975;21(12):1754-60.

29. Sheiko, MC; Burkhardt, RT, Batsakis, JG. Glucose measurements. A 1977 CAP Survey analysis. Am J Clin

Pathol 1979;72(2 Suppl):337-40.

30. Oster JR, Rietberg B, Taylor AL, Perez GO, Chandra R, Gardner LB. Can beta-hydroxybutyrate be detected

at the bedside by in vitro oxidation with hydrogen peroxide. Diabetes Care 1984;7:80-22.

31. Cartwright J, Richard M. Green RM. Tyrosine-derived 4-hydroxyphenylpyruvate reacts with ketone test

fields of 3 commercially available urine dipsticks. Vet Clin Pathol 2010;39:354–357.

32. Häring HU, Gallwitz B, Müller-Wieland D, Usadel KH. Diabetologie in Klinik und Praxis: Das

Referenzwerk für die alltägliche Praxis [Diabetology in Clinic and practice. A reference for the daily

routine]. Thieme 2011.

33. Zeugswetter F, Pagitz M. Ketone measurements using dipstick methodology in cats with diabetes mellitus. J

Small Anim Pract. 2009;50(1):4-8.

34. de Schepper J. Degradation of haemoglobin to bilirubin in the kidney of the dog. Tijdschr Diergeneeskd.

1974;99:699-707.

35. Tang X, Xia Z. An experimental study of hemolysis induced by onion (Allium cepa) poisoning in dogs. J

Vet Pharmac Therap, 2008;31:143-149.

36. Pagès JP, Trouillet JL. Thrombocytopénie dans la babésiose du chien. A propos de 153 observations.

Pratique médicale et chirurgicale de l’animal de compagnie 1984;19: 222–227.

94

37. Thrall MA, Baker D, Campell TW. Veterinary Hematology and Clinical Chemistry. Philadelphia:

Lippincott, Williams & Willkins; 2004.

HEMATOLOGY

ESVCP PLENARY LECTURE

ERYTHROCYTE ENZYME DEFICIENCIES IN DOMESTIC ANIMALS

J.W. Harvey. Department of Physiological Sciences, College of Veterinary Medicine, University of Florida,

Gainesville, FL


MAMMALIAN HEMATOPOIESIS


Gainesville, FL


Hematopoietic Microenvironment. Beginning in mid-gestation and continuing throughout postnatal life,

mammalian blood cells are produced continuously from hematopoietic stem cells (HSCs) within extravascular

spaces of the bone marrow because of the unique microenvironment present in the marrow. The hematopoietic

microenvironment is a complex meshwork composed of stromal (fibroblast-like) cells, endothelial cells,

adipocytes, macrophages, subsets of lymphocytes, NK cells, and osteoblasts; extracellular matrix components;

and glycoprotein growth factors that profoundly affect HSC and hematopoietic progenitor cell (HPC)

engraftment, survival, proliferation, and differentiation.

Stromal cells and endothelial cells produce components of the extracellular matrix (ECM), including

collagen fibers, basement membranes of vessels and vascular sinuses, proteoglycans, and glycoproteins, as well

as various growth factors. In addition to providing structural support, the ECM is important in the binding of

hematopoietic cells and soluble growth factors to stromal cells and other cells in the microenvironment so that

optimal proliferation and differentiation can occur by virtue of these cell-cell interactions.

Adhesion molecules (most importantly β1-integrins) on the surface of hematopoietic cells bind to ECM

glycoproteins such as vascular cell adhesion molecule-1 (VCAM-1). The spectrum of expression of adhesion

molecules on hematopoietic cells that will differentially bind to these ECM glycoproteins varies with the type,

maturity, and activation state of the hematopoietic cells. In addition to anchoring cells to a given

microenvironmental niche, binding of adhesion molecules on hematopoietic cells also plays a role in cell

regulation by direct activation of signal pathways for cell growth, survival, and differentiation or by modulating

responses to growth factors. Proteoglycans enhance hematopoiesis by trapping soluble growth factors in the

vicinity of hematopoietic cells and by strengthening the binding of hematopoietic cells to the stroma.

Hematopoietic cells develop in microenvironmental niches within the marrow. During steady state

conditions, quiescent HSCs are concentrated near endosteal and trabecular bone where osteoblasts help regulate

their numbers. HSC and HPGs are also located in a vascular niche near vascular sinuses where they appear more

active and likely have homeostatic roles during steady state conditions. Erythroid cells develop around

macrophages, megakaryocytes form adjacent to sinusoidal endothelial cells, and granulocyte development is

associated with stromal cells located away from the vascular sinuses.

Hematopoietic Stem Cells and Progenitor Cells. The most primitive HSC has the capacity to differentiate into

hematopoietic progenitor cells (HPCs) of all blood cell lineages and several cell types in tissue. A separate

mesenchymal stem cell has the ability to differentiate into multiple lineages including marrow stromal cells,

adipocytes, osteoblasts, chondrocytes, fibroblasts, and myoblasts. When measured in an in vitro cell culture

assay, HPCs are referred to as colony-forming units (CFUs). HPCs that rapidly proliferate, retain their ability to

migrate, and form multiple subcolonies around a larger central colony in culture, are called burst-forming units

(BFUs). The HSC produces a common lymphoid progenitor (CLP) and a common myeloid progenitor

(CMP).The CLP is believed to give rise to B-lymphocytes, T-lymphocytes, and NK cells. The CMP is believed

to give rise to all nonlymphoid blood cells, as well as macrophages, dendritic cells, osteoclasts, and mast cells.



95

The CMP (also called CFU-GEM) gives rise to the megakaryocyte-erythrocyte progenitor (MkEP) and

the granulocyte-monocyte progenitor (GMP). The MkEP produces megakaryocyte progenitors (MkP) and

erythrocyte progenitors (EP). The GMP produces the granulocyte progenitor (GP), the monocyte-dendritic cell

progenitor (MDP), the basophil-mast cell progenitor (BMaP), and the eosinophil progenitor (EoP).

Hematopoietic Growth Factors. Proliferation of HSCs and HPCs cannot occur spontaneously, but requires the

presence of specific hematopoietic growth factors (HGFs) that may be produced locally in the bone marrow

(paracrine or autocrine) or produced by peripheral tissues and transported to the marrow through the blood

(endocrine). All cells in the hematopoietic microenvironment, including the hematopoietic cells themselves,

produce HGFs and/or inhibitors of hematopoiesis. Some HGFs have been called poietins (erythropoietin [EPO]

and thrombopoietin [TPO]). Other growth factors have been classified as colony stimulating factors (CSFs)

based on in vitro culture studies. Finally, some HGFs have been described as interleukins (ILs).

Hematopoietic cells coexpress receptors for more than one HGF on their surface. The number of each

receptor type present depends on the stage of cell activation and differentiation. Binding of an HGF to its

receptor results in a series of enzymatic reactions that generate transcription factors that promote the synthesis of

molecules that inhibit apoptosis, the formation of cell-cycle regulators (cyclins), and the synthesis of additional

HGFs and their receptors. The pathways involved in generating lineage restricted transcription factors is

complex and beyond the scope of this presentation.

HGFs vary in the type(s) of HSCs and/or HPCs that they can stimulate to proliferate. Factors are often

synergistic in their effects on hematopoietic cells. In some instances, an HGF may not directly stimulate the

proliferation of a given cell type, but may potentiate its proliferation by inducing the expression of membrane

receptors for HGFs that do stimulate proliferation. Some glycoproteins such as IL-1 and tumor necrosis factor-α

(TNF-α) can modulate hematopoiesis indirectly by stimulating marrow stromal cells, endothelial cells, and T-

lymphocytes to produce HGFs. Different combinations of HGFs regulate the growth of different types of HSCs

and/or HPCs.

Early-acting HGFs are involved with triggering dormant (GO) primitive HSCs to begin cycling. Stem

cell factor (SCF), flt3 ligand (FL), and TPO are important early factors. Intermediate-acting HGFs have broad

specificity. IL-3, granulocyte-macrophage-CSF (GM-CSF), and IL-4 support proliferation of multipotent HPCs.

These factors also interact with late-acting factors to stimulate proliferation of a wide variety of committed

progenitor cells. Late-acting HGFs have restricted specificity. Macrophage-CSF (M-CSF), G-CSF, EPO, TPO,

and IL-5 are more restrictive in their actions. They have their most potent effects on committed progenitor cells

and later stages of development when cell lines can be recognized morphologically. TPO appears to an

exception. In addition to stimulating platelet production, it is important in maintaining a population of HSCs in

their osteoblastic niche.

Erythropoiesis. The earliest EP is the burst-forming unit-erythrocyte (BFU-E) which differentiates into the

colony forming unit-erythrocyte (CFU-E). EPO is the principal growth factor that promotes the viability,

proliferation and differentiation of EPs expressing specific cell surface EPO receptors. EPO receptor copies on

cell surfaces increase to maximum values in CFU-E cells and then decline in rubriblasts and continue to decrease

in later stages of erythroid development. Because of their EPO receptor density, CFU-E cells readily respond to

EPO, promoting their proliferation, differentiation and transformation into rubriblasts, the first morphologically

recognizable erythroid cell type.

Erythroid precursors develop bound to macrophages which may promote basal erythrocyte production

by producing positive growth factors, including EPO; however, they may inhibit erythropoiesis by producing

negative factors such as IL-1, TNF- tions. Although some degree of

basal regulation of erythropoiesis occurs within the marrow microenvironment, humoral regulation is important,

with EPO production occurring primarily within peritubular interstitial cells of the kidney and various inhibitory

cytokines being produced at sites of inflammation throughout the body. EPO production is stimulated by tissue

hypoxia which is mediated by hypoxia-inducible factors that control the transcription of the EPO gene in EPO

secreting cells.

Rubriblasts are continuously generated from marrow EPs. The production of a rubriblast initiates a

series of approximately four divisions over a period of 3 or 4 days to produce about 16 metarubricytes that are no

longer capable of division. An immature erythrocyte, termed a reticulocyte, is formed following extrusion of the

metarubricyte nucleus. Reticulocyte maturation into erythrocytes begins in the bone marrow and is completed in

the peripheral blood and spleen in dogs, cats, and pigs.

96

Leukopoiesis. A variety of early and intermediate acting cytokines with overlapping specificities are important

in neutrophil production (also called granulopoiesis). GPs are stimulated to proliferate and differentiate into

myeloblasts by G-CSF. This cytokine appears to play a role in the basal regulation of granulopoiesis, as well as

function as a primary regulator of the neutrophil response to inflammatory stimuli. G-CSF increases the number

of cell divisions and reduces the time for granulocytic progenitors to develop into neutrophils. It also promotes

the release of neutrophils from bone marrow into blood. Activated helper T-lymphocytes produce various growth

factors including IL-3 and GM-CSF. Mononuclear phagocytes, fibroblasts, and endothelial cells can produce

GM-CSF and G-CSF when appropriately stimulated.

Neutrophilic cells within the bone marrow can be included in two pools. The mitotic pool includes

myeloblasts, promyelocytes, and myelocytes. Approximately four or five divisions occur over several days. The

postmitotic pool includes metamyelocytes, bands, and segmented neutrophils. The marrow transit time from

myeloblast to release of mature neutrophils into blood varies by species, but is generally between 6 and 9 days.

This time can be shortened considerably when inflammation is present.

Mature neutrophils may provide negative feedback inhibition on their own production by releasing

substances locally in the marrow that inhibit granulopoiesis, clearance of circulating G-CSF following binding to

surface receptors on neutrophils, and by phagocytosis of invading microorganisms that would otherwise result in

the production of HGFs by tissue cells.

Eosinophil production in marrow parallels that of neutrophils. Activated Th2 lymphocytes produce IL-

5, which promotes the proliferation of EoPs and production of mature eosinophils.

The BMaP gives rise to the basophil progenitor and the mast cell progenitor (MaP). A specific growth

factor for the production of basophils has not been identified. IL-3 appears to be the major growth and

differentiation factor for basophils, but other growth factors including GM-CSF, IL-5, TGF-β, and nerve growth

factor also promote the production of basophils.

MDPs give rise to monocytes and common dendritic cell progenitors (CDPs). Monocytes are produced

through the combined effects of IL-3, GM-CSF, M-CSF, and IL-34 on proliferation and differentiation of bone

marrow progenitor cells. Monocytes are important effector cells during inflammatory conditions. They exit the

blood, respond to the tissue environment, and differentiate into subsets of macrophages and inflammatory

dendritic cells.

B-lymphocyte progenitors produce mature, naive B-lymphocytes in the marrow in most mammals, in

specialized ileal Peyer's patches in dogs, pigs, and ruminants, and the bursa of Fabricius in birds. Although a

number of cytokines are involved in B-lymphocyte production in marrow, IL-7 appears to be an especially

important positive growth factor. B-lymphocyte lymphopoiesis is inhibited by several factors including TGF-β,

IFN-α, InFN-β, IFN-γ.

T-lymphocyte progenitors leave the marrow and migrate to the thymus where they develop into T-

lymphocytes under the influence of the thymic microenvironment and growth factors (including FL and IL-7)

produced in the thymus.

Most NK cells are produced from progenitor cells in the bone marrow. Growth factors controlling their

production need further characterization, but SCF, IL-2, IL-7, and IL-15 can stimulate NK cell development

from progenitor cells in vitro.

Thrombopoiesis. Blood platelets in mammals are produced from multinucleated giant cells in bone marrow

called megakaryocytes. The earliest MkP is the burst-forming unit-megakaryocyte (BFU-Mk). When

appropriately stimulated, this progenitor cell divides and differentiates into colony-forming unit-megakaryocyte

(CFU-Mk) progenitor cells, which divide and differentiate into megakaryoblasts. Mitosis stops at this stage and

endomitosis (nuclear reduplication without cell division) begins. Generally 2 to 5 nuclear reduplications occur

resulting in 8 to 64 sets of chromosomes (8N-64N) in mature megakaryocytes. Cell volume increases with each

reduplication, resulting in the formation of giant cells.

Megakaryocytes develop just outside vascular sinuses. Protrusions of cytoplasm (proplatelets) from

megakaryocytes form and extend into sinuses where they can be sheared off by the force of flowing blood. These

beaded-appearing proplatelets eventually fragment into individual platelets within the sinuses and general

circulation.

A number of cytokines can stimulate or enhance the proliferation and expansion of megakaryocyte

97

progenitor cells; however, TPO is the key regulator of platelet production. It increases platelet production by

stimulating megakaryocyte proliferation, survival, and size (ploidy). The major sites of TPO production appear

to be the endothelial cells of the liver. The amount of TPO produced in the body appears to be relatively

constant. TPO receptors (c-Mpl receptors) on blood platelets and maturing megakaryocytes can bind, internalize

and degrade TPO, providing negative feedback on platelet production. IL-6 stimulates thrombopoiesis by

increasing the production of TPO by the liver, which contributes to the thrombocytosis seen in some

inflammatory conditions.

Reference. Harvey JW. Veterinary Hematology. A Diagnostic Guide and Color Atlas. St. Louis, MO: Elsevier

Saunders; 2012:33-48.

PLATELET COUNTING IN THE CAT

C. Trumel., F. Granat , N. Bourgès Abella, A. Geffré. Laboratory of Clinical Pathology of the National

Veterinary School of Toulouse, France.


The measurement of platelet concentration is important for many diagnostic and therapeutic purposes. Its

accuracy can be markedly affected by platelet aggregation which generally decreases the count and can thus

result in apparent thrombocytopenia, regardless of laboratory method or species 1, 2

. Platelet clumping is often

observed in feline blood specimens, even in normal cats 3. Furthermore, feline platelet and red blood cell (RBC)

sizes often overlap because of the large size of platelets and small size of RBCs in this species 3, 4, 5

. As a

consequence, 1/ the platelet count for feline specimens with aggregates is often inaccurate regardless of the

laboratory method used; 2/ the mean platelet volume (MPV) and mean corpuscular volume (MCV), white blood

cell count (WBC) and differential can also be inaccurate in feline whole blood specimens when impedance-based

analyzers are used 1, 6, 7

. Although size overlapping problems can be resolved by flow cytometry, the occurrence

of platelet aggregates needs to be reduced at the preanalytical step by use of platelet inhibitors.

1/ Platelet activation is a complex mechanism which need to be understood before choosing the most adequate

inhibitor efficient in all species in which CBC are routinely performed. 2/ To be used for routine hematology,

this should be stable, able to be associated with an anticoagulant, should not modify the CBC results and allow

delayed analysis.

After needle introduction for blood sampling, tissue factor (TF), collagene, ADP from altered endothelial cells

and epinephrine initiate thrombin formation and platelet activation via membrane receptors. The most important

pathways activated are mediated via ADP receptors, thromboxane A2 formation and contribute to fibrinogen

fixation on glycoprotein IIb/IIIa by activation of IIb/IIIa via an inside out signaling pathway 8. The most potent

inhibitors used in clinical situations against thrombosis are ADP, TXA2, IIb/IIIa inhibitors, whereas the most

frequently used in vitro inhibitors act via AMPc elevation and indirect inhibition of the previously mentioned

pathways.

The anticoagulant CTAD (Citrate, Theophylline, Adenosine, and Dipyridamole) used in human medicine for

platelet assessment has been reported to reduce feline platelet aggregation , and in one study in humans, a

combination of EDTA and CTAD was more efficient than CTAD alone at 4°C. 10, 11

. EDTA and CTAD limited

or even broke up platelet clumping up to 48 hours after blood collection and enabled to obtain more accurate

platelet and white blood cell counts in feline blood samples, without altering the other variables of the complete

blood count 12

. Other reagents have also been used to obtain resting platelets, such as kanamycine 13, 14

, and

PGE1 15, 16

but are less easy to handle, whereas CTAD tubes are commercially available.

1. Norman EJ, Barron RC, Nash AS, Clampitt RB. Prevalence of low automated platelet counts in cats:

comparison with prevalence of thrombocytopenia based on blood smear estimation. Vet Clin Pathol.

2001;30:137-40.

2. Koplitz SL, Scott MA, Cohn LA. Effects of platelet clumping on platelet concentrations measured by use of

impedance or buffy coat analysis in dogs. J Am Vet Med Assoc. 2001;219:1552-1556.

3. Zelmanovic D, Hetherington EJ. Automated analysis of feline platelets in whole blood, including platelet

count, mean platelet volume, and activation state. Vet Clin Pathol. 1998;27:2-9.

4. Weiser MG, Kociba GJ. Platelet concentration and platelet volume distribution in healthy cats. Am J Vet

Res. 1984;45:518-22.

5. Boudreaux MK, Osborne CD, Herre AC, Rivera ER, Spangler EA. Unique structure of the Mloop region of

b1-tubulin may contribute to size variability of platelets in the family Felidae. Vet Clin Pathol 2010;39:417-423.

6. Dawson H, Hoff B, Grift E, Tvedten H, Shoukri M. Validation of the Coulter AcT Diff hematology

analyzer for analysis of blood of common domestic animals. Vet Clin Pathol. 2000;29:132-6.

98

7. Dewhurst EC, Crawford E, Cue S, Dodkin S, German AJ, Papasouliotis K. Analysis of canine and feline

haemograms using the VetScan HMT analyser. J Small Anim Pract. 2003;44:443-

8. Goggs R, Poole AW. Platelet signaling-A primer. J Vet Emerg Crit Care (San Antonio). 2012

9. Norman EJ, Barron RC, Nash AS, Clampitt RB. Evaluation of a citrate-based anticoagulant with platelet

inhibitory activity for feline blood cell Counts. Vet Clin Pathol. 2001;30:124-32.

10. Mody M, Lazarus AH, Semple JW, Freedman J. Preanalytical requirements for flow cytometric evaluation of

platelet activation: choice of anticoagulant. Transfus Med. 1999;9:147-54.

11. Macey M, Azam U, McCarthy D, Webb L, Chapman ES, Okrongly D, et al. Evaluation of the anticoagulants

EDTA and citrate, theophylline, adenosine, and dipyridamole (CTAD) for assessing platelet activation on the

ADVIA 120 hematology system. Clin Chem. 2002;48:891-9.

12. Granat F, Geffré A, Braun JP, Trumel C. Comparison of platelet clumping and complete blood count results

with Sysmex XT-2000iV in feline blood sampled on EDTA or EDTA plus CTAD (citrate, theophylline,

adenosine and dipyridamole). J Feline Med Surg. 2011;13:953-8.

13. Ahn HL, Jo YI, Choi YS, Lee JY, Lee HW, Kim SR, et al. EDTA-dependent pseudothrombocytopenia

confirmed by supplementation of kanamycin; a case report. Korean J Intern Med. 2002;17:65-8.

14. Sakurai S, Shiojima I, Tanigawa T, Nakahara K. Aminoglycosides prevent and dissociate the aggregation of

platelets in patients with EDTA-dependent pseudothrombocytopenia. Br J Haematol. 1997;99:817-23.

15. Gryglewski RJ, Korbut R, Ocetkiewicz A. De-aggregatory action of prostacyclin in vivo and its enhancement

by theophylline. Prostaglandins. 1978;15:637-44.

16. Tvedten H, Johansson P. Feline platelet counting with prostaglandin E1 on the Sysmex XT-2000iV. Vet Clin

Pathol. 2010;39:190-2.

BONE MARROW EXAMINATION IN DOGS AND CATS


Gainesville, FL


Reasons for Bone Marrow Examination. Bone marrow evaluation is indicated when peripheral blood

abnormalities are detected. The most common indications are persistent neutropenia, unexplained

thrombocytopenia, poorly regenerative anemia, or a combination thereof. Examples of proliferative

abnormalities in which bone marrow examination may be indicated include persistent thrombocytosis or

leukocytosis, abnormal blood cell morphology, or the unexplained presence of immature cells in blood (e.g.,

nucleated erythroid cells in the absence of polychromasia or a neutrophilic left shift in the absence of

inflammation).

Bone marrow is sometimes examined to stage neoplastic conditions (lymphomas and mast cell tumors);

to estimate the adequacy of body iron stores; to evaluate lytic bone lesions; and to search for occult disease in

animals with fever of unknown origin, unexplained weight loss, and unexplained malaise. Bone marrow

examination can also be useful in determining the cause of a hyperproteinemia when it occurs secondarily to

multiple myeloma, lymphoma, leishmaniasis, and systemic fungal diseases. It may also reveal the cause of a

hypercalcemia when associated with lymphoid neoplasms, multiple myeloma, or metastatic neoplasms to bone.

Bone marrow aspirates are done more frequently than core biopsies in veterinary medicine. Aspirates

are easier, faster, and less expensive to perform than are core biopsies. Bone marrow core biopsies require

special needles that cut a solid core of material which is then placed in fixative, decalcified, embedded,

sectioned, stained, and examined microscopically by a pathologist. Core biopsy sections provide a more

accurate way of evaluating marrow cellularity and examining for metastatic neoplasia than do aspirate smears,

but cell morphology is more difficult to assess.

Organized Approach to Bone Marrow Evaluation. Complete differential cell counts are not usually done on

marrow smears or core biopsies, but rather a number of judgments are made and recorded as presented below.

Smears and core biopsy sections should be scanned with low-power objectives to gain an appreciation of the

overall cellularity and to determine the adequacy of megakaryocyte numbers. Normal marrow appears

heterogenous. If some, or all, of a marrow smear or core section appears homogenous, an abnormal population of

cells is probably present. Regional infiltrates of neoplastic cells are more easily appreciated in core sections than

in aspirate smears.

As a general rule, erythroid precursors are smaller, have more nearly spherical nuclei with more

condensed nuclear chromatin, and have darker cytoplasm than do granulocyte precursors at similar maturation

stages. Consequently, smaller and darker cells, observed by scanning marrow smears at low power, are usually

erythroid precursors (unless lymphocytes are increased in numbers), and the larger, paler cells are usually

granulocyte precursors. Identification of specific cell type is more difficult in core biopsy sections compared to


99

aspirate smears; consequently, sections may be stained with Giemsa and Periodic acid-Schiff (PAS), in addition

to H&E, in an attempt to identify cell types present.

Cellularity. The cellularity of bone marrow is estimated by examining the proportion of cells versus fat

present in particles. Normal cellularity varies between 25% and 75% cells, depending on age. The overall

cellularity of bone marrow decreases with age because of a loss of skeletal bone mass, resulting in increased

marrow space. If the particles are composed of more than 75% cells, the marrow is interpreted as hypercellular,

and if the particles are composed of more than 75% fat, the marrow is interpreted as hypocellular. If few or no

particles are present on smears, it is not possible to accurately estimate the marrow cellularity.

Marrow can become hypercellular when one or more cell types exhibit increased proliferation in

response to peripheral needs, such as occurs in response to anemia (erythroid hyperplasia) or purulent

inflammation (granulocytic hyperplasia). The marrow may also become hypercellular secondary to dysplastic or

neoplastic proliferations of marrow cells or from infiltration of neoplastic cells from peripheral tissues (e.g., a

metastatic lymphoma). Defects in either progenitor cells or the bone marrow microenvironment necessary for

their survival and proliferation can result in hypocellular marrow.

Megakaryocytes. The frequency and morphology of megakaryocytes should be evaluated by scanning

areas of aspirate smears containing particles at low power using a 10x objective. Most large particles in aspirate

smears should have several associated megakaryocytes, and normally 80% to 90% of megakaryocytes are of the

granular, mature type. Multiple particle-rich fields should be scanned to determine an average number of

megakaryocytes per low power field. If less than 3-5 megakaryocytes are seen per field, megakaryocyte numbers

are considered low and, if greater than 15 megakaryocytes are seen per field megakaryocytic hyperplasia is

present. Abnormal megakaryocyte morphology (e.g., increased numbers of promegakaryocytes or the presence

of dwarf megakaryocytes) should be noted when present.

Erythroid Cells. The maturation and morphology of the erythroid series should be evaluated to

determine if it is complete (frequent polychromatophilic erythrocytes should be present) and orderly. There is

generally a progressive increase in numbers with each stage of development, from low numbers of rubriblasts

(generally less than 1%) to high numbers of polychromatophilic rubricytes, which may account for one quarter

of all nucleated cells in the bone marrow. Metarubricytes are numerous, but generally not as numerous as

polychromatophilic rubricytes.

Rubriblasts and prorubricytes usually do not exceed 5% of all nucleated cells. If the proportion of these

immature cells is increased, this finding should be noted. An increase in mature as well as immature cells of the

erythroid series is expected in response to anemia. If immature erythroid cells are increased and later stages are

not, it suggests a proliferative abnormality is present. Additional abnormal morphologic findings that should be

recorded when present include megaloblastic cells, frequent binucleated cells, and pleomorphic nuclei.

Granulocytic Cells. The distribution of granulocytic cells should be evaluated to determine whether the

series is complete (i.e., a normal number of mature granulocytes are present) and orderly. There is generally a

progressive increase in numbers with each stage of development, from low numbers of myeloblasts (often less

than 1%) to high numbers of mature neutrophils, which may account for nearly one quarter of all nucleated cells

in the bone marrow. Myeloblasts and promyelocytes generally do not exceed 5% of all nucleated cells. If the

proportion of these immature cells is increased, this finding should be noted. An increase in mature as well as

immature cells of the myeloid series is expected in inflammatory disorders resulting in granulocytic hyperplasia.

If immature granulocytic cells are increased and later stages are not, it indicates that either more mature cells

have been depleted in the marrow granulocyte pool, as occurs in acute inflammation, or that a proliferative

abnormality is present. Morphologic abnormalities such as large cell size and vacuolated cytoplasm should be

reported when present.

Total eosinophilic cells usually account for less than 6% of all nucleated cells in the marrow, with

basophilic precursors generally accounting for less than 1% of all nucleated cells. The increased representation

of the eosinophilic or basophilic series should be recorded. An increase in one or both of these cell lines is

usually associated with inflammatory conditions that result in increased numbers of eosinophils or basophils in

blood and/or tissues. They may also be increased in association with some myeloproliferative disorders.

Myeloid to Erythroid Ratio. A myeloid to erythroid (M:E) ratio (also referred to as a granulocytic to

erythroid ratio) is calculated by examining 500 cells and dividing the number of granulocytic cells, including

mature granulocytes, by the number of nucleated erythroid cells. Alternately, this ratio may be estimated by

experienced professionals. Normal M:E ratios are reported to be between 0.75 and 2.5 in dogs and between 1.2

and 2.2 in cats. Dilution of a bone marrow aspirate with blood can result in a falsely high M:E ratio, especially if

a substantial neutrophilia is present in blood.

Lymphocytes. Small lymphocytes generally account for less than 10% of all nucleated cells in normal

animals, but they may reach 14% in some healthy dogs and 20% in some healthy cats. Increased numbers of

mature lymphocytes may be seen in bone marrow from animals with chronic lymphocytic leukemia (CLL), and

increased number of small lymphocytes have been reported in the bone marrow of cats and dogs with erythroid

aplasia and immune-mediated nonregenerative hemolytic anemia. Increased numbers of small lymphocytes have

100

also been reported in cats with a thymoma or cholangiohepatitis. Increased numbers of prolymphocytes and/or

lymphoblasts suggest the presence of either acute lymphoblastic leukemia or a metastatic lymphoma.

Plasma Cells. Plasma cells are generally present in low numbers (<2% of all nucleated cells). When

plasma cells exceed 3% of total nucleated cells, they are considered to be increased. In addition to multiple

myeloma, increased numbers of plasma cells can occur in the bone marrow in dogs and cats with immune-

mediated hemolytic anemia, immune-mediated thrombocytopenia, and erythroid aplasia. Plasma cells are also in

increased in bone marrow in response to some infectious agents, including E. canis and visceral leishmaniasis in

dogs and feline infectious peritonitis in cats.

Mononuclear Phagocytes. Monocytes and their precursors comprise a small portion (<3%) of bone

marrow cells. They are difficult to differentiate from early granulocytic cells. Increased numbers of monocyte

precursors may be present in response to inflammatory conditions, but the occurrence of high numbers of

monoblasts indicates that a myeloproliferative disorder is present.

Macrophages generally do not exceed 1% of total nucleated cells present in the bone marrow of normal

animals. They may be increased in the bone marrow with necrotic, granulomatous inflammatory, and neoplastic

(malignant histiocytosis) conditions. Infectious agents may be present within macrophages in conditions such as

leishmaniasis, histoplasmosis, mycobacteriosis, and cytauxzoonosis. Prominent phagocytosis of nucleated and/or

anucleated erythrocytes by macrophages may occur in association with primary or secondary immune-mediated

anemias, blood parasites, malignant histiocytosis, the hemophagocytic syndrome, post-blood transfusion, and

dyserythropoiesis. Prominent leukophagocytosis is rare, but may be seen in association with immune-mediated

neutropenia or when increased marrow apoptosis is present, as occurs in myeloproliferative disorders.

Phagocytized leukocytes may also be seen with the hemophagocytic syndrome.

Other Cells Types. Comments are generally only made concerning cell types listed below when they

are present in increased numbers. Mitotic figures are increased when the proportion of cells undergoing

replication in the bone marrow is increased. They may be somewhat increased in myeloid or erythroid

hyperplasia, but they are more consistently increased in acute myelocytic and acute lymphoblastic leukemias.

Dramatically increased numbers of mitotic erythroid cells occur within the first few hours after the

administration of vincristine.

Osteoclasts and osteoblasts are commonly seen in bone marrow aspirates of young growing animals, but

are rarely seen in normal adult animals. Increased numbers of osteoclasts and osteoblasts may be seen in adult

animals when bone remodeling is increased, such as in disorders with increased parathyroid concentrations, and

in association with osteogenic sarcomas.

Mast cells are rarely seen in the bone marrow from normal domestic animals, but they may occur in

dogs with aplastic anemia of various etiologies. They may also be present in some inflammatory conditions.

Mast cells may occur in the marrow of animals with metastatic mast cell tumors. Metastatic cells from

nonhematopoietic sarcomas or carcinomas are rarely recognized in bone marrow biopsies, but should be reported

when present.

Stainable Iron. Hemosiderin appears gray to black material within our outside macrophages when

stained with routine blood stains. It appears blue following staining with the Prussian blue stain. Determination

of stainable iron in bone marrow is used as a measure of total body iron stores in dogs. A lack of stainable iron is

consistent with iron deficiency; however, negative iron staining is not necessarily predictive of iron deficiency.

Stainable iron may be absent in dogs with polycythemia vera. Cats normally lack stainable iron in the marrow.

Stainable iron in bone marrow is generally increased in dogs with hemolytic anemia and dyserythropoiesis, in

which phagocytosis of erythroid cells is increased, and in animals with anemia resulting from decreased

erythrocyte production, including the anemia of inflammatory disease. The presence of stainable iron is

considered an abnormal finding in cats and may be detected in some animals with myeloproliferative disorders,

hemolytic anemias, or after blood transfusions.

Interpretation. The final step in evaluating a bone marrow aspirate is to provide an interpretation of the

cytologic findings in light of the history, clinical findings, CBC, and results from other diagnostic tests and

procedures. For example, a high M:E ratio could indicate the presence of either increased granulocytic cells or

decreased erythroid cells. Examination of CBC results from blood collected at the same time, as well as an

estimate of the overall cellularity, usually allows the correct interpretation to be made. Bone marrow

examination generally provides information concerning the pathogenesis of abnormalities recognized in blood,

and sometimes a specific diagnosis can be made.

Reference. Harvey JW. Veterinary Hematology. A Diagnostic Guide and Color Atlas. St. Louis, MO: Elsevier

Saunders; 2012:234-359.

101

RETICULOCYTE COUNTS – WHAT IS TRUTH AND WHAT DO WE NEED TO KNOW FOR

INTERPRETING RETICULOCYTOSES?

D. DeNicola. IDEXX Laboratories, Inc., Westbrook, United States of America


LABORATORY MANAGEMENT

TOTAL ALLOWABLE ERROR: A UNIFYING CONCEPT IN THE VETERINARY CLINICAL

PATHOLOGY LABORATORY

K. P. Freeman, IDEXX Laboratories, Grange House, Sandbeck Industrial Estate, Wetherby, West Yorkshire

LS22 4YY, United Kingdom.


In most veterinary reference laboratories, considerable effort is invested in assuring the quality of laboratory

results, including statistical quality control, external quality programme participation, and medical review and

validation of laboratory results. However, despite the time and cost invested in these exercises, there are few

laboratories that DEFINE the level of quality desired for various tests and USE the defined levels as a basis for

quality planning, monitoring and improvement. Guidance regarding the need for quality standards is clear in

documents provided for quality planning by CLSI and by ISO 15189, which states ‘performance specifications

for each procedure shall relate to the intended use of that procedure’ and ‘the laboratory shall design internal

quality quality control procedures that verify the attainment of the intended quality of results’.

Total error may be a confusing term since it may refer to (1) total analytical error (2) calculated or observed total

error obtained in instrument/method performance studies or (3) quality specifications based on clinical use of the

results. Therefore, clarification of the context of the terminology and the variations which exist in the human

and veterinary literature is important. The Stockholm conference hierarchy of models for quality specifications

indicates that these may be based on clinical use/outcome, biological variation, expert opinion, or current

technological state-of-the-art. Although distinctions are often made between analytical quality and pre-analytical

or post-analytical quality, the clinician tends to lump all possible sources of error in the interpretation of results.

Once the quality specification has been determined, then total allowable error can be used in a number of ways,

including (1) instrument procurement - by evaluation of manufacturer’s claims for bias and CV for various

instruments/methods in order to determine if instrument performance will be likely to achieve the standard of

quality required; (2) instrument/method validation - to ensure that the performance is of the standard desired and

that calculated or observed total error does not exceed the total allowable error at various clinical decision levels;

(3) QC validation - in determining the QC rules and number of materials needed to ensure a high probability of

error detection and low probability of false rejection in detection of problems that may result in excessive total

error in laboratory results and (4) ongoing monitoring and quality improvement efforts - through sigma metric

determinations and other tools, such as quality goal indices, to determine priorities for analytical improvements.

If we are to truly practice evidence-based laboratory medicine, we need to be able to provide evidence that we

provide reliable testing services based on defined quality standards. Total allowable error for laboratory testing

provides the central pillar upon which evidence is based. Our duty in veterinary medicine lies in applying our

knowledge of species-specific pathology and physiology and good laboratory practice to providing quality

specifications for veterinary testing.

ALLOWABLE TOTAL ERROR AND ITS USE IN VETERINARY PRACTICE

K. E. Harr DVM, MS, DACVP URIKA, LLC, Seattle, WA, USA


COPYRIGHT STATEMENT AND ACKNOWLEDGEMENT

The following abstract is partially excerpted from draft ASVCP Allowable Total Error Guidelines

which are under review and will be finalized in 2012. The author acknowledges the collective knowledgebase

and time commitments of contributors to this document.



102

INTRODUCTION

Quality assurance (QA) is the overall system for assuring the quality of laboratory test results. QA

includes monitoring and assessment of all laboratory systems and procedures, with the objective of identifying

problems, making corrections, and continuously improving the quality of testing service such that pre-analytical,

analytical, and post analytical error are minimized. (http://www.westgard.com/assuring-quality-through-total-

quality-management.htm Accessed January 30, 2012) Internal or in-clinic quality control (QC) validation and

reference interval generation are part of the instrument/method validation and verification process. (See ASVCP

POCA, General QA guidelines, and reference intervals found here http://www.asvcp.org/pubs/qas/index.cfm )

Once instruments/methods are judged to be suitable for routine testing, QA ensures that ongoing instrument

performance is stable and that the errors inherent to the instrument/methods do not exceed levels that would

invalidate the interpretation of test results. Important QA tools include regular quality control (QC) procedures

and participation in an external quality assurance (proficiency testing) program. (Lester, et al., 2012)

The concept of quality requirements is the foundation for quality planning. Quality requirements can

help guide interpretation of laboratory test results because they provide perspective about variability of results

within an acceptable interval and potential significance of abnormal findings.(Hoffman et A hierarchy of quality

requirements has been proposed (Kenny, 1999), and the most stringent quality requirements are based on clinical

outcomes and clinical decision thresholds. Quality requirements may also be based on data about biologic

variation of an analyte, analytical performance criteria (e.g., as mandated by CLIA for human medicine4), expert

opinion and/or technological state-of-the-art. (Kenny, 1999)

As the presence of in-clinic analyzers has expanded significantly in the past 15 years, the need for well-

tailored quality assurance programs has expanded. In a recent Veterinary Information Network poll in the US,

greater than 10% of veterinary clinics admitted that they did absolutely no quality control on their units which

they had had for more than one year. When asked why, numerous comments basically stated that they didn’t

know how. Clinicians need to be taught a simple statistic that can be used to perform periodic external quality

assurance as well as in-clinic daily QC as dictated by the instrument’s stability and internal QC monitoring.

BIAS (inaccuracy)

Total systematic error which includes constant and proportional bias. Presence of bias may be due to

multiple factors. It is the difference between the measured result and the concentration of a known standard.

Generally used to describe the inaccuracy of a method relative to a comparative method in a method comparison

experiment. The term bias has a specific meaning in the statistical t-test and in difference plot analysis, where

bias (expressed in analyte units) equals the difference between the mean values of the two methods being

compared or the average of all the differences between the paired sample values, i.e. the intercept on a Bland

Altman graph. Bias may also be expressed as a percentage according to the formula bias(%) = mean target –

mean measured/meantarget x 100.

IMPRECISION (random error)

Lack of repeatability or reproducibility of the same result; represented by the standard deviation (in

units of the test) or coefficient of variation (percent). Standard Deviation (SD) is a measure of variability or

diversity and shows how much variation or dispersion there is from the average (mean or expected value). A

small SD indicates that the data points tend to be very close to the mean, whereas large SD indicates that the data

points are spread out over a wide range of values. SD is the square root of a dataset’s variance. Coefficient of

Variation (CV) is standard deviation divided by the mean and expressed as a percentage. CV for purposes of

determination of calculated TE should be determined from precision studies using quality control materials at

various levels of analytes, known standards and/or patient samples.

ALLOWABLE TOTAL ERROR

Westgard was the first to introduce the concept of total error in 1974.(Westgard, 1975) Analytical

imprecision (reproducibility of the result) and bias (systematic error) were combined into a single measure of the

uncertainty of a test result. The ideal situation is to have highly accurate and precise measurement, i.e. low bias

and low CV or SD, respectively. Westgard originally used TE = bias(%) + 1.65CV, but a coefficient (z value) as

high as 6 has been used by some authors for method validation studies.(Westgard, 1996)

The ASVCP guideline defines total allowable error as bias(%) + 2CV which is consistent with CLIA

recommendations. (CLIA '88 Proficiency Testing Limits, U.S. Federal Register). If units of the test are used,

then the equation, bias (expressed in units of the test) + 2SD, is used to calculate allowable total error.

Observed or calculated total error (TEobs) is the sum of random error (imprecision) and systematic error

(bias can be calculated from instrument performance data according to the formula), as defined in this guideline,

2CV + bias(%) or 2SD + bias (quantity of analyte). The calculated TE is specific for a single

instrument/method. Allowable total error (TEa) or desirable total error is an analytical quality requirement that

sets a limit for both the imprecision (random error) and inaccuracy (systematic error or bias) that are tolerable in

103

a single measurement or single test result to insure clinical usefulness. Tables for allowable total error are found

at the end of this document.

Instrument performance evaluation procedure and calculations

The following procedure may be performed when, a new instrument is being considered for purchase, a

new instrument is evaluated to ensure that it performs according to manufacturer’s claims, routine internal

quality control of an instrument - conducted to ensure adequate ongoing performance, evaluating performance

during an external quality assurance (proficiency) program.

Three methods that may be used:

1. Comparison with peer group means in an external QA program participation. This typically must be done

using an external quality assessment program that is employed to help insure quality laboratory results. While

some external quality assurance programs use assayed materials in human medicine, typically in veterinary

medicine, unassayed materials are used and there is reliance on the peer group mean. Peer group is defined by

same instrument and/or method as that upon which the result is obtained. An external quality assessment using

comparison to a peer group is dependent on the numbers of instruments included in the peer group as well as

other laboratories’ maintenance of equipment and quality control.

2. Comparison with target values provided by manufacturers of assayed quality control materials.

An assayed quality control material (known standard) may be repeatedly measured for 5 days to

determine mean, bias, SD and coefficient of variation. In this situation, the mean of the results should be

compared to the mean of the assayed mean to determine bias. These data can then be used to calculate total error

(TEobs) of the analyte. The assayed QCM should be specific for the equipment and methods being evaluated;

the instrument manufacturer should be consulted if there is any doubt regarding QCM suitability. Please consult

the manufacturer to insure that it is appropriate for the equipment and methods.

3. Based on comparison with known gold standards for various analytes (standards provided by external

regulating or governmental organizations and agencies)

As external quality control programs currently in existence are method specific and methods used by in-

clinic laboratories are frequently not represented, option 1 is often not available to the in-clinic laboratory. The

following steps are designed for quality assurance assessment for the in-clinic laboratory but may also be used

by reference laboratories. All steps should be carried out by appropriately trained personnel who are

knowledgeable regarding the analyzer’s operation and the facility’s quality assessment program. Calculations

can easily be carried out using commercially available software programs. Calculations should be performed for

each analyte and each QCM.

Measure each QCM daily for a minimum of five days. (Rishniw, et al., 2012) Using these data, for each

QCM and each analyte, calculate: Mean (average), Standard deviation (SD), and Coefficient of variation (CV).

This value represents between-day (interassay) imprecision of the analyzer. The mean, SD, and CV of the

analyzer derived from these QC data are referred to as the calculated or observed SD, and CV.

Calculate the analyzer’s measured bias using the measured mean and the QCM manufacturer’s reported

mean for the assayed control material (using same instrument and/or method as that used by the analyzer)

according to the formula. QCM manufacturer’s reported means are commonly found in the QCM package insert,

categorized according to the instrument and method producing the assayed values.) Measured bias may be a

positive or a negative number, depending upon whether the analyzer’s results are lower or higher than the

manufacturer’s reported mean. If bias is a negative number (e.g., 5.0%), then the absolute number (5.0%)

should be used below.

Calculate the analyzer’s observed total error (TEobs), using measured CV and measured bias. Compare

measured TEobs to TEa. If TEobs < TEa (or very close to it), then the quality requirement is met and instrument is

considered suitable for measurement of that analyte. If TEobs > TEa, then several options exist.

TEobs Interpretation and Assessment of External Quality Assessment Results

Calculated total error (TEobs) for all analytes determined on in-house or reference lab equipment

should be compared to the ASVCP total allowable error guidelines found in Table 1. If calculated total

allowable error (TEobs) is greater than that which is acceptable (TEa), attempts should be made to identify and

correct causes of imprecision (high CV) and inaccuracy (high bias).

104

Table 1. Total Allowable Error as defined in the ASVCP draft guidelines.

Analyte Low Analyte

Values

Within RI High Values CLIA Value

Albumin 15% 15% 15% 10%

Alkaline

Phosphatase

NCR 25%

(20% desirable)

25%

(20% desirable)

30%

Alanine Amino

Transferase

NCR 20% 20% 20%

Ammonia NCR 20% 20% Not found

Amylase NCR 20% 20% 30%

Aspartate Amino

Transferase

NCR 25% 25% 20%

Bicarbonate 20%

(15% desirable)

20%

(15% desirable)

20%

(15% desirable)

10% (RCPA) to 20% (CAP)

Bile Acids1,2,3

20% 20% 20% None found

Cholesterol 20% 20% 20% 10%

Chloride 5% 5% 5% 5%

Creatine Kinase NCR 30% 30% 30%

Creatinine4 20% 20% 20% 15%

Gamma Glutamyl

Transferase

NCR 20% 20% 15% (RCPA) to 30% (CFX)

Glutamate

Dehydrogenase

NCR 30% 25%, >90IU 20% None found

Glucose 10% 20% 20% 6% Low, 10% High

Iron 30% (15% desired) 30% 30% 20%

Potassium4 5% 5% 5% 0.5mmol/L

Lactate5,6,7

NCR 40% 40% 10 (RCPA ) to 30% (CFX)

LDH NCR 20% 20% 20%

Magnesium8,9,10

15% desirable,

20% acceptable

15% desirable,

20% acceptable

15% desirable,

20% acceptable

25%

Sodium4 5% 5% 5% 4mmol/L

Phosphorus4,11

20% 15% 15% 10-23% (CAP)

Sorbitol

Dehydrogenase

NCR 20% 20% None found

Total Bilirubin NCR 30%

(25% desirable)

30%

(25% desirable)

0.4 mg/dl, 20%

Total Calcium4,9,11

10% 10% 10% 2% (BV) to 8% (CFX)

Total Protein 10% 10% 10% 10%

Triglyceride NCR 25% 25% 25%

Troponin12

NCR 70% 70% 20%CV maximal with

around 50% TEa if

calculated

Urea4 15% 12% 12% 2mg/dl, 9%

Uric acid13

10% 10% 10% 17%

If these sources of error cannot be corrected or if problems occur repeatedly, the manufacturer of the

instrument and/or a boarded clinical pathologist with expertise in QA should be called upon for further

assessment. Further assessment may include attempts to improve performance capability by analyzer

adjustments, operator training, replacement of reagent with new reagent or different manufacturer product, or,

potentially, analyzer replacement. Alternately, the initial quality requirements may be relaxed. This is not

recommended but is possible only if potential additional error can be tolerated in diagnostic judgment. This

requires education of ALL clinicians using the analyzer regarding amended total allowable error of the analyte(s)

in question. Any changes outside of the recommended TEa in this document must be justified and documented

in a laboratory handbook. This should be done only upon consultation with a boarded veterinary clinical

pathologist.

105

Three to five boarded clinicians (ACVIM or ECVIM with various specialties) gave opinions upon

clinically desired TEa at low, mid, and high analyte concentrations and activities, except for troponin where the

opinion of a single cardiologist was used. Total allowable error was calculated directly from reference equipment

used by QALS members using the equation 2CV + bias% = TEa% to insure that TEa was possible. NCR=Not

Clinically Relevant. CAP College of American Pathologists Participant Summary, April 2004. CLIA - CLIA '88

Proficiency Testing Limits, U.S. Federal Register. BV -Spanish Society of Clinical Chemistry and Molecular

Pathology (SEQC) table of Desirable Quality Specifications based on Biological Variation, 2004 Update. For

details, visit www.westgard.com/guest26.htm (accessed 27 Sept 2011) and

http://www.dgrhoads.com/db2004/ae2004.php (accessed 19 November 2011) CFX "Canadian Fixed Limits",

The College of Physicians and Surgeons of Saskatchewan RCPA Royal College of Pathologists of Australasia

and the Australasian Clinical Biochemist Association Quality Assurance Program

TABLE 1 REFERENCES 1Reed, SM and Bayly, WM. Equine Internal Medicine. London, England:WB Saunders; 1998.

2Hoffman, WE, Baker, G, Rieser S et al. Alterations in selected serum biochemical constituents in equids after

induced hepatic disease. Am J Vet Res 1987;48:1343.

3Mendle, VE. Pyrrolizidine alkaloid-induced liver disease in horses: An early diagnosis. Am J Vet Res. 1988;

49: 572 4Savage , C. Urinary Clinical Pathologic Findings and Glomerular Filtration Rate in the Horse. Vet Clin Equine.

2008;24:387–40 5Moore JN, Owen R ap R, Lumsden JH. Clinical evaluation of blood lactate levels in equine colic. Equine Vet J.

1976;8:49-54.

6Lagutchik MS, Ogilvie GK, Hackett TB, Wingfield WE. Increased Lactate Concentrations in Ill and Injured

Dogs. JVECCS. 1998;8:117-127. 7de Papp E, Drobatz KJ, Hughes D. Plasma lactate concentration as a predictor of gastric necrosis and survival

among dogs with gastric dilatation-volvulus: 102 cases (1995-1998) JAVMA. 1999;215(1):49-52. 8Khanna C, Lund EM, Raffe M, Armstrong PJ. Hypomagnesemia in 188 Dogs: A Hospital Population-Based

Prevalence Study. JVIM. 1998;12:304-309. 9Martin LG. Hypercalcemia and Hypermagnesemia. In Advances in Fluid and Electrolyte Disorders/Vet Clinics

of North America. 1998;28:565-585. 10

Cortes YE, Moses L. Magnesium Disturbances in Critically Ill Patients. Compendium. 2007;7:420-427. 11

Toribio, RE. Disorders of Calcium and Phosphate Metabolism in Horses. Vet Clin Equine. 2011;27:129–147 12

Porciello, F., Rishniw, M., Herndon, W., Birettoni, F., Antognoni, M. and Simpson, K. Cardiac troponin I is

elevated in dogs and cats with azotemia renal failure and in dogs with non-cardiac systemic disease. Australian

Veterinary Journal. 2008;86: 390–394. 13

Harr KE. Clinical Chemistry of Companion Avian Species: A Review. Vet Clin Pathol. 2002;31(3):140-151.

References In Text

Kenny D, Fraser CG, Petersen PH and Kallner A. Consensus agreement. Scand J Clin Lab Invest. 1999;59:585.

Lester, S., Harr, K. E., Rishniw, M, Pion, P.D. A Review Of Current Quality Assurance Concepts And

Considerations For Quality Control Of In Clinic Biochemistry Testing. 2012;In press – Accepted. JAVMA.

Rishniw M, Pion PD, Maher T. The quality of veterinary in-clinic and reference laboratory biochemical testing.

Vet Clin Pathol. 2012;41(1):92-109.

Westgard JO, Carey RN, Wold S. Criteria for judging precision and accuracy in method development and

evaluation. Clin Chem. 1974;20:825-833.

Westgard JO. Error budgets for quality management: Practical tools for planning and assuring the analytical

quality of laboratory testing processes. Clin Lab Manage Rev. 1996;10(4):377-81, 384-93, 396-403.

Westgard QC. Desirable specifications for total error, imprecision, and bias, derived from intra and inter-

individual biologic variation. http://www.westgard.com/biodatabase1.htm (Accessed September 2011).

http://www.westgard.com/guest26.htm

http://www.dgrhoads.com/db2004/ae2004.php

106

QC VALIDATION – AN INTRODUCTION

E. Leidinger. Invitro Diagnostic Lab. Rennweg 95, Vienna, Austria.


WHY DOING QC-VALIDATION?

Quality control (QC), to be more precisely statistical QC, is done in (almost) every lab with a more or less effort

and considerable costs - I once calculated the overhead for QC roughly with 15% in our lab. In human labs there

are usually national or international regulations (like CLIA in the US) which force the laboratories to perform a

minimum of QC, but there are no such regulations for veterinary clinical pathology labs.

But even regulations to meet QC goals do not automatically mean that QC is done right; i.e. doing it to improve

the lab performance continuously and sustainably. That’s the point were QC validation should start: it is an

approach to make the most from a bulk of QC data you produce every day in your lab.

HOW-TO-DO QC-VALIDATION

QC validation is the approach of going through the whole process of (statistical) QC. The purpose of QC

validation is to determine a) whether statistical QC procedures are appropriate for detecting medically important

errors and b) the quality of performance required by different tests. This implies that there is no QC rule which is

applicable for all tests! QC validation is a 5-step procedure, starting with:

1. Define the quality required for a test based on the analyser performance

2. Determine method imprecision and bias for the test

3. Identify candidate internal quality control (IQC) procedures

4. Predict IQC performance

5. Set performance goals and select an appropriate IQC procedure

1. Define the quality required for the test

Quality requirements, expressed as total allowable error (TEa) may be taken from the literature, determined

based on interpretation of test results, determined from knowledge of biologic variation and/or from information

from external proficiency testing/quality assurance schemes.

The total allowable error (TEa) has been defined as the amount of error that can be tolerated without invalidating

the usefulness of a test result. It is calculated by combining imprecision (random error or CV) and inaccuracy

(bias) of the measurement procedure. For details refer to the formula collection provided in the course library.

Specifications for TEa for testing in humans can be found in the CLIA (Clinical Laboratory Improvement

Amendments, current version from 1988) and in the listings of biologic goals provided by a European group

(Ricos et al, 1999; reprinted in Westgard, J.O.: Basic Planning for Quality, appendix 3). Some of the criteria; e.g.

for cholesterol, might be too stringent for veterinary laboratory settings, but may be useful for orientation.

Some quality requirements (TEa) for veterinary testing are available at:

http://www.westgard.com/quality-requirements-for-dogs-cats-and-horses.htm

In the same way as it is recommended to set up lab-specific reference values, it is possible and recommended to

install a set of lab (analyser) specific TEa by using internal and external QC data and – if available – clinically

significant decision levels. For example, the clinically significant decision level for WBC count in cancer

chemotherapy would be 2000/µl (therapy is stopped if WBC fall below this cut-off). The lab has to define the

total allowable error for WBC count in a way that the clinician’s requirement is met. It is obvious that the

meeting of the goal heavily depends on the analyser’s performance.

2. Determine method imprecision and bias

If starting with introduction of a new method/instrument, estimates of precision may be obtained method

validation studies and bias from comparison of methods.

Imprecision can also be determined by calculation the standard deviation (SD) and coefficient of variation (CV)

from QC data of a 2-3 month period.

According to Six Sigma QM the desirable precision is 1/6 of the tolerance specification (the TEa) One way of

applying this to laboratory tests is to divide the CLIA proficiency testing acceptability criteria by 6 to establish

specifications for the precision of lab methods.

http://www.westgard.com/quality-requirements-for-dogs-cats-and-horses.htm

107

Bias can be determined based on external proficiency testing/quality assurance data or by comparison of the

laboratory-established mean for a QC material with the ‘target value’ provided by the manufacturer of the QC

material. If bias determined from several sources is similar, this is a good indication that the estimation of bias is

accurate.

The fundamental principle of statistical QC is to characterise performance under stable operating conditions in

order to identify changes due to unstable operation, i.e. when problems occur. Thus, in principle, the control data

from any out of control run should not be included in the calculation of control limits because it does not

represent stable operation.

This procedure often is complicated in some analyses by the shifting of the means of QC data over time as the

control material aged or deteriorated respectively (Freeman, 2000 and own observations). This is frequently seen

in haematology QC; the shelf-life stated by the manufacturer usually refers to unopened bottles and is much

shorter as soon as the material is in use. Another (quite frequent!) problem is that the target values provided by

the manufacturer’s data sheet simply may vary for same analytes.

Both, CV and bias can be calculated manually or by the analyser software lab statistic programs (such as

Analyse-It® or by simple spread-sheets). I’ll provide you an example of such a spread-sheet in the course library.

Note: imprecision and inaccuracy are performance characteristics, not quality requirements. However, a certain

level of quality can be archived by combinations of those characteristics.


Candidate IQC procedures are the control (Westgard) rules and the characteristics of probability of error

detection and false rejection that they provide based on the performance of your instrument/method.

A control rule is a decision criterion for interpreting quality control data and making a judgement on the control

status of an analytical run. An analytical run is rejected when the control measurements does not fulfil the stated

conditions, i.e., when a control value exceeds the specified control limits.

The most common practice in clinical laboratories is the use of Levey-Jennings type control charts with limits set

as either the mean plus and minus 2 standard deviations or plus and minus 3 standard deviations. The control

rule in these cases would be 12s or 13s, meaning a run is rejected of 1 control run exceeds the 2 (or 3) standard

deviations. As you will learn in this course, the 12s rule though very commonly used, frequently is not the best

choice.

Another possibility to derive control rules is the use of OPSpecs charts. OPSpecs charts are plots of the

inaccuracy and the imprecision. You learned about those charts in the previous course section.

Briefly, you use the calculated CV (on the x-axis) and bias (y-axis) for a defined test at a given TEa to get an

operating point. The position of the operation point on the chart defines the control rule(s) and number of control

runs needed. This can be easily (a bit expensively, though) achieved by Westgard’s software EZ-Rules3®. We’ll

also provide you with online forms and fill-in charts to perform the QC validation manually (downloaded from

the Westgard.com website).


Three definitions are needed to predict the IQC performance:

1. The probability for error detection (Ped) is a performance characteristic of a QC procedure that describes how

often an analytical run will be rejected when results contain errors in addition to the inherent imprecision (CV)

of the procedure. Ideally, the Ped should be 1.00 (100%); however, a Ped >90% is considered acceptable in

practice.

2. The probability of false rejection (Pfr) describes how often an analytical run will be rejected when no errors

occur in addition to the inherent imprecision (CV) of the procedure. Ideally, the Pfr should be 0.00 (0%);

however, a Pfr <5% is considered acceptable in practice.

Ped refers to the case where a real error occurs, but the QC procedure is unable to detect it (too low Ped)

Pfr refers to the case where the applied QC procedure indicates that an error has occurred, but in realty

there is no problem (too high Pfr)

Ped is analogous to sensitivity of a diagnostic test, and that Pfr is analogous to false positive rate (which is [1-

specificity]) of a diagnostic test.

108

Ped and Pfr should be dealt with by proper selection of the QC procedures that are implemented in the laboratory.

The power of a QC procedure depends on the size of change or error occurring; therefore, it is useful to know the

probability of rejection as a function of the size of either the systematic or random error that is occurring.

3. Sigma-metric: the six sigma (for σ, the mathematical symbol for standard deviation, SD) is another approach

to predict IQC. It will be covered in detail at the end of this course. Briefly, Six Sigma focuses on controlling a

process to 6 SD, a higher sigma metric means the systematic error that must be detected to help ensure accurate

results by the use of statistical QC is large and should be more easily detected. A lower sigma metric means QC

must detect smaller systematic errors.

TEa .............. quality requirement in terms of TEa (defined by CLIA or Ricos et al, 1999)

biasmeas ............. method bias

CV ............... Coefficient of variation (CV); measurement of imprecision

The calculation of Ped and Pfr, and σ can be done by:

1. Calculation of the critical systematic error or

2. Critical-error graph or

3. EZ-Rules® (Westgard QC)

1. Calculation of the critical systematic error

The critical-sizes of the systematic error (SE) and random error (RE) that need to be detected by the QC

procedure can be calculated from the quality requirement and the observed imprecision and inaccuracy by the

following equations:

TEa .............. quality requirement in terms of TEa (defined by CLIA or by Ricos et al, 1999)


smeas ............. standard deviation (method precision)

1.65 z-value, corresponding to 5% of the area in one tail of a Gaussian distribution (1.65

standard deviations, one-sided)

TEa quality requirement in terms of TEa


smeas ............. standard deviation or CV if expressed as % (method precision)

1.65 ............. z-score, corresponding to 5% of the area in one tail of a Gaussian distribution (1.65

standard deviations, one-sided)

Large values for the critical systematic error (>4.0) indicate large errors that should be easily to detect by the QC

procedure. Small values for critical size systematic error (<4.0) are more difficult to detect, and may indicate the

need for improvement in the QC method.

QC for manual assays is usually optimized for detection of random error, whilst QC for automated instrument

assay is usually optimised for detection of systematic error.

2. Critical-error graph

The critical-error graph (a type of power function graph) shows the probability of rejection on the y-axis versus

the size of error on the x-axis. It gets its name from the statistical concept of ‘power’ which refers to the

probability of detecting a change.

A critical-error graph is simply a power function graph that also displays the size of error that is medically

important and needs to be detected by the QC procedure. Remember that a power function graph shows the

probability of rejecting a run on the y-axis versus the size of error on the x-axis. Like power function graphs,

critical-error graphs can be prepared for both systematic and random errors.

109


Select control rules and the number of control runs needed to keep the error detection high (minimum Ped > 90%)

and the probability of false rejection low (maximum Pfr <5% or better <1%). Simple rules are preferred over

multi-rules because of the ease and speed of application.

Example quality validation for WBC

The formulas needed for the calculations are shown in the formula collection

1. Determine the quality required for the test

Example: Required TEa for WBC (CLIA) +/- 15%

TEa recommended for veterinary use at low WBC (decision level of 2,000 cells/µl) = +/- 20% (Freeman and

Gruenwaldt (1999), Vet.Clin.Pathol. J.)

2. Determine method imprecision, bias, and total error

Target mean (Abbott, lot #L0140) 2.70 G/l

Measured mean (n=32, IQC) 2.69 G/l

Measured standard deviation (SD) 0.089

Measured method imprecision (CV) 3.3%

Measured inaccuracy (bias) 0.4%

Calculated TE (TEmeas = biasmeas + 3CV) 10.3%

The calculated TE is smaller than the allowed TE (TEa) of 15-20% as determined above, so this

method/instrument is suitable for this application.


Use the CV (x-axis) and bias (y-axis) to define the operating point. This can be done manually using a

normalised OPSpecs Chart (http://westgard.com/normcharts.html) or with the software EZ-Rules3®

(Westgard QC).

AQA: analytical quality assurance (synonymous with probability of error detection > or = 90%).

http://westgard.com/normcharts.html

110


The above OPSpecs Chart tells you that at an error detection level of 90% (=AQA(SE): there is a 90%

chance to detect a critical systematic error) and a TEa of 15% you can use a simple 12.5s control rule with 3

controls materials (N=3; let’s say low/normal/high control blood) to be on the safe side The rule means that

the run is rejected if 1 out of 3 controls exceeds +/- 2.5 standard deviations.


At a 12.5s control rule the probability of false rejection (Pfr) is 3%. The 2.00 sigma performance-line marks

the limit of stable performance. A small improvement of the imprecision (to 3.1%) would allow you to use

only 2 instead of 3 controls; then you are left of 12.5s/2N line. This would save you one control run a day!

The critical systematic error (SE) is: [(15-0.4)/3.3]-1.65=2.77

This means that a systematic shift equivalent to 2.77 times the CV of the method needs to be detected by the

QC procedure.

The sigma metric for WBC in this example is 4.4; 3 would be the minimal required quality; 4-5 is quite

typical for many processes.

TOXICOLOGY

GLP PRINCIPLE IN PRECLINICAL TESTING

M. Černe, E. Lovšin Barle. Lek Pharmaceuticals d.d., Verovškova 57, Ljubljana, Slovenia


Background: The issue of data quality has an important international dimension. The Good Laboratory Practice

(GLP) regulations for non-clinical laboratory studies published by the US Food and Drug Administration in 1976

provided the basis for the work of the Expert Group, which comprised experts from the different countries and

organizations as the Commission of the European Communities, the World Health Organization and the

International Organization for Standardization.

Objective: The purpose of these principles of good laboratory practice is to promote the development ofquality

test data. Comparable quality of test data forms the basis for the mutual acceptance of data amongcountries. If

individual countries can confidently rely on test data developed in other countries, duplicative testing can be

avoided, thereby saving time and resources. The application of these principles should help to avoid the creation

of technical barriers to trade, and further improve the protection ofhuman health and the environment.

These principles of good laboratory practice should be applied to the non-clinical safety testingof test items

contained in pharmaceutical products, pesticide products, cosmetic products, veterinary drugs as well as food

additives, feed additives, and industrial chemicals. These test items are frequentlysynthetic chemicals, but may

be of natural or biological origin and, in some circumstances, may be livingorganisms. The purpose of testing

these test items is to obtain data on their properties and/or their safetywith respect to human health and/or the

environment.Non-clinical health and environmental safety studies covered by the principles of goodlaboratory

practice include work conducted in the laboratory, in greenhouses, and in the field.

Unless specifically exempted by national legislation, these principles of good laboratorypractice apply to all non-

clinical health and environmental safety studies required by regulations for thepurpose of registering or licensing

pharmaceuticals, pesticides, food and feed additives, cosmeticproducts, veterinary drug products and similar

products, and for the regulation of industrial chemicals.

Methods: Organization for Economic Co-operation and Development (OECD) member countries has

established criteria for the performance of these studies. To avoid different schemes of implementation that

could impede international trade in chemicals, OECD member countries have pursued international

harmonization of test methods and good laboratory practice.


111

OECD Series on Principles on Good Laboratory Practice and Compliance Monitoring (as revised in

1997, ENV/MC/CHEM (98)17).

OECD methods, each for different purpose as it is prescribed in ICH guidelines.

ICH (International Conference on Harmonization) guidelines from M (multidisciplinary), S (safety) and

Q (quality) series as a regulative for registration purpose of medical product.

Additionally the local legislation on GLP compliance, local laws and by-lows and guidance on animal

welfare.

Results: GLP system facilitates the exchange of information and protects the human health and environment.

Duplicative testing can be avoided and costs saved to government and industry. The confidence on results should

be greater.

Mutual acceptance of data in an OECD member country acquired in accordance with OECD test guidelines and

OECD Principles of Good Laboratory Practice shall be accepted in other member countries for purposes of

assessment and other uses relating to the protection of man and the environment.

Conclusion: Good Laboratory Practice (GLP) is a quality system concerned with the organizational process and

the conditions under which non-clinical health and environmental safetystudies are planned, performed,

monitored, recorded, archived and reported.OECDmember countries have established criteria for the

performance of these studies.To avoid different schemes of implementation that could impede international trade

inchemicals, OECD member countries have pursued international harmonization of test methods and

goodlaboratory practice.

VETERINARY TOXICOLOGY: AN INTEGRATED APPROACH

G. Counotte. Animal Health Service, P.O. Box 9, NL-7400 AA Deventer, The Netherlands.


Introduction

Veterinary toxicology can be very complex as there are a wide variety of poisons and chemicals, minerals

(including trace and heavy elements), toxins from biological origin (plant, fungal, animal) and also feed

additives like vitamins or antibiotics. The field of toxicology can be divided in several ways. For example:

research, teaching, diagnostics and advices. The focus of the Animal Health Services (AHS) is mainly on the

field of diagnostics and advices. Toxicology is not a field where you can work on your own: you have to

cooperate with veterinarians, farmers, experts on other fields, and so on. In this presentation, some examples will

be given of this integrated approach.

Organizationand Analytical capacityof Veterinary Toxicology in the Netherlands

At the AHS we have a telephone service for veterinarians. If they suspect something abnormal, they can ask for

help. The subjects of most telephone calls are infectious diseases. But sometimes infectious diseases are ruled

out and then toxicology comes in focus. Together with the clinical specialized veterinarians of the AHS, the

problem is discussed and a way to come to a diagnosis and/or solution is set. When animals died, they can be

transported by our own pick up service and brought in for post mortem examination. Veterinary pathologist does

the necropsies and if they suspect intoxication, the liver, kidney, stomach / rumen content is stored, eventually

with vitreous humor.

The laboratory of the AHS is equipped for analytical toxicology: ICP-OES (currently changing to ICP-MS), GC-

MS, LC-MS/MS and HPLC (with various detectors). But also clinical chemical methods for blood and urine

analysis and equipment for drinking water analysis. Analytical capacity is only a part of veterinary toxicology:

interpreting the results, combing with other findings, clinical investigations, all must work together to solve the

problem.

The screening methods are validated and sometimes specially developed to detect toxicological relevant

compounds (volatile, non-volatile, elements). Detecting the toxin alone is not enough: the detected toxin must

give the symptoms that have been seen by the local veterinarian.A further step is to advise the farmer and local


112

veterinarian what to do to prevent further intoxications. For Dutch Veterinarians we wrote a booklet about

veterinary toxicology.

When there are more intoxications of the same kind or if the food safety is endangered, the government is

alarmed.

Silo gas intoxication caused by maize-silage

Silo gas intoxication is also known as Silo Filler’s Disease:it is an occupational disease that results from

pulmonary exposure to oxides of nitrogen (NOx). The first time we were involved was when at a farm with dairy

cattle, maize was harvested and a commercial starter culture containing lactobacillus bacteria was mixed with the

maize during harvesting. The farmer noticed a gas pocket underneath the plastic sheet on the next morning and

more sand was put on the silo and the gas was released from under the plastic sheet. In the evening the gas

pocket had reappeared and it was noticed that the cows avoided the stable section adjacent to the silo. The gas

pocket pushed the sand away from the surface of the plastic sheet and a reddish-brown vapor escaped. The

farmer tried to put the cows out to pasture but they were reluctant to go out into the darkness. There was no wind

on these days so the gas could slowly enter the stable. Because the farmer was worried about the situation, he

inspected the herd during the night and noticed that some cows were restless and dyspnoeic. One 7-year-old cow

was in the stable section adjacent to the silo but looked completely normal; the other cows were on the other side

of the stable. One cow died early in the morning and the local veterinarian was called. Colored vapors were

again seen coming from the edge of plastic sheet and the veterinarian called the Animal Health Service and

reported that he saw a brownish colored gas. We directly advised to call the fire brigade in order to measure NOx

in the air. We also advised to stay out of the gas and transfer the cows in open areas. The measurement of the fire

brigade showed that the NOx was above their maximum detection limit (2150 ppm). But also CO (more than

1400 ppm) was measured. The fire brigade removed the gasses from the stable with large ventilators and after 4

hours checked the air again.Two cows died and in the animal that was necropsied, lesions resembling those of

SFD (Silo Filler’s Disease) were observed: a foamy content in the tracheal lumen, petechiae in the tracheal wall

and on the heart, and severe pulmonary oedema. No further abnormalities were found. The quick cooperation

between farmer, local veterinarian, AHS and fire brigade prevented more dead cows and health problems of the

farmer and his family.

Nitrite intoxication in pigs through wastewater from air washers

A case of several nitrite intoxications in swine. In a short interval, the AHS received several pigs and swine’s

that suddenly died after drinking or eating. The post mortem findings were a (dark) brown carcass without any

other abnormalities. Farmers and veterinarians told us that only two, three or four animals suddenly died and all

other animals didn’t show any signs. Laboratory findings showed that the methemoglobin concentration of the

hart blood of the swineswas between 21 and 50 %, indicating a nitrite intoxication. Questioning the farmers

about potential sources of nitrite, revealed that water from air washers was leaking in feed or drinking system.

Analysis of the feed and/or drinking water showed that the ammonium content was about 2 to 3 gram per liter,

nitrate between 0,3 and 0,6 gram per liter but the nitrite content was between 2,5 and 6,8 gram per liter. This

amount of nitrite in water is enough to kill a pig of 50 kg after drinking 100 to 150 milliliters of water. Air

washers are used to prevent air pollution with ammonium. They use water and bacteria (biological air washers as

they are called) to convert ammonium via nitrite and nitrate to nitrogen. Air washers are always on the top of a

building: so leaking water can enter the stables. We contacted the producers of air washers to make sure that they

install the washers correctly and control them on leaking.

Monitoring Heavy Elements

When animals are send in for post mortem examination and the pathologist cannot find a specific cause, the liver

is screened for trace and toxic elements (As, Cd, Co, Cu, Cr, Fe, Mo, Ni, Pb, V, Zn). From these elements and

the ration between the levels in the liver, we can sometimes give additional suggestions explaining the dead of

the animal. We established reference values based on age (for cows) and we are now working on reference

values in liver of swine and poultry.

High levels of Zinc in liver of calves During screening on trace elements, we observed a high frequency (more than 30 %) of high zinc levels in livers

of very young calves. Therefore we decided to do a project on the origin of these relative high levels (more than

1000 mg/kg dry matter zinc). Especially very young animals (1 to 75 days old) had sometimes rather elevated

zinc levels while all other elements (such as copper and iron) are normal. 68 Farms were incorporated in this

study: 34 that had calves with normal zinc levels (below 500 mg/kg dm) and 34 that had calves with elevated

levels (more than 750 mg/kg dm). After statistical analysis of all factors and measurements, we concluded that

age of the animal, feeding concentrates to calves and the ration of the mother were the most important risk

113

factors for a high zinc level of the calve. But until now, we don’t believe we have the most important factor. But

from calculations we revealed that the amount of zinc fed to animals is about twice as high as it should be.

Chronic Copper intoxication in horses

A local veterinarian called the AHS: he did some laboratory tests in blood of horses. The reason was that the

horses didn’t perform very well. He found nothing abnormal, except a rather elevated gGT. In 19 of the 20

horses, he had an elevated gGT. Elevated gGT can be a symptom of DILI (drug induced liver injury). The

problem is than to find the cause of this intoxication. The horses were housed in an old stable that was recently

completely renovated. We analyzed all feed on heavy elements, plant toxins, pesticides and so on. But nothing

was found. After again asking the owner about details, it was elucidated that she installed a new drinking water

system. All made of copper and with a heating device to prevent freezing of the water during the winter. It also

was a system where the water was pumped around. Only one horse (that was housed in a different part of the

building) received water from another drinking water system. And this horse had a normal gGT. We analyzed

the water and found that the water contained about 170 µg/L opper. The upper limit of copper in drinking water

is set to 1300 µg/L (for humans) by the WHO and 500 µg/L for livestock by the FAO. But for certain sheep

breeds we set the maximum of copper in drinking water at 50 µg/L because of copper accumulation in the liver.

Horses are fairly tolerant of high copper levels in feed. In studies were horses were given copper orally, no

adverse symptoms were detected. Therefore, the maximum tolerable concentration of copper for horses has been

set at 250 mg/kg of ration. But nothing is published about the effect of copper in drinking water on horses.

Copper is stored in the liver and the copper content of the liver is used to diagnose (chronic) copper intoxication.

No horse died, so we didn’t have liver. Blood is nog a good matrix for diagnosing chronic copper intoxication.

Dr. Tresemiek Picavet (Dipl ECEIM) and dr. Hilda de Cock (DiplACVP)were also involved in this case. The

outcome of theire histological examination of a liver biopsy was that an intoxication is a possible cause of the

liver damage. Tresemiek Picavet took also a liver biopsy for us from one horse for analysis on copper. The result

was that the copper level in the liver of the horse was 63 mg/kg dm. Normally the copper in liver of horses is

between 10 and 25 mg/kg dm. Therefore we concluded that the construction of the drinking water system

resulted in elevated copper concentrations in the water which resulted in elevated copper in the liver of the

horses, resulting in liver damage. After changing the drinking water system, the gGT returned almost to normal

after 6 months.

Plant toxins

There are many different kind of plant toxins and they are mostly difficult to analyze. The four most important

pyrrolyzidine alkaloids from senecio plants can be detected with GC-MS. But many other toxins not. In our

group we have a specialist on toxic plants. But then there must be something to determine. If the plants are cut

and mixed, it is sometimes very difficult to determine which plant is involved. We are now working on a

screening method (LC-MS/MS) for the 10 most important plant toxins in the Netherlands.

Cooperation

Colleagues working and cooperating during these cases:

Jet Mars (plant toxins)

Jan Vos / Thijs Roumen (veterinary pathologist)

Menno Holzhauer / Ad Moen / Maarten Weber / Koos Verhoeff/ Ruben Tolboom (ruminant specialist)

Jobke van Hout (Swine Specialist)

Tresemiek Picavet (diplEquine Internal Medicine Specialist), Hilda de Cock (diplAmerican College of

Veterinary Pathology)

Ingrid Santman-Berends (statistical analist)

Literature

A list of significant references is available upon request.

114


THE GULF OIL SPILL: PETROLEUM INTOXICATION

K. E. Harr. URIKA, LLC, Seattle, WA, USA


The Gulf Oil Spill

On April 20, 2010, an explosion occurred on the Deepwater Horizon oil rig harvesting the Moncando

well about 50 miles east of Louisiana in the Gulf of Mexico. 11 people were killed in the initial explosion but the

number who have lost their lives since is not directly attributed to the disaster. The largest accidental oil spill on

the planet now cited as the largest environmental disaster in U.S. history resulted. Oil flowed into the Gulf for 87

days straight. Initial amounts of spill were gross underestimations and the US Coast Guard now estimates that

5,000 barrels or 200,000 gallons of oil per day flowed into the gulf. Over200 million gallons of oil total were

released into the Gulf of Mexico, and 1 million gallons of dispersants were used. The Deepwater

Horizon/Moncando spill was almost 20 times the size of the Exxon Valdez spill, previously the largest in U.S.

history.

Over 1,000 miles of beaches and wetlands were oiled. More than 8,000 birds, 1,000 sea turtles and 600

dolphins and whales have been collected either dead or injured near the site of the spill. This is required to be a

direct count of number of animals found and is a fraction of the real numbers. Due to the deepwater, high

pressure nature of the spill, all levels of the water column, not just surface regions where oil typically floats,

were affected. Ancient deepwater corals that can live more than 2,000 years were killed by oil from the spill. In

some areas of the Gulf, 50% of the fish caught had lesions. At the height of the spill, approximately 40% of

federal waters in the Gulf of Mexico were closed to fishing, an area of almost 90,000 square miles. The closures

may represent an annual loss of $250 million. Over 20% of the critical spawning habitat of Atlantic bluefin tuna

was covered with oil during the peak spawning months; their populations have declined by about 80% since

1975. Total economic damages from impacts to fishing-dependent industries could cost $8.7 billion and 22,000

jobs over the next 7 years. Some historically abundant shrimp fishing grounds are 80% or more below normal

catch levels in 2011. Lesions and deformities are beginning to be documented on fish, crabs and other wildlife.

At least 32 dolphins in Barataria Bay, Louisiana, one of the hardest hit spots by the Deepwater Horizon oil spill,

have been given physicals and are reported as severely ill according to NOAA officials. A range of symptoms

includingemaciation, anemia, hypoglycemia with hepatic enzyme increases and evidence of respiratory disease

are reported. One of the studied dolphins has already been found dead. In 2011 there were 159 strandings just in

Louisiana, almost 8 times the historical average in previous years. (NOAA, per communication,

http://response.restoration.noaa.gov/deepwaterhorizon)

BPs partners are Transocean, owner of the rig, and Haliburton, who contracted work such as concrete

and other product work. Deepwater Horizon is a mobile exploratory rig, not a production rig, so it moves around

the sea floor, is not as securely fastened, and may not be exactly suited for the underwater terrain. Drilling had

been unusually troubled in the region by “kicks” or frequent bursts of natural gas upon drilling which cause

emergency shutdown. While warned by federal regulators that this was problematic and caution should be used,

BP persisted in drilling. “The day of the explosion, engineers reportedly argued over whether or not to remove

dense drilling mud from the well bore, replacing it with much lighter sea water. Normally this step is taken only

after a second cement plug is hardened in the piping, a process that takes several hours.” Until this plug is fully

installed, heavy mud is the first line of defense against kicks when oil and natural gas surge up the bore to the rig

platform.“A worker told the Wall Street Journal that the crew was preparing to drop the cement plug down the

riser, standard procedure, when the order came to instead pump out the mud. “Usually we set the cement plug at

that point and let it set for six hours, then displace the well,” he said. The worker told the Journal that this

dangerous step was first cleared with the Minerals Management Service. The MMS has since refused comment.”

(CNN archive)

BP and Transocean executives admitted to the House Energy Committee that this decision was made to

replace the mud before plugging the well, even though this would increase the chances of an explosion, and even

though the operation failed a critical pressure test the same day. This clearly reckless decision to press forward

was very likely done to protect BP’s profit interests, both because it paid rig owner Transocean an estimated

$500,000 per day for use of Deepwater Horizon and its crew, and wanted to bring the new well into active

production.It is likely that this decision combined with the failure of two other lines of defense: cement outside

the well bore’s piping under the ocean floor, which is designed to prevent natural gas from moving up the bore

and the riser to the rig; and the blowout preventer, a massive piece of equipment that sits on the ocean floor and

is equipped with powerful hydraulic shears whose task is to sever piping in the event of a blowout. (CNN

archive)


http://response.restoration.noaa.gov/deepwaterhorizon

115

Halliburton, which contracted for the cement and mudding work on the rig, had deployed a new

chemical cement that it said would be resistant to structural damage caused by methane hydrates, which were

present in the undersea rock in high quantities. Many experts in the field disagreed as there were temperature

effects in the surrounding rock that were not considered. When the cement failed, gas began to force its way up

the riser. At this point, concrete well plugs in the pipe should have blocked the gas. But contrary to normal

practice, the final plug had not been installed, and the salt water was not heavy enough to stop the high pressure

gas from rising. (https://s3.amazonaws.com/pdf_final/DEEPWATER_ReporttothePresident_FINAL.pdf,

accessed May 31, 2012)

The Deep Water Horizon Settlement has significantly decreased access to any of the minimal clinical

pathology data that was collected during this event. The US government issued BP, Transocean and Halliburton

with citations for violating oil industry regulations ahead of what are expected to be massive fines over the

Deepwater disaster. BP has set aside $40 billion for payments expected and their stock share, having rebounded

is still one third less than that in April 2010. In addition to fines, private class action lawsuits are pending.

Economic and Property Damages Settlement is expected to amount to astronomical sums of money and call for

this class action law suit closes in August. This lawsuit includes the Seafood Compensation Program, Individual

and Vendor Economic Loss, Startup and Failed Business Economic Loss, Coastal Real Property Damage,

Wetlands Real Property Damage, Real Property Sales Loss, Subsistence Loss, Vessel Physical Damage, and

more filing categories.

( http://www.boemre.gov/DeepwaterHorizon.htm - Bureau of Ocean Energy Management settlement website

and http://www.deepwaterhorizonsettlements.com/ - official website))

The oil has been fingerprinted in the Coast Guard Lab in New London, CT. It is expected that there will

be significant increases in regional carcinogenicity and likely forthcoming differences in fecundity of the species

in the region. Many of the compounds identified as components of this spill are confirmed

carcinogens.Polycycloaromatichydrocarbons, benzene, 1,3-Butadiene, and many more have been established

without question as a human carcinogen by IARC, EPA, and WHO.

As current data is not available, this talk will focus on experimental and field data collected over the

past 25 years since the Exxon Valdeez oil spill in Prince William Sound in Alaska. Though this may not

represent the exact features that will be observed over time in the Gulf Region, as the composition of the crude

oil is different, it does represent a reasonable approximation.

Clinical signs typically associated with petroleum intoxication vary with the composition of the

petroleum product. That containing predominantly gasoline and aerosolized compounds will have different

clinical signs than fuel oil used for heating. Concentrations of benzenes and polycyclic aromatic hydrocarbons

directly correlate with carcinogenicity. Additionally, route of exposure will effect clinical signs. Clinical signs

of oil spills include aspiration pneumonitis (most common complication with volatile compounds), local

irritation to extensive chemical burns (direct contact), GI, CNS and cardiovascular complications, bone marrow

toxicity and hemolysis resulting in decreases in one or multiple cell lines, hepatic and renal failure (especially

the chlorinated hydrocarbons), renal tubular acidosis (toluene toxicity). While this is a natural disaster, there is

regular petroleum intoxication which occurs at a background level. Approximately 60,000 cases of petroleum

intoxication are reported in the US annually which are generally accidental in children under 5 years. (CDC,

http://www.atsdr.cdc.gov/substances/toxsubstance.asp?toxid=75)Human epidemiologic studies show important

increases in cancers of the kidney, stomach, brain, pancreas, prostate, lung, and skin as well as hematopoietic

and lymphatic leukemias as a result of exposure to gasoline, its components, and its vapors. (Mehlman, 1992)

In general, these are hydrophobic compounds that therefore penetrate transdermally as well as through

other contact tissues such as the respiratory tract. In the lungs, volatile compounds may directly displace oxygen

and result in hypoxia, encephalopathy, seizures and death. Damage to tissue membranes results in hemorrhage,

hyperemia, edema, surfactant inactivation, and then leukocyte infiltration and vascular thrombosis. All of this

may contribute to a hypoxic state which may resolve in 2-8 days dependent upon duration and severity of

exposure. One can literally appear intoxicated when exposed to petroleum. Disinhibition, narcotic-like

depression, and euphoria as in alcohol or narcotic toxicity may be observed. Eventually, lethargy, headache,

obtundation, and coma may result. Dysrhythmias are commonly observed. Underlying etiologies include

hypoxia, myocardial sensitization to catecholamines, and direct myocardial damage. Sudden death is uncommon

but has been reported as a result of coronary vasospasm due to hydrocarbon inhalation.

After an oil spill, bioaccumulation occurs over time, potentially continually, dependent on sediment

concentrations and natural history of animal populations.Total oil concentration in blue mussels

(Mytilustrossulus) was 1.2mg/g 6 months after the Exxon Valdeez spill with no change in concentration when

resampled 18 months after the spill. (Hartung, 1995)As these compounds are hydrophobic, bioaccumulation

occurs in fatty tissues and, as they are poorly metabolized, typically increases as one ascends the food

chain.Hematologic and biochemical changes at onset may be severe, resulting in death and include pancytopenia,

(or individual cell lines such as aplastic anemia, thrombocytopenia), hepatic enzyme increases 5 or more times

https://s3.amazonaws.com/pdf_final/DEEPWATER_ReporttothePresident_FINAL.pdf

http://www.boemre.gov/DeepwaterHorizon.htm

http://www.deepwaterhorizonsettlements.com/

116

normal, dermal and ocular ulceration, and acute renal shutdown. Over time clinical changes may be more

nondescript including numerous forms of hematologic neoplasia including myelodysplastic syndrome, acute

myelogenous leukemia, acute blastic lymphocytic leukemia, lymphoma, and many more. While international

epidemiological studies documenting causative etiology of petroleum components (benzene, PAH, etc.) abound,

it may be difficult to prove that individual cases are a direct result of petroleum exposure and so the full effect of

the natural disaster is not known.(Rinsky et al., 1987, Wong, 1987, Golomb et al., 1982, and Dryver et al., 2004)

Changes in chromatid fragmentation, structural and numerical chromosomal aberrations, and sister chromatoid

exchanges (SCEs) as well as other DNA mutations have been used as supportive evidence that the neoplastic

event is indeed due to petroleum intoxication. (Zhang et al., 2002)

Changes in labwork may also be effected by mode of exposure and duration. In an experimental study

using mink (Mustelavison) as an animal model for endangered sea otters (Enhydralutis), acutely GGT and ALP

were markedly increased - (300 (GGT) and 6 (ALP) times controls or greater) - in dermally exposed animals

while animals fed the oil with food had minimal increase - (8 times controls GGT) or no statistically significant

increase ALP) . (Mazet, 2000)Over a 6 month time period this reversed itself, with the orally-dosed animals

having higher ALP and GGT values than those with dermal exposure which had samples that approached normal

values. All liver enzymes had significant differences in means from controls but varied according to time and

route exposure. Additionally, hepatic enzyme values were not always outside of reference intervals in all

exposed animals. Two types of crude oil were used and produced different biochemical changes specific to the

oil composition. Interestingly in this study, LDH concentration was found to be predictive of reproductive

success 6 months after a single acute exposure. All groups had 50-80% reduction in fecundity in the next

breeding season which directly contributes to population decreases post spill. Other notable changes were

decreased sodium, chloride, and calcium attributed to diarrhea in orally dosed animals and dermally exposed

animals. Further studies on laboratory data will be discussed.

REFERENCES

1. Dryver E, et al. Occupational Exposures and Non-Hodgkin's Lymphoma in Southern Sweden. Int J

Occupation Environ Health. 2004;10:13-21.

2. Golomb HM, et al. Correlation of Occupation and Karyotype in Adults With Acute NonLymphocytic

Leukemia. Blood. 1982;60(2):404-411.

3. Hartung, R. Assessment of the potential for long-term toxicological effects of the Exxon Valdez oil spill

on birds and mammals. In: Well PG, Butler JN, Hughes JS, eds. Exxon Valdez oil spill:fate and effects

in Alaskan sea waters. Philadelphia, PA:American Society for Testing and Materials; 1995:693-725.

4. Mazet J K, et al. Evaluation of changes in hematologic and clinical biochemical values after exposure

to petroleum products in mink (Mustela vison) as a model for assessment of sea otters (Enhydra lutris).

AJVR. 2000;61:1197-1203.

5. Mehlman MA. Dangerous and cancer-causing properties of products and chemicals in the oil refining

and petrochemical industry. VIII. Health effects of motor fuels: Carcinogenicity of gasoline. Environ

Res. 1992;59(1):238-249.

6. Rinsky RA, et al. Benzene and Leukemia: An Epidemiologic Risk Assessment. NEJM. 1987;316:1044-

1050.

7. Wong O. An Industry Wide Mortality Study of Chemical Workers Occupationally Exposed to Benzene.

I. General Results. Brit J Indust Med. 1987;44:365-381.

8. Zhang L, et al. The nature of chromosomal aberrations detected in humans exposed to benzene. CRC

Critical Reviews in Toxicology. 2002;32(1):1-42.

117

SERUM IRON ANALYTES IN HEALTHY AND ILL FLORIDA MANATEES


Gainesville, Florida, USA.


Background: Serum iron and transferrin concentration, measured as total iron binding capacity (TIBC), provide

information concerning iron metabolism in animals. Objective: To measure serum iron and TIBC and calculate

the percent saturation of transferrin with iron in serum from healthy and ill Florida manatees. Methods: Serum

samples were obtained from clotted blood collected from 226 healthy and 81 ill manatees. Iron analytes were

measured on a Hitachi 911 (Boehringer Mannheim/Roche Applied Science). Serum amyloid A (SAA) was

measured using a manual anti-bovine SAA ELISA method (Tridelta Development Limited). The two-way

ANOVA was used to compare test results from manatees by location (wild versus captive) and age class (adults,

subadults, large calves), as well as location and sex in adults. Bivariant simple linear regression was used to

compare iron analytes to SAA concentrations in ill animals. Results: Serum iron concentrations were not

significantly different by location in healthy manatees, but tended to be lower in younger animals. TIBC values

were over 20% higher in healthy captive versus wild manatees. The percent saturation with iron was slightly

lower in captive versus wild manatees and slightly lower insubadults and large calves versus adults. No

significant sex differences were identified in iron analytes in healthy adults. Significant negative linear

correlations were identified between SAA and serum iron, TIBC, and the percent saturation with iron in ill

adults. Conclusions: Small differences in serum iron analytes are measured in healthy manatees depending on

location and age, but not sex. Inflammation, as evident from increased SAA values in ill animals, results in

significant decreases in serum iron analytes.


HAEMATOLOGICAL CHANGES IN HORSES WITH ENDOTOXAEMIA

I. Lilliehook1, H.W. Tvedten

2, J. Brojer

2, A. Edner, K. Nostell

2. 1

University Animal Hospital and 2Dept of

Clinical Sciences, University of Swedish Agricultural Sciences, Uppsala, Sweden. Correspondence: [email protected]

Endotoxinaemia is common in horses with colitis, peritonitis, neonatal septicaemia, laminitis, metritis and

inflammatory airway disease. Horses are particular sensitive to the effects of endotoxins. Endotoxin is a heat-

stable lipopolysaccharide (LPS) from the cell wall of Gram-negative bacteria. A soluble binding protein (LPS-

BP) transports LPS from blood to the CD14 receptor on the macrophages, which induces activated mononuclear

cells to produce inflammatory cytokines such as; tumor necrosis factor (TNFα) and interleukin-1 (Werners et al.

2005). This may lead to cytokine mediated systemic inflammation (SIRS) and tissue damage. Complications

include hypovolemic shock, DIC and multiple organ failure. High concentrations of LPS are found in the lumen

of the normal equine intestine, which could leak into circulation or the peritoneal cavity during gastrointestinal

disease (King and Gerring 1988). LPS induces pronounced neutropenia by up-regulation of adhesion molecules on both neutrophils and

endothelial cells. This causes margination of the leukocytes in the blood vessel and this probably one of the

major causes of pronounced neutropenia seen after experimental induced endotoxaemia (Weiss and Evanson

2002). After intravenous infusion of endotoxin, the leukocyte numbers immediately start to decrease (Kiku et al.

2003). Lowest leukocyte counts are seen after 1-2 hours. Platelets also rapidly decrease, while haematocrit

increases (Burrows 1979, Ewert et al 1985). With repeated treatment with endotoxins every 6 hours neutrophils

numbers drop and then rebound within 6 hours and drop again after next infusion (Ward et al. 1987).

Intraperitoneal injection causes less pronounced haematological changes (Burrows 1979). Most studies of experimental endotoxaemia in horses have been completed within 6-24 hours. We monitored 8

horses for 3-6 days after a six hour long intravenous infusion with endotoxin (total 500µg/kg). In all horses a

severe neutropenia was seen for 4-6 hours. This was followed by a moderate neutrophilia which persisted more

than 3 days. The clinical signs (fever, colic, increased heart and respiration rate) subsided within 2 days. Based

on the appearance of peroxidase cytograms of Advia 2120 (Siemens Healthcare Diagnostics), the neutrophils

gradually appeared larger and less peroxidase positive and the most prominent changes were seen after more

than 3 days. The horses also developed a mild to moderate thrombocytopenia that persisted for more than 3 days.

References:

118

Burrows GE. Equine Escherichia coli endotoxinemia: Comparison of intravenous and intraperitoneal endotoxin

administration. Am J Vet Res 1979;40:991-998

Ewert KM, Fessler JF, Tempelton CB, Bottoms GD, Latshaw, HS, Johnson MA. Endotoxin-induced

hematologic and blood chemical changes in ponies: Effect of flunixin meglumine, dexamethasone and

prednisolone. Am J Vet Res 1985;46:24-30.

Kiku Y, Kusano K, Miyake H, Fukuda S, Takahashi J et al. Flow cytometric analysis of peripheral blood

mononuclear cells induced by experimental endotoxemia in horse. J Vet Med Sci 2003;65:857-863.

King JN, Gerring EL. Detection of endotoxin in cases of equine colic. Vet Rec1988;123:269-271.

Ward DS, Fessler JF, Bottoms GD, Turek J. Equine endotoxemia: Cardiovascular, eicosanoid, hematologic,

blood chemical and plasma enzyme alterations. Am J Vet Res 1987;48:1150-1156.

Weiss DJ, Evanson OA. Evaluation of lipopolysaccharide-induces activation of equine neutrophils. Am J Vet res

2002;63:811-815

Werners AH, Bull S, Fink-Gremmels J. Endotoxaemia: a review with implications for the horse. Equine vet J

2005;37: 371-383.

WORKSHOPS

TOXICOLOGY

OVERVIEW OF ROLES AND RESPONSIBILITIES, LABORATORY PERSONNEL AND THE

EXPANDING ROLES OF THE CLINICAL PATHOLOGIST IN DRUG DEVELOPMENT

L. Tomlinson, Pfizer Inc., Exploratory Drug Safety, Cambridge, MA, USA


Veterinary clinical pathologists have a thorough understanding of comparative biology, physiology,

pharmacology, and pathology which positions them well to contribute to drug discovery and development in

industry. We will explore current practices for the roles and responsibilities of veterinary clinical pathologists

and laboratory personnel at surveyed companies and identify best practices for industry. There is increased

recognition that clinical pathologists are necessary for the efficient operation and interpretation of clinical

pathology in standard drug discovery and development studies. Beyond this recognition, there is a growing

awareness of the breadth and versatility of these individuals throughout industry. However, there are still many

situations in which veterinary clinical pathologists could be further utilized, such as in translational medicine.

Translational medicine, which includes animal model work and biomarkers translatable to the clinic, is an area in

which veterinary clinical pathologists can have substantial impact. Nontraditional roles in industry will be

explored with examples of clinical pathology leaders in biomarker development, drug safety, immunotoxicology,

pharmacovigilance and as liaisons to medical and research institutions.

DATA ANALYSIS / INTERPRETATION OF DATA

B. Von Beust, Labor am Zugersee, Switzerland


As opposed to diagnostic clinical pathology in connection with clinical veterinary patients the analysis and

interpretation of data in preclinical research and safety attempts to answer a different set of questions.

Specifically, instead of asking whether treated animals suffer a specific disease the interest is whether there is a

compound-related adverse effect, and if yes, what it means for the compound in question. This requires the

119

integrated assessment of clinical pathology data with in-life observations, concurrent anatomic pathology

findings, exposure and metabolism data, and anticipated pharmacological effects of the test compound. As will

be presented in this session, we recommend general guidelines for interpreting and reporting clinical pathology

data. This includes the significance of statistical analysis and the use of reference intervals. However, the

approach to the interpretation of nonclinical data cannot be prescribed to cover all potential scenarios. Rather,

depending on the species involved and based on the focus of the study the clinical pathology data may have to be

evaluated on a case-by-case basis using a subjective “weight of evidence” approach. As this requires a profound

understanding of the context around each individual clinical pathology parameter we consider clinical

pathologists to be the best qualified individuals for the interpretation and reporting of the clinical pathology data

in preclinical research and safety.

BEST PRACTICES IN ROUTINE LABORATORY INVESTIGATIONS: HEMATOLOGY,

COAGULATION, CLINICAL BIOCHEMISTRY, AND URINALYSIS

L. Tomlinson, Pfizer Inc., Exploratory Drug Safety, Cambridge, MA, USA


The core clinical pathology panel recommended for laboratory animal toxicity and safety studies consists of

well-characterized hematologic, blood chemistry, and urine analyses that inform on general body homeostasis

and organ function and/or injury. Minimal testing recommendations have historically been broad or

intentionally vague, which provides flexibility for appropriate and specific study design and withstand the rapid

pace of growth in biomarker capabilities. Our recent poll of routine testing practices in companies across

industry suggests that standard testing panels have evolved along with technologic advances. Current standard

panels, as indicated by survey respondents, generally followed or exceeded current guidelines. This presentation

will initially focus on survey data for current standard panels, then will move to the appropriateness of current

practices and guidelines in the context of existing knowledge and testing capabilities, and finally there will be a

proposal of the need to update guidelines.

NON ROUTINE LABORATORY INVESTIGATIONS: BONE MARROW, CYTOLOGY,

ENDOCRINOLOGY AND OTHERS

D. Bounous, Bristol Myers Squibb Co., Discovery Toxicology – Clinical Pathology


Clinical Pathologists and clinical pathology laboratories in pharmaceutical companies are increasingly expanding

to include more than the routine biomarker panels in their repertoires. Bone marrow cytology, fluid cytology,

and new biomarkers and platforms are utilized in many investigative studies. These are areas in which clinical

pathologists are traditionally trained, but in pharmaceutical and toxicology studies this requires a different

perspective than the more traditional diagnostic role. This expansion of traditional training further integrates the

clinical pathologist into a role of consultant and advisor for interpretation of data in investigative studies for drug

development.

INSTRUMENT AND METHOD VALIDATION

B. Von Beust, Labor am Zugersee, Switzerland


Standard procedures for instrument and method validation are well established and described in text books and

online references. In well established industry laboratories under the supervision of specialized clinical

pathologists state of the art procedures are practiced and documented for all instruments and methods. However,

in smaller laboratories without previous exposure to standard validation procedures or in a non GLP environment


120

it may not always be possible to perform and document a complete package of validation procedures due to

shortages in resources and personnel. The presentation will discuss basic validation procedures based on a

number of real life problems encountered in environments without standardized instrument validation. In

addition, practical examples will propose possible approaches for method validations in toxicologic clinical

pathology.

POINTS TO CONSIDER FOR NEW BIOMARKER QUALIFICATION

D. Bounous, Bristol Myers Squibb Co., Discovery Toxicology – Clinical Pathology


Interest in biomarker development in both the clinical and nonclinical setting has led to the increasingly

widespread use of novel biomarkers for evaluation of target organ injury or drug target pharmacology. While

the process for analytic validation of new parameters is both well understood and broadly implemented,

characterization of unique biological aspects of these novel markers is often less comprehensively performed.

Biomarker qualification involves biologic or physiologic characterization, with understanding of biologic

variability, pre-analytic variables, as well as consideration of species-relevance and specificity for target organ or

pharmacology. Novel biomarker qualification also involves pathophysiologic characterization, with

consideration of the relationship of novel marker changes to histopathologic findings or disease state, chronology

and reversibility. Evaluation of diagnostic performance across a variety of treatments or experimental conditions

is a critical aspect of the biologic qualification of novel biomarkers that is increasingly being performed.

HOW TO PUBLISH

WRITING FOR PUBLICATION IN VETERINARY MEDICINE: KEY TO SUCCESS

M. Christopher1, K. Young

2.

1University of California-Davis, Pathology, Microbiology, and Immunology,

Davis, CA, USA; 2University of Wisconsin-Madison, Department of Pathobiological Sciences, Madison,

WI, USA

Booklet available on link: (http://www.wiley.com/WileyCDA/Section/id-612222.html)

PROTEOMICS AND ANIMAL HEALTH (COST- Action Workshop)

PROTEOMICS IN VETERINARY SCIENCE: AN OVERVIEW

P. D. Eckersall. Institute of Infection, Immunity & Inflammation and the School of Veterinary Medicine,

University of Glasgow, Bearsden Rd., Glasgow G61 1QH, UK


Proteomics is an investigatory approach in which ideally all of the proteins or peptides in a biological

sample are separated, identified and quantified. However, there are limitations due to technology and resources

which limit this aim. Nevertheless over the last few years the technology to enable the simultaneous

investigation of the complex mix of proteins found in a biological sample has evolved considerably and there has

been a rapid increase in proteomic investigation in basic and clinical sciences. Unfortunately, this expansion of

proteomics has largely by-passed veterinary and animal science with only around 3% of published proteomic

papers being related to studies on domestic animals. These pioneering investigations demonstrate the potential

for future expansion of research into the protein composition of tissues and biological fluid in veterinary science

(Eckersall and Whitfield, 2011; Rodrigues et al., 2012).

A measure of the progress which has been achieved by advancing proteomic technology is illustrated by the

history of studies on the bovine milk proteome. In 2001, the most abundant milk proteins, caseins, -

lactalbumin and -lactoglobin were the only proteins to be found following 2 dimension electrophoresis (2DE)


http://www.wiley.com/WileyCDA/Section/id-612222.html

121

(Galvani et al., 2001). Advances in the technology have subsequently allowed the identification of 22 of the less

abundant proteins in milk, of which 17 were found to be up-expressed in milk from cows with mastitis (Boehmer

et al., 2008). Most recently a quantitative proteomic investigation of milk from dairy cows in an experimental

model of mastitis (Danielsen et al., 2010) has revealed 80 milk proteins of which 49 were shown to change in

concentration following lipopolysaccharide challenge.

Similarly early investigations of the serum or plasma proteome of domestic animals produced proteome

maps by the use of 2DE (Talamo et al., 2003). Subsequently alterations to the serum proteome has been used to

monitor pathophysiological responses for instance during the acute phase response (Thongboonkerd et al., 2009)

or in response to anabolic steroid treatment (Draisci et al., 2007). There have now been initial studies of a large

variety of tissue from the major domestic species in health and disease or experimental investigation (Eckersall

and McLaughlin, 2011). Thus the altered expression of proteins the liver during bovine ketosis in relation to

carbohydrate, lipid and protein metabolism have been identified (Xu and Wang, 2008). The proteome

responses of monocytes to bovine viral diarrhoea virus (Pinchuk et al., 2008) and of alveolar macrophages to

porcine reproductive & respiratory virus (Zhang et al., 2009) illustrate the potential for increasing our

understanding of the immune response in farm animals. Thus detailed examination of the temporal effects of

viral infection on the intracellular proteins in their responses to these and other pathogens of veterinary

importance can be made.

Demonstration that proteomics can have a valuable role in veterinary research needs to be supplemented

with mechanisms whereby this can be achieved. Major impediments to the application of these technologies are

the cost of equipment and expertise available. The COST Action on Farm Animal Proteomics (www.cost-

faproteomics.org) has been established in Europe to overcome these obstacles by creating links between

established centres with the necessary proteomic technology and researchers who desire to apply proteomics to

their own areas of endeavour. The Working Group meetings, Training Schools and Short Term Scientific

Missions of this COST Action are open to those who contemplate the use of proteomics and especially to early

stage researchers in farm animal science.

References

Boehmer, J., Bannerman, D., Shefcheck, K., Ward, J., 2008. Proteomic Analysis of Differentially Expressed

Proteins in Bovine Milk During Experimentally Induced Escherichia coli Mastitis. J. Dairy Sci. 91,

4206-4218.

Danielsen, M., Codrea, M.C., Ingvartsen, K.L., Friggens, N.C., Bendixen, E., Rontved, C.M., 2010. Quantitative

milk proteomics - Host responses to lipopolysaccharide-mediated inflammation of bovine mammary

gland. Proteomics 10, 2240-2249.

Draisci, R., Montesissa, C., Santarnaria, B., DAmbrosio, C., Ferretti, G., Merlanti, R., Ferranti, C., De Liguoro,

M., Cartoni, C., Pistarino, E., Ferrara, L., Tiso, M., Scaloni, A., Cosulich, M.E., 2007. Integrated

analytical approach in veal calves administered the anabolic androgenic steroids boldenone and

boldione: urine and plasma kinetic profile and changes in plasma protein expression. Proteomics 7,

3184-3193.

Eckersall, P.D., McLaughlin, M., 2011. Proteomics in Animal Health and Disease. In: Eckersall, P.D., Whitfield,

P.D. (Eds.), Methods in Animal Proteomics. John Wiley & Sons, Chichester, pp. 243-318.

Eckersall, P.D., Whitfield, P.D., 2011. Methods in Animal Proteomics. Wiley-Blackwell, Chicester, UK.

Galvani, M., Hamdan, M., Righetti, P.G., 2001. Two-dimensional gel electrophoresis/matrix-assisted laser

desorption/ionisation mass spectrometry of commercial bovine milk. Rapid Communications in Mass

Spectrometry 15, 258-264.

Pinchuk, G.V., Lee, S.R., Nanduri, B., Honsinger, K.L., Stokes, J.V., Pinchuk, L.M., 2008. Bovine viral diarrhea

viruses differentially alter the expression of the protein kinases and related proteins affecting the

http://www.cost-faproteomics.org/

http://www.cost-faproteomics.org/

122

development of infection and anti-viral mechanisms in bovine monocytes. Biochimica et Biophysica

Acta-Proteins and Proteomics 1784, 1234-1247.

Rodrigues, P., Eckersall, P.D., Almeida, A., 2012. Farm Animal Proteomics. Wageningen Academic Publishers,

Waginengen.

Talamo, F., D'Ambrosio, C., Arena, S., Del Vecchio, P., Ledda, L., Zehender, G., Ferrara, L., Scaloni, A., 2003.

Proteins from bovine tissues and biological fluids: Defining a reference electrophoresis map for liver,

kidney, muscle, plasma and red blood cells. Proteomics 3, 440-460.

Thongboonkerd, V., Chiangjong, W., Mares, J., Moravec, J., Tuma, Z., Karvunidis, T., Sinchaikul, S., Chen,

S.T., Opatrny, K., Matejovic, M., 2009. Altered plasma proteome during an early phase of peritonitis-

induced sepsis. Clinical Science 116, 721-730.

Xu, C., Wang, Z., 2008. Comparative proteomic analysis of livers from ketotic cows. Veterinary Research

Communications 32, 263-273.

Zhang, H.M., Guo, X., Ge, X.N., Chen, Y.H., Sun, Q.X., Yang, H.C., 2009. Changes in the Cellular Proteins of

Pulmonary Alveolar Macrophage Infected with Porcine Reproductive and Respiratory Syndrome Virus by

Proteomics Analysis. Journal of Proteome Research 8, 3091-3097.

PROTEOMICS IN VETERINARY SCIENCES: A CASE STUDY IN TICK-BORNE DISEASES

A.M. Almeida1,2,*

, M. Ventosa2,3

, D. Meyer4, D. Martinez

5, T. Lefrançois

4, N. Vachiéry

4, A.V. Coelho

2, I.

Marcelino2,3,4;* 1

IICT/CVZ & CIISA, FMV, Lisboa, Portugal; 2ITQB-UNL, Oeiras, Portugal;

3IBET, Oeiras,

Portugal; 4CIRAD, UMR, Petit-Bourg, Guadeloupe, FWI; and

5CIRAD, UMR Montpellier, France. *Both

authors contributed equally


Introduction

Tick-borne diseases (TBDs) affect 80% of the world’s cattle population, severely hampering livestock

production. Some of these diseases are caused by obligate intracellular tick-borne pathogens (TBPs) such as

Theileria spp, Babesia spp, Anaplasma marginale and Ehrlichia ruminantium (Marcelino et al., 2012).

Immunization strategies against TBDs are currently available, although with a varied extent of efficiency

(Marcelino et al., 2012a); the identification of new antigens is of capital importance to the development of novel

vaccines. Presently, vaccine research and development relies increasingly on the novel “Omics” approaches:

genomics, proteomics, transcriptomics, and metabolomics, in order to understand key metabolic processes that

lead to protective immunity (see a schematic representation in Figure 1). Despite the recent availability on

databases on extended information on TBPs genomes, the increased knowledge on their biology has proven to be

difficult since they have very complex life cycles, with several developmental forms and holding the ability to

infect and multiply within different types of cells and hosts. Albeit limited genetic tools are currently available,

complementing genomic information, post-genomics strategies such as proteomics are nowadays being used

more frequently.

Proteomics and cattle TBDs: an overview

Available proteomic studies on the mentioned cattle TBDs are related to one or the several components of the

infection cycle: pathogen, host (usually cultivated cells) and the tick vector. In this section we will briefly

mention these studies. Readers are however recommended to read our recently published review (Marcelino et

al., 2012a) for further details.

Proteomic studies on Anaplasma marginale have been targeted to identify outer membrane proteins (OMPs), as

these proteins are known to induce protective immune response in cattle and to understand the transition from

the host to the tick vector. Several studies were performed to analyze differentially regulated proteins between

A.marginale-infected and uninfected host and tick cells (Lopez et al., 2005; Noh et al., 2008). These studies

allowed identifying several immunogenic proteins from the OMP family and from the Type Four Secretion

System; vaccination experiments were used to validate their use as potential vaccine candidate. A Proteomics


123

study also revealed that A. marginale surfome in vector cells is less complex that the one in host cells (Noh et al.,

2008).

Figure 1 Omics and vaccine discovery. The development of new vaccines against TBDs such as Heartwater,

imply implies the profound knowledge of the intimate relations between host-vector-pathogen. Vaccine

candidates can be identified by analysis of the pathogen’s genome, transcriptome, proteome, or metabolome.

Systems biology will integrate the overall data to build prediction methods to identify protective epitopes.

(Artwork by Simão Mateus)

In Theileria spp., 2D electrophoresis and MALDI-TOF has been used to analyze the protein expression pattern

of different T. parva strains (Sugimoto et al., 1989a) and the schizont-protein spot patterns of the same Theileria

stabilate cultivated in two different infected cell lines. While no significant difference in protein expression

pattern was observed between strains, the author observed a differential protein expression pattern depending on

host cells. To detect immunodominant schizont surface proteins (surfome), Theileria spp surface antigens were

characterized using 2D and Western blot (Sugimoto et al., 1992). As Theileria spp. transform host cell

(leucocytes) inducing uncontrolled proliferation, differential protein expression patterns in infected bovine

lymphoblastoid cells was also performed (Sugimoto et al., 1989b); the results showed that ten proteins were

found in infected cells but not in uninfected cells, and seven of these were detected in preparations of purified

schizonts.

Most studies available on Babesia spp. are from tick cell models. The identification of these proteins may

provide new insights into the molecular interactions between Babesia spp. and the tick vector. Using 2D

followed by MS or/and capillary-HPLC-electrospray tandem mass spectrometry (HPLC-ESI-MS/MS),

differences in expression of proteins from ovaries (Rachinsky et al., 2007) and midgut tissue (Rachinsky et al.,

2008) of infected versus non-infected Rhipicephalus. (Boophilus) microplus ticks were assessed. The results

showed that infection with Babesia induced up-regulation of five metabolic enzymes related to electron and

proton transport, protein processing and retinoic acid metabolism.

Few proteomics studies are currently available for E .ruminantium, being mainly related to the differential

expression of the immunodominant E.ruminantium MAP1 family proteins in infected bovine endothelial

(Marcelino et al., 2012b; Postigo et al., 2008), and tick cell cultures (Postigo et al., 2008). These studies revealed

that this MAP1 protein is differentially expressed not only in both tick and host cells but also along the

developmental cycle of the bacterium inside the host cells. In 2011, our group presented the first partial

proteome map of E.ruminantium (Marcelino et al., 2010); 25% of the identified were found to be isoforms,

revealing that post-translational modifications can be of significant importance for E.ruminantium.

Interestingly, for some TBPs, the proteome seems to be more studied than the proteome. This is the case for

Ehrlichia ruminantium; indeed several manuscripts described the global gene expression profiling of Ehrlichia at

different stages of development or are specifically related to map1 gene expression either in host or vector cells

(Bekker et al., 2002; Postigo et al., 2007; Pruneau et al., 2011). Whole transcriptome analysis was also

performed for Theileria spp in host cells, namely from two breeds of cattle (resistant and susceptible to disease)

but also during the complex developmental cycle (Oura et al., 2006; Jensen et al., 2008). In Babesia spp.,

transcriptomics studies were performed to study antigenic variation but also to study peptidases expression in

attenuated versus virulent strain (Mesplet et al., 2011); an expression oligonucleotide microarray was also

124

developed (Lau et al., 2007). For A.marginale, differential gene expression was studied in different tick and host

cells gene expression in different tick and host cells (Agnes et al., 2010; Mercado-Curiel et al., 2011).

Final remarks

It is clear that “Omics” in the TBDs context are still in their infancy. Still, we believe that these cutting edge

technologies will now significantly contribute to overcome knowledge gaps on the role of parasite molecules

involved in cell invasion, adhesion, and transmission, surely revolutionizing the time and capacity for

discovering potential candidate vaccines. Joining all the Omics data using an integrative systems biology

approach, the development of novel and effective vaccines against several TBDs will likely be a reality.

Acknowledgements

Authors acknowledge funding from project ER-TRANSPROT (PTDC/CVT/114118/2009), Post-doc grant

SFRH/ BPD/ 45978/ 2008 (I. Marcelino) and the Ciência 2007 program (AM Almeida), all by FCT(Lisboa,

Portugal).

References

- Agnes JT, Herndon D, Ueti MW, Ramabu SS, Evans M, Brayton KA, Palmer GH. Association of

pathogen strain-specific gene transcription and transmission efficiency phenotype of Anaplasma

marginale. Infect Immun. 2010;78: 2446-2453.

- Bekker CP, Bell-Sakyi L, Paxton EA, Martinez D, Bensaid A, Jongejan F. Transcriptional analysis of

the major antigenic protein 1 multigene family of Cowdria ruminantium. Gene 2002;285: 193-201.

- Oura CA, McKellar S, Swan DG, Okan E, Shields BR, Infection of bovine cells by the protozoan

parasite Theileria annulata modulates expression of the ISGylation system. Cell Microbiol. 2006;8:

276-288.

- Jensen K, Paxton E, Waddington D, Talbot R, Darghouth MA, Glass EJ. Differences in the

transcriptional responses induced by Theileria annulata infection in bovine monocytes derived from

resistant and susceptible cattle breeds. Int J Parasitol. 2008;38: 313-325.

- Lau AO, Tibbals DL, McElwain TF. Babesia bovis: the development of an expression oligonucleotide

microarrayExp Parasitol. 2007;117: 793-798.

- Lopez JE, Siems WF, Palmer GH, Brayton KA, McGuire TC, Norimine J, Brown WC. Identification of

novel antigenic proteins in a complex Anaplasma marginale outer membrane immunogen by mass

spectrometry and genomic mapping. Infect Immun. 2005;73: 8109-8118.

- Marcelino I, de Almeida AM, Brito C, Meyer DF, Barreto M, Sheikboudou C, Franco CF, Martinez D,

Lefrançois T, Vachiéry N, Carrondo MJ, Coelho AV, Alves PM. Proteomic analyses of Ehrlichia

ruminantium highlight differential expression of MAP1-family proteins. Vet Microbiol 2012b; 156:

305-314.

- Marcelino I, de Almeida AM, Ventosa M, Pruneau L, Meyer DF, Martinez D, Lefrançois T, Vachiéry

N, Coelho AV. Tick-borne diseases in cattle: Applications of proteomics to develop new generation

vaccines. J Proteomics (2012a; in press).

- Mesplet M, Palmer GH, Pedroni MJ, Echaide I, Florin-Christensen M, Schnittger L, Lau AO. Genome-

wide analysis of peptidase content and expression in a virulent and attenuated Babesia bovis strain pair.

Mol Biochem Parasitol. 2011;179: 111-113.

- Noh SM, Brayton KA, Brown WC, Norimine J, Munske GR, Davitt CM, Palmer GH. Composition of

the surface proteome of Anaplasma marginale and its role in protective immunity induced by outer

membrane immunization. Infect Immun. 2008;76: 2219-2226.

- Postigo M, Taoufik A, Bell-Sakyi L, Bekker CP, de Vries E, Morrison WI, Jongejan F. Host cell-

specific protein expression in vitro in Ehrlichia ruminantium. Vet Microbiol 2008;128: 136-147.

- Postigo M, Taoufik A, Bell-Sakyi L, de Vries E, Morrison WI, Jongejan F. Differential transcription of

the major antigenic protein 1 multigene family of Ehrlichia ruminantium in Amblyomma variegatum

ticks. Vet Microbiol. 2007;122: 298-305.

- Pruneau L, Emboulé L, Gely P, Marcelino I, Mari B, Pinarello V, Sheikboudou C, Martinez D, Daigle

F, Lefrançois T, Meyer DF, Vachiery N. Global gene expression profiling of Ehrlichia ruminantium at

different stages of development. FEMS Immunol Med Microbiol (2011).

- Rachinsky A, Guerrero FD, Scoles GA. Differential protein expression in ovaries of uninfected and

Babesia-infected southern cattle ticks, Rhipicephalus (Boophilus) microplus. Insect Biochem Mol Biol.

2007;37, 1291-1308.

- Rachinsky A, Guerrero FD, Scoles GA. Proteomic profiling of Rhipicephalus (Boophilus) microplus

midgut responses to infection with Babesia bovis. Vet Parasitol. 2008;152: 294-313.

125

- Sugimoto C, Conrad PA, Mutharia L, Dolan TT, Brown WC, Goddeeris BM, Pearson TW. Phenotypic

characterization of Theileria parva schizonts by two-dimensional gel electrophoresis. Parasitol Res

1989a;76: 1-7.

- Sugimoto C, Mutharia LM, Brown WC, Pearson TW, Dolan TT, Conrad PA. Analysis of Theileria

parva immunodominant schizont surface antigen by two-dimensional polyacrylamide gel

electrophoresis and immunoblotting. Parasitol Res. 1992;78: 82-85.

- Sugimoto C, Mutharia LM, Conrad PA, Dolan TT, Brown WC, Goddeeris BM, Pearson TW. Protein

changes in bovine lymphoblastoid cells induced by infection with the intracellular parasite Theileria

parva. Mol Biochem Parasitol. 1989b;37: 159-169.

INCREASED OXIDATIVE STRESS AND PEROXISOMAL FATTY ACID DEGRADATION IN

EARLY LACTATING HOLSTEIN COWS DEVELOPING FATTY LIVER SYNDROM - LESSIONS

FROM PROTEOMIC ANALYSIS

B. Kuhla1, C. Schäff

1, D. Albrecht

2, M. Röntgen

1, C. C. Metges

1, H. M. Hammon

1.

1 Research Unit

Nutritional Physiology “Oskar Kellner”, Leibniz Institute for Farm Animal Biology (FBN), Dummerstorf,

Germany; 2 Institute of Microbiology, Ernst-Moritz-Arndt-University, Greifswald, Germany.


The early lactation period of high-yielding dairy cows is characterized by a tremendous mobilization of body

reserves including glycogen, protein and fat depots and a drop into negative energy balance (NEB). With

increasing extent of body fat mobilization, concentrations of plasma non-esterified fatty acid (NEFA) and

deposition of triacylglycerides (TAG) in the liver are increasing during early lactation (Hammon et al., 2009).

However, dairy cows respond to NEB at the onset of lactation with only a small increase in hepatic fatty acid

oxidation (Grum et al., 1996) accompanied by the very limited hepatic VLDL export of TAG in the bovine, both

contributing to the development of compromised liver health such as hepatic steatosis. The underlying metabolic

and molecular mechanisms contributing to liver disease due to an altered hepatic lipid metabolism, however, are

not well understood but the involvement of oxidative stress may contribute to it.

Therefore, the aim was to investigate the expression of hepatic enzymes involved in fatty acid oxidation in

parallel with those belonging to the antioxidative stress response system in the liver of cows with a different

extent of body fat mobilization during the periparturient period.

To this end, 19 multiparous German Holstein cows (2nd

to 4th

lactation, >10,000kg/305d in at least one previous

lactation) housed in a tie stall from week 7 before until 5 weeks after calving. Cows were fed three different total

mixed rations according to their physiological state, during the far-off dry period (week -7 to -4, 5.87 MJ NEL

and 128 g nXP/kg DM), the close-up dry period (week -3 until calving, 6.49 MJ NEL and 137 g nXP/kg DM),

and lactation (week 1 to 5, 7.06 MJ NEL and 163 g nXP/kg DM). While individual feed intake was measured

daily, body mass (BM), body condition score (BCS), back fat thickness (BFT), and milk yield were determined

weekly. Blood samples were taken as soon as cows entered the far-off dry period once weekly and around

parturition twice weekly to measure plasma concentrations of glutamate dehydrogenase (GLDH) and aspartate

transaminase (AST). The liver of the animals was biopsied at five consecutive time points at days -34, -17, 3, 18,

30 relative to calving. A further liver sample was obtained from slaughter at day 40 after calving. Tissue samples

were immediately frozen in liquid nitrogen. The total liver fat content (LFC) was analysed, and based on the

average amount of LFC post partum, cows were retrospectively allocated to high (H; LFC >24.4% DM; n=10))

and low (L; LFC <24.0% DM; n=9) fat mobilizing groups.

Also, 50 mg of finely ground frozen liver was extracted and prepared for proteomic analysis as described earlier

for feed restriction-induced fatty liver (Kuhla et al., 2009). Briefly, extracts were applied to 2-dimensional gel

electrophoresis followed by colloidal Coomassie staining. For spot detection and quantitation 2D gels were

processed using Decodon Delta 2D software. Normalized spot volumes were analysed by the Mixed Model of

SAS (version 9.2) with fixed effects for group and time relative to parturition. All spots that were differentially

expressed between groups (p<0.05) were picked, tryptic digested and analysed on a 5800 MALDI TOF/TOF


126

analyser. The subsequent NCBInr and Swiss-Prot database search yielded 170 identified protein spots. Among

them we were able to identify several proteins involved in fatty acid transport, mitochondrial and peroxisomal

fatty acid degradation as well as in defence against oxidative stress.

Dry matter intake increased after calving but was lower in H than in L cows throughout the whole experimental

period. Liver fat content was highest at day 18 post partum in both groups but was significantly higher in H than

in L cows. Plasma GLDH and AST were not different between groups but were approximately 1.4 fold higher in

H than in L cows at day 18 post partum.

The expression of medium chain acyl-CoA dehydrogenases (the first enzyme of the beta-oxidative pathway) and

peroxisomal enoyl-CoA hydratase were higher after parturition as compared to the prepartal period but was also

higher in H than in L cows. Enoyl-CoA hydratase and 3-hydroxy acyl-CoA dehydrogenase, the next two

downstream enzymes of the beta-oxidative pathway, were also higher expressed post partum, but lower

expressed in H than in L cows. Moreover, 3-ketoacyl-CoA thiolase, the 4th

enzyme of the beta-oxidative

pathway, was higher expressed post partum only in L but not in H cows. The amount of catalase, which

detoxifies H2O2 generated during peroxisomal fatty acid degradation, continuously decreased from d -17 to d +30

relative to parturition in both groups but remained higher in H than in L cows. A similar course of expression

was observed for peroxiredoxin-6 which also degrades H2O2 and for Cu/Zn superoxide dismutase, which

degrades peroxide radicals to H2O2. Moreover, DJ-1 protein, which protects against oxidative stress by

scavenging hydroperoxyl radicals continuously increased after calving and remained higher in H than in L cows.

On the other hand, expression of glutathione-S-transferase also decreased from d -17 to d +30 relative to

parturition but was lower in H than in L cows.

Our results suggest that in early lactation the first step of the beta-oxidative pathway is activated particularly in

the liver of H cows which presumably is due to the higher plasma NEFA concentration. The lower expression of

further downstream located enzymes of the beta-oxidative pathway in H cows suggests incomplete fatty acid

degradation by this pathway which might have contribute to the development of fatty liver. The reduced

expression of downstream located beta-oxidative enzymes may either occur as a negative feed-back regulatory

response to prevent excessive heat production or due to a limited availability of coenzyme A. Furthermore, H

cows seem to degrade more fatty acids via the peroxisomal pathway than L cows. This implies that H cows are

more subjected to oxidative stress, in particular elicited by H2O2 and peroxide and hydroperoxyl radicals.

In summary, our results clearly show that cows highly mobilizing body fat in early lactation are not able to

completely oxidize fatty acids. This is associated with an increased oxidative stress and a higher extent of fatty

liver.

The study was supported by Deutsche Forschungsgemeinschaft (DFG; KU 1956/4-1).

References:

Bernabucci U, Ronchi B, Lacetera N, Nardone A. Influence of body condition score on relationships between

metabolic status and oxidative stress in periparturient dairy cows. J Dairy Sci. 2005;88:2017-2026.

Grum DE, Drackley JK, Younker RS, LaCount DW, Veenhuizen JJ. Nutrition during the dry period and hepatic

lipid metabolism of periparturient dairy cows. J Dairy Sci. 1996;79:1850-1864.

Hammon HM, Stürmer G, Schneider F, Tuchscherer A, Blum H, Engelhard T, Genzel A, Staufenbiel R, Kanitz

W. Performance and metabolic and endocrine changes with emphasis on glucose metabolism in high-yielding

dairy cows with high and low fat content in liver after calving. J Dairy Sci. 2009;92:1554-1566.

Kuhla B, Albrecht D, Kuhla S, Metges CC. Proteome analysis of fatty liver in feed-deprived dairy cows reveals

interaction of fuel sensing, calcium, fatty acid, and glycogen metabolism. Physiol Genomics. 2009;37:88-98.

127

POST TRANSLATIONAL MODIFICATIONS OF ACUTE PHASE PROTEINS IN VETERINARY

MEDICNE: A PROTEOMIC APPROACH

F.Ceciliani. Department of Animal Pathology, Hygiene and Veterinary Public Health, School of Veterinary

Medicine, University of Milan, Milan, Italy.


There is a need for accurate biochemical markers to aid in early diagnosis, staging and prognosis of diseases. In

most of the cases, the simple serum increase of specific acute phase proteins does not provide this information.

Therefore, many researchers turned to different strategies, focusing not only on the modification of the

expression of a given protein, but exploiting also other chemical properties, such as post-translational

modifications, including phosphorylation degree, carbohydrate moiety or different aggregation status. In cancer

for example, many of the existing biomarkers of disease are, in fact, glycoproteins or carbohydrate moieties

(such as CA 19–9 in pancreatic cancer), and probing glycoproteins for potential biomarker discovery may

provide better diagnostic ability.

Serum Amyloid A (SAA) and α1-acid glycoprotein (AGP) are acute phase proteins, the increase of their serum

concentration being a hallmark of a systemic reaction of inflammation (Petersen et al., 2004).). In some cases,

the detection of high concentrations of these APP is regarded as pathognomonic of specific diseases, such as, for

example, the increase of AGP during Feline Infectious Peritonitis (Duthie et al., 1997). Both these proteins

feature wide post-translational modifications. AGP is a major acute phase protein in cats and rats, and a

moderate one in most of the species investigated so far, excluding pig. Beside its simple increase in serum

concentration, one of the most remarkable features of AGP is the extent of its carbohydrate moiety, which

accounts for approximately the 45% of its molecular weight (Ceciliani and Pocacqua, 2007). The

oligosaccharides micro-heterogeneity of AGP is profoundly affected by pathologic conditions. Different

glycoforms appear in plasma during systemic inflammation or diseases, and their modifications are important for

the fine tuning of several AGP immunomodulatory activities. Post-translational modeling of SAA is interesting

as well. SAA is not glycosylated, but it is present in serum in different multimers, that are likely to have

physiological significance, at present completely unknown. The unstable secondary structure of SAA is also the

main responsible of its precipitation as insoluble beta-sheet rich fibrils, which is the molecular basis of

amyloidosis (Chiti and Dobson, 2006). Given the background of its highly informative capability for both

glycosylation pattern and multimerization status, proteomic analysis is an ideal tool to study of the relationship

between structure and function of both proteins.

Proteomics can be defined as the study of protein expression, protein-protein interactions, or post-translational

modifications. Proteomic approach ranges form a simple small scale analysis, which analyze one or few proteins

at a time, such as those investigations aimed to map post-translational modifications, to those focused on larger-

scale analysis, targeting hundreds to thousands of proteins in a single experiment. In many cases, the term

proteomics has replaced the use of the term protein science.

The capability to analyze several proteins at once gives to proteomics the possibility to demonstrate how cells

can dynamically respond to changes in their environment. Therefore, proteomic techniques can identify new and

potentially unexpected changes in protein expression, interaction, or modification as a result of experimental

treatment, or different physiological status, or diseases.

This communication focuses on the relationship between the structure and the functions of AGP and SAA in

domestic animals, including cow, goats and cats. Carbohydrate moiety of bovine AGP plays important roles on

monocyte apoptosis, as well as on neutrophil degranulation, demonstrating that the sialic acid external residues

of the protein are extremely important to maintain AGP functions (Ceciliani et al., 2007; Miranda et al., 2010).

The phosphorylation degree is important as well, and its modification can further modulate the phagocytosis of

both monocytes and granulocytes (Lecchi et al., submitted).

Post translational modification of the protein has been also determined in goats. While the increase of AGP

during acute phase response is modest, yet we demonstrated that a systemic inflammation, such as Caprine

Arthritis Encephalitis Viral (CAEV) can sensibly modify AGP’s carbohydrate moiety (Ceciliani et al., 2009).

Taking advantage of the importance of AGP as major acute phase protein in cats, its post-translational

modification has been studied in several disease models, including Feline Infectious Virus and Feline Leukemia

Virus (Pocacqua et al., 2005) affected animals and, most interesting, in Feline Infectious Peritonitis, where a

direct involvement of post-translational modification of the “pathological” glycosylation pattern has been

128

recently related to immunomodulatory functions of the proteins(Ceciliani et al., 2004; Rossi et al., manuscript in

preparation) .

Post-translational modification analyses can be also useful to determine where the protein comes from, which is

particularly important for its local detection. Different tissues produce different AGP glycoform, with a specific

sialylation degree. This information could be very important in order to ascertain which is the origin of the

protein, and possibly which is the glycoform that is most increased during the inflammatory reaction.

Serum Amyloid A glycosylation is not an issue. What is interesting in SAA is its trend to multimerize.

Dimerization and polymerization status has been studied by means of proteomics in both colostrum and serum in

pig species, where several multimers have been identified.

In human amyloidosis and Alzheimer disease proteomic techniques have been successfully employed in the

characterization of the circulating amyloidogenic precursors and the analysis of affected tissues, for the

diagnostic identification of the fibril components and for characterizing disease-related changes in protein

expression (Stoppini et al., 2009).

Proteomics has the ability to discover potentially new and unexpected connections

between a change in physiological status and a change in protein expression, thus playing an important role in

determining functions for many acute phase proteins.

In recent years, proteomics-based discoveries have contributed tremendously to elucidating molecular alterations

in human diseases. Veterinary medicine and animal sciences are catching up. Although only a small subset of the

human proteome is currently used in a diagnostic setting, it is almost certain that the use of proteomic signatures

as biomarkers will gain popularity in the coming years. In the future, proteomic technologies are likely to

become components of most clinical laboratories and will provide diagnostic information that drives clinical

decision making.

References:

Ceciliani F, Grossi C, Giordano A, Pocacqua V, Paltrinieri S. Decreased sialylation of the acute phase protein

alpha1-acid glycoprotein in feline infectious peritonitis (FIP). Vet Immunol Immunopathol. 2004;99(3-4):229-

36.

Ceciliani F, Pocacqua V. The acute phase protein alpha1-acid glycoprotein: a model for altered glycosylation

during diseases. Curr Protein Pept Sci. 2007;8(1):91-108.

Ceciliani F, Pocacqua V, Miranda-Ribera A, Bronzo V, Lecchi C, Sartorelli P. alpha(1)-Acid glycoprotein

modulates apoptosis in bovine monocytes. Vet Immunol Immunopathol. 2007;116(3-4):145-152.

Ceciliani F, Rahman MM, Lecchi C, Maccalli M, Pisoni G, Sartorelli P. Systemic and in vitro expression of goat

alpha(1)-acid glycoprotein during Caprine Arthritis-Encephalitis Virus infection. Vet Immunol Immunopathol.

2009;131(1-2):50-58.

Chiti F, Dobson CM. Protein misfolding, functional amyloid, and human disease. Annu Rev Biochem.

2006;75:333-366.

Duthie S, Eckersall PD, Addie DD, Lawrence CE, Jarrett O. Value of alpha 1-acid glycoprotein in the diagnosis

of feline infectious peritonitis. Vet Rec. 1997;141(12):299-303.

Lecchi C , Scarafoni A,Bronzo V, Martino P.A., Cavallini A,Sartorelli P, Ceciliani F Alpha1 acid glycoprotein

modulates Escherichia coli phagocytosis and killing by bovine neutrophils and monocytes. Submitted

Miranda-Ribera A, Lecchi C, Bronzo V, Scaccabarozzi L, Sartorelli P, Franciosi F, Ceciliani F. Down-regulatory

effect of alpha 1-acid glycoprotein on bovine neutrophil degranulation. Comp Immunol Microbiol Infect Dis.

2010; 33(4):291-306.

Petersen HH, Nielsen JP, Heegaard PM. Application of acute phase protein. measurements in veterinary clinical

chemistry. Vet Res. 2004; 35(2):163-187.

129

Pocacqua V, Provasi E, Paltrinieri S, Gelain E, Comunian C, Ceciliani F. Glycan moiety modifications of feline

alpha1-acid glycoprotein in retrovirus (FIV, FeLV) affected cats. Vet Immunol Immunopathol. 2005;107(1-

2):17-26.

Rossi G, et al., Hyposialylated alpha1-acid glycoprotein depress phagocytosis of feline Neutrophils, manuscript

in preparation.

Stoppini M, Obici L, Lavatelli F, Giorgetti S, Marchese L, Moratti R, Bellotti V, Merlini G. Proteomics in

protein misfolding diseases. Clin Chem Lab Med. 2009;47(6):627-635.

SEPARATION TECHNIQUES AND THE USE OF DIGE FOR PROTEOMIC ANALYSIS OF

VETERINARY SAMPLES

A. Marco-Ramell, A. Bassols. Departament de Bioquímica i Biologia Molecular. Facultat de Veterinària.

Universitat Autònoma de Barcelona. Cerdanyola del Vallès. Spain.


Proteomics is a rapidly emerging technology with a large range of applications, including veterinary sciences.

Surprisingly, there are still few applications of serum proteomics in the animal field. The bovine and porcine

serum 2-DE maps have been published and some applications to clarify physiopathological questions have been

described.

The identification of new biomarkers for diagnosis or for monitoring the general status of an individual is one of

the main potential applications for proteomics and already new potential markers have been identified in human

medicine. In this kind of analysis, serum is potentially the most valuable biological sample, because it contains

thousands of different proteins and peptides and it is the most easily accessible, noninvasive, and widely

collected sample. But, unfortunately, working with serum has a very important disadvantage: the protein content

of serum is dominated by a handful of high-abundance proteins, with their estimated concentration exceeding the

low-abundance proteins highly by 10 orders of magnitude. To detect these proteins present in low levels using

currently available technologies, it is advisable to remove the most abundant proteins. Pros and cons of all these

approaches should be taken into consideration when dealing with pretreatment of complex samples as serum.

Many strategies have been developed for the selective removal of albumin and other high-abundance proteins to

facilitate the analysis of other, less abundant proteins, in serum. Thus, albumin and other major proteins can be

removed by commercial or home-made reagents based in immunoaffinity approaches; IgGs may be removed by

using immobilized protein A or protein G and lectin affinity columns can be used to capture or enrich serum

glycoproteins. However, many of the most used methodologies for depletion of abundant proteins are based in

an immunological approach and, thus, they usually have a high specificity for human or laboratory animal

proteins. The removal of highly-abundant proteins raises another problem: it is well known that albumin and also

other proteins may act as transport proteins and thus are likely to bind many species of interest, such as peptide

hormones, cytokines, and chemokines. As such, the affinity-based depletion systems may also eliminate some

other proteins bound to those highly-copied proteins simultaneously, in a process of co-depletion. Thus, it has

been described that during human serum albumin depletion, another 815 species were co-depleted and that, when

capturing IgGs, another 2091 species were co-depleted.

Another different strategy is based, not in the depletion of high-abundant proteins, but in the enrichment of the

sample in low-abundance proteins. This technology is commercially available under the trade name of

ProteoMiner™ and it is based on the use of a combinatorial peptide binding library, which affinity-captures and

amplifies the low abundance proteome. This technology, since it is not based on an immunological approach,

should be species independent, and thus of potential application to samples of veterinary interest.

We have optimized the use of ProteoMiner™ for its application to porcine and bovine livestock, since these are

the most interesting species due to their economical interest.

Most probably, this sample preparation methodology may be also applied to other sample types and other animal

species. Veterinary applications will thus benefit from a simple method to enrich for low-abundance proteins.

We have analyzed its efficiency by comparing the results of using ProteoMiner™ with a commercial Albumin

and IgG Removal kit, which is based on the immunoaffinity depletion using high affinity antibodies for the

human proteins. This reagent was not useful for the depletion of these two main proteins, especially for bovine

130

samples due to low cross-reactivity of the antibodies to the animal proteins. ProteoMiner™ showed also a good

reproducibility in both species.

The other main question when looking for biomarkers in serum or other biological samples is to quantitatively

differentiate between the protein content in the diseased versus the healthy specimens. Although quantitation

may seem an obvious question that some may undervalue, it becomes a crucial point when comparing

unidentified spots. Several responses have been given to this question (quantitation by labeling, label-free

techniques, etc) and, more recently the SRM/MRM technology that has been applied to human medicine as a

very potent tool for biomarker identification. These techniques, though, are technically complex and, in some

cases, difficult to apply to biological fluids. Furthermore, all these methods are gel-free and based on some kind

of MS read-out, which is usually best done in the low molecular mass range of tryptic peptides. Consequently,

much information that is important to distinguish protein isoforms can be lost.

A very good alternative is to use differentially labeled samples and to separate them by bidimensional

electrophoresis together in the same gel. Labeling is performed with a fluorescent tag, that allows optical

detection of proteins with an excellent dynamic range and sensitivity (1 fmol of protein). On the other hand, gel

electrophoresis, in particular 2-DE, is well-suited to separate and analyze proteins and their isoforms. The

combination of these two technologies was applied for the first time in 1997 and named DIGE (difference gel

electrophoresis). One of the main aims for the development of DIGE was also to overcome the inherently poor

reproducibility of 2-DE, which makes accurate quantitative comparisons between proteins on different gels

difficult. In DIGE, differentially labeled samples are combined and co-separated on the same gel, thus being

subjected to the same handling procedures during 2-DE separation and raising the confidence of the results.

Furthermore, an internal control labeled with a third fluorescent reagent is often used, improving the accuracy of

relative quantitation of protein spots across gels and aiding in spot matching across gels. This technique is also

compatible with downstream identification of protein spots from gels by MS. For all these reasons, DIGE has

become popular for reproducible, large-scale expression profiling.j

We have applied both techniques (ProteoMiner™ for low-abundant protein enrichment and DIGE for differential

protein expression analysis) to the search of new biomarkers for stress and welfare in large animals.

In the study presented here, we used a proteomic approach to understand the mechanism of adaptation of cattle to the

environment and to identify new markers for stress or welfare in cows. Our rationale was to compare cows from the

same breed living in different conditions, in such a way that differences due to the genetic background could not

account for changes in the proteomic profile. The Bruna dels Pirineus breed is a beef type selected from the old

Brown Swiss and used for extensive beef cattle production. Two groups of cows were maintained during the winter

in different living conditions representing different degrees of challenging environments. One group was maintained

on a diet of cultivated pastures in a valley in the pre-Pyrinean Mountains with daily contact to humans (control/good

conditions), whereas the other group was living in the Alberes massif (Eastern Pyrenees) in semiferal conditions

(stress/hard conditions). For biological validation of the results, we included a third group of cows from the Alberes

breed. The Alberes is a native Catalonian cattle breed living in semiferal conditions in the Alberes massif, far from

human contact and mainly browsing shrubs and branches, thus providing a living situation that may be considered

the most challenging one. In this way, the three groups of cattle represent three different management systems that

keep the animals under several degrees of nutritional and environmental challenges.

Serum from Bruna cows living in both environments was subjected to Proteominer enrichment of low-abundance

proteins, followed by DIGE labelling, two-dimensional electrophoresis and MALDI-MS or ion trap MS. A positive

identification was achieved for 15 proteins, which mainly belonged to three biological functions, the oxidative stress

pathway (glutathione peroxidase (GPx) and paraoxonase (PON-1)), the acute phase protein family (Heremans

Schmid glycoprotein alpha2 (α2-HSG)) and the complement system.

Biological validation included the Alberes breed. GPx and PON-1 were validated by an enzymatic assay and found

to be higher and lower, respectively, in cows living in hard conditions. α2-HSG was validated by ELISA and found

to be reduced in hard conditions. Other biomarkers of the redox status were also altered by living conditions: protein

carbonyl content, superoxide dismutase (SOD) and glutathione reductase (GR).

Our results show that changes in the redox system are the main adaptation of cows living in challenging

environmental conditions.

In conclusion, pretreatment of samples, especially in the case of serum, is an essential requirement for obtaining

useful results. Choosing the right procedure should follow a critical design of the objectives of the work, and the

pros and cons of each approach. Furthermore, DIGE is a powerful technique with good reproducibility and

quantitative accuracy and that provides information on isoform-specific expression. The study presented here for

131

Bruna cows is an example of how proteomic techniques can be applied to veterinary or animal production

problems.

References:

1) Boschetti E, Righetti PG. The ProteoMiner in the proteomic arena: a nondepleting tool for discovering low-

abundance species. J Prot. 2008;71:255– 264.

2) Marco-Ramell A, Bassols A. Enrichment of low-abundance proteins from bovine and porcine serum samples

for proteomic studies. Res Vet Sci. 2010;89:340–343.

3) Marco-Ramell A, Arroyo L, Saco Y, García-Heredia A, Camps J, Fina M, Piedrafita J, Bassols A. Proteomic

analysis reveals oxidative stress response as the main adaptative physiological mechanism in cows under different

production systems, J Prot. 2012; doi:10.1016/j.jprot.2012.04.002.

4) Timms JF, Cramer R. Difference gel electrophoresis. Proteomics 2008;8:4886–4897.

132


ORAL PRESENTATIONS

EFFECT OF TIME RELATED TO CALVING AND RUMEN PROTECTED CHOLINE (RPC)

SUPPLEMENTATION ON BLOOD ANALYTES AND ON LIVER LIPID AND GLYCOGEN.

T. Gaal1, P. Elek

2, F. Husveth

3.

1School of Veterinary and Biomedical Sciences, Faculty of Health Sciences,

Murdoch University, Perth, Australia; 2Provimi Hungary Co. Zichyujfalu, Hungary;

3Department of Animal

Science, Georgikon Faculty of Agriculture, University of Pannonia, Keszthely, Hungary.


Background: High producing dairy cows have temporary changes in blood analytes and may develop fatty liver

after calving. The latter might be prevented by supplementation of the diet with RPC. Objective: The aim of this

study was to compare some blood and liver parameters in both control and RPC-supplemented transition cows to

their pre-calving values and text-book reference ranges. Methods: Blood and liver biopsy samples were obtained

from 16 Holstein Friesian cows supplemented with RPC and from 16 control animals 21 days before and 7, 21,

35, and 60 days after calving. Blood glucose, triglycerides, cholesterol, non esterified fatty acids, beta-hydroxy-

butyrate, urea, ammonia, and AST with liver total lipids, triglycerides and glycogen were measured. Effects of

time-to-calving interval and RPC supplementation on all analytes were statistically evaluated. Results:

Significant time-related changes in all blood parameters were found in both groups after calving with the highest

increase in blood NEFA, BHB, AST and ammonia (8x, 3.5x, 2x, and 1.7x, respectively) on d7/d21 when

compared to pre-calving values. RPC cows had relatively lower NEFA, BHB, and ammonia with higher TG post

partum than controls. Liver TL and TG increased and liver glycogen decreased in both groups after calving, with

milder fatty infiltration of the liver in RPC-cows. Liver glycogen was not influenced by RPC supplementation.

Conclusion: Due to significant changes post partum, text book reference ranges for adult cows can only be used

with care in transition cows. Choline supplementation of the diet may also influence these results.

ASSOCIATION BETWEEN HEMATOLOGICAL AND ELECTROPHORETIC PROFILES AND

PREVALENCE OF POST-PARTUM DISEASES IN DAIRY COWS.

S. Paltrinieri1, P. Moretti

2, M. Venturini

2, N. Morandi

3, A. Giordano

4. 1

University of Milan, Milan, Italy; 2Central laboratory, Large Animal Hospital, Milan, Italy;

3Parco Tecnologico Padano, Lodi, Italy;

4Department

of Veterinary Pathology, Hygiene and Public Health, Milan, Italy.


Background: Opportunistic bacteria can induce post-partum diseases (e.g. metritis, mastitis, digital dermatitis,

retained placenta, etc.) in dairy cows. Metabolic disturbances can predispose to these diseases and the

association between the prevalence of disease and changes in chemical metabolites is well known. Conversely,

little is known about the association between the prevalence of disease and haematological or electrophoretic

profiles. Objective: To investigate the possible presence of haematological and electrophoretic profiles

associated with subclinical inflammation potentially predisposing to post-partum diseases. Methods: The health

status of 3 dairy herds was monitored over a 3 month period. Routine hematology and serum protein

electrophoresis were performed on samples collected 3 days after parturition from 110 animals (herd 1 = 40;

herd 2 = 36; herd 3 = 34). Clinical events occurring in the transition period were recorded. Haematological and

electrophoretic results recorded in the 3 herds from animals that remained clinically healthy were statistically

compared to each other. Results: The prevalence of post-partum diseases in herd 2 (22.2%) was significantly

lower (P<0.05) than that recorded in herd 1 (42.5%) and in herd 3 (47.1%). No significant differences between

leukograms and electrophoretograms recorded in herd 2 compared with herds 1 and 3 were found. Conversely,

RBC, Hb and Ht were significantly higher in herd 2 compared with other herds. Conclusions: The prevalence of

post-partum disease is not associated with leukogram abnormalities or protein profiles potentially predisposing

to infections. Conversely, the different erythrograms suggest that lower prevalence of disease is associated with a

better metabolic profile.


133

HEMATOLOGICAL CHANGES ASSOCIATED WITH RETAINED PLACENTA IN CATTLE.

P. Moretti1, A. Giordano

2, M. Venturini

3, N. Morandi

4, S. Paltrinieri

2.

1Large Animal Hospital, Lodi, Italy;

2Department of Veterinary Pathology, University of Milan, Milan, Italy;

3Central laboratory, Large Animal

Hospital, Lodi, Italy; 4Parco Tecnologico Padano, Lodi, Italy


Background: Retained placenta (RP) occurs frequently in cattle but little is known about the pathogenic or

prognostic role of hematological changes in this disease. Objective: To describe hematological changes

associated with RP and to assess their possible prognostic relevance. Methods: Blood samples were collected 2

to 4 days after calving in cattle from two different herds (A: n=45; B: n=45). Seventeen cows were affected by

RP in herd A and 27 cows in herd B; Results from RP-affected cows were compared with those of clinically

healthy controls (28 from herd A; 18 from herd B). The follow up of RP-affected cows was monitored during the

whole lactation period and they were then subgrouped based on the presence (8 in herd A; 8 in herd B) or

absence (9 in herd A; 19 in herd B) of further health problems. Results: In both herds, RP-affected cows had

lower neutrophil counts (associated with lower total leukocyte counts in herd B) and higher RBC, Ht and Hb

values.Cattle showing complications after pregnancy (metritis, mastitis, digital dermatitis) had significantly

lower RBC, leukocyte, and lymphocyte counts compared with cattle that did not have complications.

Conclusions: Leukopenia and neutropenia is associated with RP; Leukopenia and lymphopenia in the early

postpartum period could predispose to further complications during transition and lactation. The possible

association between leukopenia and RP will be further investigated within the ProZoo project, focused on the

identification of the genetic basis of resistance/susceptibility to diseases.

SURVEY OF VITAMIN A, BETA-CAROTENE, CALCIUM AND PHOSPHORUS CONCENTRATIONS IN

SERUM OF EWES WITH RETAINED PLACENTA.

A. Davasaztabrizi. Department of Clinical Sciences, Tabriz Branch, Islamic Azad University, Tabriz, Iran.


Background: Retained placenta (RP) may result from a number of factors, such as abortion, induced labor,

delayed gestation, early parturition, uterine atony, infections, and seasonal and hormonal disorders. In addition, it

is well known that deficiencies of some vitamins and minerals induce or predispose animals to RP. Objective:

The objective of the present study was to investigate the relationship between blood serum concentrations of

vitamin A, Beta- Carotene, Calcium and Phosphorus and the development of RP in ewes. Method: 25 ewes were

retained placenta; they have not excreted their placenta after parturition. 20 others have excreted their placenta

normally after parturition. Blood samples were obtained from all understudying animals from jugular vein by

venoject. Samples were centrifuged and sera were prepared and amounts of vitamin A, Beta- Carotene, Calcium

and Phosphorus were measured. Results: Data showed that there is no significant difference among vitamin A

and β-carotene between two groups (P>0.05). While, revealed that there is a significant difference in mean value

of Calcium and Posporous among two groups. Conclusion: This research indicated that imbalance of calcium

and phosphorous and their deficiency can induced retained placenta in ewes in Tabriz region of Iran.


1. THE EFFECT OF PARITY ON BONE FORMATION DURING LATE PREGNANCY AND EARLY

LACTATION IN SAANEN GOATS

M. Belić1, V. Kušec

2, M. Robić

1, J. Grizelj

1, A. Svetina

1, Z. Vrbanac

1, R. Turk

1.

1Veterinary Faculty, Zagreb,

Croatia; 2Clinical Institute of Laboratory Diagnosis, Clinical Hospital Centre, Zagreb, Croatia.


Background: Bone alkaline phosphatase (BALP) activity changes in different physiological conditions of the

organism, so in pregnancy and lactation it can have a significant role in evaluation of the rate of bone turnover.

There are gaps in our knowledge about changes in bone metabolism during pregnancy and lactation in small

ruminants. Objective: The purpose of this research was to assess the effect of parity on bone formation during

pregnancy and lactation in goats. Methods: The research was conducted on 35 goats Saanen breed which were

divided into 3 groups regarding the number of previous deliveries (primiparous, biparous and multiparous goats).

Blood samples were obtained 15 days before and 15 days after delivery. The activity of BALP was measured

with a commercial enzyme immunoassay kit. Differences between study groups were tested by Student’s t-test.

Results: In primiparous goats BALP activity was significantly lower (p<0.001) 15 days after delivery compared

to the activity measured 15 days before delivery. In biparous and multiparous goats, BALP activity was higher


134

post delivery than in late pregnancy but not significantly. There was statistically significant (p<0.001) negative

correlation between BALP and age in biparous and multiparous goats. Conclusions: Results demonstrated an

active bone formation in late pregnancy and early lactation in goats indicating an increased bone turnover during

transition period.

2. AGREEMENT BETWEEN ELECTROCHEMILUMINESCENCE AND CHEMILUMINESCENCE

IMMUNOASSAY METHODS FOR DETERMINATION OF SERUM FREE TETRAIODOTHYRONINE

VALUES IN HOLSTEIN COWS.

B. Eshratkhah1, A.R. Mirzaei

2, M. Mostafaei Bigham

2.

1Islamic Azad University, Shabestar branch, Tabriz,

Iran; 2Department of Veterinary Clinical Pathology, Faculty of Veterinary Medicine, Sha, Shabestar, Iran


Background: The radioimmunoassay (RIA) method is the most common assay for determination of free-

tetraiodothyronine (fT4) concentration in ruminants. However, RIA has some disadvantages compared to the new

determination immunoassay methods such as Chemiluminescence (CLIA) and Electrochemiluminescence

(ECLIA). The CLIA and ECLIA methods have been validated for human samples only. Objective: The aim of

this study was to determine serum fT4 values and its correlation between the CLIA and ECLIA methods in

Holstein cows. Methods: Blood samples were collected from the jugular vein of 45 clinically healthy and non-

pregnant animals, aged from 2 to 4 years in August. fT4 concentration was measured in separated sera using

both CLIA and ECLIA methods. Results: For determination of fT4, the intraassay coefficient of variation (CV

%) of CLIA and ECLIA were 6.5% and 7.6% respectively.The interassay CV % were 9.6 % and 8.9%,

respectively. Our results indicate that there was no significant difference between the two methods though the

mean was higher using the CLIA method. The regression analysis revealed a significant correlation between two

methods (Y = 2.151 + 0.944 X, r2 = 0.594; r = 0.771, p < 0.001). Conclusion: The CLIA and ECLIA can be

used as an alternative to the RIA method for measurement of the fT4 concentration in veterinary diagnostic

laboratories and exhibited acceptable of sensivity and precision for the serum fT4 determination in Holstein

cows.

3. SUBCLINICAL MASTITIS IN SHEEP: NOVEL DIAGNOSTIC FINDINGS.

A. Miglio1, M.T. Antognoni

1, L. Moscati

2, C. Maresca

2, A. Valiani

2, E. Scoccia

2, V. Mangili

1, G. Fruganti

1.

1University Of Perugia, Faculty Of Veterinary Medicine, Perugia, Italy;

2Istituto Zooprofilattico Sperimentale

Dell'Umbria E Delle Marche, Perugia, Perugia, Italy.


Background: Subclinical mastitis (SM) negatively affects yield and milk quality. The udder of sheep with SM is

clinically healthy, but milk presents an increased Somatic Cell Count (SCC) (>500.000cells/ml) and mastitis

bacteria. Objective: To compare conventional methods of diagnosis of SM (physical examination of udder and

cyto-bacteriological analysis of milk) with markers of inflammation measured in blood, serum and milk.

Furthermore, we investigated changes of some laboratory parameters in sheeps affected by SM. Methods:

Udder-half milk-samples and blood-samples were collected from 132 clinically healthy lactating Lacaune ewes.

We performed SCC, bacteriological analysis, neutrophil-granulocyte count (MNG) and concentration of Milk-

Amyloid-A (MAA) in milk as well as blood leukocyte count (WBC) and concentrations of blood-serum total

proteins and electrophoretic protein fractions (PT), Iron (Ir), Zinc (Zn), Urea (Ur), Lysozyme (Lys), Haptoglobin

(Hp) and Serum-Amyloid-A (SAA). The data were submitted to retrospective epidemiological study and

descriptive and univariate analysis. Results: MNG and MAA were positively correlated with SCC and also

significantly higher (P≤0.05) in mastitic samples (MNG: mean±SD=4.236.505±764/ml and 95%CI=1.079.448-

7393.562/ml; MAA: 114,37±41,14µg/ml and 98,24-130,50µg/ml) compared with healthy samples (MNG:

8.070±434/ml and 6.358-9.783/ml; MAA: 29,68±27,98µg/ml and 23,84-35,52µg/ml). Blood parameters were

not correlated with SM and SCC>500.000cells/ml. Conclusions: This report, the first to evaluate MAA in ewe

milk with a milk ELISA-kit, suggests MNG and MAA as potential markers of SM in sheep. Future studies,

including more sheep breeds, are needed to confirm the reference limits detected. WBC, PT, Ir, Zn, Ur, Lys, Hp

and SAA are not useful to identify the SM.

135

4. OVINE MALIGNANT THEILERIOSIS: THE STATUS OF ANTIOXIDANT VITAMINS, SERUM LIPID

PROFILE, LIPID PEROXIDATION AND ERYTHROCYTE ANTIOXIDANT DEFENSE.

R. Sadoughifar1, S. Nazifi

2, Razavi

2. 1

Iran, Shiraz university, Veterinary Medicine School, Isfahan, Iran; 2Shiraz university, Shiraz, Iran.


Objective: In order to investigate more aspects of the erythrocytic antioxidant defense and the lipid content of

the serum during ovine malignant theileriosis, 50 1-2 year old Iranian fat-tailed sheep that were naturally

infected with Theileria lestoquardi were selected and divided into three subgroups according to their parasitemia

rates (< 1%, 1–3%, 3–5%).Also, 10 non-infected sheep served as controls. Blood samples were collected, and

hematological parameters, the activities of antioxidant enzymes including superoxide dismutase (SOD),

gluthatione peroxidase (GPX) and catalase, osmotic fragility of the RBCs, the level of malondialdehyde (MDA)

as an index of lipid peroxidation, phosphatidylserine, antioxidant vitamins (A, E and C) and the concentrations

of serum lipid composition (cholesterol, triglyceride and lipoproteins including HDL, LDL and VLDL

cholesterol) were measured. A significant decline in the activities of antioxidant enzymes and also remarkable

increases in the concentration of MDA, osmotic fragility and phosphatidylserine were evident in infected sheep,

along with significant correlations with the hematological parameters. The results showed that the antioxidant

agents could be important factors in the prevention of erythrocytic membrane damage during parasitemia. In

addition, the unaltered level of antioxidant vitamins in the infected animals showed that such vitamins did not

elicit effective responses to the oxidative damages. The level of the major lipid components of the serum

remained unchanged during infection, which implied that the infection with T. lestoquardi did not induce

important changes in lipid metabolism, and these components are unlikely to have a major role in the process of

anemia.

5. MEASURING GLUCOSE CONCENTRATION IN BLOOD OF COWS WITH THE OPTIUM XCEED TM

METER AND THE BIOCHEMICAL ANALYSER – COMPARISON OF METHODS.

J. Ježek, J. Starič, M. Klinkon, M. Nemec. University of Ljubljana, Veterinary faculty, Clinic for ruminants,

Ljubljana, Slovenia


Background: Measuring blood glucose concentration under field conditions is useful in farm animal practice.

Objective: The aim of our research was to compare two different methods for measuring blood glucose

concentration, a reference biochemical analyser and an on-site method for measuring glucose concentration.

Methods: The measurements were performed on 29 bovid blood samples. An appropriate blood volume for

antiocoagulant was drawn into sodium fluoride and potassium oxalate evacuated tubes. The glucose

concentration was measured with the Optium Xceed TM

meter in whole blood, directly on-site. The results were

compared with the measurements preformed by the biochemical analyser, Cobas Mira. The data were processed

with SPSS (Ver. 15). The correlation coefficient by Pearson was calculated and for assessment of agreement the

method by Bland and Altman (1986) was used. Results: The mean concentration of glucose established with

Optium Xceed TM

was (3.61 ± 1.47 mmol/L). It was slightly lower than the mean concentration established by

the Cobas Mira biochemical analyser (3.62 ± 1.17 mmol/L). The correlation coefficient was 0.984. The

calculation of the agreement confirmed that, on average, Optium Xceed TM

gave for 0.02 mmol/L (SD = 0.41

mmol/L) lower values than the biochemical analyser. Conclusion: Based on our results, we think that this hand-

held meter is appropriate for measuring glucose concentration in the field conditions.

6. CHEEK TOOTH ABNORMALITIES IN SHEEP FLOCKS.

V. Erjavec. Veterinary faculty, Ljubljana, Slovenia


Background: Dental problems are one of the main causes of premature culling of sheep, i.e. long before they

have reached the end of their natural reproductive life. Objective: To determine the range of dental pathology

occurring in both clinically healthy sheep and those showing loss of condition within a flock of sheep.

Specimens submitted for investigation of unexplained death at a pathology centre were also examined. The

literature tends to concentrate on a syndrome, often referred to as 'broken mouth', affecting the incisor teeth.

However, the posterior or cheek teeth are rarely considered and their disease, wear and function during

mastication is poorly understood. Methods: The farm records were studied to determine losses from known or

suspected dental disease. Post-mortem examinations were performed when feasible on slaughtered animals and

any that died naturally to try to determine whether dental disease was a factor. When lesions were found, the


136

skulls were prepared to show dental abnormalities. Results: Over the 5 years of the study, 34% of the flock were

found to have visible incisor abnormalities (gingival recession, excessive mobility and tooth loss). 10% of the

flock showed non-specific signs such as weight loss, breeding failure or unexpected death. All animals of this

latter group were confirmed to have cheek tooth disease in addition to their incisor abnormalities. Advanced

cheek tooth disease was found in 84% of the sheep submitted to the pathology laboratory whilst only 28% had

advanced incisor disease. Conclusion: The results show that dental disease is a serious problem for sheep flocks.

7. GLUTAVAC® TEST SET (GLUTARALDEHYDE BLOOD TEST) – A DIAGNOSTIC SUPPORT FOR

FOREGIN BODY DISEASE.

T. Zadnik1, S. Rakulič Zelov

2.

1Veterinary faculty, Ljubljana, Slovenia;

2Veterinary station, Žužemberk,

Slovenia


Background: Traumatic peritonitis and reticulopericarditis are consequences of perforated reticula with sharp

objects. The glutaraldehyde test is used on whole blood for the diagnosis of acute and chronic inflammation and

infection. Objective: We have used the Glutavac ® test as a diagnostic support in cows which showed symptoms

of rumen stasis. Methods: 70 cows with acute rumen stasis were analyzed using ready-to-use vacutainers with

the Glutavac® test. Venipuncture was perfromed at the vena coccygica. The vacutainer was inverted3-times and

then left for 15 minutes. If the blood coagulates in 5 minutes after it has been mixed with reagents, the result is

highly positive (+++). If the coagulation is finished in 10 minutes, the result is positive (++). If the coagulation is

finished in 15 minutes, the test is poorly positive (+). In all cows which showed positive test reaction,

exploratory laparorumenotomy was performed. Results: Of Glutavac® assayed blood of 70 cows with acute

rumen stasis, 54 (77.2 %) bovid samples had highly positive reactions (49 samples were associated with

traumatic inflammation and 5 with liver fluke infestation. 9 (12.8 %) cows showed positive (++) reaction (7

cows had reticuloperitonitis traumatica and 2 had liver fluke infestation). 7 (10.0 %) cows showed poorly

positive (+) results (1 cow had traumatic indigestion and 6 had liver fluke infestation). Conclusion: Based on

results, we recommend the use of Glutavac® test as a simple valuable paraclinical confirmative diagnostic

method to be used in field conditions.

8. MONITORING SUBCLINICAL KETOSIS IN TRANSITION COWS PREGNANT WITH A SINGLE

CALF OR TWINS.

T. Zadnik1, R. Lombar

2. 1Veterinary faculty, Ljubljana, Slovenia;

2Veterinary station, Tenetiše, Slovenia


Background: The 7-day close-up period and 15-day fresh period is a critical transition for optimal health in

dairy cow. The gold standard test for monitoring subclinical ketosis is serum beta-hydroxy butyrate (BHB).

Clinical ketosis is typically defined as 2.6 mmol/L BHB or more. At concentrations above 1.40 mmol/L BHB,

cows are at risk for metabolic disorders. Objective: The aim of this study was to compare BHB concentrations in

the whole blood of cows in the early pre- and early post-calving period and pregnant with a single calf or twins.

Methods: The blood BHB concentration was measured with the Optimum XceedTM

meter. The results were

compared within groups of transition cows (n = 80) which were pregnant with a single calf (group 1) or with

twins (group 2) (n = 42). All data were statistically analyzed with SPSS 17.0 (one-way ANOVA). Results:

Statistical analyses showed that mean (1.12 ± 0.93 mmol/L) BHB concentration in group 1 (single calf) and

mean (1.19 ± 1.23 mmol/L) BHB values in group 2 (twins) were not statistically significantly different (P>0.05).

However, cows with twins were 3-times more prone to fetal membrane retention, metritis, mastitis, and

abomasal dislocation. Conclusion: Based on the results, we think this BHB method is appropriate for the

detection of ketosis in field conditions. The advantage of the method is that it is simple and quick. This test is a

precise tool to monitor subclinical ketosis and is useful for periodic assessment of subclinical ketosis in a herd.

137

9. LOGISTIC REGRESSION MODELING FOR THE PREDICTION OF LEFT DISPLACED ABOMASUM

IN DAIRY COWS.

S. Safi1, N. Basiri

1, A. Rahimi foroushani

2.

1Faculty of Veterinary Medicine, Science and Research Branch,

Islamic Azad University, Tehran, Iran; 2Department Of Epidemiology and Biostatistics, Faculty of Public

Health, Tehran U, Tehran, Iran


Left displaced abomasum (LDA) is one of the major metabolic diseases in dairy cattle, which causes alarge

economic loss in dairy industry. Thus, predicting the occurrence of LDA, especially in early weeks post-partum

is very important. In the present study, 14 biochemical analytes of cows with LDA before and after parturition

were compared to those of healthy cows (the control group) using the logistic regression statistical model.

Changes in six analytes, including beta-hydroxybutyrate (BHB), non-esterified fatty acids (NEFA), aspartate

aminotransferase (AST), calcium, sodium and potassium, were found to have statistically significant differences

(p<0.05) between groups and were included in the prediction model. The model was proposed as: Log (odds) =

12.778 + 0.053 (NEFA) - 0.031(Na) - 0.550 (Ca) - 0.721(K) + 0.988 (BHB) + 0.008 (AST) = X; Odds = exp (X)



10. CANINE CUTANEOUS HISTIOCYTOMA: A RETROSPECTIVE STUDY OF 55 CASES (2002-2011).

C. Pressanti, F. Granat, A. Geffre, M.C. Cadiergues. Toulouse Veterinary School, Toulouse, France


Background: Canine cutaneous histiocytoma (CCH) is a common benign neoplasm, usually found in young

dogs and presenting as a solitary lesion that undergoes spontaneous remission. Objective: The purpose of this

study was to compare features of the disease in a specific population to the few data available in the literature.

Methods: The medical records of dogs diagnosed with CCH in Toulouse Veterinary School between 2002 and

2011 were reviewed. Collected information included breed, age, sex, location and size of the tumor, clinical and

cytological examination, and progression. Diagnosis was made at minimum by cytological examination. The

data obtained were compared to a reference baseline population (Ƙhi-2 test, p 0.05). Results: Fifty-five dogs

were included. Boxers, Bulldogs and crossbred dogs were significantly overrepresented compared with the

reference population. Mean age was 2.4 years. The average size was 1.1 cm. Preferential distribution of the

tumor was extremities (19/55), head (17/55), trunk (14/55), and neck (3/55). Cytological characteristics were

quite homogeneous: clumps of round cells with a variable amount of pale, often vacuolated basophilic

cytoplasm; round nuclei, occasionally indented, usually eccentric with finely granular chromatin. Twenty tumors

regressed spontaneously and 12 were surgically excised. Discussions: CCH affects all breeds with

overrepresentation of Boxers and Bulldogs. It affects most commonly dogs less than 3 years old although in this

study 5 dogs were over 6. The extremities, the face and particularly the pinnae and eyelids were most often

affected. All cases were benign neoplasms. Neither recurrent/persistent CCH nor the severe langherhans cell

histiocytosis was reported.

11. CANINE NEOPLASTIC EFFUSION: MORPHOMETRIC ANALISYS OF CYTOLOGICAL SAMPLES.

G. Ghisleni, V. Pizzatti Sertorelli, A. Forlani, V. Bronzo, M. Caniatti. DIPAV University of Milan, Milan,

Italy


Background: Cytology is the best way to detect the presence of malignancy in body cavity fluids. The

differential diagnosis beetween mesothelioma and carcinomas is difficult and may require additional diagnostic

techniques, such as immunocytochemistry. Objective: The purpose of this study was to evaluate the morphology

of neoplastic cells in body cavity fluids using image analysis software, to identify their mesothelial or epithelial

origins. Materials and methods: Ten canine neoplastic effusion (5 carcinomas and 5 mesotheliomas), were

included in this study. Immunocytochemistry with a panel of 6 antibodies (CKAE1/AE3, CK20, vimentin,

desmin, HBME1, CK5/6) was used to support the cytologic diagnosis of mesothelioma vs. carcinoma. For image

analysis, 50 neoplastic cell and 10 clusters of cells were studied on May Grünwald-Giemsa stained smears. Both

cellular and nuclear area, diameter (maximum, mean, minimum), perimeter and roundness of neoplastic cells

were calculated. Area, diameter (maximum, mean, minimum), perimeter and roundness of larger clusters of cells

138

were also measured. Statistical analysis of data was made. Results: On morphometry, values for mean area of

cellular clusters in carcinomas were found to be 66.234,68 µm2, while for mesothelioma were 21.813,93 µm

2, (P

< 0.006, highly significant). Significant differences were also noted among carcinomas and mesotheliomas for

all other measurements, except roundness. Conclusion: These results suggest that automated cytodiagnosis may

aid in carcinoma and mesothelioma diagnosis. Further study evaluating stained archived cytology samples

(retrospective studies) and development of systems of automated cytodiagnosis are warranted.

12. EFFECT OF NEEDLE GAUGE ON THE QUALITY OF CANINE SPLEEN FINE NEEDLE

ASPIRATION SPECIMENS – A PRELIMINARY STUDY.

N. Bourgès-Abella, C. Layssol, A. Geffré, C. Trumel. Institut National Polytechnique-Ecole Nationale

Vétérinaire de Toulouse, TOULOUSE, France


Background: Fine-needle aspiration biopsy (FNAB) of the spleen is a valuable tool to assess hemolymphatic

disorders, infectious disease and tumoral infiltration. Needle diameters used for spleen FNAB are variable in

literature and have never been compared nor has animal pain been assessed. Objectives: The aim of this study

was to compare the pain and quality of spleen FNAB specimens obtained with three different needle diameters:

23, 25 and 27gauges on dogs with spleen abnormalities for which clinicians had prescribed FNAB. Methods: 21

dogs were included in the study and spleen FNAB was performed according to the procedure followed routinely

with the three needles in randomized order for each dog. The 63 slides were independently, randomly and

blindly studied by two clinical pathologists (CT and AG) and graded with a subjective scoring system. Animal

pain was subjectively evaluated by the operator who performed spleen FNAB. Results: Agreement between the

two observers was good. Samples obtained with 27G needle method had lower cellularity (p = 0.046), poorer

stroma (p = 0.02) and less mesothelial cells (p = 0.001). No difference was found between the three needle

gauges for the 17 other criteria nor for animal pain. Conclusion: These preliminary results suggest that 27G

needle may be less valuable than 23 and 25G needle for spleen FNAB.

13. DETERMINATION OF PROLIFERATIVE ACTIVITY IN CANINE LEUKEMIAS BY FLOW

CYTOMETRY.

A. Poggi1, B. Miniscalco

1, L. Aresu

2, M.E. Gelain

2, S. Comazzi

3, V. Martini

3, E. Morello

1, F. Gattino

1, F.

Riondato1.1Dep. of AnimalPathology, University of Torino;

2Dep. of Public Health, Comparative Pathology and

Vet. Hygiene, University Of Padua; 3Dep. of Veterinary Pathology, Hygiene and Health, Unit of General

Pathology, University of Milan; Italy.


Background: Nuclear antigen ki67 is widely used as prognostic indicator in many human tumors, while in

veterinary medicine only few studies on solid tumours exist and no dataconcerningleukemias are

present.Objective: To evaluate Ki67 positive cells in peripheral blood samples fromleukemic dogs by flow

cytometry. Methods: 21 canine leukemias were evaluated. Based on clinical aspects, CBC, immunophenotype

and cytologic features, leukemias were divided in acute lymphoblastic (ALL, n=2), acute myeloblastic (AML,

n=4), acute undifferentiated (AUL, n=3), chronic lymphocytic (CLL, n=8) and CLL/lymphoma complex (n=3).

EDTA blood samples were labeled with Ki67-FITC (clone MIB-1) with a rapid fixation/permeabilizationmethod

using methanol.Proliferative activity was expressed as percentage of Ki67 positive cells. Results: Percentage of

Ki67 positive cellswas significantly higher in acute respect to chronic leukemias (P<0.01).In particular

decreasing values were recorded in AML, ALL, AUL and CLL with statistically significant differenceAML and

CLL (P<0.05).B-CLL presented higher proliferative activity compared to T-CLL (13% and 4,8%, respectively),

although without statistic significance.No differences between aberrant and not aberrant immunophenotypes

were detected among CLL. Conclusion: The higher proliferative activity recorded in acute canine

leukemiascompared to chronic leukemiasconfirms the role of Ki67 antigen as areliable indicator of biological

behavior.A larger series of samples is neededto check for differences between immunophenotypesamong AML,

ALLand CLL.Follow upinformationswill allow to detect if ki67 could be used to differentiate prognostic

evaluation.

139

14. PROGNOSTIC VALUE OF KI67 IN CANINE LYMPHOMA DETERMINED BY FLOW CYTOMETRY.

A. Poggi1, B. Miniscalco

1, L. Aresu

2, M.E. Gelain

2, S. Comazzi

3, V. Martini

3, E. Morello

1, F. Gattino

1, F.

Riondato1.

1Dep. of Animal Pathology, University of Turin;

2Dep. of Public Health, Comparative Pathology and

Vet. Hygiene, University Of Padua; 3Dep. of Veterinary Pathology, Hygiene and Health, Unit of General

Pathology, University of Milan; Italy.


Background: Percentage of Ki67 positive cells (%Ki67) is related to malignancy and survival in human non

Hodgkin’s lymphoma. Few data from immunohistochemical studies are available for canine lymphoma (LSA).

Objective: To investigate the prognostic significance of flow cytometric %Ki67 in canine LSA. Methods: 47

canine LSAs were classified by flow cytometry and cytology, according to updated Kiehl classification, in: 33

high grade (HG) and 1 low grade (LG) B-LSAs; 10 HG and 3 LG T-LSAs. Lymph node aspirates were labeled

with Ki67-FITC (clone MIB1) using a methanol-based method. Differences in %Ki67 were assessed by t-test.

Kaplan-Meier curves were used to evaluate prognostic significance of %Ki67 for complete remission and

survival in 16 dogs treated with Madison-Wisconsin protocol. Results: %Ki67 was significantly higher in HG

compared to LG LSAs (p<0.001). No significant difference between B- and T-LSA was detected. Treated LSAs

with Ki67>40% showed a significantly faster complete remission (CR), a significantly lower survival at the end

of chemotherapy (day 175) and overall survival compared to LSAs with Ki67<40%, regardless of grade of

malignancy and lineage. Ki67>40% was also associated to a faster CR among HG LSAs, B-LSAs and HG B-

LSAs and to a lower overall survival among HG LSAs. Conclusion: Flow cytometric %Ki67 is useful in

differentiating HG vs LG LSA using a cutoff value of 12%. Regardless of cell lineage, %Ki67 can also be

considered as a prognostic marker for time of remission and survival using a cutoff value of 40%.

NOVEL BIOMARKERS

ORAL PRESENTATIONS

URINE PROTEOME IN DOGS AFFECTED BY LEISHMANIASIS.

A. Buono1, F. Dondi

1, E. Ferlizza

1, G. Isani

1, J. Duque

2, P. Tapia

2, C. Zaragoza

2, R. Barrera

3.

1University of

Bologna, Ozzano Emilia, Italy; 2University of Extremadura, Caceres, Spain;

3Department of Internal Medicine,

University of Extremadura, Caceres, Spain


Background: Leishmaniasis is a frequent cause of nephropathy in dogs. Evaluation of the urine proteome is a

useful and non invasive method to monitor the therapy and discover novel biomarkers. Objective: Compare

different electrophoretic methods for urine protein separation and monitor therapy in dogs affected by

leishmaniasis. Methods: Urine samples were collected from 43 leishmaniotic dogs. UP/C, UA/C and high

resolution electrophoresis (HRE) were performed for all samples. Moreover, SDS-PAGE (26 samples) and a

semi-automated SDS-AGE (12 samples) were tested. Results: UP/C and UA/C median (range) values were 1.45

(0.06-20.08) and 0.38 (0.01-14.8) respectively. With HRE different electrophoretic patterns were evidenced and

urinary albumin concentrations (median 32 mg/dL; range 5-463 mg/dL) and percentage (mean±SD; 39±13.7)

were determined. With SDS-AGE different patterns of proteinuria were found: 9 dogs presented mixed

proteinuria (complete or incomplete) and 3 presented with non selective glomerular proteinuria. Upon SDS-

PAGE, 35-40 different bands were commonly visualized.The most abundant proteins were at molecular weights

of 71, 66, 55, 46, 37, 26, 22, 14 kDa. Conclusion: HRE is a useful and rapid method to monitor renal function

and therapeutic effectiveness. In addition, urinary albumin concentration can also be quantified. SDS-AGE,

according to literature, allows the localization of nephronal damage. SDS-PAGE, due to its high sensitivity,

visualizes many urine proteins, which could be studied as novel biomarkers of nephropathy.

140

EVALUATION OF THE REVISED QUANTITATIVE INSULIN SENSITIVITY CHECK INDEX IN DAIRY

COWS SHOWING VARIOUS FORMS OF KETONE PATTERN AND PUERPEAL METRITIS

M. Kerestes1, V. Faigl

1, M. Kulcsár

1, O. Balogh

1, J. Földi

2, H. Fébel

3, Y. Chilliard

4, G. Huszenicza

1.

1Faculty of Veterinary Science, Budapest, Hungary;

2Intervet, Budapest, Hungary;

3Research Institute for

Animal Breeding and Nutrition, Herceghalom, Hungary; 4INRA, Saint-Genès-Champanelle, France.


Background: In dairy cows, insulin resistance may play a pivotal role in the pathogenesis of metabolic diseases.

The revised quantitative insulin sensitivity check index (RQUICKI ) was evaluated previously in healthy cows.

There is no data on changes in RQUICKI, in periparturient or hyperketonemic cows. Objective: The aim of the

study was to evaluate the RQUICKI in hyperketonemic cows without or with puerperal metritis. Methods:

Blood samples were collected from 28 Holstein cows from -18 d before, through 70 d after calving for ß-

hydroxy butyrate (BHB), non-estherified fatty acids, glucose, and insulin. Animals were subjected to intravenous

glucose (GTT) and insulin tolerance test (ITT) at 3 time points. Animals were assigned into four groups:

Normoketonemic (NK; n=9); Transiently hyperketonemic (Transient HK; n=7); Continuously HK (Continuous

HK, n=7); and Continuously HK with puerperal metritis (Continuous HK+PM; n=6). Results: Insulin area under

the curve and insulin response to glucose were lower in early postpartum period than in late-pregnancy or mid

lactation (P<0.001) and in cows affected by continuous HK (P<0.001). Insulin stimulated blood glucose decrease

was lower in HK cows with/without PM. There was a strong time effect on RQUICKI value (P<0.001). There

was no difference in the RQUICKI in cows with different form of HK and no correlation between the RQUICKI

and tolerance tests was observed. Conclusion: The RQUICKI is affected by the considerable changes of plasma

metabolic concentrations around the calving and has low discrimination power in diagnosing decreased insulin

sensitivity in cows affected by various metabolic diseases.

DIAGNOSTIC VALUE OF ACUTE PHASE INDEX OF MILK IN DETECTION OF SUBCLINICAL

MASTITIS IN DAIRY COWS.

S. H. Shirazi-Beheshtiha1, S. Safi

2, V. Rabbani

1, M.B. Bolourchi

3.

1Karaj Branch, Islamic Azad University,

Karaj, Iran; 2Science and Research Branch, Islamic Azad University, Tehran, Iran;

3Faculty of Veterinary

Medicine, University of Tehran, Tehran, Iran.


Background: Currently, somatic cell count (SCC) and bacterial culture are considered the reference standard

methods for diagnosis of subclinical mastitis. However, SCC has a low diagnostic accuracy and bacterial culture

is time-consuming. For the identification of animals with subclinical mastitis, new biomarkers with high

diagnostic accuracy are needed. Objective: The aim of the present study was to compare the clinical accuracy of

milk amyloid A, whey proteins and acute phase index (API) with SCC and bacteriology for the diagnosis of

bovine subclinical mastitis. Methods: A total of 90 clinically healthy cows were randomly selected. Of these, 56

cows were considered to have subclinical mastitis based on a SCC higher than 100 × 1,000 cells/mL of milk and

positive bacterial culture results of milk samples obtained from at least one of the quarters. Milk amyloid A and

whey proteins were measured in the samples and API was calculated. Receiver-operating characteristic analysis

was used to assess the performance of each test using SCC and bacterial culture as the reference methods.

Results: API was the most accurate test with a diagnostic sensitivity of 98.2%, specificity of 94.1% and clinical

accuracy of 0.994 at cutoff point of > 0.105. Conclusion: Based on the high sensitivity and specificity of milk

API and milk amyloid A in diagnosis of subclinical mastitis observed in this study, we recommend these

methods be used as reliable alternative or complement tests to the routine SCC for early diagnosis of subclinical

mastitis in order to minimize the economic losses due to this disease.

141

CASE PRESENTATIONS

ORAL PRESENTATIONS

A CASE OF CANINE SURRA IMPORTED INTO GERMANY.

M. Defontis1, J. Richartz

1, N. Engelmann

1, C. Bauer

1, V.M. Schwierk

2, P. Büscher

3, A. Moritz

1.

1Justus-

Liebig University, Giessen, Germany; 2Pulheim-Stommeln, Germany;

3Antwerpen, Belgium


A 9-year old male Jack Russel Terrier with a travel history to Thailand was presented with chronic lethargy,

weight loss and unilateral ocular opacity. On blood smear evaluation numerous trypomastigotes were observed.

Molecular identification of the parasite revealed infection with Trypanosoma evansi, the causative agent of

Surra. The patient was treated with suramin and suffered a relapse with central neurological symptoms after 88

days. Histopathological evaluation revealed marked non-purulent meningoencephalitis and PCR testing in CSF

was positive for T. evansi.

CHRONIC LYMPHOCYTIC LEUKEMIA/SMALL CELL LYMPHOMA IN A HORSE.

F. Cian1, G. Tyner

2, V. Martini

3, S. Comazzi

3, J. Archer

1.

1Department of Veterinary Medicine, University of

Cambridge, Cambridge, United Kingdom; 2Chiltern Equine Clinic, Chalfont St Giles, United Kingdom;

3Department of Veterinary Pathology, University of Milan, Milan, Italy


A 16 year old, Irish Draft mare was admitted to the Chiltern Equine Clinic for an annual health check. The mare

was in excellent body condition. A mild generalized lymphoadenomegaly was identified. Rectal palpation and

transrectal ultrasonography revealed prominent mesenteric lymph nodes. A transcutaneous abdominal

ultrasonographic evaluation was unremarkable. Hematology revealed a marked leukocytosis (63.06 x10^9/l)

with lymphocytosis (58.2 x10^9/l) with the majority being small lymphocytes. No evidence of anemia or

thrombocytopenia was identified. The serum chemistry profile showed mild hyperglobulinemia. Severe

hypoglycemia and mild elevation in phosphate were likely to be due to delayed analysis. Serum protein

electrophoresis was unremarkable. Fine needle aspirates of multiple lymph nodes and a bone marrow aspirate

were performed, both revealing the presence of a monomorphic population of small lymphocytes similar to those

observed in the peripheral blood indicating the presence of a leukemic small cell lymphoma/chronic lymphocytic

leukemia (CLL). Considering the lymphoadenomegaly was identified at the same time that the lymphocytosis,

the distinction between these two conditions could not be made. The peripheral blood was examined by flow

cytometry and immunocytochemistry and the results suggested a T cell phenotype. As the horse remained

clinically normal no treatment was performed though regular re-examinations were undertaken. Small cell

lymphoma and leukemia are rare in horse and according to literature they are most commonly of T cell

phenotype. To the authors’ knowledge this is the first report of combined use of immunocytochemistry and flow

cytometry in a horse with leukemic lymphoma/CLL.

CASE REPORT: CUTANEOUS T-CELL LYMPHOMA.

B. Rütgen1, I. Flickinger

2, B. Wolfesberger

2, B. Litschauer

2, A. Fuchs-Baumgartinger

3, S.E. Essler

4, A.

Saalmüller4, I. Schwendenwein

5. 1

University of Veterinary Medicine Vienna, Vienna, Austria; 2Clinic for

Internal Medicine,University of Veterinary Medicine Vienna, Vienna, Austria; 3Institute of Pathology,

University of Veterinary Medicine Vienna, Vienna, Austria; 4Institute of Immunology, University of Veterinary

Medicine Vienna, Vienna, Austria; 5Central Laboratory, Department for Pathobiology, Univ. of Veterinary

Medicine, Vienna, Austria.


A 7 year old male Boxer with a 3.5 year history of atopy and food hypersensitivity was presented with multiple

diffuse nodules and macules of the skin and tongue and jaundiced mucosal membranes. Cytologic and histologic

examination of the skin lesions revealed cutaneous epitheliotropic lymphoma. Cells showed expression for CD3+

and CD8+ in flow cytometry. CBC showed a moderate leukocytosis with 16% atypical lymphocytes with

irregularly cleaved nuclei. In peripheral blood FCM showed an elevated proportion of the CD8+T lymphocyte

subpopulation, indicating a malignant population of T cell origin and a monoclonal band for TCRγ in the PCR

for antigen receptor rearrangement. Liver enzyme activity was markedly elevated and abdominal ultrasound

showed increased echogenicity of the liver, and lymphadenomegaly. Fine needle aspirates of the liver confirmed

infiltration with malignant lymphocytes showing the same morphology as the cells detected in skin and

142

peripheral blood. Treatment was induced with L-asparaginase, CCNU and prednisone. Partial clinical remission

of the skin and tongue lesions was achieved within 10 days and hematological abnormalities resolved. Despite

further treatment with L-asparaginase and CCNU the dog relapsed within 1 month and was euthanized.


15. “WATER DIABETES” IN A GREYHOUND ASSOCIATED WITH CORTICOSTEROIDS.

L. Blythe. Oregon State University, Corvallis, OR, United States of America


Background: Greyhounds differ from other breeds of dogs in a number of ways. Corticosteroids have been

known to cause transient polyuria and polydipsia in normal dogs, which is usually manageable by the owner.

Objective: The aim of this case study is to describe the use of corticosteroids in a Greyhound. Case Study: An

eight year neutered male Greyhound presented to the Teaching Hospital with lethargy, anorexia, and neck pain

following an episode where he chased a skunk under a building. A CT scan revealed a ruptured disc at cervical 4

and 5 vertebra with disc material present in the vertebral canal. A ventral slot surgical procedure removed the

disc material. Following surgery 30 mg/kg of methylprednisolone was given. Upon release, excessive urination

was noted. Water was restricted to 1 ½ liters per day, but polyuria continued with urination every 30 to 40

minutes. He was readmitted to the Teaching Hospital where a renal panel and urinalysis revealed urine specific

gravity of 1.007. Skin turgor showed 3% dehydration. The polyuria was treated with continuous IV therapy. By

day 4, urine specific gravity was 1.032. Results: Severe polyuria was induced in this Greyhound by a single dose

of 30 mg/kg dose of methylprednisolone. Four days of continuous intravenous fluids, water restriction, and

vasopressin was needed to return urine concentrating ability to normal. Conclusion: Greyhounds should not be

given corticosteroids whenever possible to avoid their use or, when given, the lowest possible dose should be

considered.


ORAL PRESENTATIONS

CHANGES IN THE CONCENTRATIONS OF SERUM AMYLOID A (SAA) FOLLOWING SURGERY,

TRAUMA OR SNAKE ENVENOMATION IN DOGS: A STUDY OF THE KINETICS OF SAA.

M. Christensen1, E. Moldal

1, R. Langhorn

1, A. Goddard

2, A. Tvarijonaviciute

3, M. Kjelgaard-Hansen

1.

1Department of Small Animal Clinical Sciences, University of Copenhagen, Frederiksberg, Denmark;

2Department of Companion Animal Clinical Studies, University of Pretoria, South Africa; 3Department of

Animal Medicine and Surgery, University of Murcia, Murcia, Spain


Background: Serum amyloid A (SAA) has proven useful for diagnostic purposes in dogs, but advanced

knowledge about the kinetics is needed if follow-up measurements should be used for monitoring purposes.

Objective: To investigate the kinetics of SAA in dogs by measuring the concentrations at different intervals

after castration/ovarioectomy/ovariohysterectomy, trauma (blunt accidental traumas), and snake envenomation

(vipers or cobras) in dogs. Material: Serum was collected prior to surgery and 6, 12, 24, 48, 72, and 96 hours

after (n=5-30); <12, 12-24, 36-48, and 60-72 hours post trauma (n=3-9), and <6, 6-18, 24-30, and 36-48 hours

post envenomation (n=19-40). Method: A validated latex agglutination turbidimetric assay was used for

measurements of SAA. Changes in concentrations were tested for significance using Kruskal-Wallis and Dunns

multiple comparison tests (significance level P<0.05). Results: Significant 2 to 337-fold increases of SAA were

observed 24 hours post surgery (n=21). Similar results were observed in snake envenomed dogs showing

significant 1 to 13-fold increases in SAA between <6 hours and 6-18 hours (n=13) and 24-30 hours (n=35),

respectively. Significant 7 to 50-fold increases in SAA were observed post trauma <12 and 12-24 hours (n=6).

No additional increases after 24 hours were observed in any of the groups. SAA decreased significantly 96 hours

post-surgery towards pre-surgical concentrations (n=5). Conclusion and perspectives: Significant increases in

SAA were observed in the first 24 hours post surgery, trauma and envenomation, representing three etiologically

different causes of systemic inflammation. Additional increases after 24 hours should however not be expected

in uncomplicated cases.

143

EVALUATION OF A CANINE SPECIFIC QUANTITATIVE POINT- OF- CARE (POC) IMMUNOASSAY

FOR MEASURING C-REACTIVE PROTEIN IN DOGS.

M. Costa, K. Papasouliotis. School of Veterinary Sciences, University of Bristol, Bristol, United Kingdom


Background: Measurement of C-reactive protein (CRP) aids in the diagnosis and management of various

infectious, inflammatory, immune mediated or neoplastic diseases. Objective: To evaluate a new canine specific

point-of-care immunoassay (POC) (TECOmedical, Switzerland). Methods: CRP was measured in 40 canine

serum clinical samples by a previously validated canine immunoturbidometric assay (Reactivlab, Scotland), a

new canine ELISA (TECOmedical, Switzerland) and the POC. The POC was evaluated with intra- and inter-

assay precision studies (seven to ten consecutive runs of low and high concentration samples) and with linearity

studies [equal step dilutions of a high concentration sample (111.2 mg/L)]. Results: The intra-assay coefficient

of variation (CV) was 18.2 % with a low (15.6 mg/L) and 37.8 % with a high concentration (111.2 mg/L)

sample. The inter-assay CV was 52.5 % with a low (6.9 mg/L) and 13.6 % with a high concentration (145.1

mg/L) sample. Linearity was acceptable. The Spearman’s correlation coefficient (rs) a) between the POC

(median: 13.4, range: 5.1-194.6 mg/L) and TECO ELISA (median: 8.6, range: 0–148 mg/L) was 0.89

(p<0.0001) and b) between the POC and Reactivlab (median: 21.9, range: 0.1 – 367 mg/L) was 0.81 (p<0.0001).

Agreement studies (Deming regression and Bland-Altman difference plots) between the POC and Reactivlab

methods revealed a negative bias (mean:-33, 95% limits of agreement:-169 to 103 mg/L), mainly due to samples

with high concentrations. Agreement studies between the POC and ELISA showed a positive bias (mean: 13.9,

95% limits of agreement:-46 to 74 mg/L). Conclusions: The POC has an acceptable performance for clinical

purposes, but results obtained by the POC and the other two methodologies cannot be used interchangeably.


16. ACUTE PHASE PROTEIN RESPONSE IN PIGS AFTER EXPERIMENTAL INFECTION WITH

DIFFERENT STRAINS OF PORCINE REPRODUCTIVE AND RESPIRATORY SYNDROME VIRUS.

Y. Saco1, M. Cortey

2, J. Segalés

3, R. Pato

4, R. Pena

4, F. Martínez-Lobo

5, C. Prieto

5, A. Bassols

6.

1Universitat Autonoma de Barcelona, Bellaterra, Spain;

2Unité de Virologie Moléculaire, Emergence et co-

évolution virale UMR 6578, Marseille, France; 3CReSA, UAB-IRTA. Dept. Sanitat i Anatomia Animals. Fac.

Veterinaria. UAB, Barcelona, Spain; 4Servei de Bioquímica Clínica Veterinaria. Fac. Veterinaria. UAB,

Barcelona, Spain; 5Dpto. de Sanidad Animal. Fac. Veterinaria. Univ. Complutense de Madrid, Madrid, Spain;

6Departament de Bioquímica i Biologia Molecular, Fac. Veterinaria. UAB, Barcelona, Spain.


Background: Porcine reproductive and respiratory syndrome virus (PRRSV) is a major porcine pathogen,

characterized by reproductive failure in sows and respiratory disease in growing pigs. The lack of heterologous

protection by PRRSV vaccines is currently a major problem in the field and better knowledge in the

pathogenesis of this infection is necessary for developing more efficacious vaccines. Objective: The aim of this

work was to characterize and compare the acute phase response of pigs experimentally infected with different

strains of PRRSV. Methods: 105 3-week-old piglets were divided in 7 groups of 15 animals of which, 4 groups

were exposed to genotype 1 (European) strains, 2 to genotype 2 (American) strains and 1 served as non-

inoculated, negative control. Clinical signs were recorded daily. On days 7, 14 and 21 post-inoculation (PI) five

animals per group were euthanized and pathologically investigated. Blood samples were taken before challenge

and on days 3, 6, 9, 12, 15, 18 and 21 PI. The levels of acute phase proteins (APPs) haptoglobin (Hp), Pig-MAP

and CRP were quantified. Kruskal-Wallis or Mann-Whitney tests were performed to investigate the relationship

between levels of APPs versus PRRSV strain and time after infection.Results: Hp and CRP were significantly

discriminatory between infected and control pigs, but not Pig-MAP. Pigs challenged with genotype 2 strains

showed significantly higher levels of APPs than those infected with genotype 1. Conclusions: APP levels differ

between genotypes; the higher production of APPs induced by American strains correlated with the more

pathogenic properties of this strain.

144

17. DIAGNOSTIC VALUE OF MILK ACUTE PHASE INDEX IN DETECTION OF BOVINE

SUBCLINICAL MASTITIS.

S. Safi1, S.H. Shirazi-Beheshtiha

2, V. Rabbani

2, M. Bolourchi

3.

1Science and Research Branch, Islamic Azad

University, Tehran, Iran; 2Department of Clinical Sciences, Faculty of Veterinary Medicine, Islamic Azad

University, Karaj, Iran; 3Department of Clinical Sciences, Faculty of Veterinary Medicine, Tehran University,

Tehran, Iran


Currently, somatic cell count (SCC) and bacterial culture are considered the gold standard methods for diagnosis

of bovine subclinical mastitis. However, SCC has a low diagnostic accuracy. Therefore, new biomarkers with

high diagnostic accuracy are needed for identification of infected animals. The objective of this study was to

determine the diagnostic value of milk APPs and acute phase index for the diagnosis of subclinical mastitis in

dairy cows. Ninety Holstein cows were randomly selected from 5 dairy farms in Tehran province, Iran. Milk

samples were analyzed for milk amyloid A, albumin, beta-lactoglobulin, alpha-lactalbumin, immunoglobulins

concentrations and milk acute phase index. Receiver-operating characteristic analysis (ROC) was used to assess

the clinical accuracy of each test using SCC as the reference method. At a cutoff value of 130000 for SCC, milk

acute phase index was the most accurate analysis, with a sensitivity of 96.2% and specificity of 92.1% at values

> 0.225. Furthermore the sensitivity and specificity of milk acute phase index in bovine subclinical mastitis

caused by prevalent types of microbial pathogens in Iran were 100% and its clinical accuracy was 1. In

conclusion, acute phase index has a high sensitivity and specificity in diagnosis of subclinical mastitis and may

replace SCC so early diagnosis of subclinical mastitis will become easier and economic losses will decrease.

18. SERUM AMILOID A AS A MARKER OF INFLAMMATION IN CANINE JOINT.

J. Francuski1, N. Andrić

2, M. Lazarević Macanović

3, D. Marković

4, A. Radovanović

4, M. Kovačević

Filipović1.

1Department of Pathophysiology and Biochemistry,

2Department of Equine, Small Animals, Poultry

and Wild Animal Diseases, 3Department of Radiology and Radiation Hygiene,

4Department of Histology and

Embriology, Faculty of Veterinary Medicine, University of Belgrade, Serbia.


Background: Gross and cytological synovial fluid (gcSF) evaluation enables differentiation between infectious

and non infectious clinically visible inflammation, but is not useful in distinction between subclinical

inflammatory and degenerative changes of the joints. In dogs, serum amyloid A (SAA) is a major acute phase

protein and could be a useful tool in diagnosis of subclinical inflammatory joint diseases. Objective: The aim of

this study was to compare clinical signs, gross anatomy, radiology and gcSF findings with SF SAA

concentration in canine knee joints. Methods: Examination was performed on four military working dogs (11

years old) and two non working dogs (8 months old) euthanized for different reasons. Knee joints were X rayed

and gross anatomy changes were assessed by modified Making's grading system. GcSF evaluation and SF total

protein (biuret method) were done. SAA concentration was determined by ELISA (TRIDELTA, Ireland).

Results: Three military dogs without signs of lameness, no X ray and gross anatomy changes and no gcSF

abnormalities did not have detectable SF SAA. The forth military dog had lameness and visible erosive

osteoarthritis (Manking scale 4), no gcSF abnormalities, but had detectable SAA concentration. Two young dogs

(8 months) euthanized because of idiopathic epilepsy (no signs of lameness) and the crush syndrome (no visible

knee joint changes) did not have gcSF abnormalities but had increased SF SAA concentrations. Conclusion: SF

SAA concentration might be an important parameter in diagnosis of inflammatory joint diseases even in cases

without lameness or X ray, gross anatomy visible changes and gcSF abnormalities.

145


ORAL PRESENTATION

PCR-BASED DETECTION OF A. PHAGOCYTOPHILUM DNA IN PARAFFIN EMBEDDED SKIN

BIOPSIES FROM DOGS SEROPOSITIVE AGAINST A. PHAGOCYTOPHILUM.

I. Berzina1, N. Müller

2, C. Krudewig

3, I. Matise

1, R. Ranka

4, M. Welle

3.

1Faculty of Veterinary Medicine,

Latvia University of Agr, Jelgava, Latvia; 2Institute of Parasitology, Vetsuisse-Faculty, University of Bern, Bern,

Switzerland; 3Institute of Animal Pathology, Vetsuisse-Faculty, University of Bern, Bern, Switzerland;

4Latvian

Biomedical Research and Study Center, Riga, Latvia


Background: Canine granulocytic anaplasmosis (CGA) is caused by the rickettsial microorganism A.

phagocytophilum. CGA is mostly characterized by fever, joint problems, lethargy, anorexia, thrombocytopenia

and other nonspecific clinical signs. Skin lesions have been described in naturally infected lambs and humans.

The pathobiology of CGA is not entirely clear and the persistence of the organism after the resolution of clinical

signs has been questioned. Objective: To investigate if A. phagocytophilum is associated with skin lesions in

dogs seropositive against the bacterium. Methods: Tissue blocks from skin biopsies of 12 seropositive dogs

were evaluated. Histology of the skin lesions did not indicate an apparent cause. DNA was extracted from the

paraffin embedded tissues and a conventional PCR for partial 16s rRNA gene was performed with the carry-over

elimination by the use of uracil-DNA glycosylase. Results: In skin biopsies from 3 dogs A. phagocytophilum

DNA was amplified, two of the resulting sequences were 100% identical to the prototype A. phagocytophilum

human strain (accession number at the GenBank U02521). Titers of the PCR-positive dogs were 1:320, 1:800

and 1:2048. Time period between serology and biopsy sampling ranged between 4-90 days. Histopathologically

skin lesions were characterized by a moderate to severe edema and variable hemorrhage. In addition a superficial

and periadnexal mixed cell infiltrate and multifical nodules composed of neutrophils and histiocytes were seen.

No obvious signs of vasculitis were found. Conclusion: A. phagocytophilum DNA can be recovered from skin

lesions in dogs seropositive against A. phagocytophilum and may be causative for the skin disease.


19. PRO-INFLAMATORY CYTOKINES QUANTIFICATION IN PIGS EXPERIMENTALLY

INOCULATED WITH ACTINOBACILLUS PLEUROPNEUMONIAE.

M. Quezada¹, A. Islas¹ A. Ruiz¹, T. Casanova¹, C. Lecoq ², D. Muñoz². ¹Faculty of Veterinary Sciences.

University of Concepción,Chile; 2Agricultural Service Of Agricultural Ministery of Chile


Background: .Actinobacillus pleuropneumoniae (App) is the agent of Porcine Contagious Pleuropneumonia.

App is characterized by its membrane lipopolysaccharide that is able to induce an inflammatory answer with

stimulation of pro-inflammatory cytokines. This study evaluated the inflammatory response induced by App,

trough the quantification of IL-1β, IL-6 and TNF-α in tonsils, retropharyngeal and mediastinal lymph nodes.

Methods: For this, thirty pigs of eight weeks age were inoculated with an Isolated field of App. Control group

(n=10) was inoculated with a sterile culture medium and infected group (n=20) was inoculated with the isolated

field of App. The pigs were euthanized in batches of 5 animals per group, for control group at 0 and 48 hours

post-inoculation (hpi) and for infected group at 4, 8, 24, and 48 hpi. Tissues were obtained for mRNA extraction

and cytokines quantification by qPCR. Results: Revelated significant differences in cytokines expression. IL-1β

increases its level at 4 and 24 hpi in the tonsil; at 8 and 48 hpi in the retropharyngeal lymph nodes at 8 hpi in the

mediastinal lymph nodes. For TNF-α, it was observed a significant increase in the mediastinal lymph nodes.

Conclusions. These results helped to confirm that the patterns of expression of cytokines are consistent with the

pathogenesis of PCP. Support DIUC Nº 209.152.019-1-0

146

20. EVALUATION OF LUNG LESIONS OF PIGS INOCULATED WITH A LOW PATHOGENIC

SEROTYPE 6 OF ACTINOBACILLUS PLEUROPNEUMONIAE.

A. Islas1, M. Quezada

1, A. Ruiz

1, D. Muñoz

2, C. Lecocq

2.1Faculty of Veterinary Sciences, University of

Concepcion, Chillán, Chile; 2Agricultural and Livestock Service, Ministry of Agriculture, Chile, Santiago, Chile


Objectives: To study the macroscopic and histological lesions caused by A. pleuropneumoniae (App), serotype

6, inoculated intranasally in susceptible pigs. Methods: The research was done with 40 pigs of 8 weeks old;

serologically negative to App. Pigs were subdivided in 2 groups (G1 and G2) of 20 animals each and housed in

2 different separate units. Animals were monitored daily, for 15 days, to control their temperature and clinical

signs. G1 (control group) was inoculated intranasally with 5 mL of sterile PPLO medium + NAD. Pigs in group

G2 were inoculated intranasally with 5 mL of PPLO + NAD culture medium containing 9.3 x 109 CFU/mL of

App. Five pigs from each group were sacrificed at 6, 24, 48 and 72 hours post-inoculation (hpi). Lung score was

evaluated and tissue samples were taken for microbiological culture, PCR and histopathology. Results: G1 did

not show any lung injury, lung score 0. In G2, injury was observed from 6 hpi, the lung score was 1.0, 1.4, 11.2

and 4.4 at 6, 24, 48 and 72 hpi, respectively. Histopathologically, in G2 was observed characteristic lesions of

the disease after 48 hpi, with accumulations of PMNs and macrophages in alveoli and ducts, and necrotic

lesions. The isolation of App from lung tissue was 0, 20, 80 and 100% at 6, 24, 48 and 72 hpi respectively.

Conclusions: The lesions were progressive, with characteristics lesions since 48 hrs, which was coincident with

the App isolates from lung samples.Support: FONDECYT 1111045 Project, Chile.

21. CRAYFISH PLAGUE IN SLOVENIA: FIRST REPORT OF THE PLAGUE AGENT APHANOMYCES

ASTACI.

D. Kušar1, A. Vrezec

2, M. Ocepek

1, V. Jenčič

1.

1Veterinary Faculty, Ljubljana, Slovenia;

2National Institute of

Biology, Ljubljana, Slovenia


Background: Freshwater crayfish suffer from many diseases, but the crayfish plague caused by parasitic

oomycete Aphanomyces astaci is the most detrimental for indigenous crayfish species (ICS), leading to complete

extinctions of affected populations. In Slovenia, it was reported at the beginning of the 20th century but not in

recent times; however, the invasion of resistant non-indigenous crayfish species (NICS) was first recorded in

2003. Objective: The aim of our study was to inspect all ICS and NICS populations that inhabit Slovenian

freshwaters, and show no clinical signs of infection, for the presence of A. astaci by molecular means which

represent a reliable and fast alternative to the formerly established diagnostic tools. Methods: After extraction of

DNA from crayfish cuticle using the commercial DNeasy Blood & Tissue Kit (Qiagen) preceded by the

mechanical tissue disintegration in the MagNA Lyser Instrument (Roche Diagnostics), highly specific TaqMan

MGB real-time polymerase chain reaction was performed to detect A. astaci in crayfish material. Results: Out of

67 analyzed crayfish, two specimens of Austropotamobius torrentium (stone crayfish, ICS) and four of

Pacifastacus leniusculus (signal crayfish, NICS) tested positive. Conclusions: Crayfish plague has not yet

become a considerable problem in Slovenia. However, results revealed the first presence of A. astaci in the ICS

population of A. torrentium that was presumably not in any contact with the NICS carrier and opened new

questions of the recent crayfish plague expansion in Europe.

22. PROTEINURIA IN DOGS NATURALLY INFECTED WITH THE BACTERIUM ANAPLASMA

PHAGOCYTOPHILUM.

U. Ravnik1, B. Premrov Bajuk

2, K. Babnik

2, N. Tozon

1.

1Small animal clinic, Veterinary faculty, Ljubljana,

Slovenia; 2Institute of Physiology, Pharmacology and Toxicology, Veterinary faculty, Ljubljana, Slovenia


Background: Tick-transmitted infections are frequent triggers of immune mediated diseases in animals.

Infection gives rise to immune complexes of microbial antigen – antibody – complement which may lodge in

capillary beds of different organs. Objective: The aim of this study was to investigate the occurence and origin

of proteinuria in dogs naturally infected with A. phagocytophilum. Methods: Urine samples were obtained from

45 dogs seropositive or PCR positive to A. phagocytophilum, and 17 control dogs. Microscopic evaluation of

urine sediment, urine protein to creatinine ratio (UPC) and SDS-PAGE electrophoresis of urine proteins were

performed. Results: Proteinuria was detected in 36 (58,1 %) samples. Proteins were divided in low molecular

weight proteins (LMW)(< 66 kDa), middle molecular weight (MMW) (66 – 76 kDa) and high molecular weight

(HMW) (> 76 kDa). LMW proteins with bands on 10, 15, 25 and 55 kDa were frequently observed in control

147

dogs so those were considered physiological. In 11 samples LMW and MMW or LMW, MMW and HMW

together with UPC above the reference interval and casts in urine sediment were found, all in seropositive dogs.

Conclusion: Infection with A. phagocytophilum may lead to the development of immune – mediated

glomerulonephritis characterised by the presence of glomerular non-selective and tubular proteinuria which

prompts a follow-up of seropositive patients for early signs of chronic renal disease.

23. EPIDEMIOLOGICAL SITUATION OF IBR/IPV, MB/BVD AND SOME RESPIRATORY VIRUSES IN

SLOVENIAN AUTOCHTHONOUS BREEDS

M. Nemec1, P. Hostnik

2, Z. Klinkon

3, T. Zadnik

1, M. Klinkon

1.

1Veterinary Faculty, Clinic for Ruminants,

Ljubljana, Slovenia; 2Veterinary Faculty, National veterinary institute,Virology laboratory, Ljubljana, Slovenia;

3Veterinary clinic Klinkon, Homec, Slovenia


Background: We studied the epidemiology of infectious bovine rhinotracheitis / infectious pustular

vulvovaginitis (IBR / IPV), bovine viral diarrhea (BVD), infection with the bovine respiratory syncytial virus

(BRSV), the parainfluence 3 virus (PI-3) and the adenovirus 3 ( adeno 3) in the autochthonous Cika cattle.

Objective: The purpose of the study is to collect preliminary epidemilogic data in order to maintain a healthy

herd. Methods: The study included 94 Cika cattle breed from different Slovenian regions. Blood samples for

haematology, biochemistry and serology inclduing the detection of antibodies against IBR / IPV, BVD, BRSV,

PI-3 and Adeno 3 were collected and divided according to the results of positive and negative serological

investigations. Significant differences between the studied values were determined using the Student's t-test.

Results: We found a positive serological reaction to IBR / IPV in 2 Cika cows. In 36.67 % of the tested animals

antibodies to BVD were serologically detected. 21.59% of the animals were serologically positive for the

presence of BRSV, 47.73% of tested samples were seropositive for the PI-3 virus and more than half (62.50%)

for the adenovirus type 3. The results of hematological and biochemical investigations show that the most

significant differences were observed in BVD and only one blood analysis was significantly different in

BRSV.Conclusion: The preliminary study showed minimal exposure to IBR / IPV and the greatest prevalence

of exposure to adenovirus type 3 based on serologic evaluation.


ORAL PRESENTATIONS

PRELIMINARY REFERENCE INTERVALS FOR FLUOROGENIC MEASUREMENT OF THROMBIN

GENERATION IN THE BEAGLE DOG.

M. Defontis1, S. Côté

2, M. Stirn

2, D. Ledieu

2. 1

Justus-Liebig University, Giessen, Germany; 2Basel,

Switzerland.


Background: The serine protease thrombin plays a crucial role in the coagulation cascade. Fluorogenic

measurement of its formation after tissue factor stimulation and using the Calibrated Automated Thrombogram

(CAT) is a more sensitive tool to detect thrombotic or hemorrhagic tendencies compared to the plasmatic

coagulation times PT and aPTT. Objective: The aim of this study was to determine preliminary reference

intervals for the CAT variables in platelet poor plasma collected from healthy Beagle dogs. Methods: Citrated

blood was taken from 20 healthy Beagle dogs (10 males and 10 females between 12 and 20 months of age) by

jugular venipuncture and platelet poor plasma was immediately prepared and stored at -80°C for maximum 2

weeks. The animals were kept under controlled housing conditions and had not received any medication for the

last 15 days. For each animal, CAT measurements were performed in triplicate. The results were displayed on

histograms for visually inspection and a 90% double-sided reference interval was calculated for the following

parameters: Lagtime, Time to peak, Peak height, ETP (Endogenous Thrombin Potential) and Start Tail using the

MedCalc® software and a robust method (CLSI Guidelines C28-A3). Results: The calculated 90% double-sided

reference intervals were as follow: Lagtime= 1.1-1.9 min, Time to peak= 2.5-3.6 min, Peak height=83.9-

155.4nM, ETP= 241.5-375.3nM/min and Start Tail: 5.0-15.2min. Conclusion: The Calibrated Automated

Thrombogram can be used in the Beagle dog to quantify thrombin generation.

148

FLOW CYTOMETRIC EVALUATION OF DRUG RESISTANCE IN CANINE LYMPHOMA SHORT TERM

CELL CULTURES.

P. Vajdovich. Szent István University, Faculty of Veterinary Science, Budapest, Hungary


Background: Chemotherapy resistance is an essential factor in reponse to treatment. Prediction of response of

the neoplastic cells to various cytostatic drugs can help in choosing the appropriate therapy. Objective: The aim

of the study was to evaluate the apoptotic response of the cells derived from lymph nodes of dogs with

lymphoma to cytostatic drugs. Methods: Six dogs with diffuse large B-cell lymphoma were included in the

study. Lymphatic cells were separated, cell suspension was made for immunophenotyping and a short term cell

culture was also prepared. Immunophenotype of cells was evaluated by flow cytometer. The following markers

were examined: CD3, CD4, CD5, CD8, CD14, CD21, CD34, CD45, MHC II. Lymph nodes were evaluated by

immunohistochemistry. Proliferation ability was evaluated by Ki67 marker. Cell cultures were treated by the

following drugs: vincristine (Vin), cyclophasphamide (Cyc), doxorubicin (Dox), methylprednisoslone (Mpred),

etoposide (Eto). Etidium bromide treatment of cells was used to differentiate the apoptotic or viable cells.

Results: The apoptotic rate increased in cells according to the following course Cyc, Vin, Mpred, Dox, Eto.

Apoptotic rate in cell cultures correlated between Dox and Cyc (r=0.974), and between Vin and Mpred

(r=0.968). The cell suspensions rich in CD14 positive cells (monocyte marker) and CD34 positive cells

(haemopoietic stem cell marker) negatively correlated with the apoptotic rate in cell cultures treated by Vin

(CD14, r=-0.994, CD34, r=-0.995) and Mpred (CD14, r=-0.980, CD34, r=-0.981). Conclusion: Flow cytometric

evaluation of cytostatic resistance can help in proper drug selection for the treatment of canine lymphoma.

MEASUREMENT OF SOLUBLE MESOTHELIN RELATED PEPTIDES (SMRP) IN CANINE BODY

CAVITY EFFUSIONS.

M. Costa1, K. Murphy

2, D. Wilson

1, K. Papasouliotis

1.

1School of Veterinary Sciences, University of Bristol,

Bristol, United Kingdom; 2Bath Veterinary Referrals, Bath, United Kingdom


Background: In humans, measurement of Soluble Mesothelin Related Peptides (SMRP) has been considered

useful in differentiating mesothelioma from other malignant or benign effusions. It has been shown that human

and canine mesothelins share the same open reading frame. Objective: To measure SMRP in canine body cavity

effusions by the MesoMARK™, an ELISA designed for humans that uses the monoclonal antibodies OV569

and 4H3 (Fujirebio Diagnostics, Inc, USA). Methods:Thirty two canine effusions (19 pleural, 11 peritoneal, 2

pericardial) collected over 18 months were included in the study. Final diagnosis was based on clinical,

cytological, clinicopathological, imaging and histopathological findings. Aliquots of the fluids were stored at -

20°C until measurement of SMRP concentrations with the MesoMARK™. According to manufacturer’s

instructions a calibration curve was constructed using six SMRP standards (0-32nmol/L). Results: 10 effusions

were neoplastic [mesothelioma (n=4), lymphoma (n=2), carcinoma (n=3), undifferentiated (n=1)], 16 were non-

neoplastic [chylous (n=1), septic (n=1), transudate/modified transudate (n=14)] and 6 contained suspicious or

atypical mesothelial cells on cytology. During the study, 3 calibration curves were constructed; optical densities

(absorbance) for the 0, 2, 4, 8, 16 and 32 nmol/L standards ranged from -0.003-0.003, 0.086-0.113, 0.165-0.199,

0.298-0.350, 0.493-0.594and 0.804-1.024, respectively. The absorbances generated by all clinical samples

ranged from -0.004 to 0.010. Conclusions: SMRP was undetectable in all clinical samples. Lack of cross-

reactivity of the monoclonal antibodies with canine SMRP and/or the absence of SMRP from canine effusions

may explain our findings. The MesoMARK™ is not a useful test in canine medicine.


24. FEASIBILITY OF DETECTION OF TESTOSTERONE AND ESTRADIOL IN HAIR USING

COMMERCIAL ELISA KITS.

T. Snoj1, A. Nemec Svete

2, N. Cebulj-Kadunc

1, S. Kobal

1.

1Institute of Physiology, Pharmacology and

Toxicology, Veterinary Faculty, Ljubljana, Slovenia; 2Clinic for Surgery and Small Animal Medicine,

Veterinary Faculty, Ljubljana, Slovenia


Background: Detection of steroids in hair can be performed by several methods; however the use of commercial

ELISA kits has not been described for this purpose. Objective: In the present study, the detection of hair

testosterone and estradiol after intramuscular administration of testosterone propionate and estradiol valerate

149

with commercial ELISA kits is described. Methods: Wistar rats were divided into two experimental (16

animals) and two control groups (14 animals). Eight female rats were subjected to I.M. administration of

testosterone propionate (20 mg/kg) and eight male rats to estradiol valerate (10 mg/kg). Animals in the control

groups received placebo (0.2 mL of sterile olive oil). Hair samples were collected before treatment and 3, 6 and

9 days after treatment. Extraction of steroids from hair was performed by methanol. Commercial testosterone

and estradiol ELISA kits were used to determine testosterone and estradiol concentrations in hair. Results: A

significant increase (P<0.05) in hair testosterone was observed 3, 6 and 9 days after the administration of

testosterone propionate. Estradiol valerate administration resulted in a significant increase (P<0.05) in hair

estradiol concentrations on days 3 and 9 after treatment. In comparison with the control group, hair testosterone

and estradiol concentrations were significantly higher (P<0.01) at all three sampling times after treatment.

Conclusion: Results indicate that detection of testosterone and estradiol in hair can be achieved by commercial

ELISA kits used in the present study. Up to 0.80 % of the administrated testosterone and 0.06 % of the

administrated estradiol were present in hair.

25. ELECTRON MICROSCOPIC EXAMINATION OF BUFFY COAT: SIMPLE METHOD USING PLASTIC

MEMATOCRIT TUBE AND APPLICATION FOR DIAGNOSIS OF LYSOSOMAL STORAGE DISEASES

IN DOGS AND CATS.

A. Yabuki, S. Yoneshige, K. Mizukami, M.M. Rahman, M.M. Uddin, O. Yamato.

Kagoshima University,

Kagoshima, Japan.


Background: Electron microscopic (EM) observation of the buffy coat helps for diagnosis of the lysosomal

storage diseases, but it is not easy to prepare the EM samples from the buffy coat. Objective: The aim of the

present study was to develop a simple method to prepare the EM sample from the buffy coat and evaluate the

diagnostic efficacy of this method. Methods: Blood samples were obtained from dogs and cats including clinical

cases with lysosomal storage diseases. Bloods were drawn into the plastic hematocrit tubes and centrifuged.

Short pieces containing buffy coats were cut off and fixed with glutaraldehyde and osmium tetraoxide. Buffy

coats were pushed out from the tubes in a rehydration step and embedded in epoxy resin using flat molds. After

polymerization, sample blocks were attached to the other blocks formed with a capsule mold so that longitudinal

section can be obtained. Thin sections were stained with toluidine blue, and ultrathin sections cut from leucocyte

layers were stained with uranyl acetate and lead. Results: The present method provided microscopic view of the

arrangement of the layers which consist of platelets, leucocytes and red blood cells, and target leucocytes can be

easily found out within the layers. In the clinical cases, ultrastructural features of the inclusion bodies in

leucocytes were identified without difficulty. Morphological quality of the cells was successfully preserved.

Conclusion: Using the present method, buffy coat is conveniently prepared for EM, and this method is useful for

ultrastractural diagnosis of the buffy coat in lysosomal storage diseases.

26. LYMPHOCYTE ANTIGENS: STABILITY OF EXPRESSION AMONG HEALTHY DOGS AND UPON

STORAGE.

F. Riondato, A. Poggi, A. Rota, B. Miniscalco. Dep. of Animal Pathology, Grugliasco (TO), Italy.


Background: Reliable detection of fluorescence intensity (FI) by flow cytometry is fundamental. FI is

dependent on instrument settings and measurement should be done using controls with known FI, preferably

microbeads or cell subsets with stable mean FI (MFI) within the population. No data about the stability of

antigen expression in healthy dogs exist, both among different subjects and after sample storage. Objective:

Evaluate MFI stability of main lymphocyte antigens among different subjects and after storage (fresh vs 24h).

The final aim is to detect antigens to be used as internal controls in flow analyses of FI. Methods: Fresh (n=14)

and stored (n=20) EDTA peripheral blood samples from healthy dogs were submitted to flow MFI analysis of

CD3, CD5, CD4, CD8, CD21, cyCD79b using conjugated MAbs. Adding FlowCheck microbeads (Coulter) MFI

was calculated as MFIratio (CD/beads). Stability among subjects was assessed as CV of MFIratio (fresh

samples). For each CD, CV of MFIratios and mean CV of fluorescence CVs were considered. Comparison

between MFIratio and CV of fresh vs stored samples was carried out (t-test). Results: Smaller variation between

subjects was reported by CD79b, CD4 and CD21hi. Minor variation between lymphocytes of single subjects was

reported by CD21hi and CD4. No CDs reported significant MFI differences between fresh and stored samples;

significant differences in CV was detected for CD5 and CD21. Conclusion: Considering that CD79 needs

permeabilization and that a consistent CD21hi population is not constantly recognizable, CD4 is the best internal

control for CDs MFI evaluation.

150


ORAL PRESENTATIONS

PREDICTIVE VALUE ADVISOR: A SOFTWARE FOR THE EVALUATION OF THE IMPRECISION OF

PREDICTIVE VALUES OF TEST RESULTS ACCORDING TO IMPRECISION OF SENSITIVITY,

SPECIFICITY AND PRE-TEST PROBABILITY.

N. Bourgès-Abella1, P. Bourdaud'hui

2, A. Geffré

1, D. Concordet

2, J.P. Braun

1, O. Dossin

1, C. Trumel

1.

1Institut National Polytechnique-Ecole Nationale Vétérinaire de Toulouse, Toulouse, France;

2UMR331

Toxalim, Institut National polytechnique-Ecole Nationale Vétérinaire, Toulouse, France


Background: It is commonly accepted that the imprecision of criteria of diagnostic efficiency needs to be

reported as the 95% confidence interval of sensitivity, specificity, likelihood ratio or odds ratio. Moreover, the

imprecision in evaluation of pre-test-probability is not precisely quantifiable. The effects of all these

imprecisions are not taken into account in the estimation of predictive values. Objective: To evaluate the

possible effects of the all imprecision factors on predictive values and design a freeware allowing clinicians to

make simulations. Methods: A web-based calculator was devised to compute the 95% CIs of PVP & PVN as a

function of pre-test probability, according to the number of animals plus either Se and Sp or the numbers of true

and false negatives and positives. Moreover, based on the uncertainty of pre-test-probability proposed by the

user, histograms representing the range of PVP and PVN are displayed. A test version is available at :

http://www.biostat.envt.fr/ppvnpv/. Evaluation: This software allows clinicians to test different

hypotheses/imprecisions for a given test and to appreciate the uncertainty of the PVs which can be estimated for

the tests they have performed. Conclusion: Imprecision of PVP and PVN aids to support the clinician’s medical

decision giving fast quantification of uncertainty which is too long to calculate without specialized tools and is

usually underestimated. It cannot be used for each case but in some instances. Even retrospectively, it can help to

make a correct use of the data published, which are too frequently not used except for meeting publication

requirements.

REFERENCE VALUE ADVISOR (V.2) NOW PERFORMS REGRESSION-BASED REFERENCE

INTERVALS WITH CONTINUOUS COVARIABLES.

N. Bourgès-Abella1, A. Geffré

1, D. Concordet

2, J.P. Braun

1, C. Trumel

1.

1Institut National Polytechnique-

Ecole Nationale Vétérinaire de Toulouse, Toulouse, France; 2UMR331 Toxalim, Institut National polytechnique-

Ecole Nationale Vétérinaire, Toulouse, France


Background: The reference intervals determined using Reference Value Advisor v.1, a freeware running with

Excel 2003, were obtained rapidly but did not allow testing possible effects of covariates. Objective: To propose

a tool that helps identify effects of continuous factors of variation (e.g. age, weight) and represents regression-

based reference limits with their 90% confidence intervals (CIs) as reference bands on graphs. Methods: A set

of macros was designed for Excel 2007 and 2010 to compute regression-based reference limits and their 90%

CIs as graphs. These reference bands can be built using homoscedastic and heteroscedastic polynomial

parametric models for a single or several covariables. The computation of the optimal Box-Cox transformation

of the analyte concentration is proposed. Nonparametric reference bands can also be computed when the sample

size is large enough. Results: This software allows clinical pathologists to easily check whether or not a

covariate can be considered as a valid factor of variation and to automatically derive the corresponding reference

limits. Conclusion: Most of the factors of variation are demographic characteristics. Taking these factors into

account can substantially reduce the width of standard reference intervals. This is helpful in veterinary clinical

pathology as it improves the information for the medical decision when reference limits are used as decision

threshold.

151

BIOCHEMISTRY

ORAL PRESENTATIONS

ACTIVATION OF LIPID METABOLISM IN CANINE BABESIOSIS CAUSED BY BABESIA CANIS

N. Kučer1, V.Mrljak

1, R. Rafaj Barić

2, J.Kuleš

2, I.Šmit

1, J. Selanec

1, M. Crnogaj

2.

1Clinic for Internal

Diseases, Faculty of Veterinary Medicine University of Zagreb, Croatia; 2Department for Chemistry and

Biochemist, Faculty of Veterinary Medicine, Zagreb, Croatia.


Background: Development of medicine, biochemistry and immunology has provided a more complete insight

into factors that influence the pathogenesis of canine babesiosis. Today it is known that inflammation has a

major role in complicated and severe forms of disease. Lipid metabolism is of importance in babesiosis

involving the acute phase response as part of a complex process regulated by nervous and endocrine systems.

Because the majority of hemoparasites including babesia cannot synthesize their own lipids, they take them up

from host plasma, predominantly from high density lipoproteins fraction. Objective: The aim of this research

was to determine if there are changes in lipid metabolism during babesiosis and when they occur during the

course of disease. Methods: Experiments were conducted on 17 healthy and 26 naturally infected dogs. Blood

samples were taken on 1st day, (at admission), and on 3

rd and 7

th day after admission. Biochemical parameters

were obtained using an automated biochemical analyzer (Olympus AU 600) according to standard methods with

original reagent kits. Biochemical profile included triglycerides, high density lipoproteins, low density

lipoproteins and cholesterol. Results: Our data show that the cholesterol concentration has significantly

increased on day 3 in sick dogs, while the high density lipoproteins concentration had significantly decreased at

day 1. There were no major alterations in the triglyceride and low density lipoproteins concentration.

Conclusion: This study confirms that lipid metabolism is being activated during babesiosis.

LACTATE DEHYDROGENASE (LDH) ISOENZYMES IN DOGS AFFECTED BY LYMPHOMA.

A. Giordano, D. Stefanello, M. Pastore, R. Ferrari, L. Giori, P. Moretti, S. Paltrinieri. University of Milan,

Milan, Italy.


Background: Lactate dehydrogenase (LDH) is a marker of cell damage of many tissues. Total LDH is used for

the diagnosis of skeletal and cardiac muscle damage, or of hepatic and renal disorders. In people, LDH has also

been used as marker of neoplasia, since the isoenzymatic pattern of LDH changes in neoplastic tissues. An

increased total LDH activity has been observed in dogs with lymphoma, but little is known about the possible

diagnostic or prognostic role of LDH isoenzymes in canine lymphoma. Objectives: To provide information

about total activity and isoenzymatic patterns of LDH in dogs with lymphoma at first diagnosis and during the

course of disease or following chemotherapy. Methods: After a preliminary validation, 108 sera from 31 dogs

(20 with neoplasia and 11 healthy controls) were examined. Neoplastic dogs were sampled at first presentation

and during the follow up. Based on clinical and laboratory findings, sick dogs were grouped as follows: L=

lymphoma (n=13); Lth= lymphoma already undergoing therapy (n=3); O= neoplasia other than lymphoma

(n=4). Results: The percentage of isoenzymes was less influenced than total LDH by anticoagulant and storage.

At first presentation, dogs with neoplasia showed higher LDH activity than controls. Dogs with lymphoma

showed the highest values of LDH-2 and LDH-3 with different patterns between T-and B-lymphoma. No

specific changes were recorded during the follow up. Conclusions: Increases of total LDH activity and of LDH-

2 and LDH-3 could support a diagnosis of lymphoma. Further data are needed to verify the prognostic role of

LDH isoenzymes.

EFFECTS OF PLATELET-ACTIVATING FACTOR RECEPTOR ANTAGONIST (PAFRA) ON SELECTED

INFLAMMATORY AND BIOCHEMICAL PARAMETERS IN A RAT ENDOTOXEMIA MODEL.

R. Col, E. Keskin. Selcuk University, Konya, Turkey


Background: Platelet-activating factor (PAF) is a potent autacoid mediator produced by a veriety of cells

involved in septic inflammatory reactions. PAFRA was demonstrated to have potent anti-inflammatory activity

and survival benefit. During endotoxemia, Lipopolysaccharide (LPS, endotoxin) and proinflammatory cytokines

are involved in the pathogenesis of sepsis and plays a pivotal role in the initiation of tissue damage, multiple

152

organ failure (MOF) and haemostatic disturbances. Objective: In this experimental study, our aim was to

investigate the role of PAFRA on inflammatory and biochemical disturbances in LPS-treated rats. Methods: A

total of 32 adult male Wistar rats (weight range: 200– 250 g) were divided into four equal groups: Group 1

served as negative control (C). Animals in Group 2 were given lipopolysaccharide (Escherichia Coli LPS,

0.111:B4) intravenously (1.6 mg/kg). In Group 3, PAFRA (10mg/kg) was injected intraperitoneally. In Group 4,

PAFRA (10mg/kg IP) and LPS (1.6 mg/kg IV) were administrated simultaneously. Blood samples were

collected 6 h after treatment. Results: LPS caused statistically significant increases in serum TNF-a, IL-6 and

IL1β levels, C - reactive protein, AST, ALT, LDH, creatinine, BUN, cholesterol, triglyceride concentration, and

caused statistically significant decreases in glucose, total protein and albumin levels, compared to control group.

In group 4, PAFRA inhibited serum TNF-a and IL1β levels compared with the group 2 (P<0.05). However, there

were no significant differences in the other values between the two groups. Conclusions: At the administered

dose and route, PAFRA has a slight effect in the pathogenesis of endotoxemia.

URINE PROTEOME FROM HEALTHY DOGS, CATS, HORSES AND COWS.

E. Ferlizza, E. Armuzzi, A. Buono, E. Carpenè, G. Andreani, F. Dondi, G. Isani. University of Bologna,

Ozzano Emilia, Italy


Background: Data on the urinary proteome of domestic animals are limited, as a consequence the study of

urinary proteome of healthy domestic animals is the first step to discover new biomarkers of nephropathy.

Objective: The study of the urine proteome from healthy dogs, cats, horses and cows. Methods: Urine samples

were collected from clinically healthy dogs (15), cats (10), horses (37) and cows (10). For all samples, UP/C was

calculated and SDS-PAGE with silver staining was performed. Results: All samples had UP/C lower than 0.2.

After SDS-PAGE, all samples showed many bands, which were undetectable by routine methods. Dogs had 15-

19 bands, cats: 17-20, horses: 13-15 and cows: 15-18. On the basis of the apparent molecular weight (MW)

reported in kDa and comparing with human urine proteome, we tried to identify the most common proteins: P90

(Tamm-Horsfall), P76 (transferrin), P64 (albumin) P54 (immunoglobulin heavy chain) P27 (immunoglobulin

light chain) and P14 (lysozyme). All samples contained putative albumin, transferrin, IG heavy and light chains;

Tamm-Horsfall was clearly evident in dogs, horses and cats. Dogs and cows presented proteins mainly at MW

lower than albumin, cats presented proteins mainly at MW higher than albumin, while horses presented proteins

at both high and low MW. Conclusion: SDS-PAGE with silver staining is a very sensitive method to detect low

abundance proteins in urine of healthy domestic animals, allowing the identification of different species-specific

electrophoretic profiles. Work is in progress to confirm the identity of the different proteins.

EVALUATION OF THREE URINE DIPSTICKS FOR DOGS, CATS, AND COWS.

S. Klenner1, M. Defontis

1, N. Bauer

1, K. Failing

2, A. Moritz

1. 1

Central Laboratory, University (JLU) Giessen,

Hannover, Germany; 2Unit for Biomathematics and Data Processing, Giessen, Germany.


Background: Urine evaluation is performed using dipsticks usually designed for human use. Objective: Aim of

this study was to evaluate the CombiScreenVet11Plus (CS), the Combur10UX (CB) and Multistix10SG (MS) for

urinalysis in dogs, cats, and cows. Methods: Urine samples from 101 dogs, 50 cats and 100 cows were evaluated

by all dipsticks using their automated reflectometer testing devices compared to the respective reference

methods. The Spearman rank correlation coefficient and sensitivity and specificity for detection of proteinuria,

glucosuria, and isosthenuria were calculated. Results: Between automated reading and reference methods,

correlation was poor in dogs and cows for all sticks for RBC, WBC, and specific gravity as well as for glucose

(dogs), protein (cows), WBC (cats) and poor/fair for specific gravity (cats). Fair/good correlation was present for

glucose (cats, cows), protein (dogs), RBC (cats). All sticks showed good/excellent correlation for all species for

pH. Detection of glucosuria (cut-off 50mg/dl) was sensitive (dog 80(MS)-100%, cat 85(MS)-93%, cow 76(MS)-

100%) and specific (dog 96-98%, cat 96-100%, cow 87(CB)-96%). CS and CB were sensitive (dog 83+92%, cat

67+100%, cow 91%) but dominantly nonspecific (dog 65+39%, cat 93+53%, cow 47+20%) for detection of

isosthenuria while specificity of the MS excellent in all species (97-98%). Evaluation of proteinuria (cut-off

30mg/dl) lacks specificity (CS+MS dog 81+38%, cat 79+32%, cow 43+27%) except for CB (dog 91%, cat 95%,

cow 89%). Sensitivity for detection of proteinuria lacks in dogs (CS 74%) and cows (CB 37%). Conclusion:

Detection of WBC and specific gravity are hampered in all dipsticks and all species.

153

CLINICAL RELEVANCE OF TGF-ß1 IN DOGS

S. Neumann, S. Gaedcke. Institute of Veterinary Medicine, Goettingen, Germany


Background: Cytokines are mediators of disease mechanism, so there serum concentration could be of clinical

relevance. Objective: Aim of this study was to proof the stability of TGF-ß1 in different sample collections, to

observe the clinical relevance in different internal diseases of dogs and to assess on this way the impact of TGF-

ß1 as diagnostic tool. Methods: TGF-ß1 concentration was assessed using enzyme linked immuno sorbant assay

(ELISA) from 169 dogs. Stability under controlled storage conditions as well as correlation of serum and plasma

levels from 55 dogs was performed. Results: TGF-ß1 was stable at a storage temperature of -70°C for minimum

of three month in plasma and serum. The serum concentration is in accordance to the platelet count. Plasma

concentrations have no relation to the platelet count. Different reference values could be fond for plasma and

serum TGF-ß1. Acute inflammatory diseases have no influence on the TGF-ß1 concentration in serum or

plasma. Diseases, which have in influence, were those with fibrosis during its course. Dogs with lung fibrosis,

hepatic fibrosis and Diabetes Mellitus have significantly increased concentrations of TGF-ß1 in serum or plasma

compared to healthy controls. Conclusion: Assessment of TGF-ß1 in a daily clinical practice is feasible and

offers potential diagnostic utility as marker for fibrosis in dogs.

LDH IN BODY CAVITY FLUIDS: IS IT DIAGNOSTICALLY USEFUL?

C. Smuts, J. Mills, T. Gaál. Murdoch University, Perth, Australia

AuthorDr Smuts, Celia , Murdoch University, Perth, Australia (Presenting author) Co-author(s) Mills, Jennifer,

Murdoch University, Perth, Australia Gaál, Tibor, Murdoch University, Perth, Australia TopicBiochemistry

(including endocrinology, acute phase proteins, urine and novel biomarkers)


Lactate dehydrogenase (LDH), an enzyme of the anaerobic glycolytic pathway with several isoforms, is found

intracellularly in most tissues. Increased LDH is frequently used in human medicine to distinguish pleural

exudates from transudates (Light’s criteria), and therefore may also be useful to help differentiate body cavity

effusions in animals. Because LDH is a large molecule that does not pass readily though intact endothelial

surfaces, concentrations in pleural or peritoneal fluid do not equilibrate rapidly with plasma values, and high

effusion LDH is therefore likely to originate from inflammatory or damaged cells in that cavity. We tested

pleural, peritoneal and pericardial effusions from dogs, cats and horses (n=50) using three different LDH tests –

RANDOX wet chemistry testing both the lactate-to-pyruvate (L-P) and pyruvate to lactate (P-L) reaction and dry

chemistry (IDEXX) measuring P-L. Although there is a good correlation between all the tests (r2= 0.98), there

are significant differences between their absolute values. IDEXX chemistry produces the highest values

(approximately 4x Daytona L-P) followed by Daytona P-L (approximately 2x L-P values) and Daytona L-P

usually the lowest. Therefore it is necessary to establish different cut-off values for differentiating between

transudates and exudates depending on which method is used. Results to date show that LDH activity tends to be

highest in exudates and modified transudates in all methods evaluated and lowest in transudative effusions. This

test may be useful for general practice where fluid analysis is not immediately available.

CANINE PANCREATIC LIPASE (SPEC CPL) AND C-REACTIVE PROTEIN (CRP) IN DOGS TREATED

WITH ANTICONVULSANTS (PHENOBARBITAL AND POTASSIUM BROMIDE).

J. Pastor1, V. Albarracin

1, A. Meléndez-Lazo

1, J. Rodón

2.

1Universitat Autònoma de Barcelona, Bellaterra,

Spain; 2IDEXX Spain, Barcelona, Spain.


Background: Animals with anticonvulsant treatments (potassium bromide and / or phenobarbital) may be

predisposed to pancreatitis. Canine pancreatic specific lipase (Spec cPL) is a sensitive test for pancreatitis

diagnosis. Elevations in C-reactive protein CRP (CRP) are associated with the clinical severity of pancreatitis.

Objective: To evaluate Spec cPL, C-PR and liver enzyme concentrations in dogs treated with phenobarbital and

/ or potassium bromide. Methods: Serum samples from dogs (n = 337) with anticonvulsant treatment (potassium

bromide and / or phenobarbital) were included in the study. Serum concentrations of potassium bromide,

phenobarbital, Spec cPL, triglycerides, cholesterol, GGT, ALP, ALT, AST and CRP were determined. Results:

Animals treated with potassium bromide and phenobarbital (n=55) have stastistically higher values of ALP

compared with animals with single treatments (bromide n=21 and phenobarbital n=257). Animals treated with

potassium bromide had statistically significant lower values of CRP compared with other groups. 6.8% (23/337)

of the animals showed an increase of Spec cPL over the reference range and showed a statistically significant

154

correlation between this parameter and concentration of triglycerides, ALP and ALT. There were no statistically

significant differences in CRP values between three groups of Spec cPL (less than 200 µg/L, 201-399 µg/L and

> 400 µg/L). Only 2 of 337 animals (0.6%) showed a Spec cPL > 400 mg/L and CRP above reference range

suggesting pancreatitis. Conclusions: Animals treated with phenobarbital and / or potassium bromide may have

an increase in serum concentrations of Spec cPL, however pancreatitis is rare in these animals.

IMMULITE 1000 MEASUREMENTS IN VETERINARY SAMPLES.

R. Pato1, R. Pena

2, Y. Saco

2, A. Bassols

2.

1Universitat Autonoma de Barcelona, Bellaterra, Spain;

2Servei de

Bioquímica Clínica Veterinaria, Departament de Bioquímica, Bellaterra (Barcelona), Spain


Background: The measurement of hormones by immunoanalysis in serum of veterinary species is a common

problem due to the lack of specific reagents. IMMULITE® Enzyme-Amplified Chemiluminescence methods

have been validated basically in human samples, but not for veterinary use in the majority of the available

assays. The aim of this study was to test cross reactivity of the specific IMMULITE® 1000 reagents for human

samples in veterinary species. Methods: Veterinary serum samples were tested using an IMMULITE® 1000

system (Siemens Healthcare Diagnostics) and the following human kits: Total T4 and Third Generation TSH kits

for pig serum; ACTH kit for horse and rat serum; Estradiol and Progesterone kits for female Catalan donkey

serum and Progesterone kit for bovine serum. Results: In serum obtained from umbilical blood of newborn

piglets (n=35), total T4 values ranged from <1-11 µg/dL and TSH values ranged from 0.006-0.097 µUI/mL. In

horse serum of newborn foals (n=27), ACTH values of control group ranged from 15.3-31.1 pg/mL. ACTH was

also determined in healthy rats, and basal values ranged from 10.7-64.8 pg/mL (n=16). In female Catalan

donkey, serum obtained in different moments of sexual cycle, 17-β-estradiol values ranged from <20-126 pg/mL

(n=64) and progesterone values ranged from 0.421-36.8 ng/mL (n=16). In bovine serum (n=60), progesterone

ranged from <0.2 to 10.9 ng/mL. Conclusions: Values obtained were similar to those described in bibliography.

A good linearity under dilution was obtained for each assay. No interferences were detected.

USE OF BIOMARKERS IN ANIMAL MODELING OF HEART FAILURE AND CARDIORENAL

DYSFUNCTION.

C. McClay, J. Urban, R. Gerhart, N. Papenfuss. Medtronic, Minneapolis, United States of America


Background: Development of animal models for congestive heart failure (CHF) and cardiorenal dysfunction

(CRD) permits trialing of novel medical devices and/or drugs for safety and efficacy as potential therapies. Many

biomarkers are used for diagnosis, prognosis, and assessment of therapeutic response in humans afflicted with

CFH and CRD. Utilization of assays that are cross-reactive or newer species-specific assays has been adopted in

veterinary medicine and preclinical safety evaluation. Objective: Clinical laboratory tests were performed to

characterize progression of CHF and CRD by two different canine models. Methods: For the CHF model, serial

injection of polystyrene microspheres was performed to induce global myocardial ischemia. Atrial natriuretic

peptide (ANP), brain natriuretic peptide (BNP), N-terminal pro brain natriuretic peptide (NT-proBNP), cardiac

troponin I (cTnI), and C-reactive protein (CRP) were assayed at selected time points. For the CRD model, high-

rate ventricular pacing was initiated at 240 beats per minute for 10 days. Concentrations of urine and plasma

chemistry analytes (including ANP, BNP, NT-proBNP) and glomerular filtration rates were compared before

and after pacing. Results: For the CHF model, CRP and cTnI levels increased significantly 24 hours post-

embolization, while NT-proBNP increased once hemodynamic compromise was attained. For the CRD model,

GFR decreased while ANP and NT-proBNP increased significantly after pacing. Conclusions: Temporal levels

of biomarkers are useful at different milestones of model development. However, several biomarkers failed to

correlate with clinical symptoms, physiological functions, or diagnostic imaging. Care must be exercised before

translating human tests to animal species.

155


27. DIAGNOSTIC MEASUREMENTS OF CANINE SERUM AMYLOID A USING AUTOMATED LATEX

AGGLUTINATION TURBIDIMETRY: AN INTERLABORATORIAL COMPARISON

M. Christensen1, J. Ceron

2, A. Tvarijonaviciute

2, M. Kjelgaard-Hansen

1.

1Department of Small Animal

Clinical Sciences, University of Copenhagen , Frederiksberg , Denmark; 2Department of Animal Medicine and

Surgery, University of Murcia, Murcia, Spain

Background: Canine serum amyloid A (SAA) has proven useful as an inflammatory marker and a latex

agglutination turbidimetric immunoassay (LAT) (Eiken, Japan) has been validated for use in dogs. If an assay

should be useful for routine diagnostic purposes it should however be applicable across different laboratories.

Objective: The aim of the study was to evaluate the performance of the LAT across two laboratories. Method:

An automated clinical chemical analyser was used at the University of Murcia (Olympus AU600) for

comparison of the initial evaluations previously obtained at the University of Copenhagen (Advia 1800).

Intraassay coefficients of variations (CV) were calculated from replicate determinations of serum pools. The

lower limit of quantification (LLOQ) was the concentration where CV exceeded 20%. Detection limit (DL) was

determined from replicate determinations of blanks (n=7). Inaccuracy was investigated by dilution of pooled

serum with high SAA concentration (linear regression). An interlaboratorial method comparison was made by

measuring SAA in 20 samples in both settings (linear regression and Bland-Altman plot). Results: Imprecision

was 1.4% when measuring high concentrations of SAA and increasing CVs were observed towards

LLOQ=18mg/L. DL was 0.0mg/L. Acceptable linearity under dilution was observed from 0-2700mg/L. A

systematic disagreement was observed between the laboratories (Y-intercept 22.45). Conclusion: Comparable

performance was obtained in the two laboratories and comparable overlap performances can be expected.

However, clinical decision levels should be estimated locally.

28. ELEVATED PLASMA ADIPONECTIN LEVEL AND PERIPHERAL BLOOD LEUKOCYTE

ADIPONECTIN RECEPTOR EXPRESSION IN INSULIN DEFICIENT DOGS.

N. Mori. Nippon Veterinary and Life Science University, Tokyo, Japan


The aim of this study was to investigate the effects of obesity or insulin deficiency on plasma adiponectin level

and peripheral blood leukocyte (PBL) adiponectin receptor 1 and 2 (ADIPOR1 and –R2) expression in dogs.

Obese and insulin deficient (ID) dogs demonstrated a significant reduction (60% lower) and significant increase

(2x higher) in circulating adiponectin level, respectively, as compared with normal controls. PBL ADIPOR1 and

-R2 mRNA expression was also significantly higher in obese (R1=333 mean fold higher, R2=64 mean fold

higher) and ID dogs pre-insulin treatment (R1=48 mean fold higher, R2=16 mean fold higher) as compared with

normal control PBL. Insulin treatment reduced ADIPOR1 (3 mean fold higher) and -R2 (1.5 mean fold higher)

expression to near control PBL levels in ID animals. The upregulation of adiponectin receptor expression might

reflect an increased need for adiponectin signaling; however, the increase may have a different implication

between obese and ID dogs. This may be due to adiponectin’s contradictory paradoxical dual role, having both

anti-inflammatory and pro-inflammatory effects on PBL, especially on monocytes, depending on the biological

context and adiponectin isoform. Adiponectin receptors have the potential to become important metabolic

parameters for dogs.

29. COMPARING THE USE OF BODY FAT PERCENTAGE VERSUS 5-POINT BODY CONDITION

SCORE FOR ASSESSING OVERWEIGHT STATUS IN DOGS BY SCREENING PLASMA METABOLITE

PROFILES.

G. Li, P. Lee, N. Mori, I. Yamamoto, K. Koh, T. Arai. Nippon veterinary and life science university, Tokyo,

Japan


Currently, the 5 point BCS system is commonly used by veterinarians and clinicians to assess adiposity in dogs

in Japan. However, assigning a BCS score to an animal is subjective in nature. The use of body fat percentage

(BF %) is more objective and may be more relevant for assessing increasing adiposity, especially in overweight

animals. The sensitivity of BF % versus 5-point BCS, for detecting increasing adiposity in overweight dogs, via

alterations to plasma metabolite values were compared against healthy animals. Overall, healthy BF % range was

determined to be 15-20% for non-neutered male dogs, and 15-25% for female (non-spayed/spayed) and neutered

male dogs. BCS categorized overweight animals displayed significantly higher levels of non-esterified fatty

acids (NEFA; p=0.005), whereas significantly higher levels of NEFA (p=0.006), Total cholesterol (T-Cho;

156

p=0.029), and Triglycerides (TG; p=0.001) were observed in BF % categorized overweight animals as compared

to healthy animals. BF % positively correlated with plasma insulin, NEFA, T-Cho, TG, Blood Urea Nitrogen,

creatinine, and Total Protein levels which all tend to increase as a result of increasing adiposity. BF % appears to

be more sensitive than 5-point BCS to detect for alterations in plasma metabolite values, especially lipid

metabolites, induced as a result of increasing adiposity in dogs. Therefore, 5-point BCS and BF % should

complement one another and be used in tandem, in order to better detect and diagnose overweight status in dogs,

allowing for possible early intervention and prevention of development of more advanced stages of obesity.

30. cDNA CLONING AND mRNA EXPRESSION OF DOG CDKAL1.

I. Yamamoto, A. Mori, H. Takemitsu, N. Mori, G. Li, K. Kawasumi, T. Arai. Nippon Veterinary and Life

Science University, Tokyo, Japan


Background: CDK5 regulatory subunit-associated protein 1 - like 1 (CDKAL1) gene encodes a protein that

regulates insulin secretion through the regulation of CDK5 activity. Objective: In this study, we performed

cDNA cloning of dog CDKAL1 to determine the gene structure and mRNA expression profile in tissues.

Methods: Total RNA was extracted from dog tissue using TRIzol reagent. The full cDNA cloning was

performed using 5’ and 3’ RACE PCR method. The mRNA expression profile was performed by Realtime PCR

analysis. Results: The dog CDKAL1 cDNA was consisted with 160 bp of 5’ – UTR, 1728 bp of ORF and 953

bp of 3’ – UTR. The predicted dog CDKAL1 amino acid sequence was compared to other animals and it

revealed high sequence similarity to cow (92.2 %), human (91.7 %), mouse (88.5 %), frog (83.6 %), salmon

(80.3 %) and chicken (80.2 %). Based on BLAST Genome analysis, dog CDKAL1 gene consisted with 15 exons

and revealed the presence of approximately 630 kb intron. The mRNA expression was detected in all examined

tissues and high levels in heart, skeletal muscle and testis in 2 year olds male beagle. Conclusion: These results

suggest that dog CDKAL1 gene is well conserved and may have tissue specific mRNA expression mechanisms

in dog.

31. DETECTION OF PROTEINURIA IN DOGS AND CATS – AGREEMENT BETWEEN AN IN HOUSE

DRY CHEMISTRY SYSTEM (AUTION MICRO ™) AND CONVENTIONAL WET CHEMISTRY

B. Rütgen, I. Schwendenwein. University of Veterinary Medicine Vienna, Vienna, Austria.


Background: Proteinuria is an early symptom of renal disease and is crudely assessed by dipstick or

precipitation methods. Determination of the urinary protein creatinine ratio (UPCR) allows quantification of

urinary protein losses without 24h urine collection. In house ssays for assessment of UPCR are not widely

available. Objective: Purpose of this study was to determine agreement between the UPCR results derived by

the Aution Micro™an in house dry chemistry system to conventional methods on a Cobas 501c autoanalyzer.

Methods: The Aution Micro™ (Menarini Diagnostics) measures urine creatinine and urine albumin by

reflectance photometry and calculates UPCR. Creatinine is determined by a palladium-dye complex reaction and

albumin by a dye binding method (TCTIF) which forms a pale pink complex. The reference methods were an

enzymatic method for creatinine and a turbidimetric method with benzethoniumchloride for protein on a Cobas

501c. Simultaneous analysis of 100 fresh canine urine samples and 81 feline samples was performed. Statistical

evaluation of agreement was performed by a kappa test as only semiquantitative results are obtained by the test

system. The cutoff for the reference method in dogs was 0.3 and 0.5in cats. Results: 87% of dogs and 81% of

cats were correctly classified by the test, which had a sensitivity of 85% in dogs and 80% in cats. Specificity was

89% in dogs and 81% in cats. Conclusions: The Aution Micro shows adaequate agreeability, sensitivity and

specificity to conventional methods for the semiquantitative assessment of proteinuria in dogs and cats.

32. HEME-MEDIATED BINDING OF ALPHA-CASEIN TO FERRITIN: EVIDENCE OF ALPHA-CASEIN

BINDING TO FERROUS IRON.

K. Orino, Y. Yoshikawa, K. Watanabe. Kitasato University, Towada-shi, Japan


Background and Objective: Iron-storage protein, ferritin, is not only present in all kinds of cells but also in

extracellular fluids such as serum, synovia, and milk. Ferritin binds heme as well as iron. Bovine α-casein was

identified as a ferritin-binding protein. This study was done to reveal the binding mechanism of α-casein with

ferritin. Methods: Casein protein coated-plates (20 pmol/well) were incubated with bovine spleen ferritin (1

157

pmol/well) followed by detection using biotinylated anti-bovine spleen ferritin antibody and ALP-labeled avidin.

To examine the binding mechanism of α-casein with hemin and metal ions, biotinylated hemin was used. Casein

protein coated-plates (4 pmol/well) were incubated with biotinylated hemin (32 pmol/well) followed by

detection using ALP-labeled avidin. Results: The binding of α-casein to bovine spleen ferritin was blocked by

hemin, but not by protoporphyrin IX or zinc-protoporphyrin IX. The binding of α-casein to ferritin and

biotinylated hemin was inhibited by ferrous ammonium sulfate (FAS). FAS-mediated inhibition of α-casein to

biotinylated hemin was neutralized with Ferrozine, but not with NTA, while FAS- as well as ferric chloride-

mediated inhibition was neutralized by NTA. The following ions also inhibited α-casein-biotinylated hemin

binding in order of potency of inhibition: Fe2+

, Fe3+

, Cu2+

, Zn2+

, Mn2+

, Ca2+

, Mg2+

. Conclusion: These results

suggests that the binding of α-casein to ferritin is heme-mediated through direct binding of α-casein to heme iron

on the surface of the ferritin molecule, and that α-casein preferentially binds Fe2+

compared with any other metal

ions, including Fe3+

.

33. C-REACTIVE PROTEIN AND SOME BIOCHEMICAL PARAMETERS IN FEMALE DOGS AFTER

OVARIOHYSTERECTOMY.

N. Hadzimusic. Veterinary faculty Sarajevo, Sarajevo, Bosnia-Herzegovina


Background: An increase in serum CRP concentration as well as changes in some biochemical parameters have

been described after different surgical procedures in dogs. Ovariohysterectomy is widely used as a method of

contraception and a large number of female dogs are exposed to surgery for neutering each year. Objective: The

aim of this study is to investigate CRP concentrations before and after ovariohysterectomy, as well as correlation

between CRP concentrations and some biochemical parameters (total protein, albumin, alkaline phosphatase,

alanine transaminase, aspartate transaminase and lactate dehydrogenase). Methods: Ten clinically healthy

female dogs were admitted to elective ovariohysterectomy. Blood samples were collected from distal cephalic

vein just before ovariohysterectomy (base values) and after 24 hours (Day 1), 48 hours (Day 2), 72 hours (Day 3)

and seven days (Day 7). Results: ThemeanserumlevelsofCRPonDay1(85.15 ±16.2 μg/mL) and Day 3 (31.27 ±

12.06 μg/mL) were significantlyhigher (P<0.001 for Day 1 and P<0.05 for Day 3)than the base value

(15.35±13.77μg/mL). No statistical difference was found between the base value and Day 7 (12.08 ± 4.91

μg/mL). There was no significant relationship between CRP levels and investigated biochemical parameters.

Conclusions: Present study shows that CRP changes rapidly, whereas maximum CRP concentration was

detected on the first day after surgery, while on the seventh postoperative day serum CRP concentration was

within physiological ranges. During postoperative period changes in CRP concentration and investigated

biochemical parameters showed no significant correlation.

34. COMPARATIVE STUDY OF PLASMA LIPIDS IN HEALTHY DOGS AND DOGS WITH SKIN

DISEASES.

R. Barrera1, P. Ruiz

1, C. Zaragoza

1, F.J. Duque

1, A. Buono

2.

1University of Extremadura, Cáceres, Spain;

2University of Bologna, Bologna, Italy


Background: The diagnostic protocol in canine dermatology includes determination of plasma lipids, which is

also used in the diagnosis of endocrine diseases. However, these parameters can also increase in dogs with

nonendocrine skin disorders. Objective: The aim of this study was to contribute to the interpretation of the levels

of plasma lipids in the diagnosis of skin diseases in canine medicine. Methods: 142 dogs were studied including

Group I (20 healthy dogs) and Group II (122 dogs with skin disease). Diseases included: hyperadrenocorticism

(16), hypothyroidism (7), demodectic mange (18), leishmaniasis (28), primary pyoderma (14) and allergy (39).

The plasma concentrations of total cholesterol, HDL-cholesterol, LDL-cholesterol and triglycerides were

determined. Results: When we compared the results obtained in the different diseases with the observed in

healthy dogs we found statistically higher concentrations (P < 0.001) in all parameters in both endocrine

diseases, a significant increase (P < 0.05) of LDL-c in the subgroup with demodectic mange, a significant

increase (P < 0.05) in the concentrations of total cholesterol, triglycerides and LDL-c in dogs with leishmaniasis

and, finally, a significant increase in the concentrations of total cholesterol (P < 0.001), triglycerides, HDL-c and

LDL-c (P < 0.05) in the dogs with allergy. Conclusions: The increase of the plasma concentrations of lipids and

lipoproteins is very frequent in skin diseases and not only in diseases of endocrine origin. The study of these

lipid alterations is very useful to diagnose the diseases affecting the skin. Study funded by Junta de Extremadura

(Spain).

158

35. RELIABILITY OF A SINGLE COLLECTION OF URINE SAMPLE IN DETERMINATION OF URINE

PROTEIN TO CREATININE RATIO.

G. Rossi1, A. Zatelli

2, S. Paltrinieri

3.

1University of Milan, Milano, Italy;

2Clinica Veterinaria Pirani, Reggio

Emilia, Italy; 3Department of Veterinary Pathology, Hygiene and Public Health, Milan, Italy


Background: Several studies stressed the importance of correctly identify and treat proteinuria in dogs. The

persistence of proteinuria is a key elements in the assessment of proteinuria. Therefore, proteinuric dogs should

be repeatedly tested to be classified as “persistently proteinuric” Nevertheless, no studies about the biological

variability of proteinuria in dogs are available. Objective: To assess the biological variability of proteinuria and

its possible influence on the classification of patients based on the International Renal Interest Society (IRIS)

staging. Methods: Urine samples were collected from 36 dogs. Exclusion criteria were: poorly controlled renal

disease, recent changes of treatments or diet; presence of concurrent diseases. Urine samples were collected

twice, at least two weeks apart, and centrifuged. Sediment analysis and measurement of the urinary

protein:creatinine (UPC) ratio were performed. Results of first and second sampling were compared to each

other using a Wilcoxon test either on the whole population or after grouping dogs according to the IRIS stage.

Results: Dogs were substaged as N.23=non-proteinuric, N.6=borderline proteinuric, N.7=proteinuric. No

significant differences were detected between the UPC ratio recorded at first and second sampling either in the

whole population (P=0.905) or in groups based on IRIS staging. (Stage I: p=0.821; Stage II: p=0.687; Stage III:

0.468). Nevertheless, in 8/36 cases the biological variability was high enough to induce a shift from non

proteinuric to borderline proteinuric. Conclusions: the biological variability of the UPC ratio in dogs with stable

renal conditions is low but it could affect the IRIS classification of patients.

36. MEASUREMENT OF SERUM PROGESTERONE IN MARES: A METHOD COMPARISON STUDY.

G. Lubas, A. Gavazza, A. Rota, D. Panzani, S. Demi. Dept Veterinary Clinic, University of Pisa, San piero a

Grado, Pisa, Italy


Background: Serum progesterone (P4) concentration in the mare can be used to determine the phase of the

estrous cycle and adds useful information in the establishment and maintenance of early pregnancy. Objective:

A comparison study used several methods/techniques to assess P4 values, including fluorescence enzyme

immuno assay (FEIA) (2 assays), chemiluminescence (2 assays), and liquid chromatography tandem mass

spectrometry (LC/MS). Methods: Forty serum samples from mares in different stages of estrus cycle or

pregnancy were collected and stored at -20°C until evaluation. P4 recovery study measured low, medium and

high concentrations used the FEIA marketed by Tosoh Bioscience (TB). All samples were initially assayed by

FEIA-TB. Fifteen samples including low, medium and high P4 values were used for comparison studies

(regression analysis, r2). Results: Recovery study for FEIA-TB showed results over 100% especially when

testing high P4 values. FEIA-TB had a very good agreement with the two chemiluminescence assays (r2 >.95),

while the two ELFA assays showed a lower r2 (.91). The lowest r2 (.87) was found between the FEIA-TB and

LC/MS values. FEIA0TB had a positive bias in comparison to all other methods. Conclusion: The P4

measurement using FEIA-TB showed good to excellent agreement when compared with different

methods/techniques. It should be pointed out that the FEIA-TB produced results consistently slightly higher than

other methods/techniques. A test-specific reference interval is recommended for clinical use in equine

reproduction.

37. ISCHEMIA MODIFIED ALBUMIN IN HORSES EXERCISED AT DIFFERENT INTENSITIES ON A

TREADMILL.

L.A. Yonezawa1, T.S. Barbosa

2, M.J. Watanabe

2, J.L. Knaut

2, C.L. Marinho

2, L.E.S. Michima

3, A.

Kohayagawa2.

1School of Veterinary Medicine and Animal Science, Sao Paulo State University, Sao Paulo,

Brazil; 2School of Veterinary Medicine and Animal Science, Sao Paulo State University, Botucatu, Brazil;

3School of Veterinary Medicine and Zootechny, University of Sao Paulo, Sao Paulo, Brazil


Background: Ischemia modified albumin (IMA) is a marker of myocardial ischemia in humans in contrast to

other biomarkers released only after cardiac necrosis. Little is known about release conditions of IMA in

exercise and this is the first report in equine species. Objectives: This study determined IMA for evaluation of

possible myocardial ischemic conditions induced by high and low intensity exercise in horses. Methods: Ten

159

clinically healthy horses were submitted to a high intensity test (HIT) and a low intensity test (LIT). Blood

samples were taken before, during and immediately after exercise, and 15 and 30 min thereafter. Serum IMA,

lactate, albumin, and plasma malondialdehyde (MDA) concentrations were determined. Results: There were no

significant changes (P>0.05) in IMA concentration in any of exercise tests. IMA levels ranged from 0.242±0.071

to 0.292±0.056 ABSU in HIT and from 0.246±0.026 to 0.277±0.021 ABSU in LIT. There was also a negative

correlation between IMA and albumin levels in both tests, and between IMA and lactate levels in LIT,

suggesting possible assay interferences. Conclusions: HIT and LIT did not promote significant changes in IMA

concentration in horsesin this study. Though literature data suggests that exercise does not produce myocardial

ischemia, we believe that it occurs. Further studies should be undertaken to determine if IMA is sensitive for

ischemic evaluation in equids in situations of exercise. Financial support: Sao Paulo State Research Foundation

(FAPESP), Brazil.

38. HIGH MOBILITY GROUP BOX 1 AS A PREDICTOR OF GASTRIC NECROSIS.

I. Uhrikova, L. Rauserova-Lexmaulova, K. Rehakova, I. Hajek, J. Doubek. University of Veterinary and

Pharmaceutical Sciences Brno, Brno, Czech Republic


Background: Gastric dilatation and volvulus syndrome (GDV) is an acute disease with high mortality. Gastric

rotation and dilatation leads to decreased venous return with development of shock and decreased perfusion of

the stomach wall. Gastric necrosis is serious complication of the disease and one of the major negative

prognostic factors. High mobility group box 1 (HMGB1) is a nuclear protein which is released during cell

necrosis. Objective: The aim of this study was to evaluate HMGB1 as an indicator of gastric necrosis. Methods:

Blood was collected from 36 dogs with GDV before surgical treatment. Serum was stored and HMGB1 analysis

was performed according to manufacturer instructions. Results: Twenty-nine dogs with GDV survived and

seven died due to gastric necrosis. From the group of survivors two dogs with gastric necrosis underwent partial

gastrectomy. There was no significant difference in HMGB1 concentration between survivors and non-survivors,

with mean HMGB1 concentrations of 6.0 ng/ml and 35.3 ng/ml, respectively. A significant difference in

HMGB1 concentration was found between patients with gastric necrosis (30.4 ng/ml) and others (5.5 ng/ml,

p=0.05). A cutoff value of 10 ng/ml had a sensitivity of 88% and specificity of 92% for the presence of gastric

necrosis. Sensitivity reached 100% at cutoff value of 6 ng/ml but specificity was only 63%. Conversely, 100%

specificity was obtained at a cutoff value of 28 ng/ml but sensitivity dropped to 44%. Conclusion: HMGB1 is a

candidate marker for detection of gastric necrosis in patients with GDV. Study was supported by Grant No.

24/2011/IGA VFU Brno.

39. COMPARISON OF THE SCHMIDT HAENSCH AND ATAGO PAL-USG REFRACTOMETERS FOR

DETERMINATION OF URINE SPECIFIC GRAVITY.

H. Tvedten1, Å. Noren

2.

1Swedish University of Agric. Sciences, Vange, Sweden;

2Strömsholm Specialist

Animal Hospital, Strömsholm, Sweden


Background: Our veterinarians complained that urine specific gravity results from our new Atago digital PAL-

USG refractometer (Atago) were too low. Materials and Methods: Specific gravity results of 60 paired patient

urine samples of dogs and cats analyzed with the Atago were compared to those of a Schmidt + Haensch model

HR12P refractometer (S+H). Additionally specific gravity of dilutions of 10 % glucose or 10 % Na Cl solutions

or highly concentrated patient urine samples were determined with both, hand held refractometers. Dilutions

were 100% to 10 % of original concentration. Results: Both refractometers reported 1.000 with distilled water.

Atago had a proportional negative error of a mean of 0.006 units. The Atago’s negative bias at a S+H urine

specific gravity of 1.040 was 0.012 units. Of 35 urine samples with readings from both instruments, 10 samples

had a S+H reading of > 1.030 while paired Atago results were 1.023-1.028. S+H results with glucose solution

dilution matched exactly expected values but Atago results were clearly less. S+H results with saline solution

dilution were closer to expected values but lower. Atago saline results were clearly less than expected or S+H.

Discussion and conclusions: Atago had a negative proportional error which would often adversely affect

determination of a clinically concentrated urine sample (> 1.030 dog, > 1.035 cat). The highest S+H reading was

1.040, thus dilution of urine would be needed to determine higher values.

160

40. EFFECT OF PHENOBARBITAL THERAPY ON SELECTED LABORATORY PARAMETERS: A

RETROSPECTIVE STUDY (2007-2012).

I. Hajek, P. Schanilec, I. Uhrikova. Veterinary and Pharmaceutical University Brno, Brno, Czech Republic.


Background: It is well known that specific drugs alter clinical biochemistry results. Phenobarbital as an

antiepileptic medicament significantly increases levels of liver enzymes in humans as well as in dogs and cats.

Magnitude of these changes is variously described in dogs and unknown in cats. Objective: The aim of this

study was to evaluate effect of phenobarbital therapy on hematological and biochemical profile. Methods: Data

from hematology and clinical biochemistry of patients medicated with phenobarbital due to epilepsy were

collected. Total number of 32 dogs and 4 cats was divided into two groups. Group A consists of 20 dogs and 4

cats with blood withdraws at the beginning of medication and one or more repeated blood collections from 1

week to 9 months of medication and group B of 12 dogs treated with phenobarbital for more than one year. Dose

of phenobarbital was from 1.6 to 6.8 mg/kg/day. Results: In group A, significant effect of phenobarbital

medication was noticed on ALP activity (p=0.02) with mean increase of 1.4 ukat/l and platelet count (p=0.03)

with mean increase of 19.109/l. No significant change was found in ALT activity, concentration of albumin, total

protein, creatinin, urea, total leukocyte or erythrocyte counts. Mean ALT and ALP activity in dogs in group B

were 1.46 and 3.67 ukat/l, respectively. In cats only albumin significantly decreased (p=0.01), but all samples

were collected within 2 months after beginning of medication. Conclusion: Clinically relevant changes in

biochemical profile were found only in alkaline phosphatase activity in dogs.

41. PROTEOMICS APPLICATION IN SERA: DIFFERENCES BETWEEN VACCINATED AND

NONVACCINATED SHEEP.

J. Mavromati1, G. Mazzuchelli

2.

1Veterinary Medicine Faculty, Tirana, Albania;

2University of Liege, Mass

Spectrometry Laboratory GIGA, LIEGE, Belgium


Background: The proteomes of laboratory-grown strain Rev 1 and 16M of Brucella melitensis, are investigated,

but no proteomic study about sheep serum after vaccination has been conducted yet. Objective: The main

purpose of this study is to identify protein biomarker candidates of the sheep vaccination process in serum.

Methods: The study was conducted in a flock of sheep free of brucellosis and in another flock vaccinated with

Rev. 1, B. melitensis through the conjunctival route, in Albania. After the preparation of the serum samples in the

mass spectrometry laboratory–GIGA - Proteomics, University of Liege, Belgium during November 2011, a

differential proteomic study using a label free LC-MSe method was conducted (nano-2D UPLC coupled to a

Synapt TM HDMS TM MSe, Waters). Results: The numbers of identified proteins were about 50 and 110

respectively in each sample pool using sheep NCBI data base and reviewed mammalian data base. The results,

especially by using the homology identification method against the mammalian database, show interesting

findings. Proteins were identified from other taxonomies indicating that these protein sequences are not yet

available in the NCBI sheep database but most probably present in the sheep serum. Conclusion: Some protein

activity seems to have been modified between the two sheep populations. This was shown by the fact that

numbers of proteins partners from the same biochemical pathway were found to be over expressed or modulated

only in the group of vaccinated sheep. These results need to be further investigated.

42. COMPARISON OF URINE PROTEIN / CREATININE RATIO IN URINE SAMPLES COLLECTED BY

CYSTOCENTESIS AND MANUAL COMPRESSION IN CATS – PRELIMINARY RESULTS.

H. Vilhena1, R. Santos

2, T. Sargo

3, T. Lima

4, S. Dias

4, F.L. Queiroga

3, A.C. Silvestre-Ferreira

3.

1Escola

Universitária Vasco da Gama, Oliveirinha - Aveiro, Portugal; 2Escola Universitária Vasco da Gama, Coimbra,

Portugal; 3Departamento de Ciências Veterinárias - Universidade Trás-os-Montes e Alto Douro, Vila Real,

Portugal; 4Policlínica Veterinária de Aveiro, Aveiro, Portugal


Background: The urine protein/creatinine (UPC) ratio is generally considered reliable only in urine samples

collected by cystocentesis. In dogs, previous studies demonstrated the efficacy of quantification of proteinuria in

urine samples obtained by free catch when compared with samples collected by cystocentesis. Objective: To

determine if, in cats, the results for UPC ratios determined in urine samples collected by manual compression

provide similar diagnostic information to that obtained in samples collected by cystocentesis. Methods: Twenty-

five client-owned cats were included in the study. Three ml of urine from the midstream phase of micturition

were collected by manual compression, and 3 ml of urine were obtained by ultrasound guided cystocentesis in all

161

cats. A complete urinalysis was performed in all samples, and pre and post-renal proteinuria was excluded.

Determination of concentration of protein and creatinine was performed by Biuret and Jaffé methods,

respectively. Cats were classified as non-proteinuric (UPC < 0.2, n = 6), borderline proteinuric (UPC 0.2 – 0.4; n

= 9) or proteinuric (UPC > 0.4; n = 10), according to the IRIS (sub) staging system. Results: The correlation

between UPC ratios in urine samples collected by manual compression and cystocentesis was strong (0.99). All

cats had UPC ratios from both methods of collection that resulted in classification in the same IRIS substage.

Conclusions: The results obtained suggest that collection of urine samples by manual compression in cats is a

reliable alternative to cystocentesis in determination of UPC ratios. Nonetheless, more studies are needed due to

the low number of animals analyzed.

43. EVALUATION OF SOME SERUM PARAMETERS AFTER OVARIECTOMY IN THE RAT.

R. Mirzaei1, B. Amoughli Tabrizi

2, A. Balila

3.

1Official authority of Tehran veterinary network, Iran veterinary

organization, Tehran, Iran; 2Department of Pathobiology, Faculty of Veterinary Medicine, Islamic Azad

University-Tabriz Branch, Tabriz, Iran; 3Faculty of Veterinary Medicine, Islamic Azad University-Tabriz

Branch, Tabriz, Iran


Osteoporosis is a systemic skeletal disease characterized by decrease bone mass and micro architectural

deterioration of bone tissue, In this study in order to evaluate the serum concentration of calcium, phosphorus,

alkaline phosphates and magnesium during osteoporosis, 80 female Sprague-Dawley rats with approximate age

of 10 weeks were allocated to 10 groups (3 control, 3 sham, 3 treatment groups and one pure control group).The

animals in treatment group were ovariectomized under anesthesia whereas only the abdominal wall was incised

in the sham groups using the same approach of the treatment groups.Evaluation of the serum levels of calcium,

phosphorus and alkaline phosphates revealed statistically significant difference between the ovariectomized

groups and control groups and also pure control group and sham groups. The highest level of calcium and

phosphorus was observed 5 weeks after ovariectomy and the lowest level at 12 and 21 weeks after ovariectomy.

The results of this study show that osteoporosis can be created by Ovariectomy in rat.

HEMATOLOGY

ORAL PRESENTATIONS

KINETICS AND CHARACTERISTICS OF FELINE PLATELET AGGREGATION IN VITRO.

B. Riond1, A.K. Waßmuth

1, S. Hartnack

2, R. Hofmann-Lehmann

1, H. Lutz

1.

1Clinical Laboratory, Vetsuisse

Faculty, University of Zurich, Zurich, Switzerland; 2Section of Epidemiology, Vetsuisse Faculty, University of

Zurich, Zurich, Switzerland


Background: Feline platelets (PLT) are prone to aggregation in vitro after blood collection rendering difficult

the determination of accurate PLT counts in the cat. No information is available about the kinetics of PLT

aggregate formation in feline EDTA blood samples and the course of PLT counts over a clinically relevant time

period of 24 hours after collection. Objectives: The aim of this study was to investigate the kinetics of PLT

aggregate formation and the course of PLT and WBC counts in feline EDTA blood samples with respect to the

variables “time after blood collection”, “anesthesia”, “bleeding conditions” and “glucose concentration”.

Methods: From 30 clinically healthy cats blood samples were analyzed over a period of 24h on the Sysmex XT-

2000iV, the Mythic 18, by the use of a counting chamber and by blood smear evaluation. Results: Fourteen

(Sysmex XT-2000iV) and 12 (Mythic 18), respectively, of 30 samples were thrombocytopenic at one to eight

time points after collection. Subsequently all thrombocytopenic samples returned to normal PLT counts.

Seventeen of 30 samples had PLT aggregates in the counting chamber. Significant differences in PLT counts

were associated with presence and size of aggregates, time after bleeding, and glucose concentration, but not

with quality of blood collection and anesthesia. Conclusion: PLT aggregation and pseudothrombocytopenia

occurred in 57% of the samples at different time points and with different duration. Deaggregation of feline PLT

aggregates could be demonstrated as a reversible effect of PLT aggregation. Quality of blood collection seems

not to be causative for PLT aggregation.

162

EFFECTIVE PREVENTION OF PSEUDOTHROMBOCYTOPENIA IN FELINE EDTA BLOOD SAMPLES

WITH THE PROSTAGLANDIN I2-ANALOGUE ILOPROST.

B. Riond1, A.K. Waßmuth

1, S. Hartnack

2, R. Hofmann-Lehmann

1, H. Lutz

1.

1Clinical Laboratory, Vetsuisse

Faculty, University of Zurich, Zurich, Switzerland; 2Section of Epidemiology, Vetsuisse Faculty, University of

Zurich, Zurich, Switzerland


Background: Prostaglandin I2 (PGI2) is the most powerful endogenous inhibitor of platelet (PLT) aggregation

but is unstable. Iloprost is a stable PGI2 analogue. Objectives: The aims of this study were to (1) evaluate the

antiaggregatory effect of Iloprost on feline PLT in EDTA blood samples from clinically healthy cats; (2)

investigate the effect of Iloprost on hematological parameters; (3) determine the stability of Iloprost in K3-EDTA

tubes for up to 16 weeks. Methods: From 20 clinically healthy cats blood was drawn from the jugular vein and

immediately distributed into a 1.3 ml K3-EDTA tube and two 1.3 ml K3-EDTA tubes containing 20 ng and 200

ng Iloprost, respectively. A CBC was performed on the Sysmex XT-2000iV and the Mythic 18 at eight

consecutive time points after collection. Blood smears were evaluated for the presence of PLT aggregates.

Results: In the absence of Iloprost, pseudothrombocytopenia was observed in 50% of the investigated samples

that led to significantly decreased optical PLT counts by a mean of 105 x10³/µl. Pseudothrombocytopenia was

prevented by the addition of Iloprost, which led to an increase in PLT counts by a mean of 108 x10³/µl.

Conclusion: A strong antiaggregatory effect of Iloprost in feline EDTA blood was demonstrated. In all cats,

pseudothrombocytopenia was prevented by adding Iloprost to EDTA tubes prior to blood collection. Iloprost

also prevented falsely increased WBC counts in samples with PLT aggregates analyzed on impedance based

instruments. Other hematological parameters were not influenced to a clinically significant degree.

EXPLORATIVE REFERENCE INTERVALS FOR BIOCHEMISTRY AND HAEMATOLOGY DATA IN

HEALTHY PIGLETS DURING THE FIRST 27 DAYS OF LIFE.

B. Rütgen1, E. Hooijberg

2, L. Schwarz

1, H.L. Worliczek

1, I. Schwendenwein

1.

1University of Veterinary

Medicine Vienna, Vienna, Austria; 2Labor InVitro, Vienna, Austria.


Background: Pigs are kept primarily for meat production but also serve as models in medical research. Object:

Data derived from serial blood sampling of a sentinel group of piglets in an experimental setting were compiled

to establish exploratory reference intervals for healthy piglets during their first 27 days of life. Methods: Twelve

piglets from two crossbred strains were serially sampled on day 0 before first colostrum intake and then on days

1, 7, 14, 21 and 27. Bodyweight was recorded simultaneously. CBC was performed by the ADVIA120™,

complemented by microscopy. Glucose, fructosamine, urea, creatinine, TP, fibrinogen, albumin, AST, ALT,

GLDH, GGT, lipase, CK, electrolytes, minerals and iron were measured by standardized methods. Statistical

analysis was performed by analyze-it™ software. Results: Body weight followed the expected growth curve

confirming health status. RBC parameters showed a significant decrease from day 0 to day one followed by a

constant increase. TWBC showed a constant increase. A highly significant increase in glucose, fructosamine,

urea, TP, albumin and fibrinogen from day 0 to day 1 was observed. Urea decreased again from day 1 to day 7

and remained stable thereafter. Creatinine was high at birth and decreased significantly on day 1 to remain stable

until day 27. Liver and pancreatic enzymes AST, ALT, GGT, CK and lipase showed a significant peak after

colostrum intake whereas GLDH was stable throughout the study period. Conclusion: These data confirm

previously published data from young piglets and follow the expected patterns which occur due to colostrum

intake, growth and physical activity.

THE “QUATREFOIL” ERYTHROCYTES: UN-EXPECTED PATTERN IN CANINE RED BLOOD CELL

MORPHOLOGY.

A. Gavazza, G. Lubas, M. Ricci, B. Gugliucci, A. Pasquini. Dept Veterinary Clinic, University of Pisa, Pisa,

Italy


Background: A variety of causes can modify RBC shape including regenerative response, altered metabolism

(iron and lipid above all, but also electrolyte depletion), oxidative injury, immune-mediated damage, mechanical

fragmentation, sepsis, toxins and drugs. Objective: The aim of this study was to confirm and investigate the

occurrence of an unusual RBC pattern (so-called “quatrefoil RBCs” or “Pisa cross”) in dog blood smears.

Methods: 3,958 blood smears from 2,016 dogs, stained with Diff-QuickÒ, were examined microscopically from

May 2009 to October 2010. To exclude the possibility of EDTA artifact, some smears were prepared from whole

163

blood without anticoagulant. Results: “Quatrefoil RBCs” were found in 3.89% canine blood smears (154 of

3958) and 6.89% of dogs (139 of 2016). Indeed, 14 dogs out of 139 presented this RBC pattern in replicate

samples. Statistically significant differences (p< 0.05) including increased age, anisocytosis, and polychromasia

were found in dogs with quatrefoil RBCs than from dogs with normal morphology. The effective presence of

this new erythrocyte pattern was confirmed both by optical and scanning electron microscopy (SEM).

Conclusion: RBCs quatrefoil represents an adhesion between two erythrocytes, which is different from both

rouleaux (organized linear arrays or chains of RBCs) and agglutination (un-organized clumping or clusters). It

was not possible to identify the etiology and clinical relevance of “quatrefoil” morphology. Some hypotheses

were advanced to explain this particular RBC arrangement.

PLATELET INDICES IN VIRULENT CANINE BABESIOSIS AND THEIR ASSOCIATION WITH

OUTCOME.

A. Goddard1, A. Kristensen

2, A. Leisewitz

1, P. Thompson

1, J. Schoeman

1.

1University of Pretoria, Pretoria,

South Africa; 2University of Copenhagen, Copenhagen, Denmark


Background: Thrombocytopenia, without clinical evidence of hemorrhage, is a consistent finding in virulent

canine babesiosis. Objective: To investigate platelet indices in babesiosis, caused by Babesia rossi, and their

association with outcome. Method: Admission EDTA whole blood samples were analyzed on the ADVIA 2120

from 68 Babesia-infected and 6 healthy control dogs. Indices included: Platelet count (PLT), mean platelet

volume (MPV), platelet volume distribution width (PDW), thrombocrit (PCT), mean platelet dry mass (MPM),

mean platelet component concentration (MPC) and platelet component distribution width (PCDW). Indices were

also determined at 24 and 48 hours in admitted dogs that survived. Results: Of the Babesia-infected dogs, 30

were treated as outpatients and 38 admitted to the hospital of which 9 died within 24 hours (13%). The following

indices were significantly different between Babesia-infected and control dogs: median PLT (32 vs. 259 ×109/L;

p<0.001); median MPV (15.7 vs. 9.8 fL; p<0.0001); median PDW (60.9 vs. 55.9%; p=0.005); median PCT (0.05

vs. 0.24%; p<0.001); mean MPM (2.3 vs. 1.8 pg; p=0.003) and mean MPC (19.3 vs. 21.6 g/dL; p<0.0001).

There were no significant differences between outpatient and admitted dogs, or dogs that died and dogs that

survived. PLT and MPC showed significant increases at 24 hours (p=0.012 and p=0.009, respectively) and 48

hours (p<0.0001 and p=0.01, respectively). Conclusion: Large, degranulated platelets may play a significant

procoagulant role in the lack of clinical hemorrhage, despite severe thrombocytopenia, observed in canine

babesiosis. Platelet activation as measured by MPV and MPC does not appear to be associated with outcome.

D-DIMER MEASUREMENT BY LATEX AGGLUTINATION;COMPARISON

OF A SEMIQUANTITATIVE AND QUANTITATIVE METHOD AND EVALUATION OF THE CLINICAL

VALUE OF THE TEST IN DOGS.

J. Archer, A. Pastorello, P. Monti, P. Kuczybska. Department of Veterinary Medicine University of

Cambridge, Cambridge, U.K.


D dimer measurement is widely used to exclude thromboembolic disease in dogs. Recently several kits for it’s

semi quantitative measurement have been discontinued. Objectives:1) comparison of a quantitative automated

method and a manual semi quantitative method.2) restrospective study of clinical outcomes. Methods: D dimer

concentrations in the same plasma sample from 45 dogs were measured with a semiquantitative (SQ) latex

agglutination kit and an automated quantitative (Q)particle enhanced immunoturbidimetric assay Retrospective

analysis of results and clinical outcome in 700 dogs. Results: SQ values of <250ng/mL corresponded to a range

of 0.03-0.11ug/mLQ: <1000ng/mL SQ to 0.21-1.07 ug/mL Q 2000->2000 ng/mL SQ to 0.24-1.17 ug/mL Q.

demonstrating that there was correlated between SQ and Q with some outliers. Retrospective study of 700 dogs:

dogs with thromboembolic disease and IMHA had the highest D dimer values (> 1000ng/mL), 23% of those

with thromboembolic disease had values >2000ng/mL, while 33% of dogs with malignant neoplasms had values

above 2000 ng/mL. Conversely of the dogs with thromboembolic disease 18% and coagulopathies 23 % had

values<250 ng/mL.Dogs with renal and respiratory diseases 68% -73% had values < 250 ng/mL. Conclusions:

moderate correlation between SQ and Q D dimer assays. Weak correlation between D dimmers and clinical

condition.

164

DIAGNOSTIC PERFORMANCES OF FLOW CYTOMETRIC DETECTION OF BLOOD AND BONE

MARROW INFILTRATION IN DOGS WITH LARGE B-CELL LYMPHOMA.

V. Martini1, M.E. Gelain

2, S. Comazzi

1.

1University of Milan, Milan, Italy;

2University of Padua, Padua, Italy.


Background: Canine lymphoma is classified as “stage V” by WHO clinical staging system when neoplastic

cells are present in blood, bone marrow (BM) or any other non-lymphoid tissue. Cytological examination is

commonly used, but procedures are not standardized. Some authors prefer more sensitive techniques such as

flow-cytometry (FC) or molecular techniques but no official cut-off for positive samples were defined. Recently,

some authors found that the presence of >10% large lymphocytes at blood smear examination had a 60%

sensitivity and a 87% specificity in detecting >10% BM infiltration. Objective and methods: The purpose of

this work was to determine if FC is a more sensitive tool compared with morphology alone in blood and BM

involvement assessment in 60 dogs with large B-cell lymphoma. Agreement between BM infiltration assessed

via cytology and FC was evaluated and a cut-off value for cytometric blood involvement suitable for predicting

>10% bone marrow infiltration was calculated. Results: A moderate agreement between BM infiltration via

cytology and FC was found. 13% of circulating neoplastic cells was identified as the ideal cut-off with 65%

sensitivity and 100% specificity to predict BM infiltration. Conclusion: Based on these results, flow cytometry

could be considered not only a more specific but also a more sensitive tool than morphological evaluation alone

for canine large B-cell lymphoma staging. Further clinical studies are needed including more cases, other

lymphoma subtypes and follow-up information to establish the possible prognostic value of bone marrow

involvement and a cut-off clinically useful.

DOUBLE ERYTHROCYTE POPULATIONS: RETROSPECTIVE STUDY ON CASES OBSERVED WITH

SYSMEX XT-2000iV (2008-2012).

N. Bourgès-Abella, B. Cuq, F. Granat, A. Geffré, C. Hanot, C. Trumel. Institut National Polytechnique-

Ecole Nationale Vétérinaire de Toulouse, Toulouse, France


Background: The Sysmex XT-2000iV is based on an impedance cell counter to measure erythrocyte count and

indexes. The standard erythrogram is a right skewed curve. In rare cases, abnormal curves were observed with

two well separated peaks which seemed to represent two different erythrocyte subpopulations. Objective: The

aim of this study was to determine how frequent this abnormality was and the associated outcomes. Methods:

Hematology reports of the Laboratoire Central de Biologie Médicale were reviewed (four-years period) for well

separated double peaks on erythrograms as the only inclusion criteria. Smears and clinical data from the cases

were then studied from the National Veterinary School data base. Results: Nine cases were identified out of the

19266 CBC performed: five cats, three dogs and a cow. Two animals (one cat and the cow) had been previously

transfused. The remaining cats and the dogs had a marked regenerative anemia but the reticulocyte count did not

fit with the second peak. In cats, one was positive for FIV and Mycoplasma haemofelis, one had cholangio-

hepatitis, and two had an immune mediated haemolytic anemia of unknown origin. In dogs, this erythrogram

abnormality was associated with microcytosis. One was a weaning puppy and the other cases were cases of

chronic bleeding. Only one animal died: it was a cat transfused with dog blood. Conclusion: This retrospective

study shows that a two-peak erythrogram is very uncommon, associated with marked regenerative anemia or

transfusion errors, and has no prognostic significance.

LEEK DIET MAY CAUSE HEMOLYTIC ANEMIA: A CASE REPORT IN A CAT.

N. Bourgès-Abella, F. Granat, A. Geffré, C. Trumel. Institut National Polytechnique-Ecole Nationale

Vétérinaire de Toulouse, Toulouse, France


Case presentation: A 7 year-old neutered female domestic shorthair cat was presented because of the ingestion

of a bone. No abnormalities were noted at clinical examination, and an abdominal radiography showed the

presence of a bone in stomach. A medical treatment based on a leek-based high fiber diet and non steroidal anti-

inflammatory drugs was decided to try naturally eliminating it. At the following presentation, eight days later, no

abnormality in the comportment of the cat was reported by the owners. At the clinical presentation, the cat did

not present abdominal pain, but the mucosa were pale. Abdominal radiography and ultra-sound suggested that

the bone had been eliminated. Complete blood cell count, blood film, biochemical, hemostatic and urine analysis

were performed. Results showed an intense hemoglobinuria and hemoglobinemia, and an anemia associated with

the presence of many Heinz bodies on blood smear. A diagnosis of intra-vascular hemolytic anemia induced by a

165

severe oxidative stress with formation of Heinz bodies was made. Leek ingestion was stopped and a medical

antioxydant treatment was prescribed. The anemia and hemoglobinuria had disappeared fifteen days later.

Interpretation: A very high suspicion of intra-vascular hemolytic anemia induced by a severe oxidative stress

due to the ingestion of leek was made. Discussion & conclusion: Leek is a plant of the same family as onion,

known to induce an oxidative stress in animals. Practitioners should be aware of leek toxicity before long-term

prescription of a high fiber diet based on leek to facilitate the elimination of foreign bodies.

GASTRIC DILATATION AND VOLVULUS SYNDROME – ARE THERE ANY CLUES IN

HEMATOLOGIC EXAMINATION?

I. Uhrikova, L. Rauserova-Lexmaulova, K. Rehakova, I. Hajek, J. Doubek. University of Veterinary and

Pharmaceutical Sciences Brno, Brno, Czech Republic


Background: Gastric dilatation and volvulus syndrome (GDV) is a life-threatening disease associated with

gastric rotation and dilatation. Prognosis depends on duration of disease, degree of gastric rotation and presence

of complications, but we are unable to determinate final outcome before surgical treatment. Objective:

Hematologic examination is inexpensive and widely reachable laboratory method. The aim of our study was to

evaluate changes in hematologic examination in patients with GDV in connection to final outcome and selected

proteins. Methods: Blood was collected from 39 dogs with GDV before (A), after surgery (B), 6-10 hours (C)

and 20-24 hours (D) after surgery. Routine hematologic examinations with blood smear evaluations and

measurements of C-reactive protein (CRP) and high mobility group box 1 (HMGB1) were performed. Results:

Analyses were performed on 142 samples obtained from 31 survivors and 8 non-survivors. Frequent hematologic

abnormalities such as leukopenia (0-29%), leukocytosis (3-66%), anemia (28-47%) and thrombocytopenia (56-

82%) were present. Significant differences between survivors and non-survivors were found in total leukocyte

counts (A, mean 11.1 vs. 16.1.109/l), neutrophils (A, 9.6 vs. 13.7.10

9/l), eosinophils (A, B; 0.14 vs. 0.03.10

9/l,

0.17 vs. 0.06.109/l) and platelet counts (B, 144 vs. 85.10

9/l). Dakryocytes decreased significantly in survivors,

whilst in non-survivors were not significantly changed (A vs. B). HMGB1 moderately correlates with segments

(A, r=0.45, p<0.01) and CRP with bands (D, r=0.44, p<0.05). Conclusion: Hematologic examination can

provide additional information to patient prognosis. Study was supported by Grant No. 24/2011/IGA VFU Brno.

EFFECT OF STORAGE CONDITIONS ON KAOLIN/ NONKAOLIN-ACTIVATED

THROMBOELASTOGRAPHY AND HEMOSTATIC PARAMETERS IN CATS.

R. Col1, C.M. Iazbik

2, G. Couto

2.

1Selcuk University, Konya, Turkey;

2Department of Veterinary Clinical

Sciences, The Ohio State University, Columbus, OH, United States of America


Background: Fresh-frozen plasma (FFP) and frozen plasma (FP) are used therapeutically in cats; however, there

are no reports regarding the hemostatic activity of these plasma products. Objective: In this study, our aim was

to evaluate the dynamics of hemostasis in fresh and stored plasma using TEG, hemostasis assays, and activities

of factors V, VIII, IX, X. Methods: Whole blood was collected in CPDA from cats (n=45) and a plasma

extractor was used to obtain plasma. We evaluated fresh plasma (FR) (<1 hour storage), FFP (<1 year storage),

and FP (>1 year storage). TEG was done using calcium chloride or kaolin as activators in a TEG-5000. OSPT;

APTT; fibrinogen concentration (FIB); and factor V, VIII, IX, X activities were measured on an automated

coagulation analyzer. Results: The R and MA in FFP and FP were significantly different from FR when using

kaolin or CaCl2. The OSPT and APTT were significantly longer in FFP and FP than in FR, and the FIB was

significantly lower in both; interestingly, the activity of factor V (but not VIII and X) was significantly lower in

FP than in FR, and in FP than in FFP; factor IX activity was significantly lower in FP than in FR. Conclusions:

Storage of feline plasma for transfusion had a significant effect on hemostasis variables as measured by TEG and

conventional assays. Although, as expected, factor V activity decreased significantly over time, factor VIII

activity did not. Factor IX activity did not decrease significantly in FFP, but was significantly lower in FP,

suggesting this may be a labile factor in cats.

166


44. CHARACTERIZATION OF CANINE BONE MARROW MESENCHYMAL STEM CELLS

C. Tejero1, L.K. Ostronoff

2, M.L. Fermín

3, C. Fragio

3, L.G. León

4, E. Kremmer

5.

1Universidad

Complutense de Madrid, Facultad de Veterinaria, Madrid, Spain; 2Department of Biochemistry and Molecular

Biology IV, Veterinary Faculty Madrid, Madrid, Spain; 3Department of Animal Medicine and Surgery,

Veterinary Faculty Madrid, Madrid, Spain; 4Institute of Bio-Organic (IUBO-AG),La Laguna, La laguna, Spain;

5Institut für Immunologie, GSF-Forschungszentrum Umwelt und Gesundheit , München, Germany


Background: Adult Mesenchymal Stem Cells (MSC) were initially described as an adherent non-hematopoietic

cell type that could form fibroblastic colonies in vitro. Currently, these cells can be defined as multipotent cells

able to differentiate into osteogenic, adipogenic and condrogenic lineages under appropriate conditions.

Objective: Our aim was to expand, and assess cell surface markers, of canine bone marrow derived

Mesenchymal stem cells. Methods: Four healthy 2-year old female Beagle dogs were used as marrow donors.

Mononuclear cells were cultured in IMDM medium with 10% DS and incubated at 38 ºC with 5% CO2, after

48hours the nonadherent cells were discarded. After obtention of a confluent MSC layer, adherent cells were

harvested with trypsin/EDTA and replated at: 700cells /cm2 with or without SCF. Cells were harvested at first,

second and third passages, and incubated with (FITC)-conjugated CD90, FITC-conjugated CD34, FITC-

conjugated CD45 and FITC- conjugated MHCII. Results: In vitro growth of MSC proceeds as an initial lag

phase, followed by a rapid expansion. Between first and second passage the cell number was multiplied

approximately 28-fold. Dot plot of stained MSC after incubation with primary antibodies revealed that these

cells had positive results for CD90, and negative for MHCII. The lack of expression for CD34 and CD45 showed

that cells of hematopoietic origin had been excluded during the cell expansion process. Conclusion: In

conclusion, we have isolated and characterized a canine bone marrow derived mesenchymal stem cell

population. Further investigations will make them promising candidates for use in regenerative medicine.

45. FIBRINOGEN CONCENTRATION MEASUREMENT IN DOGS: A COMPARISON BETWEEN THE

QBC-VET AUTOREADER AND MC1PLUS SEMIAUTOMATIC COAGULOMETER.

L.V. Athanasiou1, T.A. Petanides

2, M.K. Chatzis

1, M.N. Saridomichelakis

1,

1Faculty of Veterinary Medicine,

University of Thessaly, Karditsa, Greece; 2Veterinary Center of Thessaloniki, Thessaloniki, Greece


Background: Fibrinogen is a glycoprotein that plays a key role in blood clotting and is a non-specific indicator

of inflammation. The most widely used techniques for measuring plasma fibrinogen concentration include heat

precipitation (Millar’s technique) and modified thrombin clotting time measurement (Clauss method). Both have

been automated in a haematology analyzer (QBC-Vet Autoreader, IDEXX; Millar’s technique) and in a newly

developed semi-automatic mechanical and optical detection coagulometer (MC1 Plus, DIASys Greiner GmbH;

Clauss method). Objective: Our aim was to compare the above mentioned techniques for the measurement of

plasma fibrinogen concentration, to establish a reference interval for the new coagulometer, and to detect canine

diseases associated with hyperfibrinogenemia. Methods: Fibrinogen concentration was measured in the blood of

85 clinically normal dogs and 43 dogs presenting clinical and/or clinicopathological abnormalities that have been

previously associated with dysfibrinogenemia. The results of the two techniques were compared using Passing-

Bablock’s regression and Bland Altman difference plots. Results: No correlation was found between the results

of the two techniques. A reference interval (112-688 mg/dl) was calculated for the Clauss method following

NCLS standards. Hyperfibrinogenemia was observed in dogs with various diseases, either alone or in

combination, including leishmaniosis, monocytic ehrlichiosis, dirofilariosis, autoimmune diseases (e.g.

pemphigus foliaceous, immune mediated hemolytic anemia), inflammatory conditions (e.g. urinary tract

infection, pyometra, discospondylitis, peritonitis, fever of unknown origin), chronic liver disease and neoplasia.

Conclusion: The two techniques cannot be used interchangeably, thus a different reference interval should be

used for assessment of fibrinogen concentration in the MC1 Plus using the Clauss method.

167

46. REFERENCE INTERVALS FOR HEMATOLOGIC PARAMETERS OF ARABIAN HORSES.

A. Rocky, S. Gooraninejad, M. Razi Jalali, M. Pourmahdi, F. Naghashpour. Faculty of Veterinary

Medicine, Shahid Chamran University, Ahvaz, Iran


Background: Laboratory results are interpretated by using species-specific reference intervals, but breed, age,

and sex could influence the interpretation of results. Objective: The aim of this study was to establish reference

intervals for hematologic parameters of clinically healthy Arabian horses and to evaluate the influence of age

and sex on these parameters. Methods: A total of 255 clinically healthy female and male Arabian horses from

Khuzestan province, Iran were included in this study. Blood samples taken from jugular vein were collected into

tubes containing EDTA and immediately transferred to the Clinical Pathology Laboratory. Blood samples were

analyzed using a BC-2800Vet hematology analyzer (Mindray, China). The animals were divided into 3 age

groups: < 2 years, 2-5 years, and > 5 years old. Reference intervals for 17 hematologic analytes were expressed

as 2.5 and 97.5 percentiles. Results: Significant differences (P<0.05) primarily between < 2 and > 5 year old

horses were seen in 9 hematologic analytes: WBC, lymphocytes, RBC, hemoglobin, hematocrit, MCV, MCH,

MCHC and MPV. Significant differences (P<0.05) were also seen between male and female horses in 4

hematologic analytes: WBC, lymphocytes, hemoglobin and MPV. Conclusion: Breed-specific reference

intervals for Arabian horses will better display normal characteristics of this breed. The results of this study

showed that age has a major influence on hematologic parameters of Arabian horses and could affect

interpretation of CBC results in this breed.

47. A NOVEL POINT MUTATION IN THE BETA1-TUBULIN GENE IN ASYMPTOMATIC

MACROTHROMBOCYTOPENIC NORFOLK AND CAIRN TERRIERS.

M.E. Gelain1, W. Bertazzolo

2, G. Tutino

2, E. Pogliani

2, F. Cian

3, M.K. Boudreaux

4.

1University of Padua,

Padua, Italy; 2Veterinary Clinic “Tibaldi”, Milan, Italy;

3Department Veterinary Medicine, University of

Cambridge, Cambridge, United Kingdom; 4Department of Pathobiology, College of Veterinary Medicine,

Auburn University, Auburn Alabama, United States of America


Asymptomatic inherited macrothrombocytopenia is a well recognized condition in Cavalier King Charles

Spaniels (CKCS), due to a point mutation in the beta1-tubulin gene which affects proplatelet formation and

platelet production. Recently, a phenotypically similar asymptomatic macrothrombocytopenia was described in

Norfolk terriers (NT) and isolated cases were also reported in Cairn terriers (CT). Our aim was to evaluate the

presence of a genetic defect in the beta1-tubulin gene in macrothrombocytopenic Norfolk and Cairn terriers.

Genomic DNA was harvested from peripheral EDTA-anticoagulated blood samples from 20 healthy NT (N=13)

and CT (N=7) collected at different institutions in Italy (N=8), the United Kingdom (N=3) and the United States

(N=9). Primers were designed to assess all coding areas and exon–intron splice sites on the canine beta1-tubulin

gene. PCR products were electrophoresed on agarose gels and target bands were sequenced. None of the samples

had the CKCS mutation. Twelve dogs (9NT and 3CT) showed a single nucleotide polymorphism (SNP) in exon

1 at nucleotide position 5 (G5A) that would result in the change of an arginine to a histidine at amino acid

position 2 (R2H). Four dogs (3NT and 1CT) were heterozygous for the SNP and 4 (1NT and 3CT) showed a

normal sequence. All dogs with the SNP were macrothrombocytopenic, while heterozygous and normal dogs

had normal platelet counts and morphology. The tubulin N-terminal amino acids form the nucleotide binding

domain: it’s possible that the amino acid change at position 2 (R2H) could affect GTP binding enough to

influence platelet formation only in homozygous dogs.

48. DETERMINATION OF COMPLETE BLOOD COUNT REFERENCE INTERVALS IN PIRENEAN

MOUNTAIN DOGS.

A. Pasquini, I. Annibali, G. Biagi, B. Gugliucci, G. Lubas, A. Gavazza. University of Pisa, San Piero a Grado

(PI), Italy


Background: Though important diagnostically, few studies of the different haematological characteristics in

different dog breeds exist. The purpose of this study was to examine the complete blood count (CBC) of

Pyrenean Mountain Dog and to establish breed-specific RIs as recommended in the Clinical and Laboratory

Standards Institute (CLSI) guidelines. Materials and methods: 133 CBCs of healthy subjects were examined.

The analyses were carried-out using impedance cell-counter (HeCoVet, SEAC, Firenze, Italy), the leukocyte

differential count and morphological evaluation were performed by microscopic examination of modified

168

Wright–stained blood smears. RBC, HGB, HCT, MCV, MCHC, RDW, PLT, WBC, differential leukocyte count,

RBC morphology was quantitatively measured and PLT count estimated). Data were analyzed by multifactorial

ANOVA post hoc and Bonferroni multiple comparison, including the sex and age effects. The haematological RI

values for the Pyrenean Mountain Dogs were obtained in accordance with the CLSI recommended validation

procedure.Results: The CBC of PMD showed some peculiarities concerning a few haematologic measurements

and erythrocyte morphology (poikilocytosis). The standard RIs were validated for all CBC parameters except

hematocrit (HCT), Mean Corpuscolar Volume (MCV) and eosinophils, (PMD vs. standard RI, respectively 34.8-

49.5 vs 37-55 (%), 59-70 vs 60-76 (fL) and 0.0-2.1 vs 0.1-1.7 (× 109/L). Conclusions: The development of new

reference intervals partitioned by breed should be used for the correct clinical interpretation of laboratory data in

dogs.

49. CANINE BONE MARROW CYTOLOGICAL EXAMINATION: A RETROSPECTIVE STUDY OF 295

CASES.

V. Turinelli1, A. Gavazza

2, G. Lubas

2. 1

Idexx Laboratories Italy, Livorno, Italy; 2Dept. veterinary clinic,

University of Pisa, Pisa, Italy.


Background and objective: Cytological bone marrow evaluation is a simple exam, indicated when peripheral

blood abnormalities are detected, to stage neoplastic conditions, to search for parasitic infection or occult

diseases in animals with fever of unknown origin, weight loss and unexplained malaise. A suitable sample is

necessary for accurate assessment of the bone marrow. Methods: This retrospective study was conducted

between January 2010 and March 2012. Two hundred and ninety-five bone marrow smears were classified using

cytological and numerical criteria. Samples were collected from dogs referred for several disorders and coming

from Italy (71%), Germany (22%), The Netherlands, Switzerland, Spain, Poland, Norway and Sweden (7%).

Results: The cytological exam allowed classification of the bone marrow samples as: normal 19.3%; hyperplasia

25.1% (generalized 1.4%; megakaryocytic 6.8%; erythroid 33.8%; granulocytic 58.1%); hypo/aplasia 7.8%

(aplastic anemia 47.8%; megakaryocytic 8.7%; erythroid 30.4%; granulocytic 13%); dysplasia 1.7%

(myelodysplastic syndrome 20%, dysmegakariopoiesis 20%, dyserithropoiesis 40%, dysgranulopoiesis 20%);

lympho-plasmacytic hyperplasia 1.4%; hematopoietic malignancy 12.9%; metastasis 0.3%; miscellaneous 1%;

not diagnostic 30.5%. Conclusion: This study outlines the great incidence of both hyperplasia (granulocytic) and

malignant hemopathy (leukemia and lymphoma) and the rarity of metastasis. It is noteworthy to observe the high

number of non-diagnostic results due to several causes (poor quality of aspiration, hemodilution, smears

damaged by formalin fume). The cytologic bone marrow examination can provide information about

pathogenesis of abnormalities found in blood and often the etiological diagnosis can be made. Final conclusions

and interpretations must include other laboratory results and peripheral blood smear examination.

50. A RETROSPECTIVE STUDY OF INCIDENCE AND CLASSIFICATION IN FELINE BONE MARROW

CYTOLOGICAL EXAMINATION.

A. Gavazza1, V. Turinelli

2, G. Lubas

3. 1

University of Pisa, San Piero a Grado, Pisa, Italy; 2Idexx Laboratories

Italy, Milan, Italy; 3Dept Veterinary Clinic, University of Pisa, Pisa, Italy.


Background: The bone marrow cytological evaluation is often be used in cats affected by non-regenerative

anemia, persistent neutropenia, thrombocytopenia, and to assess the involvement in neoplastic conditions, or to

identify suspected infectious agents (e.g. Toxoplasma gondii). Peripheral blood cytopenias are frequently

observed in FIV-FeLV infected cats, where a loss of normal cellular composition of bone marrow is

documented. Objective and methods: The study was conducted between January 2010 and March 2012.

Seventy-one bone marrow smears were classified using cytological and numerical criteria. The majority of the

cases were from Italy (97%) and a few from Germany (3%). Results: The cytological exam allowed

classification of the bone marrow samples as: normal 5.6%; hyperplasia 36.6% (generalized 3.8%;

megakaryocytic 3.8%; erythroid 33.8%; granulocytic 58.1%); hypo-aplasia 12.7% (aplastic anemia 66.7%;

megakaryocytic 0%; erythroid 22.2%; granulocytic 11.1%); dysplasia 7% (myelodysplastic syndrome 40%,

dysmegakariopoiesis 0%, dyserithropoiesis 20%, dysgranulopoiesis 40%); lympho-plasmacytic hyperplasia

1.4%; malignant hemopathy 7%; metastasis 0%; miscellaneous 2.8%; not diagnostic 26.8 %. Conclusion: These

findings outline the wide incidence of hyperplasia (granulocytic) and of hypo-aplasia. Granulocytic hyperplasia

occurs most frequently in response to bacterial infections. Ineffective granulocytic hyperplasia, with increased

number of immature granulocyte precursors and decreased of mature neutrophils, frequently occurs in

myelodysplastic disorders especially common in neutropenic cats with FIV/FeLV infections. In order to make a

169

proper conclusive report from bone marrow examination a CBC should be performed the same day of bone

marrow aspiration.

51. USE OF THE MCDh* STAINING FOR HAEMATOLOGY OF THE DOG, CAT, HORSE AND CATTLE :

PRELIMINARY RESULTS.

N. Bourgès-Abella, C. Hanot, C. Marche, J.P. Braun, C. Trumel. Institut National Polytechnique-Ecole

Nationale Vétérinaire de Toulouse, Toulouse, France


Background: Micro Chromatic Detection for haematology (MCDh) (RAL diagnostics) is a new stain used in

human haematology. Although it is a fast-acting stain, it gives red metachromatic staining to azurophil granules.

Moreover, MCDh is methanol-free, which ensures higher safety for users. Objective: The aim of this

prospective double-blind study was to evaluate if MCDh gives a range of colors similar to May-Grünwald

Giemsa staining and permits to identify morphological abnormalities commonly encountered in veterinary

haematology. Methods: After determination of a standard staining procedure for pets, horse and cattle smears,

the validation of the MCDh staining was performed in 3 steps on animals of each species (dog, cat, horse and

cattle) by two experienced operators: 1/ Validation of staining times. Color and morphology of cellular

components were assessed by comparison with data from literature and ranked on a scale of 0 to 3. 2/ Method

comparison between MCDh and May-Grünwald Giemsa stainings for WBC differential and platelet counts,

according to CLSI recommendations. 3/ Method comparison between MCDh and May-Grünwald Giemsa

stainings for usual morphological abnormalities of blood cells (polychromasia, parasites, toxic neutrophils,…).

Results: Mean scores (n=46) were significantly higher (p<0.05) in MCDh smears for platelet morphology and

counts than in May-Grünwald Giemsa. Conclusion: Preliminary results suggest that MCDh may improve

platelet assessment.

52. INCREASED ERYTHROCYTE OSMOTIC RESISTANCE IN DOGS WITH SINGLE EXTRAHEPATIC

PORTOSYSTHEMIC SHUNT.

K. Rehakova1, I. Uhrikova

1, L. Rauserova–Lexmaulova

1, J. Lorenzova

1, L. Stehlik

1, O. Skor

2, J. Doubek

1.

1University of Veterinary and Pharmaceutical Sciences Brno, Brno, Czech Republic;

2Veterinary clinic, Bila

hora, Prague, Czech Republic


Background: Alterations in red blood cell deformability due to modification of membrane lipid composition

occurring in chronic liver disease are well known from human medicine but in dogs are not described.

Objective: The aim of the study was to investigate changes in erythrocyte osmotic resistance in dogs with

congenital portosystemic shunt. Methods: Twelve dogs with single extrahepatic portosystemic shunt confirmed

by surgical exploration were introduced. Osmotic fragility test with complete blood counts and liver

biochemistry profile including ammonia and total bile acids concentrations were compared with those of 30

healthy dogs. Absorbance in osmotic fragility test was assessed by ELISA reader ELX 808. Results: Dogs with

single extrahepatic portosystemic shunt had significantly increased (p<0.001) erythrocyte osmotic resistance

with 50% and 90% hemolysis corresponding to 0.35% and 0.3% NaCl solution, respectively. Target cells related

to more resistant cell population were seen directly on the blood smear in 50% of dogs, while increased osmotic

resistance against NaCl solution causing 50% hemolysis in 75%. Moreover, none of 12 dogs with PSS had

increased osmotic fragility in any degree of hemolysis. No correlation was found between mean corpuscular

volume, ammonia, bile acid or cholesterol levels and erythrocyte osmotic fragility. Conclusion: Due to

alterations in lipid metabolism in atrophic liver, dogs with congenital portosystemic shunt have significantly

more resistant red blood cells against hypotonic hemolysis suggesting their impaired deformability functions.

53. COMPARISON OF THROMBOTIC MICROSCOPIC PLATELET COUNTING METHOD TO THE

LEUKOPLATE MICROSCOPIC METHOD, ADVIA 2120 AND IDEXX VETAUTOREAD™ PLATELET

METHODS.

H. Tvedten1, J. Ljusner

2, I. Lilliehöök

2.

1Swedish University of Agricultural Sciences, Vange, Sweden;

2Swedish University of Agricultural Sciences , Uppsala, Sweden


Introduction: Thrombotic microscopic platelet counting method is stated to cause disaggregation and rounding

of platelets for more precise microscopic enumeration. Methods: Thrombotic platelet counts (PLT) of 21 feline

170

blood samples were compared to PLT determined with a Leucoplate stain method or Advia 2120. Additionally

PLT were determined in 8 canine bloods with IDEXX VetAutoread™. Precision was determined with 1 canine

blood sample analyzed 10 times. Results: Precision (CV %) was Advia 3.2 %, QBC 15.3 %, Thrombotic 7.8 %,

Leucoplate 9.0 %. Median PLT of 21 feline blood samples were 202 x 109/L Thrombotic, 238 x 10

9/L

Leucoplate and 156 x 109/L Advia. Median canine PLT were 277 x 10

9/L Thrombotic, 241 x 10

9/L Leucoplate,

267 x 109/L Advia and 289 x 10

9/L QBC. In only 5 of 21 feline samples were no aggregates seen in either of the

2 counting chambers or blood smears. More aggregates seen in Thrombotic counting chambers with 8 of 21

feline bloods compared with Leucoplate. In no sample were more aggregates seen in the Leucoplate method.

Conclusions: Precision was best with the Advia and acceptable for the 2 manual methods. QBC had worse

precision but actually measures platetcrit which is then converted to a platelet count. Platelet counts with the

Thrombotic method were comparable with the Leucoplate manual method. The Thrombotic method had more

feline platelet aggregates than the Leucoplate method. Manual methods gave higher PLT counts than Advia,

which suggests manual methods may be somewhat more accurate with blood containing platelet aggregates.

54. FACTOR VIII LEVELS IN A GROUP OF BELGIAN SHEPHERD MALINOIS DOGS BREED IN ITALY.

G. Lubas1, A. Gavazza

2, M. Caldin

3.

1University of Pisa, San Piero a Grado, Pisa, Italy;

2University of Pisa,

Dept. Veterinary Clinic, San Piero a Grado, Pisa, Italy; 3San Marco Veterinary Laboratory, Padova, Italy


Occurrence of Hemophilia A in the Belgian Shepherd Malinois (BSM) dog has been recently described. This is

the most common and severe inherited canine coagulopathy. Diagnosis is based on clinical signs, prolonged

aPTT, and reduction in factor VIII activity. The study aim was to assess the level of FVIII in BSM dogs in Italy.

Fifty-four studbook registered BSMs have been investigated (34 males, 20 females), in which Hemophilia A was

a possible condition. For each BSM, the genealogical data and, whenever possible, the history and other clinical

data were collected. All BSMs were tested for FVIII concentration using two reference labs. When possible, the

FvW concentration in females was also assessed (carrier FVIII/FvW <0.60). Most of the data are displayed in a

free accessible web site (www.malinemo.net). Ten male dogs had low level of FVIII (range 5-21%, consistent

with Hemophilia A), four females showed FVIII between 28-55% (carrier range), and 21 dogs resulted with

FVIII level >70%. Nineteen dogs showed FVIII level at the borderline either for carrier or clear range. The

present work supports the evidence of Hemophilia A in BSM dogs, with a relatively high frequency.

Unfortunately, the level of FVIII alone does not allow a clear set between carrier and clear dogs. Only the

complete clinical examination, together with laboratory data, should rule-out concurrent disorders (i.e. liver

failure or acquired coagulopathy). Further studies on factor VIII gene mutations using offspring to correct

identify the Hemophilia A in BSM dogs are in progress.

55. PROTHROMBINE TIME MEASUREMENTS BY A NOVEL POINT OF CARE DEVICE IN CANINE

WHOLE BLOOD.

P. Vajdovich1, J. Antal

2. 1

Szent István University, Faculty of Veterinary Science, Budapest, Hungary; 2Diagon

Kft. Hungary, Budapest, Hungary.


Background: A point of care device was developed for in house prothrombine time (PTT) measurement using

whole blood. This device works by turbidimetric method to detect PTT and whole blood can also be used.

Objective: The aim of the study was to evaluate the applicability of this point of care device in canine plasma

and whole blood samples. Methods: Samples from 15 dogs with various PTT values were analysed using the

point of care device and the results were compared to those of a 2 channel semi-automatic photo optical ball

coagulometer using Thromborel S (Behring) reagent . Results: Statistically significant correlation was found

between the reference method (mean: 11.6 sec) and the plasma results (mean: 7.5 sec, r=0.845), moreover whole

blood (mean: 8.6 sec, r=0.924) results of the point of care device. Plasma and whole blood results measured by

the point of care device also correlated (r=0.935). Precision CV is 4.2%, the bias of plasma results: -35.4%,

whole blood results -25.6%. Conclusion: Point of care devices can be used if supported by evidence-based

medicine. Whole blood analysis of coagulation is suitable before any surgical intervention or sampling method

together with bleeding time measurement. The device which we used for the analyis has to be adapted for the

required reference standard results.

http://www.malinemo.net/

171

56. RETICULOCYTE PARAMETERS IN DOGS WITH LEISHMANIASIS. A PRELIMINAR STUDY.

J. Pastor, A. Meléndez-Lazo, C. Miret, M. Planellas, M. Mesalles. Universitat Autònoma de Barcelona,

Bellaterra, Spain.


Background: The pathophysiological mechanisms of anemia in animals with clinical leishmaniasis is not

completely understood and may be multifactorial. Reticulocyte parameters such as reticulocyte Hgb content

(CHr) and reticulocyte MCV (rMCV) have been proven more useful in detecting early iron deficiency anemia

than conventional hematologic and biochemical indices. Few reports have been published studying CHr and

rMCV changes in infectious and chronic inflammatory diseases. Objetive: The aim of this study is to compare

erythrocytes and reticulocyte parameters (RBC, HGB, HCT, VCM, RDW, HDW, reticulocyte count, rMCV,

CHr) between dogs with clinical leishmaniasis and healthy dogs. Methods: EDTA blood samples were obtained

from 9 clinical leishmaniasis dogs no treated previously and 23 healthy Beagle dogs serological and PCR

negative for Leishmania sp. Blood samples were analyzed with the ADVIA 120 automated hematology analyser

within 12 hours from blood collection. Results: 5/9 ill animals presented with a non regenerative anemia

(HCT<35%). Statistically significant differences between ill and healthy animals were found for RBC, HGB,

HCT, RDW, reticulocytes count and rMCV (p<0.05). Animals with leishmaniasis had lower RBC, HGB, HCT,

reticulocyte count and higher RDW and rMCV than healthy dogs. Conclusion: Animals with leishmaniasis have

statistically significant increases in red blood cell anisocytosis and have inhibition of erythropoiesis that cause a

decrease in reticulocytes release and reticulocyte-related macrocytosis. This may be due to the effect of

inflammatory cytokines on erythropoiesis.

57. BLOOD PARAMETERS OF CIKA CALVES AND BULLS.

M. Klinkon1, G. Gorjanc

2, Z. Klinkon

3, J. Ježek

1, M. Simčič

2.

1University of Ljubljana, Veterinary faculty,

Ljubljana, Slovenia; 2University of Ljubljana, Biotechnical Faculty, Department of Animal Science, Domžale,

Slovenia; 3Veterinary clinic Klinkon, Radomlje, Slovenia


Background: Cika is the only indigenous cattle breed in Slovenia that was in the past mainly used for milk

production. Nowadays, its milk yield is too low compared to modern milk breeds. Therefore, farmers rear Cika

cows mainly in the cow-calf system. Objective: The aim of this study was to compare blood valuesof Cika cattle

partitioned by age. Methods: Study included 36 blood samples of 18 Cika bulls. Each animal was sampled twice

in its life; at weaning (calf) and at slaughter (fattened bull). Calves originated from different farms, but were

fattened together in the Educational research centre Logatec. The average age of calves at sampling was 7.5

months while the average age of bulls at slaughter was two years. The number of erythrocytes (RBC), leucocytes

(WBC), and platelets (PLT) and values of haemoglobin (Hb), haematocrit (Ht), mean corpuscular volume

(MCV), mean corpuscular haemoglobin (MCH), and mean corpuscular haemoglobin concentration (MCHC)

were measured with the haematological analyser, ABC Vet. Collected data were analyzed with MIXED

procedure (SAS) considering age as a linear regression and animal to account for repeated measurements.

Results: The mean values of blood values showed that Hb, Ht, MCV, and MCH significantly increased, while

RBC and PLT significantly decreased from weaning (calves) to slaughter time (bulls). WBC and MCHC stayed

unchanged. Conclusion: Despite the fact that age significantly affected some blood values, the values were

within laboratory reference values for cattle.

58. COMPARISON OF THE ABC VET AND BC 2800 VET VETERINARY HAEMATOLOGICAL

ANALYZERS.

M. Nemec, M. Klinkon, J. Ježek. Veterinary Faculty, Clinic for Ruminants, Ljubljana, Slovenia


Background: We have compared two veterinary haematological analyzers made by different manufacturers and

with the same principles of operation. Objective: The aim of the study was to evaluate the usefulness of the BC

2800 Vet analyzer in veterinary medicine. Methods: The ABC Vet was used as a reference analyzer; it is in

routine use, regularly calibrated and controlled, and included in the international inter-laboratory comparison.

Five consecutive analyses of control samples were performed at low, normal and high levels to determine the

repeatability of measurements on the BC 2800 Vet analyzer. To determine the reproducibility of the analyzer, we

used daily measurements of control blood samples. To compare analyzers, we took 76 blood samples from

different animals (13 cows, 12 sheep, 13 pigs, 11 horses, 11 dogs, 9 cats and 7 rabbits) and comparative analyses

were performed. The agreement between the results was assessed with the statistical method developed by Bland

172

and Altman. Results: In BC 2800 Vet good repeatability and reproducibility was established at all levels for

most parameters (CV <5 %). Applying the statistical method of agreement, we found appropriate comparability

between analyzers in the normal operating range for most parameters; less comparable results were found for

haemoglobin and platelets. On average, the ABC Vet analyzer gives slightly higher results for most parameters

while the WBC count is slightly lower than with the BC 2800 Vet. Conclusion: The BC 2800 Vet has proved to

be suitable for use in veterinary medicine for domestic species.

59. CANINE CBC ANALYSIS WITH THE LASERCYTE AND PROCYTE DX AUTOMATED

HEMATOLOGY ANALYZERS.

D. DeNicola1, M. Haynes

1, C. Lee

1, J. Hammond

1, M. Wellman

2, G. Couto

2.

1IDEXX Laboratories, Inc.,

Westbrook, United States of America; 2The Ohio State University, Columbus, United States of America


Background: The IDEXX LaserCyte and ProCyte Dx Hematology Analyzers provide CBC results including a

5-part differential and reticulocyte count. As a component of constant product development/improvement,

LaserCyte performance enhancements related to optimized fluidic sequence modifications have been developed.

Objective: The aim of this study was to evaluate the LaserCyte optimized sequence enhancements. Methods: 30

fresh canine samples were analyzed on the LaserCyte and ProCyte Dx within four hours of collection and within

one hour between instruments. Results: Correlation factors (R) and slopes (m) were calculated for each least

squares regression as follows: RBC (R=0.99; m=0.99), HGB (R=0.97; m=1.00), HCT (R=0.98; m=0.99), PLT

(R=0.98; m=1.00), WBC (R=0.99; m=1.00) and NEU (R=0.99; m=0.88). Remaining parameters were evaluated

by % bias and results are as follows: MCV (1%), RETIC (6%), LYM (8%), MONO (12%) and EOS (3%).

Conclusion: Strong correlations were shown for RBC, HGB, HCT, PLT, WBC and NEU. Calculated slope

values indicate accurate results between instruments. MCV, RETIC, LYM, MONO, and EOS all had data sets

with small dynamic range. Least squares regression analysis performs poorly with this type of input; therefore %

bias calculations were performed showing strong performance for MCV, RETIC and EOS, and a slight bias for

LYM and MONO. Further analysis with a larger data set will provide greater insight into each of these

parameters.

60. CANINE RETICULOCYTE ANALYSIS WITH THE LASERCYTE AND PROCYTE DX AUTOMATED

HEMATOLOGY ANALYZERS.

D. DeNicola1, C. Lee

1, M. Haynes

1, J. Hammond

1, J. Christian

2, J. Knoll

3, M. Scott

4, H. Wamsley

5.

1IDEXX Laboratories, Inc., Westbrook, United States of America;

2Purdue University, West Lafayette, United

States of America; 3Tufts University, North Grafton, United States of America;

4Michigan State University, East

Lansing, United States of America; 5University of Florida, Gainesville, United States of America


Background: Automated in-house hematology analyzers have been used to enumerate reticulocytes for many

years. The IDEXX LaserCyte analyzer relies on stained cells with precipitated RNA to selectively scatter light

while the ProCyte Dx analyzer utilizes a specific RNA fluorescence stain for reticulocyte identification. Each

method requires specific algorithm optimization. Objective: The aim of this study was to compare a newly

developed canine reticulocyte algorithm for the LaserCyte to the ProCyte Dx florescence method. Methods:

Two LaserCyte analyzers and one ProCyte Dx analyzer were installed at each of the following veterinary

colleges: Tufts University, University of Florida, Purdue University, and Michigan State University. 100 fresh

canine samples were analyzed at each location within four hours of draw and within one hour between

instrument runs. A least squares regression analysis, fixed at the origin, was performed to determine correlation

between the two analyzer responses. Results: Integrating the LaserCyte response for all eight LaserCyte

analyzers and the correlated ProCyte responses from the four ProCyte analyzers yielded an aggregated

correlation coefficient, R=0.86. The newly optimized LaserCyte canine reticulocyte algorithm yields results that

closely match ProCyte Dx with an aggregate regression slope of 0.9 across all analyzers. Conclusion: The newly

optimized LaserCyte canine reticulocyte algorithm provides results that correlate well to the fluorescent RNA-

specific methodology of the ProCyte Dx.

173

61. PORCINE HEMATOLOGY EVALUATION ON THE IDEXX PROCYTE DX HEMATOLOGY

ANALYZER.

D. DeNicola, J. Chase, J. Hammond, J. Russell. IDEXX Laboratories, Inc., Westbrook, United States of

America


Background: The IDEXX ProCyte Dx Hematology Analyzer is approved for the following species: Canine,

Feline, Equine, Bovine, and Mustaline. This study aims to support the addition of the mini-pig as a newly

supported species. Objective: This study was to evaluate the results of 100 Yucatan mini-pigs on IDEXX

ProCyte Dx as compared to the Sysmex XT-V analyzer. Methods: 100 porcine EDTA anticoagulated samples

were evaluated on four ProCytes and one XTV-V. In addition, manual packed cell volume measurements were

performed and compared to LaserCyte results. Results: Correlation coefficients were calculated from least

squares regression analysis. Correlation factor (R) and Slope (m) results from one representative LaserCyte

compared to the XT-V were as follows: RBC (R = 0.98; m= 1.01), HCT (R= 0.98; m=1.01), HGB (R=0.99;

m=0.99), MCV (R= 0.99; m=1.05), WBC (R=0.98; m=0.82), and PLT (R=0.96; m=0.89), PCV (R=0.83;

m=1.00.). Conclusion: Correlations greater than 0.95 for CBC parameters demonstrate strong correlation

between ProCyte DX and XT-V as well as ProCyte Dx HCT to manual PCV. Based on slope results, no

significant biases were noted for any of the parameters evaluated. These findings support the addition of this

species on the ProCyte Dx analyzer.

62. AGE-ASSOCIATED ALTERATION IN PERIPHERAL LEUKOCYTE SUBPOPULATION AND ITS

CYTOKINE SYNTHESIS RESPONSIBILITY TO INSULIN IN BEAGLE DOGS.

M. Fujiwara1, H. Otsuka

2, T. Yonezawa

3, I. Yamamoto

1, T. Arai

1.

1Nippon Veterinary and Life Science

University, Musashino-shi, Tokyo, Japan; 2Hiromichi, Kitasato University, Musashino-shi, Tokyo-to, Japan;

3Kitasato University, Towada-shi, Aomori-ken, Japan


Background: The immune system is considered to be affected by aging, which is linked with various immune

pathogenesis. Insulin plays an important role to maintain energy metabolism in T lymphocytes. It might have

some effect on the age-related change of immune dynamics. Objective: We observed changes in cytokine

production and insulin reactivity of PBMCs to determine the responsibility to insulin on the age-associated

immune alterations. Methods: We analyzed the relationship between peripheral leukocyte subpopulations and

their ages. Dogs were divided into three groups; young (< 4 y), middle-aged (4-8 y) and older dogs (> 8 y). After

the incubation of PBMCs with concanavalin A (Con A, 5.0 mg/ml) and/or insulin (1 mg/ml), the mRNA levels

of various cytokine genes were analyzed using a real-time PCR. Results: There were significant negative

correlations between their ages and the number of PBMCs. Notably, the number of naïve lymphocytes decreased

significantly with age. When Con A was added, the mRNA level for IL-2, IL-2Rα and IL-4 was significantly

increased in young group, whereas only IFNg and/or IL-4 were increased with aging. When insulin and Con A

were added, IL-2 mRNA was decreased in young group. Furthermore, IL-4 mRNA increased and IFNg/IL-4

ratio was decreased with aging. Conclusion: Taken together, this study demonstrated that the number of naïve

lymphocytes decreased and the cytokine synthesis from Th1 to Th2 age-dependently. Moreover, insulin-induced

Th2 conversion becomes to be more effective with aging, and these changes could be involved in age-dependent

immune degradation in dogs.

63. HEME-MEDIATED BINDING OF ALPHA-CASEIN TO FERRITIN: EVIDENCE OF ALPHA-CASEIN

BINDING TO FERROUS IRON.

K. Orino, Y. Yoshikawa, K. Watanabe. Laboratory of Veterinary Biochemistry, School of Veterinary

Medicine and Animal Sciences, Kitasato University, Aomori, Japan.


Background and objective: Iron-storage protein, ferritin is not only present in all kinds of cells but also in

extracellular fluids such as serum, synovia, and milk. Ferritin binds heme as well as iron. Bovine α-casein was

identified as a ferritin-binding protein. This study is to reveal the binding mechanism of α-casein with ferritin.

Methods: Casein protein coated-plates (20 pmol/well) were incubated with bovine spleen ferritin (1 pmol/well)

followed by the detection using biotinylated anti-bovine spleen ferritin antibody and ALP-labeled avidin. To

examine the binding mechanism of α-casein with hemin and metal ions, biotinylated hemin was used. Casein

protein coated-plates (4 pmol/well) were incubated with biotinylated hemin (32 pmol/well) followed by the

detection using ALP-labeled avidin. Results: The binding of α-casein to bovine spleen ferritin was blocked by

174

hemin, but not by protoporphyrin IX or zinc-protoporphyrin IX. The binding of α-casein to ferritin and

biotinylated hemin was inhibited by ferrous ammonium sulfate (FAS). FAS-mediated inhibition of α-casein to

biotinylated hemin was neutralized with Ferrozine, but not NTA, while FAS- as well as ferric chloride-mediated

inhibition in their interaction was neutralized by NTA. The following ions also inhibited α-casein-biotinylated

hemin binding in order of potency of inhibition: Fe2+<<Fe3+<Cu2+<Zn2+<Mn2+<Ca2+<Mg2+. Conclusion:

These results suggests that the binding of α-casein to ferritin is heme-mediated through direct binding of α-

casein to iron in the heme on the surface of ferritin molecule, and that α-casein preferentially binds Fe2+

compared with any other metal ions, including Fe3+.


ORAL PRESENTATIONS

PRELIMINARY REPORT OF HEMOSTATIC PARAMETERS IN THE EXPERIMENTAL GÖTTINGEN

MINIPIG.

M. Defontis1, S. Côté

2, M. Stirn

2, D. Ledieu

2.

1Justus-Liebig University, Giessen, Germany;

2Basel,

Switzerland


Background: The animal model, Göttingen minipig, is gaining popularity in toxicity testing, however, more

information concerning hemostatic parameters is needed. Objective: The aim of this study was to optimize

whole blood Multiplate® aggregometry and to provide preliminary values for TEG variables in the Göttingen

minipig. Methods: Citrated and heparinized whole blood samples were obtained from 20 healthy male Göttingen

minipigs (5-8 months of age) who were kept in controlled housing conditions and had not received any

medication known to have an effect on platelet function for 15 days. For Multiplate® aggregometry, the agonists

ADP, collagen, arachidonic acid (AA), Par-4 agonist and TRAP-6 were used in a wide concentration range to

determine their EC50 in heparinized blood. TEG measurements were performed in duplicate on calcium-activated

citrated blood. Mean and range values (minimum-maximum) are reported for the following TEG variables: R, K,

MA and G. For the parameter angle, median and range values are given. Intra-assay repeatability was calculated

as the overall coefficient of variation. Results: The concentrations of TRAP-6 and Par-4 agonists tested failed to

induce an aggregation response in all animals. The calculated EC50 in heparinized blood were 0.8µM ADP,

0.25µg/ml collagen and 0.06mM AA. Preliminary values for the TEG variables were: R= 5.3 (2.0-9.9) min; K=

1.35 (0.8-2.5) min; angle= 72.6 (49.8-78.4) degrees; MA= 74.6 (66.5-80.9) mm and G= 15.2 (9.9-21.2) d/sc.

Overall CVs ranged between 1.8% and 13.6%. Conclusion: The platelet agonists TRAP-6 and Par-4 agonist

cannot be used for whole blood aggregometry in the Göttingen minipig.

AFLATOXINS IN SLOVENE MILK AND FEED SAMPLES.

B. Jakovac-Strajn, G. Tavčar-Kalcher, I. Ujčič-Vrhovnik, K. Pavšič-Vrtač, K. Fon-Tacer, A. Vengušt.

Veterinary faculty, Ljubljana, Slovenia


Background: Aflatoxins are secondary metabolites of moulds, predominantly Aspergillus sp., contaminating

diverse food and feed materials. Among the aflatoxins (B1, B2, G1,G2) aflatoxin B1 is the most toxic, both for

humans and animals, and is a potent carcinogen. Its metabolite aflatoxin M1 appears in milk and milk products as

the result of intake of aflatoxin B1- contaminated feed. Objective: The aim of this work is to present the results

of analyzes, where for the first time the Slovene feed and milk samples contained aflatoxins in the concentrations

above limits given in the legislation. Methods: For the determination of aflatoxin B1 and M1, the procedures

consisting of sample immunoaffinity column cleanup and liquid chromatography were used. 243 milk samples

and 53 feed samples were investigated. Results: The maximum level for aflatoxin M1 in consumable milk given

in the legislation (50 ng/kg) was exceeded in 23 samples of milk. The concentrations were from 50 to 3554

ng/kg. 47 samples contained aflatoxin M1 in the concentrations from 5 to 50 ng/kg. Among 53 different feed

samples analyzed, 20 samples contained aflatoxin B1 in concentrations higher than maximum permitted levels

(from 0.02 to 0.663 mg/kg). 24 samples contained from 0.0002 to 0.020 mg/kg aflatoxin B1. Conclusion:

Although the exposure to aflatoxins is generally considered to occur mainly from imported materials, the

possibility of contamination of European agricultural products cannot be entirely excluded. In consideration of

the carcinogenic properties of aflatoxin B1, human exposure should be reduced to levels as low as reasonable

achievable.

175

IS ALKALINE PHOSPHATASE ACTIVITY IN BOVINE NASAL SECRETION A RESULT OF LOCAL

SYNTHESIS?

M.F. Ghazali, N.N. Jonsson, P.D. Eckersall. University of Glasgow, Glasgow, United Kingdom


Background: Investigation of bovine nasal secretion has revealed a higher activity of alkaline phosphatase

(ALP) than that found in serum, but it is not clear whether ALP is synthesised in the nasal tissue. Objective: The

aim of this study was to measure the level of ALP activity in bovine nasal secretion and in extracts of relevant

bovine tissues. Methods: Nasal secretions were collected on to absorbent material from thirty eight clinically

healthy Holstein-Friesian cows aged 2-5 years and were extracted from the absorbent by centrifugation. The

nasal secretions were examined for ALP activity and other analytes using standard clinical biochemistry

methods. Samples of heart, liver, kidney, small intestinal mucosa and nasal epithelium were obtained from cows

during necropsy. Two grams of each sample were washed with isotonic saline solution and homogenized

mechanically in 5ml saline containing 20% (v/v) n-butanol. The ALP activity in the aqueous extract was

determined as above. Results: ALP activity was higher in the nasal secretion samples, with a mean (+SD) of

1193 ± 500 U/L, compared to the laboratory reference range for bovine serum (20-280 U/L). Other biochemical

analytes were within or near or below the reference range for serum. The nasal epithelium extract had the highest

ALP activity of 1855 U/L followed by kidney (1086 U/L), small intestinal mucosa (832 U/L), liver (668 U/L)

and heart (377 U/L). Conclusion: Bovine nasal secretion has a higher ALP activity than bovine serum. The nasal

ALP results from local synthesis and secretion of the enzyme.


64. AMELIORATIVE EFFECTS OF DIETARY ALUMINOSILICATES ON BIOCHEMICAL CHANGES IN

BROILER CHICKENS DURING AFLATOXICOSIS.

D. Prvulovic1, D. Kojic

2, M. Popovic

1, G. Grubor-Lajsic

2.

1Faculty of Agriculture, Novi Sad, Novi Sad,

Serbia; 2Department of Biology and Ecology, Faculty of Science, Novi Sad, Serbia


Background: Aflatoxicosis in poultry causes histopathological and biochemical changes in major organs that

can assist in the diagnosis of toxication. Producers and researchers desire to develop an effective

decontamination technology dealing with this feed-borne toxin. Non-nutritive aluminosilicates such as clays and

zeolites are preferred because of their high binding capacities against aflatoxin and their ability to reduce

aflatoxin absorption from the gastrointestinal tract. Objective: The purpose of the present study was to evaluate

the toxic effects of aflatoxin B1 (AFB1) by biochemical examination of liver, kidney, and pancreas of broilers,

and to determine the possible preventive role of dietary aluminosilicates (mixture of clinoptilolite and bentonite)

on these biochemical values. Methods: In total, 84 broiler chicks were divided into two treatment groups:

control-basal diet and basal diet plus 5 g aluminosilicate kg-1

diet. After 21 days, twelve hours prior to sacrifice,

21 chicks from each group received one dose of AFB1 orally. Enzymes and parameters of lipid peroxidation

were analyzed. Results: The activities of gamma glutamyl transferase and glutathione-S-transferase in liver, and

superoxide dismutase, guaiacol peroxidase, and pyrogallol peroxidase in kidneys decreased after oral

administration of single dose of AFB1 to broilers. Lipid peroxidation was significantly increased in both organs.

The supplementation with aluminosilicates partially decreased these effects. No effects due to AFB1 were

observed in enzyme activities in the pancreas. Conclusion: These data suggest that a single dose of AFB1 causes

a toxic effect on biochemical indices of liver and kidney in broilers. Aluminosilicates alleviated these effects and

protects broilers against the harmful effects of AFB1.

65. DETERMINATION OF ATRAZINE IN SERUM AND URINE OF CATTLE BY HPLC, USING

MOLECULAR IMPRINTED POLYMERS (MIP).

S.Z. Peighambarzadeh 1, S. Safi

2, S.J. Shahtaheri

3, M. Javanbakht

4, A.Rahimi Forushani

5.

1Department

of Veterinary Medicine, Islamic Azad University, shoushtar Branch, Shoushtar, Iran. 2 Department of Clinical

Pathology, Faculty of Specialized Veterinary Sciences, Science and research branch, Islamic Azad University,

Tehran, Iran. 3Department of Occupational Health, School of Public Health, Center for Environmental Research,

Tehran University of Medical Sciences, Tehran, Iran. 4Department of Chemistry, Amirkabir University of

Technology, Tehran, Iran. 5 Department of Biostatistics, School of Public Health, Tehran University of Medical

Sciences, Tehran, Iran


176

Background: Atrazine is a major herbicide of s-triazine family which has been heavily used, especially in corn-

growing areas. Molecular imprinted solid phase extraction method has been developed to measure the residue of

pollutants in biological samples. Objective: The objective of the present study was to determine the residual

concentrations of atrazine in serum and urine samples of cattle fed by atrazine treated feed using MIP. Methods:

Blood and urine samples were taken from 45 Holstein cows. The cows were fed with corn silage from farms in

which atrazine was used at 3 kg/hectare. The Supel MIP Triazine10 was used as a Solid Phase Extraction media

and atrazine was detected in samples by HPLC. Results: The mean ± SD concentrations of atrazine in serum and

urine samples of the study group (0.739 ± 0.567 ppm and 1.389±0.633 ppm, respectively) were higher (P<0.05)

than the concentrations of atrazine in serum and urine samples of the control group (0.002 ± 0.005 ppm and

0.012 ± 0.026 ppm, respectively). Males and younger cows had higher concentrations of atrazine in the studied

samples (P<0.05). Conclusions: The results of this study showed that, atrazine in the feed ingredients ingested

by cattle could be transferred in to the biological samples and consequently can be considered as a potential

hazard for human beings.

66. CHARACTERIZATION OF STEM CELL MARKERS ON BONE MARROW AND ADIPOSE TISSUE-

DERIVED CANINE MESENCHYMAL STEM CELLS.

Y. Takemitsu, D. Zhao, I. Yamamoto, T. Arai. Veterinary and Life Science University, Musashino-shi Tokyo,

Japan


Bone marrow-derived mesenchymal stem cells (BM-MSCs) and adipose tissue-derived mesenchymal stem cells

(AT-MSCs) are potential cellular sources of therapeutic stem cells. MSCs are a multipotent population of cells

capable of differentiating into a number of mesodermal lineages. Treatment using MSCs appears a helpful

approach to regenerative medicine. Correct identification of these cells is necessary, but there is inadequate

information on the canine MSC profile of cell surface markers and mRNA expression. In this study, we

performed molecular characterization of canine BM- and AT-MSC using stem cell markers. Samples were

confirmed to be multipotent based on their differentiation. And these cells were checked as stem cell,

hematopoietic and embryonic stem cell markers by flow cytometry. BM- and AT-MSCs showed high expression

of CD29 and CD44, moderate expression of CD90, and others were negative. Quantitative real-time PCR

revealed expression of Oct3/4, Sox2, and Nanog in BM- and AT-MSCs. There was no significant difference in

expression of Oct3/4 and Sox2 between BM-MSCs and AT-MSCs. However, Nanog expression was 2.5-fold

higher in AT-MSCs than in BM-MSCs. Using immunocytochemical analysis, Oct3/4 and Sox2 proteins were

observed in BM- and AT-MSCs. These results provide a foundation for more reproducible and reliable quality

control in the identification of canine BM-MSCs and AT-MSCs.

67. RARELY USED METHODS FOR INVESTIGATING PORTO-SYSTEMIC SHUNTS IN DOGS.

C. Smuts1, M. Bennett

1, M. Sharman

2, J. Mills

1, T. Gaál

1.

1Murdoch University, Perth, Australia;

2Melbourne

University, Melbourne, Australia


Background: In dogs with portosystemic shunting (PSS) or chronic liver disease metabolic functions of the liver

are disrupted, resulting in increased concentrations of ammonia, amino acids and uric acid in the blood. This is

associated with hepatic encephalopathy and often excretion of ammonium-urate crystals in the urine. Objective:

To apply seldom-used methods such as electron dispersive analysis (EDA) for urinary crystal investigation,

measurement of serum aromatic amino acids (AAA) and blood ammonia concentrations to investigate a case

with PSS. Methods: Scanning electron microscopy with EDA was used to determine crystal type from urine

sediment. Blood ammonia levels were measured using a portable ammonia meter. AAA concentrations were

analysed in a human laboratory and compared to an age matched control dog. Results: AAA and blood ammonia

concentration were elevated in the patient. EDA revealed the presence of sodium and potassium urate crystals in

the urine sediment. Conclusions: Blood ammonia and serum AAA are useful tests for investigation of PSS

providing additional information for clinicians assessing dogs with hepatic dysfunction. Measurement of serum

AAA concentrations are routinely performed for people but infrequently applied in animals although testing can

be available through human laboratories. EDA as a new, semi-quantitative method for examining urinary

crystals can be effectively used to identify a morphologically unidentifiable urinary crystal type in PSS dogs.

Because many urinary crystals are morphologically similar, crystal analysis can be useful to determine their

content and hence their pathogenesis. This information is especially useful when the typical ammonium biurate

crystals are absent.

177

68. RELATIONSHIP BETWEEN COLLIE EYE ANOMALY- AND IVERMECTIN TOXICOSIS-

ASSOCIATED MUTATIONS: ANALYSIS ACROSS MULTIPLE DOG BREEDS.

K. Mizukami1, H-S. Chang

1, D. Endoh

2, A. Yabuki

1, O. Yamato

1.

1Kagoshima university, Kagoshima, Japan;

2Rakuno Gakuen university, Ebetsu, Japan.


Background: Collie eye anomaly (CEA) is an inherited ocular disorder, which is associated with a 7.8-kbp

deletion in the canine NHEJ1 gene. A 4-bp deletion in the canine MDR1 gene causes neurotoxicosis after

administration of P-glycoprotein substrates such as ivermectin. It is known that both of these genetic disorders

are common in Collie-related breeds. However, we recently demonstrated that Hokkaido dogs have a high

prevalence of the CEA-associated mutation. Object: To investigate whether the 2 mutations are actually related

or not in the population of multiple dog breeds. Materials and Methods: Collies, Border Collies, 3 Japanese

breeds (Hokkaido, Ryukyu, and Shiba dogs), Korean Jindo dogs, and 2 non-Collie breeds (Toy Poodles and

Miniature Dachshunds) were used. The genotypes of each mutation were determined using real-time PCR

methods. Results: Collies had a very high prevalence of the 2 mutations and Border Collies had a high

prevalence in CEA but a low prevalence in the MDR1 mutation. Hokkaido dogs did not have the MDR1 mutation

although this breed had a high prevalence of CEA. Ryukyu dogs did not have CEA, but some dogs had the

MDR1 mutation. The other breeds did not have the 2 mutations. Conclusion: This data suggests that these

genetic disorders arose before the establishment of Collie-related breeds but there was no apparent relationship

in non-Collie breeds. Therefore, these tests should be performed separately for each breed. Then based on the

data gathered in the present study, it should be determined which testing is indicated for individual dogs.

69. ABERRANT ANTIGEN EXPRESSION IN CANINE LYMPHOMA: A RETROSPECTIVE STUDY.

V. Martini1, A. Pasqua

1, M.E. Gelain

2, S. Comazzi

1. 1

University of Milan, Milan, Italy; 2University of Padua,

Padua, Italy.


Background: Distinguishing B- or T-cell phenotype have an important prognostic impact for dogs with

lymphoma. The presence of different aberrant phenotypes has been widely reported and recently some authors

identified a more aggressive clinical behavior for aberrant lymphomas, when compared with non-aberrant ones.

Objective: Aim of this study was to retrospectively investigate the prevalence of different aberrations in B- or T-

cell lymphomas, and their possible prognostic significance. Methods and results:Among 371 lymphoma cases

in our database, 70,1% were B-cell, 29,6% T-cell, 0,3% were biphenotypic; 15,1% had at least one aberration,

most commonly among T-cell lymphomas (35,5%). A low expression of CD45 was the most common aberration

among T-cell lymphomas, CD34 positivity among B-cell lymphomas. Although treatment was not standardized,

a significantly shorter survival was found for dogs with CD4+CD8+ T-cell lymphoma when compared to other

aberrant T lymphomas. CD34+ B-cell lymphomas had a shorter survival compared to other B cases, although the

difference was not statistically significant. Dogs with T-cell lymphoma with CD45 expression had a longer

survival, although not statistically different from the other T cases, but this aberrance was frequently associated

to a “small clear cell” morphology, that is known to bear a better prognosis. Conclusion: Although preliminary

this study suggests that some abnormalities suggest prognostic significance. Further studies are needed, with a

larger number of dogs and introducing non-aberrant control cases. Furthermore, a prospective study with

standardized chemotherapeutic treatment should be performed to assess the real prognostic value of the different

aberrations.

70. CLINICAL PRESENTATION CURRICULUM IN VETERINARY EDUCATION: A COGNITIVE

PERSPECTIVE.

S.Safi1, P. Hemmati

2, S.H. Shirazi-Beheshtiha

3.

1Department of Clinical Pathology, Faculty of Specialized

Veterinary Sciences, Science and Research Branch, Islamic Azad University, Tehran, Iran; ²Ministry of Health

and Medical Education, Center for Disease Control, Senior Officer of Center for Disease Control, Tehran, Iran;

³Department of clinical sciences, Faculty of Veterinary Medicine, Karaj Branch, Islamic Azad University, Karaj,

Iran


There is a discrepancy between the knowledge of veterinary medicine graduates and professional and society

expectations. The main reason for this discrepancy is the conventional curricula, which rely on backward

reasoning or disease-centered medical education. The aim of the present study was to develop a scheme as the

178

most important step in creation of a full Clinical Presentation (CP) module. Vomiting was chosen in this study as

a common CP in dogs. Three series of focused questions were developed. The physiological mechanisms

(visceral, cortical, vestibular, metabolic and chemical) were chosen to reconstruct the top layer of the scheme. To

reconstruct the second and third layers of the scheme anatomical concepts were applied to define the

subcategories. To design the fourth layer, the disease classes which result in vomiting in each of the

physiological schemes were considered. We believe that introducing "vomiting in dog" to veterinary medical

students as a leading presentation, meets the students' requirements because it uses clinical presentations as the

organizing core structure.

71. STATISTICAL STUDY ON PREVALENCE OF TUMORS IN INSURED DOGS IN JAPAN

A. Shimamura, T. Arai. Nippon Veterinary and Life Science University, Tokyo, Japan


Objective: Anicom, the leading pet insurer in Japan, started its operation about 10 years ago, and currently

almost 400,000 pets have insurance policies. Insurance claim data could provide epidemiological information. In

this study, we tried to investigate the prevalence of tumors with analysis of pet insurance data. Methods: All

insurance claims data for 252,414 dogs (males 133,775 and females 118,639) contracted with Anicom pet

insurance from April 1st 2008 to March 31

st 2008 were used for the research. Results: The incident rate of

tumors was 7.9% in females, higher than that of males (6.4%). Among 144 insured dogs reported as dead by 10

years of age, 16.9% claimed reimbursement because of tumor just before the death. Incident rate of tumors in

Golden Retrievers (15.4%) was the highest among the top 17 insured breeds. The reason why the rate in the

females was higher than that in the males seems to be high incidence of mammary tumors in the females. The

rate of mammary tumors was very low (almost 0) in the males but was 1.4% in the females. Conclusion: Pet

insurance data can provide information about disease trends in dogs. It is suggested that we develop more

effective sampling methods for research and introduce better treatment and prevention by more effective usage

of these data.

72. N-ACYLHOMOSERINE LACTONES OF PSEUDOMONAS AERUGINOSA IN CLINICAL SAMPLES

FROM CANINE OTITIS EXTERNA: VALIDATION OF AN ANALYTICAL METHOD USING LIQUID

CHROMATOGRAPHY WITH TANDEM MASS SPECTROSCOPY (LC-MS/MS).

K. Srimpf, J. Hosseini, T. Kotnik, D. Kušar, M. Vengušt, G. Tavčar-Kalcher. University of Ljubljana,

Veterinary Faculty, Ljubljana, Slovenia


Background: Bacteria regulate their phenotype, including virulence, in a manner of intercellular communication

(i.e. quorum sensing) via the production of autoinducers, e.g. N-acylhomoserine lactones (AHLs). Pseudomonas

aeruginosa that is associated with severe otitis externa in dogs produces mostly N-3-oxododecanoyl-homoserine

lactone (3-oxo-C12-HSL) and N-butanoyl-homoserine lactone (C4-HSL); other AHLs, e.g. N-hexanoyl-

homoserine lactone (C6-HSL), are less abundant. For detection of AHLs, highly sensitive analytical methods are

needed because concentration of AHLs in clinical samples is very low. Objective: The aim of our study was to

validate an analytical procedure for detection of 3-oxo-C12-HSL, C4-HSL and C6-HSL in ear rinses of dogs.

Methods: According to the validation scheme, ear rinses of healthy dogs were spiked with 20 ng of each of the

three selected AHLs. After dichloromethane extraction and evaporation, AHLs were re-dissolved in methanol-

water mixture and determined by LC-MS/MS. Results: The limit of detection was 0.5 ng/ml for all three AHLs.

The repeatability of results for 3-oxo-C12-HSL, C4-HSL and C6-HSL at 20 ng/ml represented by the standard

deviation (RSDr) was 10%, 13% and 15%, respectively. The relative standard deviation for the within-laboratory

reproducibility (RSDR) was 10%, 35% and 22%, and the mean recoveries were 72%, 53% and 87%, respectively.

Conclusions: Results indicated that the procedure is suitable for determination of the three selected AHLs in

canine ear rinses. Monitoring of AHLs in P. aeruginosa-affected dogs could serve as an alternative diagnostic

tool that enables clinical follow-up and appropriate therapy.

179

73. LEG WEAKNESS PATHOLOGY IN BROILER CHICKENS.

I. Dinev. Dept of General & Clinical Pathology, Faculty of Veterinary Medicine, Trakia University, Stara

Zagora, Bulgaria.


Background: The welfare of broiler chickens and breeder flocks continues to be closely related to leg skeletal

pathology. Although some reports establish that most abnormalities related to legs in chickens were largely

overcome by selection work and improvement of production systems by the end of the 20th

century, the problems

still occupy an important place in broiler production. Objective: The aim of the present work is to make a

review on the literature concerning the incidence of some major pathologies of leg skeleton in broiler chickens

and broiler breeders, related to poor animal welfare. Methods: On the basis of data on the incidence of leg

skeletal pathology published by us and by other researchers, this work goes through some reports on femoral

head necrosis (FHN), tibial dyschondroplasia (TD), rupture of the gastrocnemius tendon (RGT) and rickets.

Results: Cases of TD are related to significantly younger chickens, approximately 24 days, among modern

hybrid broiler chickens and are predominantly associated with insufficient Ca. Subclinical and clinical rickets

forms within broiler flocks are a possible prerequisite for a number of pathological conditions such as FHN,

osteomyelitis, fractures, TD, vertebral column deformations. RGT is the most common problem related to the

skeleton of the legs among parent flocks of broiler breeders. Conclusion: The analysis of the overview indicates

that the most common problems are related to skeletal leg pathology among modern hybrid broiler chickens,

namely FHN, TD and rickets. FHN was most commonly caused by osteomyelitis, which was caused

predominantly by E. coli infection.

74. PATHOMORPHOLOGICAL INVESTIGATIONS ON THE INCIDENCE OF AXIAL SKELETON

PATHOLOGY ASSOCIATED WITH POSTERIOR PARALYSIS IN COMMERCIAL BROILER CHICKENS.

I Dinev. Department of General and Clinical Animal Pathology, Faculty of Veterinary Medicine, Trakia

University, Stara Zagora, Bulgaria


Background: Most reported data are on the prevalence of lameness in broiler chickens and broiler breeders,

associated with pathology of the hind limb. Data on axial skeleton abnormalities, mostly spondylolisthesis and

scoliosis, which could be asymptomatic, can be found in separate reports. Objective: The aim of the current

pathomorphological study was to find out the prevalence of various pathological conditions of the axial skeleton

associated with posterior paralysis in commercial broiler chickens at a farm over a prolonged period. Methods:

Cases of clinical posterior paralysis at a commercial broiler chicken farm were registered over a one-year period

of research. A total of 151 vertebral columns were randomly collected from 6 isolated chicken carcasses from

each of the observed flocks at the processing plant, which had exhibited posterior paralysis. The samples were

submitted to gross, histological, radiography, and microbiological examinations. Results: Of the examined 36

broiler flocks, signs of posterior paralysis were found in a total of 1480 (0.21%) chickens from 23 flocks.

Spondylolisthesis 64 (42.3%), followed by cases of spondylosis with ankylosis, osteosclerosis and fractures

54 (35.8%), vertebral osteomyelitis 19 (12.6%), and osteochondrosis with scoliosis and fractures - 14 (9.3%)

were the most prevalent conditions. Conclusion: Summarizing the results of our research, we think that most of

the established pathological conditions, which are manifested by posterior paralysis, are related to the anatomical

features and the level of maturity of the axial skeleton among broiler chickens, while the observed vertebral

osteomyelitis was spontaneous with proven etiology.

75. EFFECTS OF ANTIPARASITIC AND IMMUNOSTIMULATIVE DOSES OF LEVAMISOLE ON

HEMATOLOGIC PARAMETERS IN DOGS.

S.H. Shirazi-Beheshtiha1, A. Shariat

1, N. Taefi-Nasrabadi

1, A. Golmohammadi

2, M.R. Taghdiri

1.

1Karaj

Branch, Islamic Azad University, Karaj, Iran, Tehran, Iran; 2Chairman of Rahpouyane Nedaye Salamat

Company, Karaj, Iran


Background: The clinical adverse effects of levamisole have been well documented. However, the effect of

antiparasitic and immunostimulative doses of levamisole on hematologic parameters in dog has not been

reported. Objective: The present study was designed to investigate the effects of orally administered levamisole

on hematology parameters of dog. Methods: A total of 18 clinically healthy dogs (2-3 years) were included in

this study. The dogs were randomly assigned into 3 equal groups. Dogs in group 1 and 2 were given levamisole

orally at a dose of 10 mg/kg of body weight for 6 days (antiparasitic dose) and 1 mg/kg of body weight 3 times


180

weekly for 2 weeks (immunostimulative dose), respectively. Dogs in Group 3 served as the controls. Blood

samples taken at various timepoints after administration of levamisole were transferred to the Clinical Pathology

Laboratory for analysis. Results: Statistically significant changes were observed in WBC count, absolute

neutrophil count, and mean cell volume (MCV) after 3, 4 and 5 days of the antiparasitic dose of levamisole

administration, compared with the control group. Statistically significant differences were observed in WBC

count, absolute neutrophil, lymphocyte, and monocyte counts, RBC count, hematocrit, hemoglobin

concentration, MCV, and mean cell hemoglobin concentration (MCHC) in dogs that received the

immunostimulative dose of levamisole, compared with the control group. Conclusion: Results of the present

study demonstrated the significant effects of administration of levamisole at both immunostimulative and

antiparasitic doses on some hematologic parameters that should be taken in consideration when interpreting

canine hematology profiles.


ORAL PRESENTATIONS

SERUM CALCIUM AND INORGANIC PHOSPHATE EVALUATION IN HERMANN'S TORTOISES

(Testudo hermanni).

M. Stvarnik1, M. Klinkon

2, A. Dovč

3.

1Veterinary faculty, Ljubljana, Slovenia;

2Clinic for Ruminants,

Veterinary faculty, Ljubljana, Slovenia; 3Institute for the Health Care of Poultry, Veterinary faculty, Ljubljana,

Slovenia


Background: Blood values of calcium and phosphorus could be influenced in Testudo hermanni by life-style,

season, and habitat diversity. T.hermanni is one of the indigenous species imported to Slovenia. Objective: The

aim of this study was to evaluate differences in serum calcium and inorganic phosphate values and Ca/P ratio in

the post-hibernation period, at different times throughout the active season, and during mating and hatching.

Methods: From a paddock, sixteen males and sixteen females were chosen for the study. Blood was taken from

the dorsal coccygeal vein. Serum was analysed with a RX Daytona biochemical analyser. Results: Mean values

for calcium were 2.64 (0.59-4.88) mmol/L and for inorganic phosphate were 0.95 (0.15-5.62) mmol/L. The Ca/P

ratio in females in the spring was 2.1:1; the ratio changed to 1:1 in May. After the egg laying period in July, the

ratio increased to 2.6:1. At the end of May the ratio was 0.97:1, which was 0.03 lower than the ratio in males.

After the egg hatching period in July the ratio increased to 2.8:1. Conclusion: We consider it important to take

samples throughout the active season to acknowledge oscillation in serum calcium and inorganic phosphate

values due to external environmental factors.

HEMATOLOGY REFERENCE INTERVALS IN CYNOMOLGUS MONKEYS.

N. Bourgès-Abella1, A. Geffré

1, E. Moureaux

2, M. Vincenti

1, J.P. Braun

1, C. Trumel

1.

1Institut National

Polytechnique-Ecole Nationale Vétérinaire de Toulouse, Toulouse, France; 2BioPRIM, Bazièges, France


Background: Currently available hematology reference values in Cynomolgus monkeys do not meet CLSI-

IFCC and ASVCP recommendations. Objective: The aim of this study was the a posteriori determination of

reference intervals in this species. Methods: Data were collected between 2009 and 2011 during routine health

examinations at BioPRIM center (AAALAC accredited). Animals originated mainly from Mauritius and also

from Philippines and Vietnam. Results were included according to health status and full control of pre-analytical

and analytical conditions. Analyses had been performed in 3mL K3-EDTA specimens with a Sysmex KX-21

analyzer at BioPRIM. Reference intervals were calculated by the nonparametric method, and effects of

covariables were tested by a general linear model. Calculations were performed with Reference Value Advisor

21 and Systat 13. Results: From the 450 cases available, 272 animals were finally included. Most (85%) were

born in captivity; age ranged from 17.3 to 120 months with a majority 2 to 3 years old. The reference intervals

were [4.85-7.00].1012

/L for RBC, [104.0-136.0]g/L for HGB, [0.365-0.470]L/L for HCT, [62.7-79.1]fL for

MCV, [17.3-23.7]pg for MCH, [264-310]g/L for MCHC, [31.9-44.0]fL for RDW, [4.7-25.7].109/L for WBC,

[2.00-20.60].109/L for Neutrophils, [1.15-5.23].10

9/L for Lymphocytes, [0.2-1.6].10

9/L for Mixed cells, [224 -

562].109/L for PLT, [8.8-12.8]fL for MPV, [10.3-18.7]fL for PDW. Conclusion: The reference limits and their

confidence intervals have been determined according to international recommendations, thus can be used for

181

transfer and/or validation. However, they are only a basis for further extensive prospective studies using more

modern analyzers.

POSTER PRESENTATION

76. HISTOPATHOLOGICAL FINDINGS IN ABOMASA OF FALLOW DEER FROM BRIJUNI REGION IN

CROATIA.

M. Robić, B. Artuković, A. Beck, R. Turk, M. Belić, Ž. Grabarević. Veterinary Faculty, Zagreb, Croatia


Background: Histopathologic examination was performed on abomasal samples of fallow deer (Dama dama)

from Brijuni region in Croatia. Objective: The aim of the study was to determine histopathological changes and

possible presence of gastric Helicobacter-like organisms in abomasa of fallow deer. Method: Paraffin

embedded, H and E stained samples of 23 abomasa taken from fallow deer were histopatholgically examined.

Samples were stained with Toluidine Blue, Giemsa and Warthin Starry method for detection of Helicobacter-

like organisms. Results: In all samples examined, chronic inflammatory changes were visible. Noteworthy,

histopathological findings were similar, indicating epizootical nature of the disease. All samples were negative

for Helicobacter-like organisms on all stains examined. Conclusion: Abomasa of fallow deer from Brijuni

region revealed chronic inflammatory changes which are morphologically similar to those described in dogs with

Helicobacter infection. However, these can not be attributed to gastric Helicobacter since such organisms were

not detected after appropriate special stains were applied. Underlying etiology of chronic inflammatory lesions in

the abomasa of these deer is still to be determined.


ORAL PRESENTATIONS

EVALUATION OF SERUM AMYLOID A, HAPTOGLOBIN, NUCLEATED CELL COUNT, TOTAL

PROTEIN AND HEMOLYSIS IN PERITONEAL FLUID FOR DIFFERENTIATION OF MEDICAL AND

SURGICAL COLIC IN HORSES.

E. Scheepers1, T. Holberg Pihl

2, M. Sanz

1, P. Page

1, A. Goddard

1, S. Jacobsen

2.

1University of Pretoria,

Pretoria, South Africa; 2University of Copenhagen, Taastrup, Denmark


Background: Horses with severe medical colic caused by duodenitis-proximal jejunitis, colitis or peritonitis that

do not need surgery, can be difficult to differentiate from horses with strangulating obstructions or displacements

that need surgery. Objective: To evaluate the usefulness of serum amyloid A (SAA) and haptoglobin (Hp)

measured in peritoneal fluid (PF) to differentiate between horses with mild medical colic (group 1, n=138),

severe medical colic (group 2, n=46) and surgical colic (group 3, n=124), compared to the classic PF markers;

nucleated cell count (NCC), total protein and hemolysis. Method: Admission PF concentrations of SAA, Hp,

NCC, total protein and hemolysis were compared between the three groups with ANOVA and chi-squared test.

The effect of duration of colic pre-admission was included in the analysis. Results: Hemolysis was present in

36% of horses in group 3, 24% in group 2 and 10% in group 1. All markers were significantly higher in group 2

and 3 compared to group 1. NCC was the only marker with a difference (p=0.015) between group 2 (mean: 5.2 x

109/L) and 3 (mean: 1.6 x 10

9/L). SAA (p<0.001) and Hp (p=0.009) were increased after 12 hours of colic

duration, whereas NCC was only increased (p=0.01) after 24 hours. Conclusion: Not all horses with hemolysis

in PF require surgery. Measuring a panel of traditional and novel inflammatory markers in PF can assist in

segregating horses with colic in three groups that require fundamentally different treatments. When interpreting

levels of a measured marker, duration of disease should be considered.

182

MEASUREMENT OF SERUM ALBUMIN BY PROTEIN ELECTROPHORESIS AND BROMOCRESOL

GREEN METHODS IN CANINE AND EQUINE PATIENTS

E. Ramery, F. Bureau. Liege University, Faculty of Veterinary Medicine, Liège, Belgium.


Background: Bromocresol green method (BCG) is routinely used in veterinary laboratories to measure albumin.

A good agreement between BCG and protein electrophoresis (SPE) in healthy domestic animals is reported.

However, in human medicine, at low albumin and high globulin concentrations, BCG also binds to α- and β-

globulins fractions. Surprisingly, there is no recent literature evaluating the BCG method in diseased domestic

animals. Objective: The purpose of the present study was therefore to compare the serum albumin values

obtained by SPE and BCG in canine and equine patients. Methods: Albumin measured by BCG and SPE was

compared in the serum from 30 canine and 30 equine patients. Results: Good correlation was found (Spearman´s

rho rs = 0.91 in dogs and 0.84 in horses). However, Altman and Bland analysis of results showed a positive bias

of BCG with SPE (3.41 g/l in horses and 0.41 g/l in dogs) that increased when only hypoalbuminemic patients

were considered (4.17 g/l in horses and 1.31 g/l in dogs). Conclusion: Results confirm that, despite good

correlation, BCG method tends to overestimate albumin, especially in hypoalbuminemic patients. This is

particularly evident in horses.


77. COMPARISON OF TOTAL LEUKOCYTES RESPONSE OF HORSES SUBJECTED TO EXERCISE ON

HIGH SPEED TREADMILL – PRELIMINARY RESULTS.

T. Barbosa, L. Yonezawa, C. Marinho, M.J. Watanabe, A. Kohayagawa. School of Veterinary Medicine and

Animal Science - Sao Paulo State University, Botucatu, Brazil.


Background and objectives: The objective was to observe differences regarding response of equine leukocytes

subjected to high intensity and short duration (HI) and low intensity and long duration (LO) exercises on high-

speed treadmill. Five Arabian horses performed two exercise tests. Individual maximum oxygen uptake (VO2max)

was determinated to stablish speeds for each horse. Methods: The HI test consisted of 5 min at 50% VO2max, 5

min at 1,5 min and 90 s at 105% VO2max set at +6% slope, and 5 min at 3,0 m/s (0% slope). The LO test

consisted of 60 min at 35% VO2max (+6% slope). Blood samples were collected before (M0), immediately after

exercise, 15 and 30 min, 6, 12, 24, 48, 72 and 96 hours after each test for determination of white blood cell

(WBC) in automated cell counter, Cell-Dyn®. Results: Only at moments M0, after 15 and 30 minutes there were

significant differences among two type of exercises (p<0, 05). Possibly the reason of higher value at M0 was due

to apprehension of horses when entered exercise place which caused neutrophils demargination, whereas at the

following moments lymphocytes had a greater mobilization from spleen to circulation, but the values did not

exceed the reference values. In both tests, up to 12 hours values were above the baseline and after 24 hours

remained below the baseline. We concluded that the high and low intensity exercises did not differ in response of

leukocytes. Financial support: São Paulo Research Foundation (FAPESP)

78. EFFECT OF EXERCISE ON HIGH-SPEED TREADMILL ON DNA DAMAGE IN PERIPHERAL

LEUKOCYTES OF HORSES.

T. Barbosa, L. Yonezawa, C. Marinho, J. Knaut, M.J. Watanabe, D. Salvadori, D. Almeida, A.

Kohayagawa. School of Veterinary Medicine and Animal Science - Sao Paulo State University, Botucatu,

Brazil.


Background: Recently, there is a growing interest in exercise-induced DNA damage due to its potential

involvement in organ dysfunction. However it has not been reported in horses submitted to strenuous exercise on

high-speed treadmill. The comet assay is a relatively new method used to measure single DNA strand breaks in

peripheral leukocytes and the tail moment is the product of tail length and percentage DNA in tail. Objectives:

The purpose of this study was to determine the effects of exercise on DNA damage in peripheral leukocytes of

horses submitted to exercise on high-speed treadmill. Methods: Ten untrained horses were subjected to exercise

at high intensity and short duration on a high-speed treadmill. The comet assay was used to assess DNA damage

in circulating leukocytes (Mononuclear cells – MN and polymorphonuclear cells - PMN) at rest before exercise

(M0), 12 h, 48 h and 72 h after test end. Results: The tail moment of DNA damage in MN was 8.27% in M0;

7.64% in 12h; 7.84% in 48h and 7.58% in 72h, while in PMN was 7.86% in M0; 6.71% in 12h; 9.17% in 48h

183

and 12.39% in 72h. There were no significantly differences (P>0.05) in all analyzed moments. Conclusions:

This protocol of exercise did not result DNA damage in peripheral leukocytes of horses as detected by the comet

assay. Financial support: São Paulo State Research Foundation (FAPESP), Brazil

79. REFERENCE VALUES FOR HAEMATOLOGICAL PARAMETERS IN LUSITANO HORSE.

A.C. Silvestre-Ferreira1, M. Cotovio

1, M. Maia

1, F. Queiroga

2, M.J. Pires

2, A. Colaço

2.

1Department of

Veterinary Sciences – Veterinary Hospital – University of Trás-os-Montes e Alto Douro, Vila Real, Portugal; 2Department of Veterinary Sciences – CECAV - University of Trás-os-Montes e Alto Douro, Vila Real,

Portugal.


Background: The Lusitano horse is an autochthonous Portuguese horse breed with a growing worldwide

expansion. Objective: The aim of this study was to establish reference values for haematological parameters

using the haematological cell counter LaserCyte (IDEXX). Methods: One-mL EDTA blood samples were

obtained from 75 healthy horses from both sexes and 2 to 23 years of age. Reference values were estimated by

bootstrap and non parametric methods with Reference Value Advisor software. Results: The reference values

were 6.3-10.7 x1012

/L for red blood cells, 30.7-46.6% for hematocrit, 12.7-17.3 g/dL for haemoglobin, 42.1-53.3

fL for mean corpuscular volume, 15.5-20.9 pg for mean corpuscular haemoglobin, 35.3-39.5 g/dL for mean

corpuscular haemoglobin concentration, 17.8-20.6% for red cell distribution width, 4.8-10.0 x109/L for white

blood cells, 2.3-6.0 x109/L for neutrophils, 0.8-4.7 x10

9/L for lymphocytes, 0.2-0.5 x10

9/L for monocytes, 0.1-

0.6 x109/L for eosinophils, 0.0-0.1 x10

9/L for basophils, 78.9-164.5 K/mmL for platelets, 4.3-9.1 fL for mean

platelet volume, 18.7-24.5% for platelet distribution width and 0-0.1% for plateletcrit. Conclusion: Except for

leukocytes and platelets, reference values obtained in this study were closer to data reported for other hot-

blooded horse breeds and provide important clinical information for clinicians to use.

80. MONONUCLEAR CELLS CONCENTRATION IN FRACTIONED SAMPLES OF BONE MARROW

ASPIRATE OF HORSE’S STERNUM.

F. Correa, J. Galecio, H. Bustamante, F. Wittwer, A. Ramirez, B. Menarim. Universidad Austral de Chile,

Valdivia, Chile.


Background: The use of bone marrow mononucleated cells (MNCs) has been described as an alternative

procedure to grafting mesenchymal stem cells (MSCs) in injured tissues. Objective: To compare the

concentration of MNCs of bone marrow samples obtained in 5 steps. Methods: Bone marrow aspirates were

obtained from the sternum of 5 Chilean Criollo horses and five sequential 5mL fractions were recovered from

each horse. Once collected, the mononuclear fraction of each sample was isolated through Ficoll density

centrifugation and sequently submitted to cell count and viability test using epifluorescence microscopy.

Results: The total number (median ± QD) of MNCs per fraction was 13.4 ± 12.6 x 106 for the first fraction, 28.6

± 5.8 x 106 for the second, 114.8 ± 51.2 x 10

6 for the third, 6.6 ± 6.1 x 10

6 for the fourth, and 4.8 ± 4.2 x 10

6 on

the fifth fraction. The first three fractions represent 93.2% of the total 25mL (the five fractions together). The

cell viability was 97.5 ± 1.2 %. Conclusions: The major finding of this study is that there is a heterogeneous

concentration of MNCs within five fractions consecutively obtained in different horses. Furthermore, we suggest

the need to obtain at least the first 15mL of bone marrow aspirate from the sternum when the sample is intended

to be used as bone marrow MNCs concentrate.

© 2012 IDEXX Laboratories, Inc. All rights reserved. ● 102190-00 ● The IDEXX Privacy Policy is available at idexx.com.

*ProCyte Dx and Catalyst Dx are trademarks or registered trademarks of IDEXX Laboratories, Inc. or its affiliates in the United States and/or other countries. Sysmex is a registered trademark of Sysmex Corporation.

Hematology and chemistry for research and routine diagnostics

ProCyte Dx® Hematology Analyzer Sysmex XT-iV Series Hematology Analyzers

More than 28 parameters to choose from:

ALB

ALKP

ALT

AMYL

AST

BUN

Ca

CHOL

CK

Cl

CREA

GGT

GLU

K

LAC

LDH

LIPA

Mg

Na

NH3

PHBR

PHOS

TBIL

TP

TRIG

UCRE

UPRO

URIC Catalyst Dx® Chemistry Analyzer

− Outstanding flexibility − − Small sample volumes −

− Multi species capabilities −

Answers for life.

Only Siemens has the innovative solutions your lab needs to reach the top and the vision to keep you there.

Planning for your future starts with choosing the right diagnostics partner today. Siemens provides comprehensive andcustomizable solutions so laboratorians and clinicians can improve productivity every day. And, with a 130-year tradition of innovation, you can trust Siemens to stay on the leading edge of emerging trends and technologies, so together wecan set a new standard in patient care for years to come. www.siemens.com/diagnostics

Who can really help me grow?

A91DX-9105-A1-4A00 © 2010 Siemens Healthcare Diagnostics Inc. All rights reserved.

9105_Siem_DX_Brand_Ad_Innovation_CAPToday:CapToday 5/3/10 11:30 AM Page 1

High quality reagents

• Extensive test menu including anti-inflammatory monitoring, pre-anaesthesia and equine health

• Wide range of specialised tests including bile acids, fructosamine, NEFA, GLDH, HDL and LDL as well as unique tests such as glutathione peroxidase and D-3 Hydroxybutyrate

RX monza

Eliminate the need for costly external testing with the RX monza; a compact clinical analyser with an extensive test menu.

Randox is an international diagnostic solutions company with 30 years experience in veterinary diagnostics, providing solutions specifically designed to accurately

diagnose and monitor the general wellbeing of a diverse range of animals.

Randox also provide bovine-based multi-analyte quality controls, EQA schemes and a web based internal quality control and peer group reporting package, ensuring quality of results.

Veterinary Diagnostic Solutions

Randox Laboratories Limited, 55 Diamond Road, Crumlin, County Antrim, United Kingdom, BT29 4QY T +44 (0) 28 9442 2413 F +44 (0) 28 9445 2912 E [email protected] I www.randox.com

SANOLABOR, d.d., Leskoskova 4, 1000 Ljubljana, Slovenia.T 00386 1 585 42 14 M 00386 41 636 650

AV000 Veterinary - Complete Diagnostic Solutions MAY12.indd 1 14/05/2012 13:25

Ko potrebujete več kot samo diagnostiko

Eurolyser SOLO POCT Testi: •  T4 •  Fructosamine •  Fibrinogen •  Haemoglobin •  cCRP

Meditrade d.o.o. Središka ulica 21 Tel: +386 1 5854 600 [email protected] 1000 Ljubljana, Slovenija Faks: +386 1 5445 401 www.meditrade.si

Hematološki analizator XT-2000iV / XT-1800iV

© 2012 IDEXX Laboratories, Inc. All rights reserved. ● 102190-00 ● The IDEXX Privacy Policy is available at idexx.com.

*ProCyte Dx and Catalyst Dx are trademarks or registered trademarks of IDEXX Laboratories, Inc. or its affiliates in the United States and/or other countries. Sysmex is a registered trademark of Sysmex Corporation.

Hematology and chemistry for research and routine diagnostics

ProCyte Dx® Hematology Analyzer Sysmex XT-iV Series Hematology Analyzers

More than 28 parameters to choose from:

ALB

ALKP

ALT

AMYL

AST

BUN

Ca

CHOL

CK

Cl

CREA

GGT

GLU

K

LAC

LDH

LIPA

Mg

Na

NH3

PHBR

PHOS

TBIL

TP

TRIG

UCRE

UPRO

URIC Catalyst Dx® Chemistry Analyzer

− Outstanding flexibility − − Small sample volumes −

− Multi species capabilities −

15th Congress of the INTERNATIONAL SOCIETY FOR ANIMAL ...

Documents

Transcript of 15th Congress of the INTERNATIONAL SOCIETY FOR ANIMAL ...