College of Veterinary Medicine Kansas State University ...€¦ · ii Annual Phi Zeta Research Day...

College of Veterinary Medicine

Kansas State University

presents

Phi Zeta Research Day

March 3, 2020

History of Phi Zeta

Phi Zeta was originated in 1925 by a group of senior veterinary students in the New York State

Veterinary College at Cornell University. With the assistance of a group of faculty members,

including the Dean of the College, Dr. Veranus A. Moore, the Society was formally organized, and

Dean Moore was elected as the first president of the Alpha Chapter. The Society of Phi Zeta was

organized in 1929 at a meeting in Detroit, Michigan, and Dean Moore became the first president of

the Society.

Also in 1929, a charter was granted to the School of Veterinary Medicine at the University of

Pennsylvania, and the Beta Chapter was established. In 1931, the Executive Committee approved

the petition of a group from Iowa State College, and the Gamma Chapter was established. Since

then twenty-four chapters have been chartered, brining the total number of chapters to twenty-

seven. Chapters of the Society may be formed at any recognized veterinary medial college or at any

other institution of higher learning.

From its beginning, it has been the aim of Phi Zeta to stand for constant advancement of the

veterinary profession, for higher educational requirements, and for high scholarship. As stated in the

Constitution, the Object of the Society shall be to recognize and promote scholarship and

research in matters pertaining to the welfare and diseases of animals.

Selection of Membership

Membership in the Society consists of two classifications, Active and Honorary. Those eligible to

election as Active Members are:

A. Any candidate for the DVM/VMD degree in a veterinary medical college where a chapter

exists, and who has completed at least two years of the professional curriculum, and who

meets the following requirements:

1. The candidate must have an acceptable personality, be of good moral character, and

possess high ideals regarding professional service conduct.

2. When elected in the junior or third year, students must rank scholastically in the

highest 10% of their veterinary medical class.

3. When elected in the senior or fourth year, students must rank scholastically in the

highest 25% of their veterinary medical class.

B. Any veterinarian who has been in possession of a veterinary medical degree for at least two

years, and who has displayed ability of high order in dealing with one or more phases of the

science of veterinary medicine, and who meets one of the following criteria.

1. The candidate is enrolled as a graduate student in a college of veterinary medicine

and has completed at least twenty semester (thirty quarter) hours of graduate credit

or has successfully passed preliminary examinations.

2. The candidate has been engaged in an intern or residency program for at least two

years or has become board certified in his/her specialty.

3. The candidate has completed two years or more on the faculty of the institution or

scientific staff of a scientific institution within commuting distance of the nearest

chapter of Phi Zeta and has been involved in veterinary research or service.

Those eligible to election as Honorary Members are:

A. Distinguished veterinarians in possession of their veterinary medical degrees for at least five

years and who have rendered notable service to their profession.

B. Persons not in possession of the veterinary medical degree, who have rendered distinguished

service in the advancement of the science relating to the animal industry and particularly of

animal diseases.

C. Only in exceptional instances shall more than two honorary members be elected by any one

chapter in any one academic year.

Active members who move from the residence of their chapter may: 1 become known as inactive

members and not subject to the payment of dues; or 2 transfer their membership to another

chapter.

Name and Symbols of the Society

The organizers of the Society, when seeking a suitable name, sought the help of a learned Greek

scholar, Professor George P. Bristol of Cornell University. Professor Bristol suggested a Greek

word, which in the Latin form is spelled PHILOZOI and means "love for animals." The

abbreviation of Phi Zeta was adopted as the name of the society. The emblem consists of a pendant

formed by the letter Phi superimposed by the letter Zeta. The design was the work of Louis Agassiz

Fuertes, the great naturalist and artist.

The Executive Committee, consisting of the president, president-elect, secretary-treasurer, and the

three most past-presidents, oversees and promotes the objectives of Phi Zeta through activities of

the various chapters. Meetings of the Society of Phi Zeta are held annually in conjunction with the

AVMA Convention. All members of the Society are invited to attend these meetings.

Each year the Society sponsors two Research Awards and helps fund lectures at various Chapters.

Chapters recognize and promote high scholarship and research through an annual initiation

ceremony, by sponsoring research days, and by inviting outstanding lecturers to speak on topics

relevant to veterinary medicine and the welfare of animals.

Chapters of Phi Zeta

Alpha ............................... Cornell University ...................................... 1925

Beta ................................. University of Pennsylvania ........................ 1929

Gamma ............................ Iowa State University ................................. 1931

Delta ................................ The Ohio State University .......................... 1934

Epsilon ............................ Auburn University ..................................... 1948

Zeta .................................. Michigan State University.......................... 1950

Eta ................................... Texas A&M University .............................. 1950

Theta ................................ Colorado State University .......................... 1950

Iota................................... Washington State University...................... 1952

Kappa .............................. University of Minnesota ............................. 1952

Lambda ............................ University of California ............................. 1953

Mu ................................... University of Illinois .................................. 1953

Nu .................................... Oklahoma State University ........................ 1958

Xi ..................................... University of Georgia ................................. 1959

Omicron........................... Purdue University ...................................... 1962

Pi ..................................... University of Missouri ............................... 1965

Rho .................................. Tuskegee University................................... 1967

Sigma .............................. Kansas State University ............................. 1969

Tau................................... Louisiana State University ......................... 1977

Upsilon ............................ University of Florida .................................. 1979

Phi ................................... University of Tennessee ............................. 1979

Chi ................................... Virginia-Maryland Regional CVM ............ 1984

Psi .................................... North Carolina State University ................. 1984

Alpha Alpha .................... University of Wisconsin ............................. 1987

Alpha Gamma ................. Oregon State University ............................. 1987

Omega ............................. Mississippi State University ....................... 1988

Alpha Beta ....................... Tufts University ......................................... 1991

Alpha Delta ..................... St. George University ................................. 2006

Alpha Epsilon .................. Western University of Health Sciences...... 2006

Alpha Zeta ...................... Ross University .......................................... 2014

Alpha Eta ......................... Midwestern University ............................... 2017

Alpha Theta ..................... Lincoln Memorial University ..................... 2018

i

The Society of Phi Zeta Sigma Chapter

presents the 2020 Kenneth D. Olson Lectureship

Adam Boyko, PhD “Dog DNA: a Journey of Discovery and

a Quest for Healthier Dogs”

Dr. Adam Boyko is an associate professor in Biomedical Sciences at the Cornell University College of Veterinary Medicine. His research focuses on genomic investigation of dogs as a model of genetic disease and evolutionary genetics. One aspect of this work is understanding the evolution and genetics of village dogs, the semi-feral pariah dogs found in much of the world today. Dr. Boyko graduated from the University of Illinois, Urbana-Champaign with a BS and received an MS in Computer Science and a PhD in Biology from Purdue University before his postdoctoral research in the Department of Biological Statistics and Computational Biology at Cornell University. He served as a Research Associate in the Genetics Department at the Stanford School of Medicine before beginning his faculty appointment at Cornell in 2011. Dr. Boyko received the Zoetis Award for Veterinary Research Excellence (2015). Dr. Boyko also serves as Founder and Chief Science Officer of Embark Veterinary, a canine genetic testing company.

ii

Annual Phi Zeta Research Day

March 3, 2020

The Sigma Chapter of Phi Zeta, est. 1969

Schedule of Events

12:00 (noon) Plenary Session Frick Auditorium, Mosier Hall Welcome by Phi Zeta President, Dr. Nora Springer, DVM, PhD, ACVP

Introduction of Keynote Speaker by Phi Zeta Vice President, Lindsay Heflin, Class of 2020

Kenneth D. Olson Phi Zeta Lectureship, Keynote Speaker Dr. Adam Boyko, Chief Science Officer of Embark Veterinary, presents: “Dog DNA: A Journey of Discovery and a Quest for Healthier Dogs”

1:15 – 2:30 pm Oral Research Presentations Basic Science Research – 301 Trotter Hall Applied/Clinical Science Research – 201 Trotter Hall 2:30 – 3:30 pm Royal Canin Poster Session with refreshments (Q&A for judging 2:00 – 3:30 pm) Mara Conference Center, 407 Trotter Hall 3:30 – 4:30 pm Oral Research Presentations Basic Science Research – 301 Trotter Hall Applied/Clinical Science Research – 201 Trotter Hall 5:00 pm Reception and Awards Ceremony Frick Auditorium and Foyer, 2nd Floor, Mosier Hall

- Initiation of New Members to Phi Zeta - Announcement and Presentation of Awards Recognizing Research

and Scholarship Accomplishments - Closing Comments

Phi Zeta Research Day 2020

generously sponsored by:

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Basic Science Research Posters Phi Zeta Research Day

March 3, 2020, 2:30 – 3:30, Mara Conference Center, 407 Trotter Hall (Posting from 1:00 – 5:00pm; Q&A for Judging 2:00 – 3:30pm)

Poster Number Presenter – Title Abstract

1. Dominica D. Genda – Inactivation of porcine reproductive and respiratory syndrome virus and Seneca virus A in cell culture using chemical feed additives ................................................... Page 1

2. Jason M. Gregory – ATP-sensitive potassium channel blockade: Mechanisms for decreased exercise tolerance ............................................................................................................................................... Page 2

3. Sarah Krueger – Evaluation of Amblyomma americanum vector competence for Anaplasma marginale ............................................................................................................................................................... Page 3

4. Keith Lewy– The Impact of Various Gut Microbiota Transfer Methods on the Development of Colitis in Response to Dextran Sulfate Sodium ...................................................................................... Page 4

5. Lauren Miller – Characterization of a Novel Tick Transmitted Ehrlichia sp. Infection in Horses .................................................................................................................................................................................... Page 5

6. Ryan Swanson – Multi-dimensional fluorescence and bioluminescence imaging and detection specificity of physiometacomposite:enzyme interfaces ..................................................................... Page 6

7. Deepa Upreti - Water extract from Euglena gracilis prevents lung carcinoma growth in mice via attenuation of myeloid-derived suppressor cells and granulocyte populations ..............

8. Brandon Verkinderen – Effect of kinase inhibitor treatment on Rift Valley Fever Virus replication .............................................................................................................................................................. Page 8

9. Yu Shin Wang – A non-destructive protocol for whole-mount bone and cartilage staining of African spurred tortoise (Centrochelys sulcata) .................................................................................... Page 9

10. Yvonne Wikander – Prevalence of Cytauxzoon felis carriers in the domestic cat population of eastern Kansas, a preliminary report ...................................................................................................... Page 10

11. Adrienne Wright – Optimization of a Protocol for the Isolation, Culture, and Cryopreservation of Umbilical Cord-Derived Canine Mesenchymal Stromal Cells for Future Clinical Use .... Page 11

12. Alexandria Zabiegala – Genetic and replication analysis of highly virulent Feline Calicivirus isolate, KS-2019 ................................................................................................................................................ Page 12

13. Michelle Zajac – Development and evaluation of a rabies enzyme-linked immunosorbent assay (ELISA) targeting IgM and IgG in human sera ......................................................................... Page 13

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Applied/Clinical Science Research Posters Phi Zeta Research Day

March 3, 2020, 2:30-3:30 pm, Mara Conference Center, 407 Trotter Hall (Posting from 1:00 – 5:00pm; Q&A for Judging 2:30 – 3:30pm)

Poster Number Presenter – Title Abstract

14. Samantha Butrico – Expression of claudin-5 and prostate-specific membrane antigen in canine hemangiosarcoma cells .................................................................................................................. Page 14

15. Macy Flowers – Investigating the occurrence of transplacental Anaplasma marginale transmission in endemic beef cattle herds … ....................................................................................... Page 15

16. Jana Gigliotti – National Equine Tick Survey: A novel method for tracking ticks on horses in the US… ................................................................................................................................................................ Page 16

17. Joshuah B. Klutzke – Evaluation of Hematocrit in Juvenile Dogs Presenting for Routine Ovariohysterectomy or Neuter .................................................................................................................. Page 17

18. J.D. Luvanga – Haematological parameters of apparently health dogs in Morogoro Municipality, Tanzania .................................................................................................................................. Page 18

19. Daniel Madden – Development of a Novel Lateral Flow Assay for Detection of African Swine Fever Virus Antigen in Whole Blood........................................................................................................ Page 19

20. Ron Orchard – Year-to-Year Comparison of a One Health Service Event to Provide Veterinary Preventative Care to Low Income Kansas Residents ........................................................................ Page 20

21. Brandt C. Skinner – Isolation and Characterization of Two Anaplasma marginale Isolates from a Kansas Beef Cattle Herd ............................................................................................................................ Page 21

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Applied/Clinical Science Oral Presentations Phi Zeta Research Day

March 3, 2020, 1:15 – 4:30 pm 201 Trotter Hall

1:15 – 1:30 Tera L. Brandt – Insulin expression patterns in canine insulinomas………………… Page 22

1:30 – 1:45 Erin C. Hennessey – Computed Tomography of Hypaxial Muscle Abscessation in 10 Dogs ..................................................................................................................................................................... Page 23

1:45 – 2:00 Mathew R. DiFazio – Introduction and Clinical Application of the “Humanoid” Ventrodorsal Thoracic Radiographic View for Improved Cranial Thoracic Visualization ..................................................................................................................................................................... Page 24

2:00 – 2:15 Zackery Bieberly – Long acting injectable methadone for post-operative analgesia… ..................................................................................................................................................................... Page 25

2:15 – 2:30 Jared Bourek – The Effect of Handling Intensity at Time of Processing on Physiological Response, Immune Response, and Vaccination Status in Beef Cattle at the Feedlot…… ..................................................................................................................................................................... Page 26

2:30 – 3:30 Break for Poster Session

3:30 – 3:45 Alyson H. Fitzgerald – Detecting and Quantifying Marijuana Metabolites in Serum and Urine of Dogs Affected by Marijuana Toxicity ......................................................................... Page 27

3:45 – 4:00 Yin Wang – Investigation of PCV3 prevalence by the peptide-based ELISA assay .. Page 28

4:00 – 4:15 Kallie Woodruff – Evaluation of Feline Urine Concentrations of Amoxicillin and Clavulanate ......................................................................................................................................... Page 29

4:15 – 4:30 Kaitlynn N. Schuck– A Tale of Three Fixatives: Detection of Middle East Respiratory Syndrome Coronavirus in Paraffin-Embedded Tissues Preserved in Three Fixatives .................................................................................................................................................................. ...Page 30

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Basic Science Oral Presentations Phi Zeta Research Day

March 3, 2020, 1:15 – 4:30 pm 301 Trotter Hall

1:15 – 1:30 Sagar Rayamajhi – Synthesis and Characterization of Liposome hybridized extracellular vesicles as a magnetic resonance imaging contrast agent ............................................ Page 31

1:30 – 1:45 Pratiksha Khanal – Vaccination with a porcine reproductive and respiratory syndrome (PRRS) modified live virus (MLV) vaccine is associated with increased gut microbiome diversity ................................................................................................................................................... Page 32

1:45 – 2:00 Ravi Thakkar – Computational design of peptides for cancer immunotherapy ..... Page 33

2:00 – 2:15 Swetha Madesh – The canine host appears to serve as a sentinel species for tick-borne diseases caused by Anaplasma, Ehrlichia and Borrelia pathogens impacting human health in the USA... ............................................................................................................................................. Page 34

2:15 – 2:30 Chandramouli Kondethimmanahalli – Proteomics analysis reveals distinct protein expression in infectious and replicative forms of Ehrlichia chaffeensis ..................... Page 35

2:30 – 3:30 Break for Poster Session

3:30 – 3:45 Edward Bird – Whole-genome characterization of two Histophilus somni isolates from the lungs of clinically diseased calves ......................................................................................... Page 36

3:45 – 4:00 Deepak Kumar – Metagenomic Next-Generation Sequencing Reveal Presence of a Novel Ungulate Bocaparvovirus in Alpacas ............................................................................................ Page 37

4:00 – 4:15 Naveen Jonnalagadda – Antibacterial Effects of Equine Adipose Mesenchymal Stromal Cell Derived Exosomes against Escherichia coli and Staphylococcus aureus ............... Page 38

4:15 – 4:30 Bianca Libanori Artiaga – Role of NKT cell agonist for influenza virus vaccines in swine ..................................................................................................................................................................... Page 39

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March 3, 2020

Dominica D. Genda

Inactivation of porcine reproductive and respiratory syndrome virus and Seneca virus A in

cell culture using chemical feed additives

Corresponding Author: Megan C. Niederwerder, DVM, PhD, Diagnostic Medicine/Pathobiology,

College of Veterinary Medicine, Kansas State University, Manhattan, KS, USA.

Co-Authors: Ana M. M Stoian2, MS, Diagnostic Medicine/Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, KS, USA. Dominica D. Genda1, BVM, Diagnostic Medicine/Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, KS, USA. Corresponding author contact information: [email protected], 785-532-4663

Keywords: Formaldehyde; Medium Chain Fatty Acids; Seneca Virus A; porcine reproductive and

respiratory syndrome virus; cell culture

Abstract

Background: Seneca virus A (SVA) and porcine reproductive and respiratory syndrome virus (PRRSV) have contributed to significant economic losses of swine production in the United States. Recently, contaminated swine feed and feed ingredients have been considered risk factors for virus transmission. The use of chemical feed additives has been discussed as one potential management strategy to mitigate this risk. The objective of this study was to evaluate the efficacy of medium chain fatty acid-based (MCFA) and formaldehyde-based (FORMALD) liquid antimicrobials against SVA and PRRSV in a cell culture model. Materials and Methods: Viral stocks of SVA and PRRSV (106 TCID50/ml) were mixed with different concentrations of MCFA or FORMALD in minimum essential media. Ten-fold serial dilutions of each virus/mitigant mixture were performed in triplicate for inoculation onto confluent monolayers of PK-15 and MARC-145 cells, respectively. The titers were determined by the serial dilution endpoint method. The differences between the titer of the control virus (no mitigant) and the mitigant-treated samples were used to measure antiviral activity. Results: FORMALD reduced the titers of both SVA and PRRSV at concentrations above 0.3% and 0.11%, respectively. MCFA reduced the PRRSV titer at concentrations greater than 0.2%. No significant reduction of SVA titers were noted after exposure to MCFA up to 5%. Conclusion: This study provides evidence suggesting that both MCFA and FORMALD may be useful as potential feed additives to mitigate the risk of SVA or PRRSV transmission through contaminated animal feed or ingredients.

mailto:[email protected]

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March 3, 2020

Jason M. Gregory

ATP-sensitive potassium channel blockade: Mechanisms for decreased exercise tolerance

Corresponding Author: Trenton D. Colburn, MS, BS, Department of Anatomy and Physiology,

Kansas State University, Manhattan, KS, USA

Co-Authors: Kiana M. Schulze, BS, Department of Anatomy and Physiology, Kansas State University,

Manhattan, KS, USA; Ramona E. Weber, BS, Department of Anatomy and Physiology, Kansas State

University, Manhattan, KS, USA; David C. Poole, Ph.D, D.Sc, Department of Anatomy and Physiology,

Kansas State University, Manhattan, KS, USA; Tim. I Musch, Ph.D, Department of Anatomy and

Physiology, Kansas State University, Manhattan, KS, USA

Corresponding Author Contact Information: [email protected], (785)-955-0146

Keywords: Glibenclamide; Exercise Tolerance; Blood Flow

Abstract

Glibenclamide (GLI), a sulfonylurea drug prescribed for Type II diabetes increases insulin release by inhibiting ATP-sensitive potassium (KATP) channels and depolarizing pancreatic beta cells. These channels are also found in vascular smooth muscle cells, whose depolarization causes vasoconstriction and reduced muscular blood flow. It was hypothesized that KATP channels support exercise tolerance by increasing and sustaining muscular blood flow and oxygen delivery. Therefore, KATP channel inhibition would reduce muscle blood flow and interstitial oxygen availability. Male Sprague-Dawley rats (~4 months old) were evaluated for submaximal exercise tolerance (critical speed; n=10), interstitial oxygen pressure (PO2is; n=9), and muscle blood flow at the site of PO2is measurement site. Critical speed was determined via 4-5 runs to exhaustion at constant speed. Under anesthesia, PO2is was measured via phosphorescence quenching during 180s electrically-induced contractions of the mixed gastrocnemius muscle during control and following muscle GLI superfusion (GLI: 5 mg/kg BW). Blood flow was determined via fluorescent microsphere technique. GLI reduced critical speed (32.27 ± 0.90 vs 29.73 ± 1.09 m/min), blood flow (32.2 ± 3.2 vs. 20.7 ± 3.7 mL/min/100g) and PO2is (Nadir: 6.60 ± 0.63 vs. 5.36 ± 0.35 mmHg; Endpoint: 8.69 ± 0.95 vs. 6.85 ± 0.58 mmHg O2; p≤0.05 for all) compared to control. These data support the hypothesis that KATP is crucial for exercise tolerance through adequate muscle blood flow and oxygen delivery. Improper use of sulfonylurea drugs when blood flow is already impaired, e.g. heart failure, can lead to an exacerbation of exercise intolerance and further impairing prognostic outcomes.


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March 3, 2020

Sarah Krueger

Evaluation of Amblyomma americanum vector competence for Anaplasma marginale

Corresponding Author: Sarah Krueger, B.S., College of Veterinary Medicine, Kansas State

University, Manhattan, Kansas

Co-Authors: Tippawan Anantatat, Department of Diagnostic Medicine/Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, Kansas

Kathryn E. Reif, MSPH, Ph.D., Department of Diagnostic Medicine/Pathobiology, College of

Veterinary Medicine, Kansas State University, Manhattan, Kansas

Corresponding Authors Contact Information: [email protected], 620-794-4255

Keywords: Anaplasma marginale, anaplasmosis, Amblyomma americanum, Dermacentor variabilis

Abstract

Background: Bovine anaplasmosis is a tick-transmitted, production-limiting disease and a major obstacle to profitable beef cattle production in the United States. Changes in climate, ecosystems, and increases in animal transport have contributed to the expansion of various disease-transmitting tick species, including the Lone Star Tick (LST) (Ambylomma americanum), a species commonly found on cattle. The intracellular rickettsial pathogen and agent of anaplasmosis, Anaplasma marginale (Am), is primarily transmitted by Dermacentor tick species in the U.S. The role of LST in transmission of bovine anaplasmosis is currently unknown; however, the frequency of LST infestation on cattle warrants examination into whether LST contributes to Am transmission. The objective of this study was to examine the vector competence of LST for Am using a controlled laboratory transmission experiment. Methods: The vector competence of LST for Am was specifically evaluated by comparing the ability of two geographically distinct LST strains to acquire and transmit Am compared to a known Am vector, Dermacentor variabilis, using an experimental tick-calf Am transmission model. The Am bacterial levels were monitored throughout the transmission process in calf blood and tick midgut and salivary gland tissues to track Am levels. Results: The Dermacentor variabilis vector tissues and calves tested positive for Am, indicating positive transmission. LST vector tissues and calves did not display detectable levels of Am. Analysis of serum samples will confirm positive or negative status of calves. Conclusion: The results provide direct evidence indicating that LST are unlikely to contribute to Am transmission in the U.S., information which in important when making disease management decisions.


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March 3, 2020

Keith Lewy

The Impact of Various Gut Microbiota Transfer Methods on the Development of Colitis in

Response to Dextran Sulfate Sodium

Corresponding Author, Keith Lewy, DVM Candidate Class of 2021, Kansas State University College

of Veterinary Medicine, Manhattan, KS

Co-Authors: Chunye Zhang, Benjamin Olthoff, Matt Birch, Aaron Ericsson, Craig Franklin,

University of Missouri College of Veterinary Medicine, Columbia, MO

Corresponding Author Contact Information: [email protected], 516-445-3623

Keywords: Microbiome; GM-transfer; colitis; histopathology

Abstract

The microbiome significantly affects the physiology and disease susceptibility of mice used in research. The gut microbiota (GM) can be transferred from one mouse to another to evaluate its role in disease susceptibility prospectively. While there are several different methods of transferring the GM, their relative transfer efficacy has not been directly compared. Moreover, it is not known whether transfer efficacy will affect the phenotype of experimental models. The objectives of this study are to determine the efficacy of transfer from donor to recipient mice using two GM transfer techniques, cross fostering and co-housing, and the degree to which transfer efficacy influences susceptibility to dextran sulfate sodium (DSS) colitis. C57BL/6 mice with different standardized gut microbiota (SGM), SGM1 (low GM richness) and SGM4 (high GM richness), were used. SGM1 mice received a GM transfer from SGM4 mice, and vice versa, via either cross-fostering (<24 hours old) or co-housing (beginning at weaning). Chronic colitis was induced via periodic administration of DSS in drinking water. Mice were weighed daily and survival of the mice was recorded. After euthanasia, histology sections of both the cecum and colon were scored for inflammatory damage. While analysis is ongoing, significant differences were found in mortality, weight loss, and histopathological disease severity between the four experimental groups. Current results suggest that the transfer method and relative richness of donor and recipient mice affect the transfer efficacy, as well as the phenotypic outcomes of that transfer, indicating the need to consider these factors in experimental design and analysis and interpretation of ‘in house’ and published data.


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March 3, 2020

Lauren Miller

Characterization of a Novel Tick Transmitted Ehrlichia sp. Infection in Horses

Corresponding Author, Brian Herrin, DVM, PhD, College of Veterinary Medicine, Kansas State

University, Manhattan KS, USA.

Co-Authors: Samantha Hancock, DVM1 , Todd Holbrook, DVM1, Susan Little, DVM, PhD1 1 Oklahoma State University Center for Veterinary Health Sciences, Oklahoma State University,

Stillwater OK, USA.

Corresponding Author Contact Information: [email protected]; 785-532-4430

Keywords: Ehrlichia ; Horse ; Tick ; Amblyomma americanum

Abstract

Although no true Ehrlichia species is known to infect horses, previous studies from our laboratory indicate that horses within the range of Amblyomma americanum can test positive for antibodies to an Ehrlichia spp. by IFA or ELISA. To determine the potential for these ticks to transmit an Ehrlichia spp. to horses, wild-caught A. americanum were placed on the naïve animals. Ticks were allowed to feed to repletion, and blood was drawn at weekly intervals for PCR and IFA testing. Additionally, Ehrlichia-negative A. americanum were placed onto the horses after the initial infestation in an attempt to confirm infection by xenodiagnosis. Post-feeding, the ticks were removed, DNA was extracted, and PCR was performed on the lab-reared ticks using nested primers targeting the 16S rRNA gene. Serologically, all horses tested antibody positive by day 21 post infestation and remained antibody positive until the end of the study period (Day 56). The highest IFA titer was 1:32768 dilution, while the other 4 horses ranged from 1:512—1:8192. No Ehrlichia spp. was ever detected by PCR of whole blood. Of the 60 ticks assessed, four tested PCR positive. All positive ticks were removed on day 21 from horse 207, the horse with the lowest initial IFA titers. Initial sequencing of positive samples most closely match with Ehrlichia chaffeensis sequences in GenBank. While clinical manifestations have yet to be demonstrated, preliminary results suggest that there is potential for horses to become transiently infected with Ehrlichia chaffeensis and infect other ticks.


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March 3, 2020

Ryan Swanson

Multi-dimensional fluorescence and bioluminescence imaging and detection specificity of

physiometacomposite:enzyme interfaces

Corresponding Author: Robert DeLong, M.S., PhD, Nanotechnology Innovation Center Kansas State, Department of Anatomy and Physiology, College of Veterinary Medicine, Kansas State University, Manhattan, Kansas, USA.

Co-Authors: Amanda Hoffman B.S.; Julie Majka, M.S.; Tej B. Shrestha, M.S., PhD,; Elza N. Mathew, M.S., Nanotechnology Innovation Center Kansas State, Department of Anatomy and Physiology, College of Veterinary Medicine, Kansas State University, Manhattan, Kansas, USA. Corresponding Author Contact Information: [email protected] Keywords: physiometacomposites; fluorescence; bioluminescence; 3-D FDS

Abstract

Introduction: We described the impact of physiologically based metal nanoparticles on Luciferase (Luc) protein enzyme functional dynamics and here Luc bioluminescence or β-galactosidase (β-Gal) enzyme fluorescence was amplified by certain physiometacomposites (PMC). It is first shown here that these properties can be used for detecting specific molecular interactions and tissue imaging and their signal is not quenched when introduced to serum or tissue homogenates. Methods: 3-dimensional fluorescence difference spectroscopy (3-D FDS), bioluminescence and MTT assays were measured on a Molecular Devices SpectraMax i3x. PerkinElmer (Caliper) LifeSciences IVIS Lumina II imager and Pearl Trilogy were used for Bio-imaging. Enzymes used were Luciferase (Luc) and beta-galactosidase (β-Gal). Results: 5% Mg/ZnO, 2% Co/ZnO and MnZnS emitted in the near infra-red range (655-665 nm) with MnZnSe 6-log order intensity. Ex vivo and in vivo performance was compared for liver bio-imaging with a 20 µg dose delivery yielding a total radiant efficiency of > 109 and average radiant efficiency of 108 which was comparable to cyanine5.5-labeled ZnO nanoparticle. Signal amplification occurred by ligation of Luc to FeZnS or MnZnS with 1.5x107 RFU at 675 nm shifting the bioluminescence also into the near infra-red (NIR). Conclusion: Radiant efficiency affords tissue imaging and the shift in the 3-D FDS pattern can be exploited to distinguish specific biomolecular interactions versus non-nonspecific interactions. These data support PMC composition optimization for enhanced fluorescence/bioluminescence, their titration with various cell types (e.g. pancreas cells) and applications including in vivo administration and real-time imaging marker-functionalization for organ-specific labeling.


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March 3, 2020

Deepa Upreti

Water extract from Euglena gracilis prevents lung carcinoma growth in mice via attenuation

of myeloid-derived suppressor cells and granulocyte populations

Corresponding Author: Deepa Upreti, Department of Anatomy & Physiology, College of Veterinary

Medicine, Kansas State University, Manhattan, KS, United States.

Co-Author: Susumu Ishiguro1, Deepa Upreti1, Nicole Robben1, Paige Cote1, Riley Burghart1, Mayme loyd1, Damilola Ogun1, Tran, Le1, Jennifer Delzeit1, Arashi Nakashima1, Ayaka Nakashima2, Kengo Suzuki2, Jeffrey Comer1, and Masaaki Tamura1 1, Departments of Anatomy & Physiology, Kansas State University College of Veterinary Medicine, Manhattan, Kansas, United States of America 2, Central Research Center, euglena Co. Ltd., Tokyo, Japan Corresponding Author Contact Information: [email protected]

Keywords: Lung cancer prevention, Euglena extract, Immunostimulation, Attenuation of MDSC,

Decrease of granulocytes

Abstract

Background: Euglena, a genus of single-cell flagellate eukaryotes, has been used as a dietary supplement for their rich nutrient content. Although recent studies suggest that Euglena extracts have anti-tumor properties, the mechanism is yet to be clarified. Methods: The anti-tumor and immunomodulatory effects of partially purified water extract from Euglena gracilis (EWE) was evaluated in cell culture and a mouse orthotopic lung carcinoma allograft model. The EWE was prepared by a PBS extraction at 37°C for 30 min, centrifugation at 2,000 g for 20 min, and filtration through a 0.22 µm membrane filter. Results: In two-dimensional cell culture, EWE treatment inhibited cell growth of both murine Lewis lung carcinoma (LLC) and human lung carcinoma cells in a dose (1–100 µg/ml) and time (24–72 h)-dependent manner. In three-dimensional spheroid culture, spheroid growth of LLC cells was significantly attenuated upon EWE treatment. In a mouse LLC orthotopic allograft model, pretreatment with EWE (100–200 mg/kg/day, via drinking water) three weeks prior to LLC cell inoculation significantly attenuated the growth of LLC tumors in the lungs of immunocompetent syngeneic mice with a significant decrease of myeloid-derived cells, primarily neutrophils. However, this attenuation was not seen for EWE treatment initiated after LLC cell inoculation. Conclusions: EWE pretreatment inhibits lung carcinoma growth mainly by stimulating host anti-tumor immunity via an attenuation of the host myeloid-derived immune suppressor cell population. Hence, the present study suggests that the partially purified extract derived from Euglena gracilis contains bioactive materials that prevent lung carcinoma growth.

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March 3, 2020

Brandon Verkinderen

Effect of kinase inhibitor treatment on Rift Valley Fever Virus replication

Corresponding Author: Juergen A. Richt, DVM, PhD, Department of Diagnostic

Medicine/Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan,

Kansas, USA

Co-Authors: David A. Meekins, PhD, Department of Diagnostic Medicine/Pathobiology, College of

Veterinary Medicine, Kansas State University, Manhattan, Kansas, USA

Corresponding Author Contact Information: [email protected], (785) 532-2793

Keywords: RVFV; NSs-protein; CK2 inhibitor

Abstract

Rift Valley fever virus (RVFV) is a mosquito-borne pathogen that can cause significant disease in humans and livestock species. It is at high risk for transboundary spread due to widespread vector competence and increased travel/trade among endemic and non-endemic countries. There are no commercially available vaccines or therapeutics to prevent or treat RVFV infection in humans. The severity of RVFV is attributed to the activity of its nonstructural protein, NSs, which is a major virulence factor responsible for inhibition of host antiviral immune responses. Previous studies show NSs is phosphorylated by casein kinase 2 (CK2), and evidence suggests that this may be necessary for NSs-interaction with specific E3-ubiquitin ligases that results in shutdown of the host cell’s interferon response. The purpose of this study was to test whether inhibition of NSs phosphorylation could be used as a treatment strategy to limit RVFV replication in vitro. Human A549 cells were treated with the drug CX4945, a potent and specific CK2 inhibitor. The effect of CX4945 on RVFV replication over 24 hours was determined via plaque assay using the RVFV MP-12 vaccine strain on treated cells compared to non-treated cells at differing MOI. Analysis of MP-12 viral replication in the presence of CX4945 show ~25-45% decrease in viral titers at 24 hours post infection. These data suggest that CK2 inhibition affects RVFV replication, but the effect is not statistically significant. Further investigations are necessary to elucidate the role of NSs phosphorylation in RVFV virulence in order to develop novel RVFV treatment strategies.


9


March 3, 2020

Yu Shin Wang

A non-destructive protocol for whole-mount bone and cartilage staining of African spurred

tortoise (Centrochelys sulcata)

Corresponding Author: Pradeep Malreddy, BVSc and AH, MS

Co-Authors: Pradeep Malreddy BVSc and AH, MS; Dept. of Anatomy and Physiology, College of

Veterinary Medicine, Manhattan, KS 66506


Keywords: Whole-mount; skeletal preparation; Alcian blue; Alizarin red; Cartilage; Bone

Abstract:

Whole-mount bone staining has been widely used as the first step in skeletal phenotype studies allowing for detection and evaluation of the changes in skeletal patterning. A number of protocols have been published for pre-natal and post-natal developmental stages of mice, chicken, zebra fish and frog, but procedures of whole-mount staining for animals with exoskeleton have not been published. In this research, a two-month-old African spurred tortoise (Centrochelys sulcata) was used as the specimen. The specimen was fixed in a solution containing formalin, Triton X- 100, and KOH as well as an enhancement solution consisting of ethylene glycol, Triton X-100, potassium hydroxide. Staining procedures were applied thereafter with Alcian blue and Alizarin red staining cartilage and bone, respectively. Trypsin was used in between cartilage and bone staining for further clearing. As expected the specimen was cleared and found no sign of destruction in soft tissue. Cartilage and bone were stained respectively. This novel procedure can serve as a starting point of the development of whole-mount staining protocol for animals with exoskeleton. With further investigation and replications, this protocol can contribute to the evaluation of malnutrition-led bone deformation in terrestrial and marine turtle.


10


March 3, 2020

Yvonne Wikander

Prevalence of Cytauxzoon felis carriers in the domestic cat population of eastern Kansas, a

preliminary report

Corresponding Author: Kathryn Reif, PhD, Department of Diagnostic Medicine/Pathobiology,

College of Veterinary Medicine, Kansas State University, Manhattan, KS, USA

Co-Authors: Tippawan Anantatat, MS Department of Diagnostic Medicine/Pathobiology, College of

Veterinary Medicine, Kansas State University, Manhattan, KS, USA


Keywords: Cytauxzoon felis; cytauxzoonosis; prevalence; tick-borne disease; Kansas

Abstract

Background: Cytauxzoon felis is a tick-borne hemoprotozoal organism of cats that causes cytauxzoonosis. Bobcats (Lynx rufus) are the natural host for this pathogen in North America. Domestic cats infected with this pathogen often present with acute illness, which is frequently fatal within 1-2 weeks. Cats that survive acute disease remain persistently infected and serve as transmission reservoirs. Studies have demonstrated that multiple C. felis strains, which may vary in pathogenicity, are circulating in the United States. Amblyomma americanum, a major transmission vector for C. felis, is the dominant tick in eastern Kansas. Therefore, cats in this area are at risk for acquiring and serving as reservoirs for C. felis. Objective: Determine the carrier (persistently infected) prevalence of C. felis in domestic cats in eastern Kansas using blood samples opportunistically collected from cats undergoing routine clinical procedures. Methods: EDTA blood samples were collected from cats undergoing routine procedures at animal shelters and veterinary clinics in eastern Kansas. DNA was extracted for real-time PCR. Specific C. felis genes were cloned and sequenced from positive PCR samples to determine their C. felis genotype. Results: Preliminary data suggests 23% (258/1121) of domestic cats in eastern Kansas are persistently infected with C. felis. Conclusions: Further investigation is warranted to determine ways to block C. felis transmission (e.g. vaccine development) and/or improve treatment options for acutely and chronically infected cats.


11


March 3, 2020

Adrienne Wright

Optimization of a Protocol for the Isolation, Culture, and Cryopreservation of Umbilical Cord-

Derived Canine Mesenchymal Stromal Cells for Future Clinical Use

Corresponding Author: Mark L. Weiss, Department of Anatomy and Physiology, Kansas State

University College of Veterinary Medicine, Manhattan, KS, United States, The Midwest Institute of

Comparative Stem Cell Biology, Manhattan, KS

Co-Authors: Larry Snyder, Department of Anatomy and Physiology, Kansas State University College of Veterinary Medicine, Manhattan, KS, United States; Kaori Knights, Department of Diagnostic Medicine / Pathobiology, Kansas State University College of Veterinary Medicine, Manhattan, KS, United States; Nora L. Springer, Department of Diagnostic Medicine / Pathobiology, Kansas State University College of Veterinary Medicine, Manhattan, KS, United States; James Lillich, Department of Anatomy and Physiology, Kansas State University College of Veterinary Medicine, Manhattan, KS, United States


Keywords: mesenchymal stromal cells; canine; clinical translation; allogenic transplantation;

Wharton’s jelly

Abstract:

Mesenchymal stromal cells (MSCs) hold great promise in the field of regenerative medicine due to their ability to create a variable localized anti-inflammatory effect in injuries such as Crohn’s disease and osteoarthritis or by incorporation in tissue engineered constructs. Currently, the MSC literature uses rodents for preclinical disease models. There is growing interest in using naturally occurring disease in large animals for modeling human disease. In review of the canine MSCs literature, it appears that canine MSCs can be difficult to maintain in culture for extended passages and this greatly varies between tissue sources, compared to human and rodent MSCs. Research using canine MSCs has been focused on cells derived from bone marrow or adipose tissue, and the differences in manufacturing MSCs between laboratories is problematic due to lack of standardization. To address these issues, here, a stepwise process was used to optimize canine MSCs isolation, expansion, and cryopreservation utilizing canine umbilical cord-derived MSCs. The culture protocol utilizes coating of tissue culture surfaces that increases cellular adherence, increases colony-forming units-fibroblast efficiency, and decreases population doubling times. Canine MSCs isolated with our protocol could be maintained longer than published canine MSCs methods before senescing. Our improved cryopreservation protocols produce on average > 90% viable MSCs at thaw. These methods enable master-bank and working-bank scenarios for allogeneic MSC testing in naturally occurring disease in dogs.


12


March 3, 2020

Alexandria Zabiegala

Genetic and replication analysis of highly virulent Feline Calicivirus isolate, KS-2019

Corresponding Author: Alexandria Zabiegala, DMP, Kansas State University, Manhattan, KS,

United States

Co-Authors: Krishani Perera, Yunjeong Kim, DVM/PhD, Kyeong-Ok Chang, DVM/PhD

DMP, Kansas State University, Manhattan, KS, United States


Keywords: Feline Calicivirus; Genetics; Replication

Abstract:

Feline calicivirus (FCV) is a non-enveloped, positive-sense RNA virus belonging to the genus Vesivirus within the family Caliciviridae. Typically, FCV infection in cats causes upper respiratory symptoms and oral ulcers with high morbidity and low mortality. However, over the past 20 years, there have been many reports of highly fatal diseases caused by virulent systemic (VS) FCV. In 2019, a feline patient exhibiting acute onset of illness with a high fever, anorexia, swollen legs and neck was presented to the Kansas State University Veterinary Health Center. Subsequent histology and laboratory testing confirmed VS FCV infection. FCV was isolated from the tissue in Crandell Reese Feline Kidney (CRFK) cells and designated as KS-2019. In this study, the full-length sequencing analysis and investigation of replication characteristics of FCV KS-2019 were conducted. The multiple sequence alignment and phylogenetic analysis using the VP1, VP2 and LC proteins of KS-2019 showed that there is approximately 80% amino acid homology between KS-2019 and other reported FCV strains and there is no evidence of phylogenetic clustering of VS FCV strains. The replication characteristics of KS-2019 in CRFK cells were determined by conducting the single-cycle replication assay and compared to those of Urbana strain, a prototype non-VS FCV strain. Single-cycle replication assay revealed that KS-2019 strain has higher virus yields in comparison to Urbana strain, which is consistent with previous reports with VS FCV strains. Further studies are undergoing to assess individual viral entry events including virus attachment, endosome entry, and endosomal escapes to determine which step is responsible for the increased replication of KS-2019 in cell culture.


13


March 3, 2020

Michelle Zajac

Development and evaluation of a rabies enzyme-linked immunosorbent assay (ELISA)

targeting IgM and IgG in human sera

Corresponding Author: Michelle Zajac, M.S. Veterinary Biomedical Science, Diagnostic Medicine

and Pathobiology, Kansas State University, Manhattan, KS, United States


Keywords: ELISA; rabies; immunology; vaccinology; immunoglobulin

Abstract:

Mesenchymal stromal cells (MSCs) hold great promise in the field of regenerative medicine due to their ability to create a variable localized anti-inflammatory effect in injuries such as Crohn’s disease and osteoarthritis or by incorporation in tissue engineered constructs. Currently, the MSC literature uses rodents for preclinical disease models. There is growing interest in using naturally occurring disease in large animals for modeling human disease. In review of the canine MSCs literature, it appears that canine MSCs can be difficult to maintain in culture for extended passages and this greatly varies between tissue sources, compared to human and rodent MSCs. Research using canine MSCs has been focused on cells derived from bone marrow or adipose tissue, and the differences in manufacturing MSCs between laboratories is problematic due to lack of standardization. To address these issues, here, a stepwise process was used to optimize canine MSCs isolation, expansion, and cryopreservation utilizing canine umbilical cord-derived MSCs. The culture protocol utilizes coating of tissue culture surfaces that increases cellular adherence, increases colony-forming units-fibroblast efficiency, and decreases population doubling times. Canine MSCs isolated with our protocol could be maintained longer than published canine MSCs methods before senescing. Our improved cryopreservation protocols produce on average > 90% viable MSCs at thaw. These methods enable master-bank and working-bank scenarios for allogeneic MSC testing in naturally occurring disease in dogs.


14


March 3, 2020

Samantha Butrico

Expression of claudin-5 and prostate-specific membrane antigen in canine

hemangiosarcoma cells

Corresponding Author: Nora L. Springer, DVM, DACVP, Ph.D, Department of Diagnostic

Medicine/Pathobiology, Kansas State University College of Veterinary Medicine, Manhattan, KS,

United States

Co-Authors: Kaori Knights, M.S., Unit(s), Department of Diagnostic Medicine/Pathobiology, Kansas

State University College of Veterinary Medicine, Manhattan, KS, United States; Raelene Wouda, BVSc

DACVIM (Oncology) MANZCVS (Small Animal Medicine), Department of Clinical Sciences, Kansas

State University College of Veterinary Medicine, Manhattan, KS, United States

Corresponding Author Contact Information: [email protected] (785) 532-4818

Keywords: hemangiosarcoma; oncology; canine; diagnostic

Abstract

Introduction: Hemangiosarcoma is an aggressive form of malignant cancer that develops from the vascular endothelial cells of various body tissues, most commonly in the spleen and heart. HSA affects dogs more than any other species and is difficult to diagnose without surgical biopsy. There is a need for canine hemangiosarcoma-specific markers that can be applied to minimally invasive diagnostic techniques such as immunocytochemistry or flow cytometry. The purpose of this study is to evaluate the expression of two candidate markers, claudin-5 and prostate-specific membrane antigen on canine hemangiosarcoma cells. Both claudin-5 and prostate-specific membrane antigen have been utilized as differential diagnostic markers of canine hemangiosarcoma via immunohistochemistry but have yet to be validated for immunocytochemistry or flow cytometry. Methods: JHE and DAL4 cell lines were used to evaluate claudin-5 and prostate-specific membrane antigen expression in canine hemangiosarcoma cells using immunofluorescence with confocal microscopy, western blotting, and flow cytometry. Canine aortic endothelial cells (CnAOEC) and human prostatic carcinoma cells (PC3) were used as positive controls for claudin-5 and prostate-specific membrane antigen, respectively. Results: Preliminary results demonstrate variable expression of both markers across all cell lines. Conclusions: Currently, claudin-5 and prostate-specific membrane antigen expression patterns are not consistently observed across cell lines or assays. Therefore, at least two additional canine hemangiosarcoma cell lines (EFS and EFB) will be assessed prior to determining if these markers are suitable for immunocytochemistry or flow cytometry.


15


March 3, 2020

Macy Flowers

Investigating the occurrence of transplacental Anaplasma marginale transmission in

endemic beef cattle herds

Corresponding Author: Kathryn E. Reif, B.A., PhD, Department of Diagnostic Medicine/

Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, Kansas, United

States

Co-Authors: Tippawan Anantatat1, B.S, Department of Diagnostic Medicine/ Pathobiology, College

of Veterinary Medicine, Kansas State University, Manhattan, Kansas, United States; Emily J.

Reppert2, DVM, MS, Kansas State University, Manhattan, Kansas, United States


Keywords: Anaplasma marginale; Transplacental transmission; Cattle

Abstract

Background and Purpose: Bovine anaplasmosis is a bacterial disease of cattle caused by the bacterial pathogen Anaplasma marginale. Anaplasmosis is endemic in Kansas beef cattle herds with approximately 47% of Kansas beef herds actively infected with A. marginale. Transmission of A. marginale to naïve animals can occur via ticks (the most efficient and only biological vector) or blood-contaminated surgical instruments and biting fly mouthparts. Anaplasmosis literature states that transplacental transmission between dam and calf may also occur, however, robust data on the frequency of transplacental transmission is lacking. This study was designed to investigate the frequency of transplacental A. marginale transmission in highly endemic cow-calf herds. Knowing how and when cattle become infected with A. marginale is important when developing anaplasmosis management strategies. Method: Two fall-calving cow-calf herds with high incidence of anaplasmosis were identified. Blood samples were collected from dams and calves and were tested for A. marginale infection using a molecular test to detect A. marginale DNA Results and Conclusion: The A. marginale infection incidence of the dams was 65.8% and 85.7 % for each cow-calf herd, respectively. The A. marginale infection rates of the calves will be examined within one month of birth and again between 5-7 months of age. Knowing how and when cattle become infected with A. marginale is important when making herd anaplasmosis management strategies, including retention of A. marginale-infected dams, use of antibiotics for the purpose of anaplasmosis control, and integration plans for new animals entering an endemic herd.


16


March 3, 2020

Jana Gigliotti

National Equine Tick Survey: A novel method for tracking ticks on horses in the US

Corresponding Author: Brian Herrin, DVM, PhD, Department of Diagnostic Medicine and

Pathobiology, Kansas State University, Manhattan, KS, USA.

Co-Author: Brian Herrin, DVM, PhD1

1: Department of Diagnostic Medicine and Pathobiology, Kansas State University, Manhattan, KS,

USA


Keywords: Equine; ticks; survey; Ixodes scapularis; Amblyomma americanum

Abstract

Introduction: Currently, there is no persistent, highly effective tick prevention commercially available for horses, leaving them vulnerable to disease and discomfort. Studying ticks on horses via the National Equine Tick Survey reveals patterns in data about tick species’ geographic distribution, seasonal prevalence, and other species characteristics as they relate to horses. Methods: Since October 2018, 49 veterinarians and owners were enrolled in the study across 22 states. In total, 1274 ticks were found on 222 horses and sent to the lab to be categorized by species, sex, and life stage. Information about the infested horses was collected as well, including geographic location, time spent in pasture, and tick location on the body. This data was then analyzed using Excel and MapViewer. Results: Tick species collected included Dermacentor albipictus, Dermacentor variabilis, Dermacentor occidentalis, Amblyomma maculatum, Amblyomma americanum, Ixodes scapularis and Otobius megnini, with I. scapularis and A. americanum representing the two most prevalent species collected year round (36% and 22%, respectively). The majority of I. scapularis ticks were collected during the fall months in the Northeast, while the majority of A. americanum ticks were collected during the spring months in the Southeast. Data also showed that the ventral head/neck area was the most common site of attachment by I. scapularis, while the inguinal area was the most common site for A. americanum. Conclusion: By documenting the burden and diversity of equine ticks, the results of this survey highlight the significance of ticks as a medical problem for horses.


17


March 3, 2020

Joshuah B. Klutzke

Evaluation of Hematocrit in Juvenile Dogs Presenting for Routine Ovariohysterectomy or

Neuter

Corresponding Author: Kate KuKanich, DVM, PhD, DACVIM (SAIM), Department of Clinical

Sciences, Kansas State University, Manhattan, Kansas, USA

Co-Authors: Nora Springer, DVM, PhD, DACVP;1 Butch KuKanich, DVM, PhD, DACVCP.2

1: Department of Diagnostic Medicine/Pathobiology, Kansas State University, Manhattan, Kansas,

USA.

2: Department of Anatomy and Physiology, Kansas State University, Manhattan, Kansas, USA.


Keywords: Juvenile; Canine; Shelter; Hematocrit; Ovariohysterectomy/Neuter

Abstract

Introduction: Hematological parameters for healthy juvenile dogs are established; however, applicability of these reference values to a shelter population of dogs with potential comorbidities is unknown. Methods: Shelter dogs (77 female, 61 male) of varied breeds presented to the Kansas State Junior Surgery Laboratory. All dogs received physical examination including dental aging, CBC, 4Dx, and flea comb, and dogs with diarrhea had fecal float +/- parvo test. Dogs were grouped into 3 categories: Puppies (P) with no adult incisors erupted (< 3 months old); Mideruption (M) with some adult incisors erupted (3-6 months old); or Adult (A) with all adult canines erupted (>6 months old). A one-way ANOVA was used to compare hematocrit (HCT) between groups. Results: There were 34 P, 22 M, and 82 A dogs, with mean calculated HCT 35.6% (range 27-46%), 37% (31-48%), 45.8% (34-59%), respectively. Three dogs were positive for Anaplasma (HCT 36-56%), 5 Ehrlichia (HCT 34-47%), and 1 heartworm (HCT 50%). Twelve dogs had fleas or flea dirt [10 A (HCT 37-47%), 1 M (HCT 37%), and 1 P (HCT 30%)]. Two dogs had hookworms (HCT 29-35%), 3 had roundworms (HCT 27-34%), 1 had tapeworms (HCT 35%), 1 had coccidia (HCT 37%), and 1 had parvovirus (HCT 40%). Hematocrit differed significantly between adults and puppies (p<0.01) and between adults and mideruption dogs (p<0.01), whether infectious cases were included or excluded. Conclusions: Juvenile shelter dogs have lower HCT than adults, similar to prior reports. Further investigation into underlying cause and influence of infectious disease is warranted.


18


March 3, 2020

J.D. Luvanga

Haematological parameters of apparently health dogs in Morogoro Municipality, Tanzania

Corresponding Author: Julius D. Luvanga, Department of Veterinary Surgery and Theriogenology,

College of Veterinary Medicine and Biomedical Sciences, Sokoine University of Agriculture, P.O. Box

3020, Chuo Kikuu, Morogoro, Tanzania.

Corresponding Author Contact Information: [email protected];

[email protected]

Keywords: dog, haematology, local breeds, Morogoro, parameters

Abstract

Reference values for haematological parameters that are normally used in Tanzanian local dogs were obtained from exotic breeds of Western countries. Based on the fact that haematological parameters varies between species and breeds, there is a need to establish standard values for local breeds of dogs which are most common in Tanzania. The present study was carried out to establish the standard haematological values of local breeds of dogs in Morogoro Municipality and compare with reference values of exotic breeds. Blood was collected from 296 local breeds of dogs in selected areas of Morogoro municipality. Blood sample was processed automatically by using haematological analyser (MS4S). There was no significant variations in the haematological values of local breeds of dogs when compared to the reference range of the exotic breed (p>0.05). In local breeds of dogs, there was no significant difference (p>0.05) in the haematological parameters between male and female animals. Significant (p<0.05) higher values of eosinophils, red blood cells count, packed cell volume and haemoglobin concentration were observed in adults than in puppies. The haematological parameters generated from this study will provide a reference for routine clinical works.



19


March 3, 2020

Daniel Madden

Development of a Novel Lateral Flow Assay for Detection of African Swine Fever Virus

Antigen in Whole Blood

Corresponding Author, Daniel Madden, DVM. Department of Diagnostic Medicine/Pathobiology,

Kansas State University College of Veterinary Medicine; Manhattan, KS

Co-Authors: Sunyoung Sunwoo, DVM, PhD. Department of Diagnostic Medicine/Pathobiology, Kansas State University College of Veterinary Medicine; Manhattan, KS, USA Natasha Gaudreault, PhD. Department of Diagnostic Medicine/Pathobiology, Kansas State University College of Veterinary Medicine; Manhattan, KS, USA Jessie Trujillo, DVM, PhD. Department of Diagnostic Medicine/Pathobiology, Kansas State University College of Veterinary Medicine; Manhattan, KS, USA Juergen A Richt, DVM, PhD. Department of Diagnostic Medicine/Pathobiology, Kansas State University College of Veterinary Medicine; Manhattan, KS, USA.

Corresponding Author Contact Information: [email protected]

Keywords: African swine fever virus; lateral flow assay; antigen detection; diagnostics

Abstract

African swine fever (ASF) is a highly lethal disease of domestic and wild pigs which has inflicted widespread devastation to swine herds in Africa, Europe, and Asia, causing millions of animal deaths and billions of dollars in economic losses. The causative agent, African swine fever virus (ASFV), is highly transmissible and environmentally resilient. No effective vaccine or treatment for ASF has been developed, which limits control efforts to movement restrictions on animals and pork products and culling affected herds. Consequently, rapid and accurate detection of ASFV is essential to combating the disease, and there is an unmet need for field-deployable pen-side diagnostic tests which possess high specificity and sensitivity to accurately inform veterinary professionals. False positive or false negative results can be severely damaging to a country and can lead to disruptive countermeasures. Using a cocktail of monoclonal antibodies recognizing a structural ASFV protein, we developed a lateral flow assay (LFA) for detecting ASFV in whole blood of swine. This assay is both field-deployable and user-friendly, requiring only water for sample dilution and providing results in 25 minutes. The LFA was capable of detecting a genotype II strain of ASFV in experimentally-infected animals at variable time-points post-infection. Importantly, the LFA possessed reliable diagnostic specificity superior to that shown by a similar, commercially-available rapid ASFV antigen detection test. Additional testing with field samples will be needed to further assess the viability of the ASFV LFA as a diagnostic assay.


20


March 3, 2020

Ron Orchard

Year-to-Year Comparison of a One Health Service Event to Provide Veterinary Preventative

Care to Low Income Kansas Residents

Corresponding Author, Ron Orchard, RVT, DVM/MPH Student, Kansas State University College of

Veterinary Medicine, Manhattan, KS, USA

Co-Authors: Kate KuKanich, DVM, PhD, DACVIM (SAIM), Department of Clinical Sciences, Kansas

State University College of Veterinary Medicine, Manhattan, KS; Alyssa Comroe, DVM, MPH,

Department of Clinical Sciences, Kansas State University College of Veterinary Medicine, Manhattan,

KS


Keywords: One-health, community service, public health

Abstract

In a One Health collaborative effort, the Kansas State University College of Veterinary Medicine joined the annual 2019 Riley County Everybody Counts service day, for the second year in a row, providing free veterinary services to community members in need. Additionally, a survey gathered information about need within the community. In 2019, 82 pets (59 dogs, 23 cats, 42 families) and in 2018 46 pets (32 dogs, 14 cats, 30 families) were examined, vaccinated, and dewormed. Twenty percent (5/20) of 2019 respondents received free healthcare services for themselves at the event. Reported annual household incomes were: <$30,000 in 65% (2019) and 73% in 2018. In 2019, 60% of families reported that if this free event did not exist, they could not afford veterinary care elsewhere, compared with 35% in 2018. Thirty-five percent (7/20) of families reported that their pets had not been examined by a veterinarian in the past 3 years. Fifty-five percent (11/20) of pets were intact compared to 38% in 2018. Cost was the most common reason reported for not having pets spayed or neutered in both years. Comparing these data over time allows identification of trends and improved targeting of resources for service opportunities within the community. Providing veterinary services at events such as Everybody Counts fulfills an essential One Health goal to provide preventative veterinary care to low-income community members, thus minimizing spread of infectious disease (rabies, parasites) between pets and people and supporting the psychological and physiological wellbeing of community members through healthy pet ownership.


21


March 3, 2020

Brandt C. Skinner

Isolation and Characterization of Two Anaplasma marginale Isolates from a Kansas Beef

Cattle Herd

Corresponding Author, Kathryn E. Reif, MS.P.H., Ph.D., Department of Diagnostic

Medicine/Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, Kansas

Co-Authors: Emily J. Reppert, MS, DVM, DACVIM, Department of Clinical Sciences, College of

Veterinary Medicine, Kansas State University, Manhattan, Kansas; Tippawan Anantatat, MS,

Department of Diagnostic Medicine/Pathobiology, College of Veterinary Medicine, Kansas State

University, Manhattan, Kansas; K.C. Olson. MS, Ph.D., Department of Animal Sciences and Industry,

College of Agriculture, Kansas State University, Manhattan, Kansas; Johann F. Coetzee, BVSc, Ph.D.,

Department of Anatomy and Physiology, College of Veterinary Medicine, Kansas State University,

Manhattan, Kansas


Keywords: Anaplasma marginale; anaplasmosis; cattle; virulence; isolate

Abstract

Bovine anaplasmosis is a bacterial disease caused by Anaplasma marginale, a pathogen that can be found worldwide and is endemic throughout the United States. Bovine anaplasmosis is conservatively estimated to cost the U.S. cattle industry >$300 million per year. To be most informative in studies evaluating different anaplasmosis control strategies, use of actively circulating strains for challenge studies is important. The objective of this study was to locate and multiply two isolates of A. marginale actively circulating in Kansas cattle and characterize the progression of infection and severity of clinical disease in adult beef cattle. Adult beef cows containing A. marginale strains not previously isolated or studied were identified from the Kansas State University Cow-Calf herd, a herd naturally-endemic for anaplasmosis. Blood samples containing these unique strains, labeled as KS1 and KS2, were collected and sub-inoculated into splenectomized calves for isolate multiplication. Once a high parasitemia was reach in the splenectomized calves, the infected blood was harvested and preserved. To determine virulence of these isolates, adult beef cows were intravenously inoculated, and progression of infection and clinical disease were monitored. KS1 challenged animals reached clinical anaplasmosis ~3 days earlier than KS2 challenged animals. KS1 challenged animals reached a peak bacteremia of 1x106.5 bacteria per millileter of blood while KS2 challenged animals reached levels of 1x108.9. Both KS1 and KS2 isolates produced clinical anaplasmosis in challenged animals that required treatment intervention. Infection kinetics differed significantly between KS1 and KS2 challenged animals with KS2 challenged animals developing significantly greater bacteremia levels.

22


March 3, 2020

Tera L. Brandt

Insulin expression patterns in canine insulinomas

Corresponding Author: Tera L. Brandt, BS, Department of Clinical Sciences, Kansas State

University, Manhattan, KS, United States

Co-Authors: Sarah M. Schneider, BS, DVM, PhD, DACVP, Diagnostic Medicine and Pathology, Kansas

State University, Manhattan, KS, Unites States; Nora Springer, BS, DVM, PhD, DACVP, Diagnostic

Medicine and Pathology, Kansas State University, Manhattan, KS, United States, Thomas

Schermerhorn, BS, VMD, DACVIM (SAIM), Department of Clinical Sciences, Kansas State University,

Manhattan, KS, United States


Keywords: Canine; insulinoma; tumor; insulin

Abstract

Canine and human insulinomas are insulin-secreting endocrine tumors of pancreatic islet beta cells that causing hyperinsulinemia and hypoglycemia. Little is known about the insulin expression patterns in canine insulinomas or how expression influences hyperinsulinemia. This study used immunohistochemistry (IHC) to compare insulin expression patterns between normal islets and neoplastic tissue in sixteen insulinomas from an archived tissue bank. IHC slides were prepared by the Veterinary Diagnostic Lab Histology Lab and insulin expression quantitated using HALO image analysis software. Visual examination showed insulin in insulinoma cells was not concentrated within discreet granules. The mean percentage (%) of insulin staining area (+) within equally-sized regions drawn around normal pancreatic islets (26.8%) and insulinoma tumor tissue (26.0%) was similar. Insulin staining was less intense in insulinomas (23.5% tumor area showed weak staining compared to 14.7% islet area) while 12% of islet area showed moderate or strong staining compared with 2.4% of tumor area. The ratio of insulin expression in tumor and islet (T:I ratio) showed that insulin expression varied between individual tumors (T:I ratio range- 0.13 to 3.5); 4 tumors had T:I>1.5 (high insulin content) and 4 had T:I<0.2 (low insulin content). The pattern and intensity of insulin staining is variable in canine insulinoma suggesting insulin content differs between individual tumors. The majority of tumors contained abundant (‘high’) insulin suggesting over expression could underlie abnormal insulin secretion. Hyperinsulinemia and hypoglycemia associated with tumors with low insulin content suggests another mechanism, like abnormal glucose sensing, could be responsible for insulin secretion.

23


March 3, 2020

Erin C. Hennessey

Computed Tomography of Hypaxial Muscle Abscessation in 10 Dogs

Corresponding Author: Erin C. Hennessey, VMD, DACVPM, Radiology, Kansas State University

Veterinary Health Center, Manhattan, KS, USA

Co-Authors: David S. Biller, DVM, DACVR, Radiology, Kansas State University Veterinary Health Center, Manhattan, KS, USA Nicky Cassel, BVCs, MMedVet, Dip ECVDI, Radiology, Kansas State University Veterinary Health Center, Manhattan, KS, USA Ellie Nuth, DVM, DACVR, Radiology, BluePearl Pet Hospital, Tacoma, WA, USA


Keywords: Computed tomography; iliopsoas; hypaxial; abscess

Abstract

Introduction: Hypaxial muscle disease is an important differential in canines presenting for back pain or pelvic limb lameness. Various imaging modalities can be employed to diagnose these cases. The current study describes computed tomography findings in dogs with known hypaxial muscle abscesses and compares the utility of computed tomography to other modalities. Methods: Ten dogs that underwent computed tomography of the lumbar spine or abdomen and had a diagnosed hypaxial abscess on surgical or microbiological examination were included. Computed tomography findings were compared with findings from other modalities. Results: Nine of the dogs were sporting breeds. Clinical signs included lethargy, fever, and abdominal pain. Abdominal and thoracic radiographs were performed in five and four dogs respectively and revealed periosteal reaction of lumbar vertebrae. Abdominal ultrasound was performed in eight dogs and showed heterogenous, hypoechoic areas in the hypaxial musculature. Ultrasound facilitated fine needle aspiration and confirmed infection in three cases. Computed tomography showed enlargement of the hypaxial muscles with fluid attenuating centers and frequent contrast enhancement. Computed tomography also revealed periosteal reaction and lysis of vertebral bodies and thickening of the diaphragm in several cases. Conclusion: Nine cases in this study had hypaxial abscesses consistent with migrating foreign material. The tenth case was diagnosed with discospondylitis. While MRI is generally considered superior for soft tissue imaging, computed tomography was able to answer the necessary clinical questions to diagnose all cases. Additionally, computed tomography is faster, more affordable, and provided more information on bone and diaphragm involvement than other modalities.


24


March 3, 2020

Matthew R. DiFazio

Introduction and Clinical Application of the “Humanoid” Ventrodorsal Thoracic

Radiographic View for Improved Cranial Thoracic Visualization

Corresponding Author: Matthew R. DiFazio, DVM, Radiology Specialty Intern, Kansas State

University, Manhattan, KS 66502, United States of America

Co-Authors: David Biller, DVM, DACVR, Section Head and Professor of Radiology, Kansas State

University, Manhattan, KS 66502, United States of America

Todd Henrikson, DVM, DACVR, Tallgrass Veterinary Imaging, Emporia, KS 66801


Keywords: thorax, radiograph, positioning, additional, non-standard

Abstract

A ventrodorsal thoracic view with maximal caudal limb traction, referred to as the “humanoid view,” is a novel positioning technique for obtaining thoracic radiographs that optimizes visualization of the cranial thorax by reducing superimposition of the scapulae. This view may aid in detection, characterization, and localization of cranially positioned lung lesions, mediastinal abnormalities, and body wall lesions. Proper positioning, advantages/disadvantages, indications, and specific clinical case examples are presented to illustrate the utility of this nonstandard view.


25


March 3, 2020

Zackery Bieberly

Long acting injectable methadone for post-operative analgesia

Corresponding Author: Butch KuKanich, DVM, PhD, DACVCP, Department of Anatomy and

Physiology, Kansas State College of Veterinary Medicine, Manhattan, KS, United States

Co-Authors: Kate KuKanich, DVM, PhD, DACVIM (SAIM)1; Kara Berke, DVM1; Emily Klocke, DVM, DACVS1; David Upchurch, DVM, MS, DACVS-SA1; Brad Crauer, DVM1; Alyssa Comroe, MPH, DVM1; Maria Jugan, DVM, MS, DACVIM (SAIM)1; Diane Mason, DVM, MS, PhD, DACVAA1; Gina Jensen, RVT1; Ron Orchard, RVT1; Kelsey Decker, RVT1; Joshuah Klutzke, BS2; Ally Fitzgerald, BS2; Kallie Woodruff, BS1; 1: Kansas State College of Veterinary Medicine, Department of Clinical Sciences, Manhattan, KS, United States; 2: Kansas State College of Veterinary Medicine, Department of Anatomy and Physiology, Manhattan, KS, United States


Keywords: Pharmacology; Analgesia; Opioids; Public Health; Dogs

Abstract

Background: Current injectable opioids for postoperative dogs require frequent injections to maintain adequate analgesia. This study was performed to assess the efficacy of injectable methadone with fluconazole as a long-acting analgesic in post-operative dogs. The hypothesis was methadone with fluconazole would provide 24 hours of analgesia with 2 doses compared to 4 doses of standard methadone injection. Methods: Forty-two healthy female dogs were enrolled, blocked based on body weight, and randomly allocated to two treatment groups. Pre-operative assessments included the Glasgow Composite Pain Scale (GCPS) and sedation assessed as none, slight, moderate, profound or unresponsive. The positive control group (methadone STD) received subcutaneous 0.5 mg/kg methadone q4h. The experimental group (methadone/fluconazole) received subcutaneous 0.5 mg/kg methadone with 2.5 mg/kg fluconazole, repeated once at 6 hours. All dogs received acepromazine, propofol induction, and isoflurane anesthesia. Routine standardized ovariohysterectomies were performed by experienced surgeons between 8 AM and 12 PM. Dogs were monitored following surgery using the GCPS and sedation scale. Results: There were no significant differences in postoperative GCPS scores between groups. One dog (methadone/fluconazole) received rescue analgesia; however, treatment failure was not significantly different between groups. All dogs were able to walk (moderate sedation or less) by 1 PM (methadone/fluconazole) and 4 PM (methadone STD) the day of surgery. Sedation was completely resolved in both groups at 5 PM the day after surgery. Conclusions: Methadone with fluconazole was able to achieve adequate analgesia with half the doses of standard methadone. This should increase compliance and feasibility in veterinary clinics.


26


March 3, 2020

Jared Bourek

The Effect of Handling Intensity at Time of Processing on Physiological Response, Immune

Response, and Vaccination Status in Beef Cattle at the Feedlot

Corresponding Author: Jared Bourek1, Kansas State College of Veterinary Medicine, Manhattan, KS

Co-Authors: S Torres2 DVM, PhD, J Welsh2 DVM, SP Terrell3 DVM, PhD, K Lukasiewicz3 DVM, Lisa

Taylor3, MS, DU Thomson1 DVM, PhD; 1:Kansas State University, College of Veterinary Medicine,

Manhattan, KS,; 2:Merck Animal Health, Madison, NJ;

Corresponding Author Contact Information: [email protected] (402)-380-1488

Keywords: Animal Welfare; Beef; Feedlot; Low Stress Cattle Handling

Abstract

Introduction & Objective: Newly received feeder calves are often experience stress upon arrival at the feedlot due to a variety of factors. The objective of this study was to determine if the effects of cattle handling intensity at processing had an effect on physiological, inflammatory, and immune stress markers in newly received feeder calves. Materials & Methods: Crossbred heifers (n=80, BW =355+/- 24 kg) from a single cohort were used for this study at a commercial cattle feeding facility in central Nebraska. These heifers were systematically allocated to treatment and used over a 42-day period to evaluate the effects of 2 handling treatments: Low stress handling (LSH): Cattle walked calmly through a crowding tub and snake processing facility up to a hydraulic chute. Electric prods and striking were not permitted. Noise from the handlers was kept to a minimum. Aggressive handling (AH): Cattle moved through the crowding tub and snake processing facility at a lope. An electric prod was applied twice (1 s per impulse) before entering a hydraulic chute. A radio was playing and yelling and whistling were encouraged. Results: Cattle administered the AH treatment had higher respiratory rates (P=0.017) than the LSH cattle immediately after treatment administration. LSH cattle tended to have lower norepinephrine plasma concentrations (P=0.05) than AH cattle immediately post-treatment. AH cattle had higher D-lactate concentrations compared to LSH cattle at post-treatment (P<0.0001). AH cattle had higher serum amyloid A concentrations at post-treatment (P=0.0002). Cattle handling did not have an effect on immunological measures (P>0.30) in cattle. These results indicate AH causes negative physiological and inflammation responses in newly received beef cattle.


27


March 3, 2020

Alyson H. Fitzgerald

Detecting and Quantifying Marijuana Metabolites in Serum and Urine of Dogs Affected by

Marijuana Toxicity

Corresponding Author: Alyson H. Fitzgerald, BS2

Co-Authors: Yuntao Zhang, PhD2, Steve Ensley, DVM, PhD2, Scott Fritz, DVM2, William Whitehouse, DVM, DACVIM (SAIM)1, Tamera Brabson, DVM, DAVECC3 1: Kansas State College of Veterinary Medicine, Clinical Sciences Department, Manhattan, KS 2: Kansas State College of Veterinary Medicine, Anatomy and Physiology, Manhattan, KS 3: Veterinary Emergency and Critical Care, Las Vegas, NV Corresponding Author Contact Information: [email protected]; 941-404-0503

Keywords: Chemistry; Toxicology; Marijuana; Canine

Abstract

Veterinarians diagnose marijuana toxicity either based on clinical signs or in conjunction with an over-the-counter human urine drug test, which has known limitations. With marijuana becoming more prevalent in the US, a more accurate test to diagnose marijuana toxicity is needed. Urine and serum samples from 16 dogs with confirmed or highly suspected marijuana toxicosis were analyzed with a novel UPLC-MS/MS method. Samples from affected dogs were collected from the VHC and outside hospitals. Aliquots of each sample were frozen at -80 C. Calibrations from 0.1-100 ng/mL and QCs were prepared using negative canine urine and serum. Samples were extracted, purified and eluted with solid-phase extraction. Waters TQS was employed for analysis. Creatinine corrections were calculated for all urine metabolites. An over-the-counter human urine drug screen test was performed on all urine samples. The LOD and LLOQ for the metabolites in serum ranged from 0.1-5 ng/mL and in urine ranged from 0.1-10 ng/mL. In serum, the following compounds were (ng/mL) detected: THC (14.1-138.1), 11-OH-Δ9-THC (1.30-23.5), 11-nor-9-carboxy-Δ9THC (0-1.80), CBD (0-20.5), THCCOOH-glucuronide (0-1.30) and THC-glucuronide (1.10-15.1). In urine, the following compounds were (ng metabolite/mg creatinine) detected: THC (.049-2.54), 11-OH-Δ9-THC (0-.569), 11-nor-9-carboxy-Δ9THC (.041-.583), CBD (0-.417), THCCOOH-glucuronide (0-.700) and THC-glucuronide (.049-4.61). All urine samples were negative on the human urine drug screen. All serum samples contained quantifiable concentrations of THC. Other metabolites were variably detected in serum and urine of affected dogs. Utilizing a validated UPLC-MS/MS method may provide a more sensitive diagnosis of marijuana toxicosis compared to a human urine drug screen test.

28


March 3, 2020

Yin Wang

Investigation of PCV3 prevalence by the peptide-based ELISA assay

Corresponding Author: Jianfa Bai, PhD, Kansas State Veterinary Diagnostic Laboratory, Kansas

State University, Manhattan, KS, USA

Co-Authors: Yin Wang, MS, Kansas State Veterinary Diagnostic Laboratory, Kansas State University, Manhattan, KS, USA; Wanglong Zheng, PhD, Kansas State Veterinary Diagnostic Laboratory, Kansas State University, Manhattan, KS, USA; College of Veterinary Medicine, Yangzhou University, Yangzhou, China; Xue Leng, PhD, Kansas State Veterinary Diagnostic Laboratory, Kansas State University, Manhattan, KS, USA; College of Animal Science & Technology, Jilin Agricultural University, Changchun, China; Elizabeth Porter, MS, Kansas State Veterinary Diagnostic Laboratory, Kansas State University, Manhattan, KS, USA; Colin Stoy, MS, Kansas State Veterinary Diagnostic Laboratory, Kansas State University, Manhattan, KS, USA; Lance Noll, PhD, Kansas State Veterinary Diagnostic Laboratory, Kansas State University, Manhattan, KS, USA; Xuming Liu, PhD, Kansas State Veterinary Diagnostic Laboratory, Kansas State University, Manhattan, KS, USA


Keywords: PCV3, ELISA, serologic prevalence

Abstract

In 2015, PCV3 was found and identified as the new porcine circovirus in the US, which could cause PCVAD-like clinical symptoms similar to that caused by PCV2. To study the serologic prevalence and evaluate the immune response, we developed the PCV3 specific ELISA assay. Experimentally inoculated animals were used to generate PCV3 positive and negative standards for the assay development. Five peptides in the capsid (Cap) protein and one from the replicase (Rep) protein in silico predicted with high immunogenicity were synthesized and evaluated with the standards. One peptide from Cap protein and one from Rep protein could be recognized by the antibody from the positive standards, but not from the negative ones. The two peptides were pooled as the coating antigen for the PCV3 antibody detection. The cutoffs were determined with the standards using the receiver operating characteristic (ROC) analysis. Validated with serial dilutions of PCV3 polyclonal antibody and clinical positive samples, the PCV3 ELISA had high sensitivity and specificity. The specificity was also verified by the negative results of PCV3 positive samples tested with the PCV2 ELISA assay. The great repeatability was assessed within plates, between plates and between runs. Further, the 367 serum samples and 276 oral fluid samples collected in KSVDL were subjected into the prevalence investigation. The PCV3 positive rates were 46% (169/367) in serum samples and 35.1% (97/276) in oral fluid samples. Therefore, we established the peptide based PCV3 specific ELISA assay to help understand the serologic prevalence and the immune response after the virus infection.


29


March 3, 2020

Kallie Woodruff

Evaluation of Feline Urine Concentrations of Amoxicillin and Clavulanate

Corresponding Author: Kate KuKanich, DVM, PhD, DACVIM (SAIM), Department of Clinical

Sciences, Kansas State University, Manhattan, Kansas, USA

Co-Authors: Butch KuKanich, DVM, PhD, DACVCP;2 Mark Papich, DVM, MS, DACVCP;3 Zackery

Bieberly;1 Joshuah Klutzke, BS;1 Ron Orchard, RVT;1 Lucy Schermerhorn1

1: Department of Clinical Sciences, Kansas State University, Manhattan, Kansas, USA

2: Department of Anatomy and Physiology, Kansas State University Manhattan, Kansas, USA

3: Department of Molecular Biomedical Sciences, North Carolina State University, Raleigh, North

Carolina, USA


Keywords: Feline; Amoxicillin; Clavulanate; CLSI Breakpoint

Abstract

Introduction: Urinary tract infections are routinely diagnosed in domestic cats, and the recommended empirical antimicrobial treatment of choice is amoxicillin or amoxicillin-clavulanate. Currently, the Clinical and Laboratory Standards Institute (CLSI) recommends the use of a conservative soft tissue breakpoint (S≤0.25 µg/mL) for feline urine culture & susceptibility reports. However, the determination of feline urine antimicrobial concentrations of these agents could provide the necessary data to establish feline urine-specific breakpoints. This study aimed to establish urine concentrations of amoxicillin and clavulanate after oral dosing. Methods: Eleven healthy young research cats were administered three 62.5 mg doses of amoxicillin-clavulanate orally at twelve hour intervals. Following the third dose, the urine of each cat was collected from litter pans or via cystocentesis over a 28 hour period, with a minimum of 3 urine samples collected per cat. The time and volume of each urination was recorded. Ultra-high pressure liquid chromatography with triple quadrupole mass spectrometry was used to determine the urine concentrations of amoxicillin and clavulanate. Results: Preliminary data analyses showed that amoxicillin concentrations were greater than 8 µg/mL in all urine samples collected prior to 12 hours, with a urine half-life of approximately 2 hours. Clavulanate was detected in all urine samples prior to 12 hours, with a urine half-life of approximately 2.5 hours. Conclusions: These results support establishing a feline urine-specific breakpoint of 8 µg/mL for amoxicillin and amoxicillin-clavulanate, similar to the CLSI established urine-specific breakpoint in dogs.


30


March 3, 2020

Kaitlynn N. Schuck

A Tale of Three Fixatives: Detection of Middle East Respiratory Syndrome Coronavirus in

Paraffin-Embedded Tissues Preserved in Three Fixatives

Corresponding Author: Dr. A. Sally Davis, B.A., DVM, PhD in Comparative Biomedical Sciences, Department of Diagnostic Medicine and Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, KS, USA; Department of Paraclinical Sciences, Faculty of Veterinary Science, University of Pretoria Onderstepoort, Pretoria, South Africa Co-author: Dr. Stephen M. Hewitt, M.D., PhD in Genetics, Laboratory of Pathology, Center for

Cancer Research, NCI, NIH, Bethesda, MD


Keywords: Middle East Respiratory Syndrome; Fixatives; Immunohistochemistry; In Situ

Hybridization

Abstract

- Middle East respiratory syndrome coronavirus (MERS-CoV) is a zoonotic pathogen endemic to dromedary camels. Since 35% of human MERS-CoV cases are fatal, inactivated tissues are a safer sample format. While 10% neutral buffered formalin (NBF) preserves histomorphology and inactivates many pathogens, it causes a rapid decline in biomolecule quality. Two novel fixatives, phosphate-buffered 70% ethanol (BE70) and BE70 with guanidium salts (BE70G), are alternative preservatives that cause less biomolecular damage. We hypothesized that existing anti-MERS-CoV immunohistochemistry (IHC) and in situ hybridization (ISH) protocols would work on tissues preserved with these fixatives. - Tracheal and nasal turbinate samples from eight alpacas experimentally infected with MERS-CoV EMC/2012 were fixed in NBF, BE70, and BE70G, paraffin-embedded, and sectioned for H&E staining and immunolabeling. Polymer detection-based anti-MERS-CoV IHC and MERS-CoV specific ISH using RNAscope chromogen-based detection were conducted. Because many ethanol-based fixatives do not require antigen retrieval (AR), ISH and IHC were run with and without AR. - MERS-CoV IHC and ISH labeled target molecules in all samples. The strongest labeling was seen in tissues preserved in NBF, followed by BE70G, and finally BE70. This was not unexpected as both assays were optimized for NBF. Both BE70- and BE70G-fixed tissues required AR to successfully visualize the targets by both methods. Histomorphology for all three fixatives without AR was very similar. However, AR reduced overall cellular definition in the ethanol-based fixed samples. These results support the use of these fixatives as alternatives to NBF, particularly when preservation of biomolecules is desired for PCR or sequencing.


31


March 3, 2020

Sagar Rayamajhi

Synthesis and Characterization of Liposome hybridized extracellular vesicles as a magnetic

resonance imaging contrast agent

Corresponding Author: Santosh Aryal, Professor, Department of Chemistry, Nanotechnology

Innovation Center of Kansas State (NICKS), Kansas State University, Manhattan, KS 66506, USA

Co-Authors: Ramesh Marasini, Ph.D. candidate, Department of Chemistry, Nanotechnology Innovation Center of Kansas State (NICKS), Kansas State University, Manhattan, KS 66506, USA; Tuyen Duong Thanh Nguyen, Department of Chemistry, Nanotechnology Innovation Center of Kansas State (NICKS), Kansas State University, Manhattan, KS 66506, USA; David Biller, Department of Clinical Sciences, College of Veterinary Medicine, Kansas State University, Manhattan, KS, 66506, USA


Keywords: Contrast agent, Gadolinium (Gd), Magnetic resonance imaging, contrast enhancement,

extracellular vesicles reconstruction

Abstract

Background: Contrast agent (CA) in magnetic resonance imaging (MRI) is now an essential add-on to get high-quality contrast-enhanced anatomical images for diagnostic imaging. However, the rapid elimination of CAs by the immune system has limited its application. As a result, the CAs dose for effective contrast is ever-increasing, resulting in toxic side effects. Considering the widespread application of Gd-based CAs, it is now very important to revisit its formulation in order to improve local concentration and minimize dose while achieving clinical goals. Methods. Therefore, we have adapted a unique strategy to maximize Gd delivery to the intended site using macrophage cell-derived extracellular vesicles (EVs) reconstructed with Gd conjugated liposomal system herein called as gadolinium infused hybrid EVs (Gd-HEV). We hypothesize that Gd-HEV, owing to the presence of EVs protein cargo, can effectively communicate with the immune system to target diseased site such as cancer. Results. Gd-HEV showed higher r1 relaxivity of 9.86 mM-1s-1 compared to 3.98 mM-1s-1 of clinically used contrast agent, Magnevist®, when measured in clinical 3T MRI. This will allow us to reduce clinically used concentration about three times while having contrast in the clinical window. Further, Gd-HEV showed preferential cellular interaction and accumulation towards cancer cells. More importantly, Gd-HEV showed excellent contrast enhancement in blood vasculature with higher retention time compared to Magnevist®. Conclusion. Our study successfully showed that the incorporation of Gd in EVs framework can help to enhance contrast ability while targeting precisely at the cancer site, and therefore can be a platform technology for the development of safer theranostic agents.


32


March 3, 2020

Pratiksha Khanal

Vaccination with a porcine reproductive and respiratory syndrome (PRRS) modified live

virus (MLV) vaccine is associated with increased gut microbiome diversity

Corresponding Author: Megan C. Niederwerder, DVM, PhD, Department of Diagnostic

Medicine/Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan,

Kansas, USA

Co-Author: Laura A. Constance, MS, Department of Diagnostic Medicine/Pathobiology, College of

Veterinary Medicine, Kansas State University, Manhattan, Kansas, USA


Keywords: Porcine reproductive and respiratory syndrome virus; Gut microbiome; Modified live

virus vaccine; Microbiome composition and diversity

Abstract

Background: Porcine reproductive and respiratory syndrome virus (PRRSV) is the most costly disease of swine production in the United States. Although PRRS modified live virus (MLV) vaccines are widely utilized to reduce PRRS-associated loses, the currently available vaccines are considered inadequate for disease control. Recently, the gut microbiome has been associated with vaccine efficacy and health outcomes following PRRSV infection in pigs. The objective of the current study was to investigate the effects of PRRS MLV vaccination on the gut microbiome composition and diversity of nursery pigs. Methods: Weaned pigs (average 23.4 ± 2.1 days) were obtained from a single commercial source and vaccinated with a commercial PRRS MLV vaccine (Ingelvac PRRS MLV; Boehringer Ingelheim Animal Health). Twenty-eight days post-vaccination, fecal samples were collected from both vaccinated (n = 12) and nonvaccinated (n = 12) pigs. Gut microbiomes were compared between groups using the Lawrence Livermore Microbial Detection Array (LLMDA). Results: Increased microbial species diversity and increased Firmicutes to Bacteroidetes ratio was found in the vaccinated pigs when compared to the nonvaccinated pigs. Significant differences were also noted in microbiome composition at the level of phylum, family and species. Specifically, Erysipelotrichaceae family was detected at a significantly higher rate in vaccinated pigs compared to nonvaccinated pigs (p = 0.003, Fisher’s exact test). Conclusions: PRRS MLV vaccination was associated with changes in the gut microbiome composition and diversity in nursery pigs. This suggests that infection with PRRS MLV may modulate the gut microbiome and that certain microbiome characteristics may contribute to vaccine efficacy.


33


March 3, 2020

Ravi Thakkar

Computational design of peptides for cancer immunotherapy

Corresponding Author: Ravi Thakkar, Department of Anatomy & Physiology, College of Veterinary


Co-Author: Prof. Jeffrey Comer, PhD, Department of, Anatomy & Physiology, College of Veterinary


Corresponding Author Contact Information: [email protected]

Keywords: Computational protein design; immune checkpoint inhibitor; cancer immunotherapy

Abstract

Cytotoxic T-Lymphocyte Associated protein-4 (CTLA-4) is an immune checkpoint protein that plays an important role in self-tolerance and deactivation of immune responses. Cancer cells often produce an abundance of CTLA-4 proteins. The association of CTLA-4 with proteins of the B7 family on T-lymphocytes, such as CD-80 and CD-86, can allow many types of cancer cells to evade destruction by the immune system. Hence, blocking of the formation of complexes between CTLA-4 and its ligands complex can reactivate the immune response against these cancers. We analyzed the X-ray crystal structure of the complex of CTLA-4 with B7-2 (CD-86) to locate key residues at the interface between the two proteins. Using the FlexPepDock module of the Rosetta molecular modeling suite, we generated de novo cyclic peptides that bind to CTLA-4. Some of the designed cyclic peptides displayed the ability to bind to the CTLA-4 protein at the same site where the CD-86/80 protein binds. In molecular dynamics simulations, some of the designed peptides stay bound with CTLA-4 for almost half of a microsecond. These computational results suggest that these designed cyclic peptides are potential candidates for immune checkpoint inhibitors.

34


March 3, 2020

Swetha Madesh

The canine host appears to serve as a sentinel species for tick-borne diseases caused by

Anaplasma, Ehrlichia and Borrelia pathogens impacting human health in the USA

Corresponding Author: Roman R. Ganta, MSc, PhD, Diagnostic Medicine /Pathobiology, 1800

Denison Ave, Manhattan KS 66506.

Co-Authors: 1) Arathy D. S. Nair, DVM, PhD, Diagnostic Medicine /Pathobiology, 1800 Denison

Ave, Manhattan KS 66506. 2) Roman R. Ganta, MSc, PhD, Diagnostic Medicine /Pathobiology, 1800

Denison Ave, Manhattan KS 66506.


Keywords: Ehrlichia; Anaplasma; Borrelia; tick-borne diseases

Abstract

Introduction: Tick-borne diseases continue to threaten the health of people and dogs. In the USA, human Lyme disease cases, caused by Borrelia burgdorferi, are the highest followed by diseases resulting from Ehrlichia and Anaplasma species. We investigated the prevalence of these diseases in dogs and then compared with human data. Methods: Blood samples collected from clinically suspected dogs from across the US were assessed for antibodies for four pathogens. An ELISA assay was performed for B. burgdorferi, while indirect immunofluorescence assay (IFA) was carried out for E. chaffeensis, E. canis and A. phagocytophilum. Results/discussion: A total of 503, 347 and 496 samples were assessed for A. phagocytophilum, Ehrlichia species and B. burgdorferi, respectively. A total of 185 (37%) samples tested positive for A. phagocytophilum, 155 (45%) for both E. chaffeensis and E. canis and 233 samples (47%) for B. burgdorferi. Some of the Ehrlichia positives are the result of extensive antigenic cross-reactions between the two species. Similarly, some A. phagocytophilum positives may represent A. platys infections. Co-infection with both Anaplasma and Ehrlichia species was observed in 21 dogs; 25 dogs were double-positive for Anaplasma and Borrelia; 11 for Ehrlichia and Borrelia; and three dogs positive for all three species. We observed a significant overlap in the geographical distribution of these diseases in dogs with that documented in people. Conclusion: Our data suggest the occurrence of tick-borne diseases in dogs is very similar to that of humans; thus, monitoring canine infections has important implications for both human and companion animal health.

35


March 3, 2020

Chandramouli Kondethimmanahalli

Proteomics analysis reveals distinct protein expression in infectious and replicative forms of

Ehrlichia chaffeensis

Corresponding Author: Roman R. Ganta, MSc, PhD; Center of Excellence for Vector-Borne

Diseases, Department of Diagnostic Medicine/Pathobiology, College of Veterinary Medicine, Kansas

State University, Manhattan, Kansas, 66506, USA

Co-Author: Roman R. Ganta, MSc, PhD.

Corresponding Authors Contact Information: [email protected], (785) 532-4612;

[email protected], (785)-473-3032

Keywords: Ehrlichia chaffeensis; Vector-Borne Diseases; Proteomics

Abstract

Background: Ehrlichia chaffeensis is a tick-transmitted rickettsial pathogen responsible for causing human monocytic ehrlichiosis (HME). The pathogen’s developmental cycle includes infectious dense-core cells (DC) and non-infectious replicating cells (RC) within an infected host. Defining proteins crucial for establish distinct growth forms is of fundamental importance in understanding the infection and replication process, which paves the way for discovering therapeutic targets against HME and other related rickettsial diseases. Methods: E. chaffeensis organisms cultivated in canine macrophages were purified as DC and RC fractions and subjected to comprehensive proteome analysis using mass spectrometry methods. From triplicate sample analysis of each fraction, we identified 201 and 385 proteins expressed in DC and RC forms, respectively. Results/discussion: The protein profiles were very distinct in the two growth forms reflecting specific functional priorities, possibly required for the infection-specific protein expression in the DC form and replication-specific protein expression in the RC form. The p28-outer membrane protein (p28-Omp) paralogs, DNA-binding proteins and metabolic enzymes were more abundantly expressed in the RC form supporting the observation that the replicative form is metabolically highly active involving physiological and morphological changes. Conclusion: This study provides a detailed proteome fundamental for the two distinct forms of E. chaffeensis and that the data are valuable for better understanding of protein expression dynamics during pathogen lifecycle stages and may also serve as a resource for identifying possible targets for diagnostics and therapeutics.



36


March 3, 2020

Edward Bird

Whole-genome characterization of two Histophilus somni isolates from the lungs of clinically

diseased calves

Corresponding Author: Edward Bird, B.S., Kansas State Veterinary Diagnostic Laboratory,

Manhattan, KS, USA

Co-Authors: Nanyan Lu, M.S., Kansas State Veterinary Diagnostic Laboratory, Manhattan, KS, USA;

M.M. Chengappa, M.S. Ph.D., Kansas State Veterinary Diagnostic Laboratory, Manhattan, KS, USA;

Jamie Henningson, DVM, Ph.D., DACVP, Kansas State Veterinary Diagnostic Laboratory, Manhattan,

KS, USA; Rachel Palinski, Ph.D., Kansas State Veterinary Diagnostic Laboratory, Manhattan, KS, USA.

Corresponding Author Contact Information: [email protected] (405)-274-3539

Keywords: Histophilus somni, Next-generation sequencing, whole-genome

Abstract

Histophilus somni inhabits the respiratory system and genital mucosal surfaces of cattle and sheep. H. somni can be a primary pathogen, opportunistic pathogen, or commensal bacterium, depending on the area of infection. Pathogenic strains are most commonly associated with bovine respiratory disease (BRD) complex and a major concern to cattle producers. H. somni can spread throughout the respiratory system and cause diseases such as thromboembolic meningoencephalitis, septicemia, pleuropneumonia, myocarditis, arthritis, and abortion. Despite the impact of this pathogen, there is limited research characterizing the genomic features that contribute to pathogenicity. In this study, two putatively pathogenic strains of H. somni were isolated from the lungs of calves co-infected with either Mannheimia haemolytica and Mycobacterium bovis or Pasteurella multocida. Both calves died of respiratory disease after multiple antibiotic treatments. Following isolation, the colonies were sequenced on an Illumina Miseq platform. An analysis of the genome completeness was performed using BOSCO, resulting in >95% of the predicted H.somni genomes recovered with an average coverage greater than 75x. Genomic features including antimicrobial resistance genes, defense mechanisms, biosynthesis, amino acid synthesis, cofactor biosynthesis, catabolism, amino acid catabolism, and metabolic pathways were compared to an H.somni genome generated from a calf without clinical signs of disease. The results of this study provide insight on the genomic contribution to pathogenicity of H. somni, an opportunistic pathogen, in the presence of other known anti-microbial resistant pathogens.


37


March 3, 2020

Deepak Kumar

Metagenomic Next-Generation Sequencing Reveal Presence of a Novel Ungulate

Bocaparvovirus in Alpacas

Corresponding Author: Deepak Kumar, MS, Diagnostic Medicine/Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, KS, USA Co-Authors: Suman Chaudhary, MS, Kansas State Veterinary Diagnostic Laboratory, College of Veterinary Medicine, Kansas State University, Manhattan, KS, USA; Nanyan Lu, MS, Kansas State Veterinary Diagnostic Laboratory, College of Veterinary Medicine, Kansas State University, Manhattan, KS, USA; Michael Duff, MS, Kansas State Veterinary Diagnostic Laboratory, College of Veterinary Medicine, Kansas State University, Manhattan, KS, USA; Mathew Heffel, BS, Kansas State Veterinary Diagnostic Laboratory, College of Veterinary Medicine, Kansas State University, Manhattan, KS, USA; Caroline A. McKinney, MPH, Department of Clinical Sciences, Cummings School of Veterinary Medicine at Tufts University, 200 Westboro Road, North Grafton, MA, USA; Daniela Bedenice, PhD, Department of Clinical Sciences, Cummings School of Veterinary Medicine at Tufts University, 200 Westboro Road, North Grafton, MA 01536, USA; Douglas Marthaler, PhD, Kansas State Veterinary Diagnostic Laboratory, College of Veterinary Medicine, Kansas State University, Manhattan, KS, USA Corresponding Author Contact Information: [email protected], (804) 503-1241 Keywords: alpaca; Bocaparvovirus; genome; next-generation sequencing; metagenomics

Abstract

Introduction: The objective of this study was to investigate the causative agent of severe enteric disease in an alpaca (Vicugna pacos) herd using Metagenomic Next Generation Sequencing (NGS). Methods: An intestinal sample from a deceased alpaca was submitted to the KSVDL for metagenomic NGS. The sample was filtered, treated with nucleases, and subjected to viral nucleic acid extraction followed by cDNA synthesis, library preparation, and sequencing on Illumina MiSeq. The raw data was analyzed using a custom bioinformatic pipeline. A complete genome of Bocaparvovirus (BoV) was de novo assembled using Ray, IVA and A5. MAFFT alignment and Maximum Likelihood phylogenetic trees were generated using Geneious with the Ungulate BoV (UBoV) sequences in GenBank (n=108). Results: The alpaca BoV genome was 5155 nucleotides and comprised of three open-reading frames (ORFs) coding non-structural proteins NS1 and NP1, and a structural protein VP1. Recombination events were lacking with the other UBoVs. Phylogenetic trees illustrated distinct branching of the alpaca BoV, sharing a common ancestor with the UBoV strains from camels (UBoV8). NS1 protein had the highest amino acid percent identity (57.89-67.85%) to the strains in UBoV8, which was below the 85% cut-off set by the International Committee on Taxonomy of Viruses (ICTV) for classifying BoVs and qualifies alpaca BoV as a tentative new UBoV species. Walker loop and Phospholipase A2 (PLA2) motifs were also identified in NS1 and VP1 genes, respectively. Conclusion: A new species of UBoV was identified in an alpaca intestinal sample by NGS. However, establishing any virus-disease association would require comprehensive PCR testing of alpaca farms and fulfilment of Koch’s postulates.


38


March 3, 2020

Naveen Jonnalagadda

Antibacterial Effects of Equine Adipose Mesenchymal Stromal Cell Derived Exosomes against

Escherichia coli and Staphylococcus aureus

Corresponding Author: Charan K. Ganta, BVSC, PhD, DACVP, Diagnostic Medicine/Pathobiology,

Kansas State University, Manhattan KS 66506

Co-Authors: T.G. Nagaraja, MVSc, PhD, Diagnostic Medicine /Pathobiology, Kansas State University,

Manhattan KS 66506; Dylan Lutter, DVM, MS, DACVS-LA, CERP, Clinical Sciences, Kansas State

University, Manhattan KS 66506; Roman R. Ganta, MSc, PhD, Diagnostic Medicine /Pathobiology,

Kansas State University, Manhattan KS 66506


Keywords: Mesenchymal Stromal Cells; Exosomes; poly I:C; Antibacterial activity

Abstract

Introduction: Defining antibacterial and immunomodulatory properties of mesenchymal stromal cells (MSC) and their secretome is an emerging area of research. The MSC-derived secretome components, exosomes, are shown to have comparable immunomodulatory properties as MSC. However, it remains unknown if the antibacterial effect of MSCs is part of the exosome cargo. In this study, we hypothesize that equine adipose MSC (aMSC)-derived exosomes show similar antimicrobial properties as aMSC against Gram negative (Escherichia coli) and Gram positive (Staphylococcus aureus) bacteria. In addition, we investigated if TLR3-stimulated aMSC exosomes enhance antibacterial properties against E. coli and S. aureus, as previously reported with MSCs alone. Methods: Equine aMSCs were first isolated and grown in low glucose DMEM medium. Exosomes were collected from both Poly I:C (TLR 3 agonist) stimulated and non-stimulated aMSCs by an ultracentrifugation method. Characterization and quantification of exosomes were done by transmission electron microscopy, nanoparticle tracking analysis and BCA protein assay. Bacterial growth was measured by optical density and colony forming unit assay at 3, 6 and 12 hour incubations with exosomes. Results: Stimulation of aMSC with poly I:C had increased the number of exosomes by 2-fold. Exosomes derived from both stimulated and non-stimulated aMSCs show inhibition of the growth of both species of bacteria. In addition, poly I:C stimulated aMSC exosomes showed enhanced inhibition of the bacterial growth in a dose dependent manner. Conclusion: Both stimulated and non-stimulated aMSC exosomes inhibit E. coli and S. aureus growth in vitro. Poly I:C stimulated aMSC-derived exosomes enhance the antibacterial activity.


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March 3, 2020

Bianca Libanori Artiaga

Role of NKT cell agonist for influenza virus vaccines in swine

Corresponding Author: J. A. Richt, DVM, PhD, Diagnostic Medicine/Pathobiology, College of

Veterinary Medicine - Kansas State University, Manhattan, Kansas, USA.

Co-Authors: B. L. Artiaga, DVM, PhD, Diagnostic Medicine/Pathobiology, College of Veterinary Medicine - Kansas State University, Manhattan, Kansas, USA; T. Kwon, DVM, MSc, Diagnostic Medicine/Pathobiology, College of Veterinary Medicine - Kansas State University, Manhattan, Kansas, USA; I. Morozov, DVM, PhD, Diagnostic Medicine/Pathobiology, College of Veterinary Medicine - Kansas State University, Manhattan, Kansas, USA; S. V. Indran, DVM, PhD, Diagnostic Medicine/Pathobiology, College of Veterinary Medicine - Kansas State University, Manhattan, Kansas, USA; V. Balaraman, DVM, PhD, Diagnostic Medicine/Pathobiology, College of Veterinary Medicine - Kansas State University, Manhattan, Kansas, USA; D. Meekins, PhD, Diagnostic Medicine/Pathobiology, College of Veterinary Medicine - Kansas State University, Manhattan, Kansas, USA; D. Bold, DVM, Diagnostic Medicine/Pathobiology, College of Veterinary Medicine - Kansas State University, Manhattan, Kansas, USA; G. Roman-Sosa, DVM, PhD, Diagnostic Medicine/Pathobiology, College of Veterinary Medicine - Kansas State University, Manhattan, Kansas, USA; W. Ma, DVM, PhD, Diagnostic Medicine/Pathobiology, College of Veterinary Medicine - Kansas State University, Manhattan, Kansas, USA; J. P. Driver, PhD, Department of Animal Sciences, University of Florida, Gainesville, Florida, USA; J. A. Richt, DVM, PhD, Diagnostic Medicine/Pathobiology, College of Veterinary Medicine - Kansas State University, Manhattan, Kansas, USA. Corresponding Author Contact Information: [email protected], (785) 323-7970

Keywords: NKT cells; influenza; vaccine; alpha-galactosylceramide; swine.

Abstract

Background: Vaccination is the base of influenza A viruses (IAV) control in pigs, but broader cross-protection is needed. Previous studies have shown that adding the natural killer T (NKT) cell agonist alpha-galactosylceramide (GC) as an adjuvant to a killed H1N1 IAV vaccine improved protection against homologous challenge, and GC added to a H3N2 live-attenuated IAV (LAIV) vaccine increased cross-reactive antibodies to a heterologous challenge. Methods: To further examine GC as a vaccine adjuvant, we vaccinated pigs with the H3N2 TX98-NS1 LAIV vaccine, with one group co-administered GC. After 21 days, pigs were challenged with wild-type H3N2 TX98 or heterosubtypic pandemic H1N1 CA04. In the follow-up study, pigs were vaccinated twice with killed H3N2 TX98 (kTX98) vaccine, with one group receiving antigen alone, another co-administered GC, and two groups vaccinated with a commercial oil-in-water adjuvant.


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After 23 days, pigs were challenged with homologous H3N2 TX98 or heterologous H3N2 CO99. Both studies included respective control groups. Pigs were euthanized 5 days post-infection. Results: FACS analysis showed increased NKT cells in blood, bronchoalveolar lavage fluid, lungs, and tracheobronchial lymph nodes of pigs that received GC in both studies. No significant differences were present in macroscopic lung lesion scores, viral shedding, and virus-specific antibody titers, independent of treatment with GC. Conclusions: Adding GC as an adjuvant to TX98-NS1 LAIV or killed TX98 vaccines produced no significant difference in vaccine efficacy against heterosubtypic CA04 or heterologous CO99 challenges, respectively. Further studies with different IAV strains and vaccines are needed to better understand how NKT activation affects influenza vaccine efficacy.

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The Sigma Chapter of the Society Phi Zeta wishes to acknowledge and thank our volunteer moderators and judges for the 2020 Annual Phi Zeta Research Day. Moderators: A. Sally Davis Brian Herrin Butch KuKanich Judges: Basic oral:

Matthew Basel, Giselle Cino, Robert DeLong Applied/clinical oral:

Natalia Cernicchiaro, Ronnie Elmore, Jessica Meekins Basic poster:

Jianfa Bai, Charan Ganta, Majid Jaberi-Douraki, Victoriya Volkova Applied/clinical poster:

Kate KuKanich, Lalitha Padireddi, Brandon Plattner, Roman Pogranichniy

College of Veterinary Medicine Kansas State University ...€¦ · ii Annual Phi Zeta Research Day...

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