HORSES - AgriFutures

RIRDC Completed Projects in 1998-1999 and

Research in Progress as at June 1999

Sub-Program 3.5

HORSES

September 1999 RIRDC Publication No 99/115

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© 1999 Rural Industries Research and Development Corporation. All rights reserved. ISBN 0 642 57953 9 ISSN 1440-6845 "RIRDC Completed Projects in 1998-99 and Research in Progress as at June 1999 - 3.5 Horses Publication No 99/115 The views expressed and the conclusions reached in this publication are those of the author and not necessarily those of persons consulted. RIRDC shall not be responsible in any way whatsoever to any person who relies in whole or in part on the contents of this report. This publication is copyright. However, RIRDC encourages wide dissemination of its research, providing the Corporation is clearly acknowledged. For any other enquiries concerning reproduction, contact the Publications Manager on phone 02 6272 3186. RIRDC Horses Research Manager Professor Reuben Rose RIRDC Level 1, AMA House 42 Macquarie Street BARTON ACT 2600 PO Box 4776 KINGSTON ACT 2604 Phone: 02 9351 6935 Fax: 02 9660 1548 Email: [email protected] RIRDC Publications Manager Eva Hickman Rural Industries Research and Development Corporation Level 1, AMA House 42 Macquarie Street BARTON ACT 2600 PO Box 4776 KINGSTON ACT 2604 Phone: 02 6272 3186 Fax: 02 6272 5877 Email: [email protected] Website: http://www.rirdc.gov.au

Published in September 1999 Printed on environmentally friendly paper by Canprint

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Foreword

This year RIRDC has produced Research in Progress, June '99, which contains short summaries of continuing projects as well as those that were completed during 1998-99 for all of the Corporation’s 20 program areas. The complete report on all the programs is only available in electronic format on our website at http://www.rirdc.gov.au The following report is a hardcopy extract covering Sub-Program 3.5. It contains all entries from continuing and completed horse research projects funded by RIRDC. Our horse program aims to assist in developing the Australian horse industry and enhancing its export potential. This report is the newest addition to our extensive catalogue of almost 400 research reports, videos and CD-Roms of projects supported by RIRDC. Please contact us for the latest publications catalogue or view it on our website: • downloads at www.rirdc.gov.au/reports/Index.htm • purchases at www.rirdc.gov.au/pub/cat/contents.html

Peter Core Managing Director Rural Industries Research and Development Corporation

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Contents

3.5 HORSES COMPLETED PROJECTS

PROJECT

No PROJECT TITLE RESEARCHER PHONE ORGANISATION PAGE

No DAV-156A Drought feeding information manual for the horse

industry Mr David Nash, Ms Angela Avery and Ms Patricia Ellis

02 60304500 Agriculture Victoria Rutherglen Rutherglen Research Institute

1

GEH-1A Further studies on abilities of progestagens to maintain pregnancy in mares

Dr. Angus McKinnon 03 5829 9566 Goulburn Valley Equine Hospital 2

HOW-1A Evaluation of the provision of training programs for the horse industry in Australia

Dr William Howey 02 9351 7979 PO Box 509 SCONE NSW 2337

3

PTP-12A Horses and land use on small properties Mr Arthur Stubbs 03 9844 1135 Primary Tasks Pty Ltd 3 PTP-13A 4th Australian Racecourse Managers’ Conference,

1999 Mr Arthur Stubbs and Mr John Neylan

03 9844 1135 & 03 9521 6982

Primary Tasks Pty Ltd and Turfgrass Technology Pty Ltd

4

SAR-14A Controlling and reducing airborne dust in horse stables and transporters

Dr Colin Cargill 08 8303 7782 South Australian research and Development Institute

5

UCS-18A Information Services for the Horse Industry Associate Professor Martin Sillence

02 6933 2205 Charles Sturt University 6

UM-23A Diagnosis of equine virus diseases using polymerase chain reactions and development of a blood test and vaccine for equine rhinovirus.

Professor M.J. Studdert, Dr C.A. Hartley, Dr H.E. Drummer.

+61-3-9344-7373 The Centre for Equine Virology, The University of Melbourne.

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UM-36A Methods of quantitative assessment for intra pulmonary blood accumulation in racehorses with EIPH

Professor Ron Slocombe, Dr. Shaun McKane

03 9742-8264 University of Melbourne 8

UMU-18A Studies on the intestinal absorption of immunoglobulins and the effects of passive immune status on neutrophil function in foals

Dr Sharanne Raidal 08 9360 2356 Murdoch University

9

UQ-59A Control of intraocular inflammation by topically applied non steroidal anti-inflammatory drugs (NSAIDS)

Dr SM Taylor & Dr IA Shiels

07 3365 3124 Department of Physiology and Pharmacology University of Queensland,

11

US-24A Studies on host-parasite interactions in disease (strangles) induced by streptococcus equi subspecies equi

A/Prof. Paul J Canfield 02 9351 2020 University of Sydney

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US-33A and US-41A

Lower respiratory tract disease in racehorses Dr R.M. Christley, Dr J.L. Hodgson, Dr J.L. Wood, Prof. S.W.J. Reid, Prof. R.J. Rose, Prof. D.R. Hodgson

02 4655 0777 University of Sydney 13

US-52A In vitro biomechanical properties of equine digital flexor tendons

Mr. Andrew Dart (02) 46 550777 University of Sydney 14

US-58A Serological investigation of Equine influenza in Australia

Dr Jennifer Hodgson (02) 4655 0745 University of Sydney

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3.5 HORSES RESEARCH IN PROGRESS

PROJECT

No PROJECT TITLE RESEARCHER PHONE ORGANISATION PAGE

No FLP-1A Learning within the Equine Industry Mr. Laurie Field 02 9498 8278; Field Learning Pty Ltd 17 DAV-118A Improved Pasture: More than just grass for horses

to exercise on? Ms Angela Avery and Mr David Nash

02 60 304500 Agriculture Victoria Rutherglen 17

UCS-22A Pasture intake and selectivity by grazing horses Dr. Michael Friend 02 6933 4006 Charles Sturt University 18 UM-29A Molecular epidemiology of Rhodococcus equi Dr. Glenn Browning 03 9344 7342 The University of Melbourne 19 UM-40A Epidemiology of equine rhinovirus type 2 (ERhV2) Dr. Carol Hartley 03 9344 7375 Centre for Equine Virology 19 UMA-15A Improved vaccine strategies for management of

equine herpesvirus diseases A/Prof. Millar Whalley 02 9850 8200 Macquarie University 20

UMA-16A Investigation of Potential EHV-1 Vaccines Using the Mouse Model of Infection.

Dr. Catherine Walker 02 9850 8219 Macquarie University 21

UMU-22A Identification of infectious respiratory disease in horses as a basis for better management of cases

A/Prof. Graham Wilcox 08 9360 2448 Murdoch University 22

UNE-62A Safe and effective grain feeding for horses Prof. James Rowe 02 6773 2225 University of New England 23 UQ-43A The influence of season on follicular dynamics and

the relationships with endocrinology and oviduct physiology in the mare

Dr. Neil McMeniman 07 3365 1108 The University of Queensland 23

UQ-45A Nutrition of grazing broodmares, their foals and young horses

Dr. Neil McMeniman 07 3365 1108 The University of Queensland 24

UQ-46A Assessment and prevention of toxicity from indospicine contaminated horsemeat

Dr. Michael Pass 07 5430 2840 University of Queensland

25

UQ-71A Novel anti-inflammatory drugs to treat inflammatory diseases in horses

Dr. Stephen Taylor 07 3365 3124 The University of Queensland 26

UQ-72A Nutritent intake and growth of young horses Dr. Neil McMeniman 07 3365 1108 The University of Queensland 26 UQ-75A The pathogenesis and prevention of equine laminitis Dr. Christopher Pollitt 07 3365 2063 The University of Queensland 27 US-53A Respiratory ventilation and tracheal sounds in

horses with airway disease Dr. David Evans 02 9351 2474 The University of Sydney 28

US-77A Training and Fitness in Athletic Horses Dr. David Evans 02 9351 2474 The University of Sydney 29 USA-6A Diagnosis of and protection against infections

caused by Rhodococcus equi Dr. Mary Barton 08 8302 2933 University of South Australia 29

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3.5 Horses Completed Projects

Project Title

Drought feeding information manual for the horse industry

RIRDC Project No.:

DAV-156A

Researcher: David Nash, Angela Avery and Patricia Ellis Organisation: Agriculture Victoria Rutherglen

Rutherglen Research Institute RMB 1145, Chiltern Valley Road, RUTHERGLEN 3685

Phone: 02 60304500 Fax: 02 60304600 Email: [email protected] Objectives

• To provide information for the horse industry on feeding and horse management in

times of drought.

Background:

Droughts are very much part of agriculture in Australian. Horse owners, like other managers of livestock, need to be prepared for times of dry conditions and drought. Other animal industries have prepared information available to assist them through drought, until the completion of this manual there was no such information available to the horse industry. A practical guide on horse feeding and management during drought was needed to help owners break the daunting planning phase of a threatening drought down to manageable steps. This manual will enable horse owners to get through drought in the most economic and stress-free way without horses, pasture or soil resources suffering.

Research

This project involved literature review and consultation with experts on drought, horse and pasture management to develop the information in the drought manual.

Outcomes

• A practical relevant manual that will assist horse owners manage dry and drought

conditions • Support for the horse industry during drought conditions

Implications

The horse industry in Australia will be better able to manage their enterprises during drought leading to reduced costs, animal health problems and environmental degradation. The wider community will also see the industry as being responsible and pro-active towards drought.

Publications

The main objective of this project was to publish information on drought for the horse industry. This information is to be printed by RIRDC as a research publication.

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Project Title

Further studies on abilities of progestagens to maintain pregnancy in mares

RIRDC Project No.:

GEH-1A

Researcher: Dr. Angus McKinnon Organisation: Goulburn Valley Equine Hospital

PO Box 2020 SHEPPARTON VIC 3630

Phone: 03 5829 9566 Fax: 03 5829 9307 Email: [email protected] Objectives

• To compare the ability of various progestagens to maintain pregnancy in mares. • To compare the ability of those progestagens to eliminate oestrous behaviour.

Background

Many or most of us (mare owners, farm managers and veterinarians) have administered progestagens (progesterone like compounds) to mares in an attempt to help them maintain a pregnancy. Years ago (published in the Equine Veterinary Journal in 1993), we found that the most common progestagens (hydroxyprogesterone caproate, also known as Depo Luteine etc.) not only failed to keep mares from showing heat (a properly acting progestagen should be able to do this) but was not able to prevent abortion when we removed the source of progesterone (the corpus luteum in the ovary). This study led us to examine further progestagens for their ability to eliminate oestrous or maintain pregnancy.

Research

What we did was take groups of mares (5 mares in each group) that were pregnant at day 16 and give them progestogen compounds. Two days later we gave prostaglandin (to bring them back into heat). If the treatments worked we would expect the mares to stay pregnant. Just to be certain we also took blood every day and measured the hormone progesterone.

Implications

The result were very interesting. Regumate worked well. All 5 mares treated maintained their pregnancies. The other four groups were a disaster. No mares in each of the other groups maintained pregnancies. All mares came into heat. The average time from injection to abortion was 3.3 (± 1.7) days. All the mares returned to oestrus and ovulated within 11 days after prostaglandin injection. The groups of mares in this study were: 1) medroxyprogesterone acetate 2) hydroxyprogesterone hexanoate 3) altrenogest 4) norgestomet and 5) megesterol acetate. These are commonly used drugs and come under a number if different names. The bottom line from this research is that you should be very careful about which drugs you chose to try and help a mare from loosing her pregnancy. Most do not appear to work.

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Project Title

Evaluation of the provision of training programs for the horse industry in Australia

RIRDC Project No.:

HOW-1A

Researcher: Dr William Howey Organisation: PO Box 509

SCONE NSW 2337 Phone: 02 9351 7979 Email: [email protected] Objectives

• To investigate and evaluate the delivery and provison of education to the

prospective and actual workforce in the horse industry in Australia. Particular attention will be paid to industry input into curriculum development, workplace experiential learning, workplace based training and assessment and employer feedback to the education providers.

Background

During the latter part of 1998 a series of visits was made to major Registered Training Organisation (RTO’s) in all States and Territories providing education and training in the horse industry. Key personnel and were identified and interviewed at each provider institute or college. A database of information relating to courses offered, course information and delivery strategy was collected. Direct or indirect contact with ‘fringe’ or non-mainstream education providers and bureaucracies was established.

Research

Access was made to a national direct mailing database established by Equine Educational of Lochinvar, NSW. Further information was gleaned from The A.E.I.S. Horse Industry Directory and the Australian Equine Education Directory (IDP Education Australia). A total of 130+ letters was sent to all Registered Training Organisations (RTO's) identified and also to a number of Workplace Training Providers and Employers. Strategic visits to the key ‘mainstream’ educational institutions were implemented.

Implications

A full report was written detailing the history and current status of the provision of education and training in the horse industry in Australia. The key Registered Training Organisations and personnel were identified. A comprehensive list of the people, colleges and courses provided throughout Australia was compiled as well as a detailed summary matrix. A work-shop will be facilitated in mid-1999 to discuss the outcomes.

Project Title

Horses and land use on small properties

RIRDC Project No.:

PTP-12A

Researcher: Arthur Stubbs Organisation: Primary Tasks Pty Ltd

Unit 58, Level 2, 247 Drummond Street CARLTON, VIC. 3053


• Publication of a booklet giving guidelines for achievement of sustainable land

use and efficient horse management on small properties in urban surroundings.

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Background:

RIRDC Project PTP-3A, “Sustainable Land Use for Depastured Horses”, identified current practices and problems associated with horsekeeping on small properties around urban areas, Guidelines were established for optimum, pasture based, grazing and nutritional strategies in such situations to ensure sustainability of land use and economy of feeding. A booklet was proposed to give more detail about the recommended guidelines, and relevant issues, and be a more effective means of their dissemination.

Research

The emphasis in the book is on sustainable land use, but includes information on matters associated with horse grazing, such as the essential facts about nutrition and aspects of health to do with feeding and grazing, eg., worm control. Chapters include: How Many Horses; Growing Pasture; Managing Pasture; How Much Feed; and Healthy Land, Healthy Horses. Assistance was sought in drafting and editing from fellow researchers, particularly on nutrition and health, and from land care people, notably those in South Australia.

Implications

The information contained will be of value to those involved with a range of horse activities who have limited land available for housing their animals. A more widespread understanding and implementation of sustainable, pasture based, grazing management for horses will have long term benefits in more effective and sustainable land use, economy of horse management and improved appearance of horse properties.

Publications

“Healthy Land, Healthy Horses – a guidebook for small properties”. RIRDC Publication No. 98/137

Project Title

4TH Australian Racecourse Managers’ Conference, 1999

RIRDC Project No.:

PTP-13A

Researchers: Arthur Stubbs John Neylan Organisation: Primary Tasks Pty Ltd Turfgrass Technology Pty Ltd

Unit 58, Level 2 9 George Street 247 Drummond Street Sandringham Vic 3191 Carlton Vic 3053

Phone: 03 9844 1135 03 9521 6982 Fax: 03 9844 4554 03 9521 6946 Email: [email protected] [email protected]

Objectives

• Improvement of inter-club communication and education on issues of racetrack

care and maintenance for the development of effective, overall strategies for racecourse management.

Background:

A survey of grass racecourses in Australia in 1995 (RIRDC Project TGT-1A) found a wide variation in the level of racecourse management expertise and technical knowledge, as well as a sense of isolation by many racecourse managers and an expressed need for more information relevant to their jobs. This led to Racecourse Managers’ Conferences being held in the following three years, at Rosehill, Moonee Valley and Doomben, each attended by many racecourse managers from most States of Australia and New Zealand. The success of these conferences led to the 4th Conference at Morphettville on April 13-15,1999, with the theme, “Managing Racecourses”.

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Outcomes

93 people attended the conference, including 70 racecourse managers and staff from throughout Australia and New Zealand, plus suppliers and consultants to the racecourse industry, research workers and turf specialists. The conference was again very well received and delegates recommended that a similar forum be organised for 2000. It was considered that the objective had been achieved through the combination of paper presentations, two workshops, input from an American racecourse manager, inspections of three local racecourses and a racehorse stud, and general liaison between racecourse managers from a wide range of situations.

Implications

The conference enabled racecourse managers to gain and exchange information on the latest developments in research, racecourse development and maintenance, people and resource management, and to learn from overseas experiences and inspection of operations at major race and training tracks. The conference will further racecourse managers’ ability to practise their profession, leading to higher standards of racecourse management with benefits accruing from improvements in track performance, appearance and life, and associated economic and environmental advantages for racing clubs and the community.

Publications

4th Australian Racecourse Managers’ Conference Proceedings,1999

Project Title

Controlling and reducing airborne dust in horse stables and transporters

RIRDC Project No.:

SAR-14A

Researcher: Colin Cargill Organisation: South Australian research and Development Institute

Roseworthy Campus University of Adelaide GPO Box 397 Adelaide, SA 5001

Phone: 08 8303 7782 Fax: 08 8303 7975 Objectives

To review published information on the generation and concentration of dust in horse stables, and to recommend strategies for dust reduction and control.

Background:

Dust is an important airborne pollutant in all forms of animal housing and although the levels of dust recorded in horse stables are lower than in intensive livestock buildings, the horse is more sensitive to dust than other species and the length of exposure is significantly greater. Dust appears to enhance both the prevalence and severity of respiratory diseases, especially conditions such as chronic obstructive pulmonary disease (COPD). It may also aid the spread of other infections.

Outcomes

Dust in animal houses is a mixture of organic material including bacteria, bacterial and fungal toxins and spores, urine, dung, undigested feed, pollen and other feed and animal components. Concentrations in stables range from 0.2 to 17.21 mg/m3 (recommended maximum value of 2.4 mg/m3) for inhalable dust and from 0.15 to 9.28 mg/m3 (recommended maximum value of 0.23 mg/m3) for respirable dust. The important airborne micro-organisms present in stables appear to be a combination of actinomycete and fungal spores. Mesophilic bacteria, such as

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Corynebacterium spp and Arthrobacter sp, and gram positive organisms such as Micrococci spp, have been isolated in significant amounts as well. Stocking density (in terms of cubic metres of airspace per horse) and building design are important aspects of dust control and reduction. Ventilation rates have a direct effect on dust levels through increased air movement, which can either dilute the concentration of airborne particles or increase re-suspension of particles and delay settling. Exhaust ventilation is a key factor in removing dust but bedding and the type of feed are more important than ventilation as a cause of dust. A number of strategies for reducing dust generation and concentration in stables were recommended. These include: • Soaking and cleaning hay, pelleting feed or using silage; • Using feeding systems where feed is not dropped into a bin; • Adding oil to pellets or coating them with oil after manufacture, (but care needs

to be taken when manipulating diets); • Using wood shavings, diced paper or synthetic material to replace straw as

bedding, and replacing bedding frequently; • Storing feed in purpose built sheds rather than in stables or barns; • Cleaning boxes and stalls daily and removing the horses during cleaning; • Designing barns and stables to maximise air exchange rates in still conditions; • Using mechanical ventilation systems designed to minimise turbulence and to

remove dust particles from around the horses breathing zone (head height); • Using positive or negative pressure systems that bring air in through a series of

holes in the roof and exhaust air at a lower level along the walls; • Developing “low dust stables” that are free of hay and straw, with wood

shavings spread over a rubber matting on the floor; and • Providing sufficient airspace for each horse.

Implications

Dust is an important factor in the health and fitness of horses. The link between respiratory disease and dust requires further study to understand the effect of bioaerosols on the physiology and immune responses of the horse. Although data is available from other countries, a survey of horse housing facilities is recommended.

Project Title

Information Services for the Horse Industry

RIRDC Project No.:

UCS-18A

Researcher: Associate Professor Martin Sillence Organisation: Charles Sturt University

School of Agriculture, Charles Sturt University PO Box 588 WAGGA WAGGA NSW 2678


• To find a way of meeting the information needs of the horse industry.

Background:

A primary objective of RIRDC is to help the Australian horse industry gain access to ‘state of the art’ information. Departments of Agriculture have not included the horse industry in their normal extension services, so there is no facility for a lay person to initiate an inquiry or find scientific information that is easily understood. This project was carried out in the context of a plan to establish an Equine Information Service.

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Research

In part 1 of this project we asked 321 members of the horse industry about their information needs, and how they thought an information service should be operated. In part 2 we identified the best sources of reliable information. In part 3 we examined information services and computer systems used by other industries. In part 4 we formed a business plan for an Equine Information Service.

Outcomes

The horse industry is strongly in favour of an Equine Information Service. Information is currently obtained though books, magazines and word of mouth, with the most popular topics being health, nutrition and training/exercise. Most of the participants would like to be able to obtain information by mail or fax, through an information service that is funded by a combined system of user pays, government subsidy and industry levy. Several industry information services are operating successfully in Australia, and these use a range of computer systems for managing information that vary in price and complexity. A large amount of horse information has been identified, and much of this is available through the library at Charles Sturt University. We propose that this University establishes an information service in partnership with the horse industry. The service would be accessible by phone, fax or internet and provide comfortable public access areas. The set up cost will be $43, 562 and running costs will be $29, 462 per year. Charles Sturt University will provide free accommodation for the service, and up to $90, 000 over three years towards the literature and running costs. Industry sponsors are being sought for the additional financial support required.

Implications

There is a demand for an Equine Information Service in Australia. Charles Sturt University will establish such a service, if additional industry support can be found.

Project Title

Diagnosis of equine virus diseases using polymerase chain reactions and development of a blood test and vaccine for equine rhinovirus.

RIRDC Project No.:

UM-23A

Researcher: Professor M.J. Studdert, Dr C.A. Hartley, Dr H.E. Drummer. Organisation: The Centre for Equine Virology,

The University of Melbourne. Cnr Park Dve and Flemington Rds, Parkville, VIC 3052

Phone: +61-3-9344-7373 Fax: +61-3-9344-7374 Email: [email protected] Internet: http://www.vet.unimelb.edu.au/research/CEV.html Objectives

• To improve existing diagnostic procedures by the development of a

comprehensive set of primers for use in polymerase chain reaction (PCR) tests for all endemic equine virus diseases in Australia, and

• By the development recombinant DNA technology to engineer antigens for use in a blood test and vaccine for ERhV1.

Background:

It is suggested that 50% of all calls veterinarians make to horses in racing are for viral respiratory disease. These diseases are responsible for many expensive treatments, poor performance and many lost training days. Improved methods that are more rapid and sensitive for the diagnosis of equine herpesvirus 1 (equine abortion virus, EHV1), EHV4 (equine rhinopneumonitis virus), EHV3 (equine coital exanthema virus) the equine gammaherpesviruses EHV2 and EHV5, equine adenovirus 1 (EAdV1), EAdV2, equine arteritis virus, equine rhinovirus (ERhV) are required.

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Research

PCR tests have been developed for EHV1, EHV4, EHV3, EHV2, EHV5, ERhV1, EAdV1, EAdV2, equine arteritis virus and equine influenza virus. Of these tests the EHV1, EHV4, EHV2, EHV5 and ERhV1 tests have been implemented into our diagnostic laboratory at the Centre for Equine Virology. The remaining tests require further rigorous validation on clinical material before they can be confidently used in this setting. Implementation of these PCR tests in the laboratory has reduced the time required to obtain and report a result once a sample has been received. Furthermore, the use of PCR has enabled the retrospective diagnosis of ERhV1 as the cause of several outbreaks of febrile respiratory illness in horses in which no virus could be isolated.

Outcomes

Substantial progress was also made in the development of recombinant DNA technology to engineer antigens for use in a blood test and vaccine for ERhV1. Insect cells were engineered to produce the structural proteins of ERhV1.

Implications

This system has been shown for other proteins to enable extremely high levels of expression of recombinant proteins which are structurally very similar to their authentic couterparts. High levels of unprocessed ERhV1 capsid proteins have now been expressed and further work is currently underway to establish expression of the processed ERhV1 capsid proteins and to establish their suitability for use as a blood test and vaccine.

Publications

1. Li, F., H. E. Drummer, N. Ficorilli, M. J. Studdert, and B. S. Crabb. 1997. Identification of noncytopathic equine rhinovirus 1 as a cause of acute febrile respiratory disease in horses. Journal of Clinical Microbiology. 35:937-943. 2. Reubel, G. H., and M. J. Studdert. 1997. Identification, cloning and sequence analysis of the equine adenovirus 1 hexon gene. Arch Virol. 142:1193-212. 3. Reubel, G. H., and M. J. Studdert. 1997. Sequence analysis of equine adenovirus 2 hexon and 23K proteinase genes indicates a phylogenetic origin distinct from equine adenovirus 1. Virus Res. 50:41-56. 4. Reubel, G. H., and M. J. Studdert. 1998. Benefits and limitations of polymerase chain reaction (PCR) in veterinary diagnostic virology. Vet Bull. 68:505-516.

Project Title

Methods of quantitative assessment for intra pulmonary blood accumulation in racehorses with EIPH

RIRDC Project No.:

UM-36A

Researcher: Professor Ron Slocombe, Dr. Shaun McKane Organisation: University of Melbourne

School of Veterinary Science, Princes Highway, Werribee, 3030.

Phone: 03 9742-8264 Fax: 03 9741-0401 Email: [email protected] Objectives

• To develop quantitative in vivo methods for measuring the volume of

extravascular blood present in the lungs of horses after a single known blood inoculums of blood into normal horse lungs and in animals with experimental EIPH.

• To determine the extent and nature of sequential structural changes to lungs after inoculation with known volumes of blood, and correlate this with changes in lung cell populations seeen typically with field cases of EIPH.

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Background:

Although the causes for EIPH are well known, this problem remains very important in the racing industry because little is known about how blood is removed from the lungs once haemorrhage develops, and the ability to identify the extent of haemorrhage after exercise remains limited. Treatment therefore remains inadequate. This project explores ways to estimate both the extent of haemorrhage after episodes of EIPH and also identify the effects of blood on lung structure and function.

Research

Studies were conducted to evaluate the sequential events important in the removal of blood after segments of lung were inoculated with blood or controls using saline. We also investigated whether mild inflammation was a risk factor predisposing lung segments to further episodes of EIPH. The usefullness of radiolabelled erythrocytes as a marker for haemorrhage was investigated using scintigraphy scans of the thorax.

Outcomes

Blood is cleared slowly from the lungs by 2 mechanisms after installation, both are not completely effective, and blood causes residual inflammation and scarring that persists for at least 21 days. Lung inflammation was shown to be a risk factor for EIPH. Scxintigraphy using technetium labelled eryhtrocytes was not found to be a practical way of determining the extent of haemorrhage in lungs after episodes of EIPH.

Implications

Our data suggests that horses at risk of severe EIPH should be allowed additional time for the lungs to heal prior to racing. In addition, inflammtion of lung tissues predisposes to EIPH and therefore efforts to minimize lung damage due to environmental and infectious factors in routine management of racehorses is warranted. Methods to estimate the severity of EIPH in living animals remain inexact and further research is required to address this issue.

Publications

McKane,S.A., Bayly, W.M., Sides, R.H., Kingston, J.K. and Slocombe, R.F. Autologous blood infusion into the lungs interferes with gas exchange and performance in exercising horses. WEAS Conference, Ontario, Canada, 1998. Slocombe, R.F. and McKane, S.A. Exercise-induced pulmonary haemorrhage - part 1. The Veterinarian May p10-12, 1998. Slocombe, R.F. and McKane, S.A. Exercise-induced pulmonary haemorrhage - part 2. The Veterinarian June, p12-15, 1998. McKane, S.A. and Slocombe, R.F. Sequential changes in bronchoalveolar cytology after autologous blood inoculation. Equine Vet J. (ICEEP 5 supplement) accepted for publication. 1999. McKane, S.A. Physiologic and pathologic effects of Exercise-Induced Pulmonary Haemorrhage on the equine lung. PhD dissertation, University of Melbourne, 1999.

Project Title

Studies on the intestinal absorption of immunoglobulins and the effects of passive immune status on neutrophil function in foals

RIRDC Project No.:

UMU-18A

Researcher: Sharanne Raidal Organisation: School of Veterinary and Biomedical Sciences

Murdoch University, WA 6150

Phone: 08 9360 2356 Fax: 08 9310 7495 Email: [email protected]

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Objectives To further understanding of immunity in the newborn foal by: • Determining whether the type of fluid fed to foals immediately after birth can

prolong the time when colostrum can be effectively administered to boost immunity.

• Evaluating the effect of age and passive immune status on the development of white blood cell function in foals.

• Examining the effects of intravenous plasma administration to healthy and septic foals on white blood cell function.

Background:

Foals usually acquire immunity from their dams by the ingestion of colostrum immediately after birth. The failure of this passive transfer of maternal immunity to the foal is the greatest risk factor for the development of infectious disease in the first weeks of life. Our understanding of foal immunity and susceptibility to disease is poor. Almost exclusively, studies to date have focused on the antibody (IgG) status of foals and little work has been completed evaluating other aspects of foal immunity, such as white blood cell function. We know that antibodies in colostrum are absorbed from the foal’s small intestine for a short time after birth. We do not know what determines when antibodies can no longer be absorbed and we do not know if the absorptive process can be prolonged. Plasma transfusion is routinely advised when foals fail to absorb an “adequate” amount of colostral antibodies. However, we do not know that this procedure is effective at reducing the incidence of septic disease or whether it has any beneficial effect for the foal other than a transient increase in circulating antibody levels.

Research

Research was conducted in two parts. Firstly the effect of delaying the ingestion of colostrum was evaluated in foals fed a glucose and electrolyte solution or a milk replacer. Colostrum was milked from each mare and fed to her foal from 12 hours after birth. The amount of IgG absorbed and aspects of white blood cell function were assessed for each foal. In the second part of the study, the effect of plasma transfusion of healthy and septic foals with failure of passive transfer of immunity was assessed, again by evaluation of circulating IgG concentrations and white blood cell function.

Outcomes

Delayed ingestion of colostrum was associated with levels of IgG that are currently considered inadequate for the prevention of septic disease. However, the administration of colostrum at 12 hours post partum was associated with the absorption of approximately 50% of administered IgG and improved cell function in adult cells assayed with foal serum. Plasma transfusion resulted in a significant increase in circulating IgG concentration, the response to treatment being better in healthy foals than septic foals. Treatment was not associated with a beneficial effect on white blood cell function for healthy foals, but there was a transient benefit on neutrophil function in septic foals. Passive immune status (IgG concentration) correlated poorly with white cell function at all sampling times in both parts of the current study, suggesting that other factors are important determinants of immunity in the foal.

Implications

The findings of the first part of the study support current recommendations that colostrum should be administered to foals within 12 hours of birth. However, even delayed ingestion of colostrum may be beneficial. If colostrum cannot be provided immediately after birth, the choice of fluid given to foals does not influence the subsequent efficiency of IgG absorption. The second part of the study did not support routine plasma transfusion of healthy foals with failure of passive transfer of immunity. Conversely, treatment of septic foals had a transient positive effect on cell function, suggesting that multiple transfusions should be considered during the course of therapy. Although the association between low circulating IgG levels and

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foal sepsis is well proved, the results of the current study suggest that consideration of IgG levels alone as a marker of foal immunity may be simplistic.

Project Title

Control of intraocular inflammation by topically applied non steroidal anti-inflammatory drugs (NSAIDS)

RIRDC Project No.:

UQ-59A

Researcher: SM Taylor & IA Shiels Organisation: Department of Physiology and Pharmacology

University of Queensland, ST LUCIA, QLD 4072


• To develop a laboratory-based test to evaluate new anti-inflammatory drugs to

treat equine recurrent uveitis.

Background:

Equine recurrent uveitis is a recurring inflammation in the eye which can cause blindness. The disease is treated with anti-inflammatory drugs, mostly steroidal and nonsteroidal compounds. The disease is difficult to control because of the ongoing pathology, and current therapy is inadequate. A range of novel anti-inflammatory drugs are being synthesised and there is a need to have a test to screen these compounds without having to use painful procedures in laboratory animals.

Research

A model of uveitis was developed to screen drugs for anti-inflammatory activity in the laboratory. The technique uses eyes removed from cadavers and their viability is maintained for a number of hours by perfusing nutrient fluid through the vasculature. The tissues respond to inflammatory substances infused into the eye. The induced inflammatory signs can then be blocked, or reversed, by drugs administered to the eye. One of the new compounds tested has been shown to have better anti-inflammatory activity than existing nonsteroidal drugs and is being prepared for clinical trials.

Outcomes

The development of this test has enabled the evaluation of a number of new drug compounds quickly and cheaply. The test does not require animal suffering.

Implications

The test should speed the evaluation of new products at less cost. Compounds with activity would then require further evaluation in animal models and clinical trials.

Publications

A manuscript has been accepted for publication in the Australian Veterinary Journal

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Project Title

Studies on host-parasite interactions in disease (strangles) induced by streptococcus equi subspecies equi

RIRDC Project No.:

US-24

Researcher: Paul J Canfield Organisation: Department of Veterinary Anatomy and Pathology,

University of Sydney, NSW 2006


• To understand the very early interactions between the horse and the causative

bacterial agent of strangles ie Streptococcus equi subspecies equi and in so doing, lay the foundation for further studies aimed at development of improved control strategies including development of newer generation vaccines.

Background:

It has been estimated that strangles accounts for a minimum direct loss to the industry of $6.5 million annually. Indirect costs in reduced export opportunities and adverse publicity associated with outbreaks of the disease in horses exported from Australia to other countries would bring the losses much higher. Thus, strangles has been rated by the industry as amongst the most important endemic disease in horses and many sectors within the industry have expressed their wish for effective control of this contagious disease. Current control involves the use of killed vaccines and management procedures that rely primarily on recognition of clinical cases of infection. Such procedures often result in horses with early strangles being introduced into groups on farms or into racing stables and subsequent outbreaks occurring. Furthermore, carrier animals are now considered to exist and there is no means available currently to identify these animals.

Research

This report describes the experimental reproduction of strangles in groups of horses and the subsequent host cell interactions with the strangles organism.

Outcomes

The experimental reproduction of the disease allowed documentation of the very early clinical signs of strangles and led to the recognition that significant changes could be detected in the lymph glands in the head before any other signs of strangles were obvious and much earlier than reported by any previous work. It also found that specific parts of the nasal passages of horse were involved in strangles infection and that changes in tissues associated with the carriage of strangles organism could be recognized at these specific sites.

Implications

These results should allow much earlier detection of horses incubating strangles and be of considerable benefit in the management of the disease. The research also suggested a means for development of a diagnostic test to detect carrier animals.

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Project Title

Lower respiratory tract disease in racehorses

RIRDC Project No.:

US-33A and US-41A

Researcher: Dr R.M. Christley, Dr J.L. Hodgson, Dr J.L. Wood, Prof. S.W.J. Reid, Prof. R.J. Rose, Prof. D.R. Hodgson

Organisation: University of Sydney University Veterinary Centre PMB #4 Narellan Delivery Centre NARELLAN NSW 2567


• To better define the risk factors associated with development of lower

respiratory tract disease in racehorses. These risk factors included the association of infectious organisms (viruses, bacteria and mycoplasma) with respiratory disease in Australian horses.

Background:

Respiratory disease continues to be a major problem in the thoroughbred racing industry. A recent survey of racehorse trainers found coughing to be one of the most serious problems facing the industry. While there have been a numbers of studies examining the possible causes of respiratory disease in racehorse, there is scant information regarding the relative contribution of each of these causes to the respiratory disease observed. This deficiency has obvious implications for the treatment, control, and management of coughing horses.

Research

We used a matched, case control study to investigate the role of specific infectious agents in causing respiratory disease in racehorses in Sydney. In this study, cases were identified by the presence of coughing. These horses were compared to a control population (non-coughing horses) in order to identify risk factors for coughing and to evaluate the association between coughing and clinical, pathological and microbiological findings.

Outcomes

A number of risk factors were identified for coughing. Young horses in early training were at greatest risk of coughing. Horses that had raced within the previous week or had been transported more than 14 days previously were also at increased risk. There was no association between coughing and recent exposure to viruses, including Equine Herpesvirus-1 and –4, and Equine Rhinoviruses-1 and –2. Approximately 40% of horses had a significant number of bacteria isolated from their lower airways which would suggest a role of these agents in lower respiratory tract disease. However, approximately 60%% of horses had no evidence of an infectious cause to the respiratory disease.

Implications

The findings from these studies should greatly change our current concepts of respiratory disease in athletic horses. The finding that viruses appear to have a minor role in causing disease in this group of horses may influence some of the current control strategies such as vaccination. In addition, while bacteria may be associated with coughing in a proportion of cases, a majority of horses had no evidence of bacterial infection. This would imply that antibiotics may be currently being over-prescribed and that steps to specifically diagnose the cause of respiratory disease in horses should be recommended.

Publications

Christley RM, Hodgson DR, Rose RJ, Reid SWJ, and Hodgson JL (1999)

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Comparison of bacteriological and cytological findings of tracheal fluid samples collected by percutaneous aspiration and via an endoscope using a plugged guarded cather. Equine Veterinary Journal, 31, 197-202. Christley RM (1998) Respiratory disease in racehorses; what is it? Equine Research Seminar, Post Graduate Foundation in Veterinary Science, University of Sydney, pp1-7. Christley RM, Wood JLN, Reid SWJ, Hodgson DR, Rose RJ, Hodgson J (1999) Risk factors for coughing in thoroughbred racehorses. In: Proceedings of the Society for Veterinary Epidemiology and Preventative Medicine, Eds: MV Thrushfield and EA Goodall. (In press).

Project Title

In vitro biomechanical properties of equine digital flexor tendons

RIRDC Project No.:

US-52A

Researcher: Mr. Andrew Dart Organisation: University Veterinary Centre Camden.

PMB 4 Narellan Delivery Centre, NARELLAN NSW 2567

Phone: (02) 46 550777 Fax: (02) 46 551212 Email: Objectives

• To evaluate the biomechanical and biochemical properties of normal and healing

equine superficial digital flexor tendons (SDFT). In addition the effect of a novel product, recombinant equine somatotropin was assessed on normal and healing tendons.

Background:

Superficial digital flexor tendon injury is a major cause of musculoskeletal injury affecting up to 30 % of horses in race training. Healing of SDFT injuries tends to be slow, with re-injury common. It has been reported that only approximately 20-60 % of affected horses may successfully return to racing with up to 80 % of horses sustaining re-injury. Recent investigations in tendon pathology have found age and exercise related changes to occur in equine tendons. These degenerative alterations may predispose the tendon to injury. The mechanisms of age related changes in musculoskeletal tissue composition and mechanical behaviour are diverse and complex. However, the role of declining circulating concentrations of various growth factors has been incriminated.

Research

Three experiments were performed examining the in vitro biomechanical properties of the metacarpal region of forelimb equine superficial digital flexor tendons. Experiment 1 was aimed at determining the in vitro biomechanical properties of forelimb SDFT from horses without evidence of clinical tendonitis. Experiment 2 examined the effect of equine recombinant somatotropin (eST) on the in vitro biomechanical properties of normal tendons. Experiment 3 examined the effect of eST administration on the in vitro biomechanical properties of healing tendons.

Outcomes

Despite the problems associated with in vitro biomechanical testing, the methodology was shown to be repeatable, and, to provide objective data on the biomechanical properties of tendons in response to various treatments. The lack of detectable effect of eST on the biomechanical properties of normal,

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mature SDFT may be related to a number of factors. The equine SDFT has been shown to reach maturity at approximately 2 to 3 years of age. It is possible that mature tendons have a reduced capacity to respond to an eST stimulus given the cellular changes that are apparent with maturity and within the metacarpal region of SDFT. In addition previous exercise history and eST dose rate may have influenced biomechanical response of the tendons. Further investigations into tendon responses to eST at different dose regimens, in immature animals, and under controlled exercise programs may be indicated. The biomechanical results support diminished structural properties in the eST treated healing tendons. This may be due, in part, to an immature or biomechanically inferior, disorganized collagenous matrix. Future histological and immuno-histochemical analysis will help to determine the exact effect of eST on collagen synthesis and ultrastructural organisation.

Implications

Despite the apparently negative findings of this study, the pharmacological basis behind the use of eST in healing of collagenous tissue injuries, including tendonitis is relatively sound. Further investigations may be warranted using the described techniques, however a longer treatment period prior to biomechanical testing may yield more favourable results.

Project Title

Serological investigation of Equine influenza in Australia

RIRDC Project No.:

US-58A

Researcher: Dr Jennifer Hodgson Organisation: University of Sydney

University Veterinary Centre Camden PMB 4 Narellan Delivery Centre NARELLAN NSW 2567

Phone: (02) 4655 0745 Fax: (02) 4655 6942 Email: [email protected] Objectives

• To determine the status of Equine Influenza in Australian horses using serological

investigations.

Background:

Equine Influenza (EI) is a major cause of respiratory disease of horses in Europe and North America where it accounts for extensive losses to equine industries both in terms of money and performance. Australia is one of the few countries in the world that is free of EI and the current quarantine protocols for entry of horses into Australia have been designed to ensure this continued freedom. In addition, exportation of horses from Australia is considerably facilitated by the absence of this viral infection. Recently, however, freedom of Australian horses from EI virus infection has been questioned by countries importing horses from Australia, and also by the Office International des Epizootiques (OIE). Consequently, demonstration of our continued freedom was of great importance for our equine import and export industries.

Research

In order to investigate the status of EI in Australia we collected two serum samples from 248 horses in the Sydney Metropolitan Region. This was part of a larger study investigating respiratory disease in racing thoroughbreds. The design of the investigation was a case control study, where identical samples were collected from cases (horses with signs of respiratory disease) and controls (horses that were clinically normal). In this way, differences between cases and controls could be assessed. By collecting samples form horses exhibiting signs of respiratory disease

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(coughing), we increased the likelihood of detecting antibodies to EI virus, as coughing is the most common sign associated with this infection. Two samples were collected from each horse in order to detect rising antibody titres which is indicative of recent infection. The collected serum samples were frozen and sent to the Animal Health Trust (AHT) in Newmarket, England for processing. The AHT is the official European Laboratory for assaying serum samples for presence of antibodies to EI virus. The assay routinely performed at the AHT is called the Haemagglutination Inhibition (HI) assay and this test is considered the gold standard for detecting antibody to EI virus with a very high sensitivity and specificity.

Outcomes

None of the serum samples assayed had evidence of antibodies to EI virus. This was in contrast to other respiratory viruses assayed (including Equine Herpesviruses-1 and –4 and Equine Rhinoviruses-1 and –2) where there was evidence of recent seroconversion in a low number of horses. By assaying serum samples from 248 horses we estimated that we would detect the presence of antibodies to EI virus in the Australian horse population with 95% confidence should these antibodies be present in as few as 1-2% of horses. However, there are several strong arguments that suggest our degree of confidence in detection of EI was in fact much higher. First, Thoroughbred race horses were the ideal population to sample as they are among the most likely candidates to have been exposed to EI virus had it been introduced to Australia. In addition, this population is extremely mobile, with horses from Sydney, Melbourne and Brisbane frequently co-mingling at race meetings. Consequently, as this virus is highly infectious and the Australian equine population is naïve, we would have expected a very rapid spread of the virus and evidence of this serologically in the Sydney horses, even if the virus were introduced in another part of the country. Finally, coughing is the most commonly reported sign associated with EI virus infection. As our study included 100 horses with respiratory disease as defined by coughing, this would increase significantly the likelihood of detection of this virus.

Implications

This study has demonstrated that there was no serological evidence of equine influenza in racehorses in the Sydney Metropolitan region, including horses demonstrating signs of respiratory disease. Due to the highly infectious nature of the virus, and the frequency with which horses in race-training con-mingle with horses both from Sydney and other parts of Australia, this data lends strong support to our continued freedom from the EI virus in Australia. These results should offer persuasive reassurances to our equine trading partners.

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3.5 Horses

Research in Progress Project Title

Learning within the Equine Industry

RIRDC Project No.:

FLP-1A

Start Date: 1/07/98 Finish Date: 30/12/99 Researcher: Mr. Laurie Field Organisation: Field Learning Pty Ltd

29 Pearson Ave GORDON NSW 2072

Phone: 02 9498 8278; 0407 459 055 Fax: 02 9498 6394 Email: [email protected] Objectives

• To develop a better understanding of the ways in which various sectors of the

equine R&D industry learn. In doing so, it will highlight opportunities for supporting learning within the industry.

Current Progress:

• Fact-finding meetings with a variety of horse industry representatives have been

held to date. • Questionnaire entitled “Getting information about horse care and the treatment of

illness” was distributed in the last issue of RIRDC Equine Research News. The deadline for return is Friday 7 May 1999. 64 completed questionnaires received in first week after distribution.

• Meetings have been held with those involved with Pony Clubs, Thoroughbred Racing and the Australian Stockhorse Association to discuss the project and the logistics of questionnaire distribution through the relevant publications. This questionnaire will be ready for desktop publishing and printing by the end of April.

A counter and visitors’ book has been placed on the RIRDC Equine Research and Development website. This has been supplying insufficient data for our needs and will be supplemented by an on-line questionnaire. This has been developed, and will be operating from early May.

Project Title

Improved Pasture: More than just grass for horses to exercise on?

RIRDC Project No.:

DAV-118A

Start Date: 30 June 1996 Finish Date: 31 December 1999 Researcher: Angela Avery and David Nash Organisation: Agriculture Victoria Rutherglen

RMB 1145, Chiltern Valley Road Rutherglen VIC 3685


• To determine the extent to which different pasture types stocked at different rates

can meet the growth requirements of young horses.

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• To compare supplementary feeding ("normal practice") with pasture-based feeding systems.

Current Progress:

From April to December, the project Improved Pastures for Horses: More than just grass for horses to exercise on, measured the growth (liveweight gain, height and various leg dimensions) of 36 filly foals from weaning. This is the largest grazing experiment ever conducted on temperate pastures in Australia. Preliminary analysis of the results has shown that fillies grazing improved pasture at moderate stocking rates can reach a heavier liveweights and greater heights than fillies raised on unimproved pasture or supplementary feeding targeted at moderate growth rates. A second year of measurement has just commenced with 36 colt foals. Pasture, grazing behaviour and soil measurements have also been taken to gain a complete understanding of the horse-grazing system. The project has received strong industry support; 72 foals have been donated by the horse industry for the duration of the experiment, supplementary feed has been donated by MITAVITE and industry has freely given their time to assess horses. Media coverage for the project has been extensive with the highlight being three screenings of a report on the ABC’s Landline series.

Project Title

Pasture intake and selectivity by grazing horses

RIRDC Project No.:

UCS-22A

Start Date: 1/07/97 Finish Date: 31/12/99 Researcher: Dr. Michael Friend Organisation: Charles Sturt University

School of Agriculture PO Box 588 WAGGA WAGGA NSW 2678


• To gain an understanding of the intake and selectivity of temperate pastures by

horses. • To undertake an assessment of the ability of pasture species and mixes to meet the

nutritional demands of young growing horses.

Current Progress:

Initial attempts during 1998 to administer alkanes to weanlings via a molasses lolly met with difficulties in acceptance. After attempting several different methods of administering the lollies the approach was abandoned in favour dosing the weanlings with an alkane suspension using the technique of Marais et al. (1996). Weanlings were successfully dosed twice daily using this method in November 1998. Four weanlings in each of the 4 pasture treatments of DAV-118 were dosed and faecal samples collected over a four day period commencing from day 5 after initial dosing. Weanlings for 1999 have arrived and introduced to the experimental plots. Intake estimates will be made in May, July, September and November of 1999. Pasture and faecal alkane concentrations will then be analysed in order to determine intake and selectivity of weanlings during 1998 and 1999.

19

Reference Marais, J.P., Figenschou, D.L., Escott-Watson, P.L. and Webber, L.N. (1996). Administration in suspension form of n-alkane external markers for dry matter intake and diet selection studies. J. Agric. Sc., Camb. 126: 207-210

Project Title

Molecular epidemiology of Rhodococcus equi

RIRDC Project No.:

UM-29A

Start Date: 1/01/97 Finish Date: 30/12/99 Researcher: Dr. Glenn Browning Organisation: The University of Melbourne

Veterinary Preclinical Centre University of Melbourne PARKVILLE VIC 3052


• To determine if there is a relationship between Rhodococcus equi strains and

morbidity and mortality patterns on different studs. • To identify protein(s) which specifically identify virulent strains and to develop a

diagnostic assay based on these protein(s).

Current Progress:

Characterisation of strains of Rhodococcus equi has continued, with comparisons made to international isolates as well as isolates made in the last stud season. There are close similarities of some Australian isolates with those from other countries. Statistical comparisons of the data indicates that there are particular strains which are most common particular studs, but we are unable to associate specific strains with more severe disease. We have been unable to identify any major antigen specifically produced by Rhodococcus equi under restrictive conditions including iron deprivation, elevated carbon dioxide concentrations and higher temperatures, and so have started construction of an expression library of genes of virulent Rhodococcus equi , which will be probed using sera from diseased foals to identify genes which are most likely to be associated with disease. We are specifically focussed on genes which occur on the virulence plasmid, which is currently being sequenced by workers in several countries. We have established active collaborations with researchers in Japan and Canada and have obtained reagents from them to further the project. The collaboration with workers in Japan has led to one joint publication which has been submitted to the Journal of Clinical Microbiology.

Project Title

Epidemiology of equine rhinovirus type 2 (ERhV2)

RIRDC Project No.:

UM-40A

Start Date: 6/07/98 Finish Date: 30/07/00 Researcher: Dr. Carol Hartley Organisation: Centre for Equine Virology

Dept of Veterinary Science PARKVILLE VIC 3052

20


To determine the epidemiology of ERhV2 in Australian horses. To design diagnostic tests for ERhV2 based on polymerase chain reaction (PCR) and enzyme linked immunosorbent assay (ELISA). To investigate the potential of an ERhV2 vaccine based on recombinant baculovirus expressed virus-like particles.

Current Progress:

In order to gain some understanding of the exposure of Australian horses to ERhV2, one hundred random samples of equine serum recently submitted to the CEV were tested in virus neutralisation assays. 42 % of the serums showed the capacity to neutralise ERhV2, indicating that these horses have had some history of exposure to this virus. 40 % of samples tested were negative (titres <10) and the neutralisation titres of 17 samples could not be determined due to the toxic effects of the serum at high concentrations. There was no evidence for cross neutralising antibody between ERhV1 and ERhV2 or vice versa. To design a PCR based diagnostic test for ERhV2, oligonucleotide primers were designed based on the genome sequence of the prototype strain of ERhV2 (Wutz et al. 1996). An RT-PCR was performed on 14 strains of ERhV2 obtained from Cornell University and The University of Kentucky. A product of the expected size (1061 base pairs) was obtained from the prototype strain, but no products were observed from any of the other strains, suggesting the sequence in the region to which the primers bind is highly variable between strains. New primers have been designed to more conserved regions of the ERhV2 genome, in order to design a diagnostic RT-PCR able to detect all strains of ERhV2.

Project Title

Improved vaccine strategies for management of equine herpesvirus diseases

RIRDC Project No.:

UMA-15A

Start Date: 1/07/98 Finish Date: 30/06/01 Researcher: A/Prof. Millar Whalley Organisation: Macquarie University

School of Biological Sciences SYDNEY NSW 2109


• Improvement of reproductive efficiency and respiratory health in horse

populations through reduction of incidence of herpesvirus abortion and respiratory infection.

• Improved preventative strategies against infectious dieases of the horse, and lower costs.

• Possible establishment of generic vaccine technology applicable to different diseases affecting horses.

Current Progress:

We have recently completed the sample collection phase of a prospective cohort study of mares and foals on a large Hunter Valley stud farm. The aims of this study were to estimate the incidence and prevalence of EHV-1 and EHV-4 infection in a group of mares and their foals and to compare these results, in a vaccinated population, with the results published in 1997 from a group of unvaccinated foals. This study will

21

allow us to examine the response to vaccination of a group of foals as well as to evaluate the effect of vaccination on the incidence and prevalence of infection. Sample testing and data collation and analysis is currently underway. As part of the vaccine development, we have developed a quantitative assay for EHV-1 glycoprotein antigens using monoclonal antibodies, and antigen prepared by CSL Ltd. is currently being tested in formulations in mice, prior to testing in horses. We have also demonstrated the potential of DNA vaccination for horses. After intramuscular injections of DNA encoding EHV-1 glycoprotein, horses developed a specific antibody response. In addition, protective responses following DNA immunisation in mice were dramatically enhanced by boosting with recombinant protein. This strategy will subsequently be investigated in horses.

Project Title

Investigation of Potential EHV-1 Vaccines Using the Mouse Model of Infection.

RIRDC Project No.:

UMA-16A

Start Date: 1/02/99 Finish Date: 31/07/99 Researcher: Dr. Catherine Walker Organisation: Macquarie University

Department of Biological Sciences NSW 2109


• To investigate the potential of vaccines using the mouse model of EHV-1

infection.

Current Progress:

We have refined the respiratory and abortion mouse models of EHV-1 disease into functional systems which can examine the effects of various immunogens prior to their being trialled in the horse. Our research group has generated a series of recombinant baculovirus subunit, deletion mutant (DISC) and DNA-based potential vaccines, which is ‘cutting edge’ technology. The mouse model experiments have determined that a protective immune response can be achieved by immunising with vaccines using a) recombinant expressed protein to just one of the envelope proteins of the virus b) DNA encoding just one of the viral envelope proteins c) a genetically engineered virus which has an envelope glycoprotein gene missing and thus cannot replicate and cause infection. An immunisation regimen which employs BOTH expressed protein and DNA as vaccines has been demonstrated to markedly enhance protection from challenge. Experiments are currently in progress to establish optimal formulations of the recombinant protein antigens.

22

Project Title

Identification of infectious respiratory disease in horses as a basis for better management of cases

RIRDC Project No.:

UMU-22A

Start Date: 1/07/97 Finish Date: 30/06/00 Researcher: A/Prof. Graham Wilcox Organisation: Murdoch University

School of Veterinary Studies MURDOCH WA 6150


• To investigate the association between viruses and respiratory disease in horses in

Western Australia by (a) clinically defining respiratory conditions of unknown or suspected viral aetiology and (b) examining the association between viruses and each of these clinical conditions. • To multiple sample affected horses to determine the effect of the stage of the

disease process and site of sampling on the ability to detect viruses associated with each clinical condition.

Current Progress:

The possible role of viral agents in horses presented to Murdoch University Veterinary Hospital for evaluation of respiratory disease and/or poor performance has been evaluated. A history was obtained for each horse and a complete clinical examination and endoscopic inspection of the upper respiratory tract were performed. Bronchoalveolar lavage (BAL), tracheal wash (TW), nasal swab and peripheral blood leucocyte samples were collected from each horse and examined for viruses using a combination of polymerase chain reaction (PCR) and conventional isolation techniques in cell culture. Cytologic examination of BAL and TW samples was a good indicator of horses with inflammatory airway disease but correlated poorly with virus isolation. The prevalence of virus infection in sampled horses was low. Affected horses could usually be identified by signalment (usually 3 year olds or younger), history (acute onset of disease and presence of in-contact affected horses) and physical examination. The sampled population has recently been extended to include younger horses commencing training and weanlings. The prevalence of latent infection of peripheral blood leucocytes with equine herpesviruses in this population was quite high.

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Project Title

Safe and effective grain feeding for horses

RIRDC Project No.:

UNE-62A

Start Date: 1/01/98 Finish Date: 30/06/00 Researcher: Prof. James Rowe Organisation: University of New England

ARMIDALE NSW 2351 Phone: 02 6773 2225 Fax: 02 6773 3275 Email: [email protected] Objectives

• To reduce the risk and incidence of foot problems, related to laminitis and

connected with feeding cereal grain, through understanding which grains are most likely to cause problems and which methods of feed treatment are most effective in reducing these risks.

Current Progress:

The review of grain characteristics and processing techniques will be published in a special edition of the Australian Journal of Agricultural Research available in July 99. We have completed two experiments to provide better estimates of nutritive value of different grains for horses. In the first experiment, with Bianca Wilson of Orange, we measured the digestibility of two cultivars of oat grain (high and low-lignin in the hull) and results will be available shortly. In the second experiment we compared oats, barley, triticale and sorghum in terms of their digestibility in different parts of the digestive tract. Analytical work on both studies is near completion. A preliminary analysis indicates the in-vitro assay for intestinal starch digestion accurately distinguishes and rank grains. The results confirm that triticale is highly digestible and sorghum is poorly digested. Barley was better than sorghum but not as digestible as oats or triticale. In the next stage we will use our in-vitro system to test grains, processed to enhance intestinal digestion of starch before checking ‘best-bet’ methods for efficacy in-vivo. We are confident that we can deliver new methods of selecting and processing grain for safer feeding of horses.

Project Title

The influence of season on follicular dynamics and the relationships with endocrinology and oviduct physiology in the mare

RIRDC Project No.:

UQ-43A

Start Date: 24/08/95 Finish Date: 1/9/99 Researcher: Dr. Neil McMeniman Organisation: The University of Queensland

School of Veterinary Science ST LUCIA QLD 4072


24

Objectives • To provide basic information on the relationship between seasonal ovarian activity, endocrinology and oviduct functon in an attempt to improve reproductive efficiency in the mare.

Current Progress:

This project is a long-term study examining the endocrinological events that occur in the mare around ovulation. The uterine tube (or oviduct) is a very complex organ responsible for the housing of sperm from the male, transporting the female egg to the site of fertilisation, coordinating the events leading to conception and nourishing the young embryo while it travels to the uterus. There is an enormous lack of basic knowledge about this organ and the events that occur within it and this study will significantly contribute to this knowledge. The information will be most applicable to scientists working in the area of infertility and breeding technologies. One spin-off from the project has been the identification of mares that respond differently to the new ovulating agent deslorelin (OvuplantTM, Peptech Animal Health, NSW). We have found that the majority of mares respond normally, by ovulating within 48 hours of receiving the drug when used as specified. However, if the mare has a functional corpus luteum producing progesterone, the response to deslorelin will be delayed. Also there is a small population of mares that fail to ovulate. These mares have a significantly different hormonal environment that may help to identify these mares. The physical changes that occur within the uterine tube are still being studied. A final report on results obtained and interpretation of their meaning will be presented in September 1999.

Project Title

Nutrition of grazing broodmares, their foals and young horses

RIRDC Project No.:

UQ-45A




• To determine the extent to which pastures can support economical production of

quality foals and young horses.

Current Progress:

The major part of this project compared the growth, from six months to two years of age, of Australian Stock horses that were either continuously or rotationally grazed with that of horses that were hand fed to maintain a liveweight gain of approximately 0.7 kg per day. The liveweights of the hand fed horses at the end of the experiment were significantly higher than those of the pasture fed horses. However, the final skeletal size of the three groups of horses, as reflected by their heights at the wither, were not significantly different. The mean body condition score and depth of fat over the rump of the hand fed horses were significantly greater than their hand fed counterparts while the muscle area of the Longissimus dorsi at the twelfth rib was not significantly different between groups.

25

This suggests that the main reason for the difference in liveweights of the hand fed and grazed horses was due to the hand fed horses being fatter than the grazed horses. These results show that satisfactory growth of horses can be obtained from pasture. Details of the nutrient content of pastures on a commercial stud in south east Queensland during the period of the trial have been determined. These data indicate that, providing adequate pasture is available, grazing brood mares and young growing hoses should obtain all of the nutrients required for satisfactory production from these pastures. Further sampling of pastures from studs in the Scone region is continuing. A final report on this project will be prepared by September 1999.

Project Title

Assessment and prevention of toxicity from indospicine contaminated horsemeat

RIRDC Project No.:

UQ-46A

Start Date: 1/07/95 Finish Date: 31/12/99 Researcher: Dr. Michael Pass Organisation: Faculty of Science

University of Queensland Lacked Bag No. 4 Maroochydore, DC, 4558


• To develop a method for detecting indospicine residues in horsemeat that is

suitable for screening large numbers of samples under commercial conditions. • To develop a definitive system for detection of indospicine residues in horsemeat

that can be used under commercial conditions for confirmation and accurate quantitation of indospicine detected by the above screening test.

• To provide information on the mechanism of indospicine toxicity that can be used by the horsemeat industry to counteract threats to our export trade by countries importing horsemeat from Australia.

Current Progress:

Progress has been made in transferring the laboratory based HPLC method for detecting indospicine in horsemeat to the commercial environment. It was used to test potentially contaminated meat destined for export. We were able to confirm within 24 hours of receipt of the meat samples that they were free of indospicine and the meat was subsequently exported. This episode raised the issue of pre-slaughter testing as a means of identifying contaminated animals. To investigate the efficacy of pre-slaughter testing, experimental animals were fed indospicine. Analysis of plasma and meat indicated that concentrations of indospicine in plasma were consistently higher than concentrations in meat. The finding is significant in that it supports the idea that pre-slaughter testing of plasma may be a more sensitive means of identifying contaminated animals. Progress has been made in developing the ELISA assay for indospicine. Despite the selectivity of the antibodies for indospicine, the sensitivity of the assay for horsemeat extracts is still inadequate due to interfering substances. However, the finding that plasma concentrations of indospicine are consistently higher than meat concentrations, indicates that testing of plasma by the ELISA method may be satisfactory for screening animals for indospicine contamination. This is currently being pursued.

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Project Title

Novel anti-inflammatory drugs to treat inflammatory diseases in horses

RIRDC Project No.:

UQ-71A

Start Date: 1/09/98 Finish Date: 1/09/01 Researcher: Dr. Stephen Taylor Organisation: The University of Queensland

Dept of Physiology and Pharmacology BRISBANE QLD 4072


• To test novel classes of anti-inflammatory drugs that will reduce the impact of

inflammatory diseases of the lung, eye and joint on the normal structure of affected tissues in horses. By reducing fibrosis, oedema, cellular hyperplasia and changes in the phenotype of resident cells, the long term impact of inflammatory diseases, like pneumonia, arthritis and uveitis, on performance and well being will be reduced.

Current Progress:

Biological activity of PLA2 inhibitors. An BSc Honours student is working on this part of the project in 1999 to characterise the pharmacology of PLA2 inhibitors and their potential for use in equine diseases. The perfused eye model and rat uterus have been used to quantify activity of the drugs and compare activity with the nonsteroidal antiinflammatory drugs ketoprofen, flunixin, piroxicam and indomethacin. Previous experiments showed PLA2 inhibitors were potent inhibitors of prostaglandin-induced contractions in the rat uterus in vitro, but it has now been shown that this activity is related to the stage of the oestrus cycle of the rats. Variable potency of the PLA2 inhibitors has been demonstrated in a perfused eye model of uveitis also, suggesting that there are other cellular mechanisms involved in this inflammatory mediator cascade. We are now investigating the events that lead to the release of prostaglandins and leukotrienes and the role of secretory and cellular PLA2 enzymes in this process. New compounds with potent inhibitory activity against secretory PLA2 have been synthesised by the Drug Design and Development Centre and will be made available for testing.

Project Title

Nutritent intake and growth of young horses

RIRDC Project No.:

UQ-72A




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Objectives • To determine whether weanling horses grazing lush spring pastures can suffer a check in growth and whether this check is due to the fact that the high soluble carbohydrate containing diets contain insufficient fibre. The consequences in terms of subsequent growth and skeletal development to a check in growth will be determined.

• The project will also provide information on the effect of pasture maturity and yield on intake and growth of young horses.

• Information on which pasture species young horses prefer to consume will also be obtained.

Current Progress:

The field work associated with the first study to investigate the effect of pasture yield on intake of young horses has been completed. Horses were grazed on three predominantly lucerne based pastures each having a different pasture yield and height. The alkane marker technique was used in this study and alkane concentrations in pasture and faecal samples from the study are being determined. Pasture dry matter and species intake will be calculated from these results. A similar study with a lush temperate grass species (rye grass) is about to commence. To further validate the alkane marker technique as a method of determining dry matter intake and digestibility and species composition of diets a series of pen studies is being conducted. Results from these studies should also provide data to allow for more accurate prediction of digestibility using the in vitro digestibility technique that is being used in our laboratory.

Project Title

The pathogenesis and prevention of equine laminitis

RIRDC Project No.:

UQ-75A

Start Date: 1/07/98 Finish Date: 30/06/02 Researcher: Dr. Christopher Pollitt Organisation: The University of Queensland



• To determine the role and control the activity of matrix metalloproteinases in the

pathogenesis of laminitis so that a product can be developed which will prevent and treat the disease.

• To determine the role of changes in the pattern of glucose metabolism in the pathogenesis of laminitis as a basis for predicting and preventing laminitis.

Current Progress:

mRNA has been isolated from hoof and used as templates to perform RT-PCRs for MMP-2. As in other mammalian MMPs, equine MMP-2 had the conserved "cysteine switch" and the "catalytic zinc binding site" and showed 92% homology with human MMP-2. Synthesised peptides of MMP-2 are being used to raise anti MMP-2 monoclonal antibodies to locate and show upregulation in laminitis. The MMP-2 primers will be used to perform in situ hybridisation and Northern analysis on laminitis affected tissues. Using our HPLC detection protocol we have completed the pharmacokinetics of the potent glucocorticoid triamcinilone in 6 horses showing persistence for 12 days after a single dose and induction of a parallel hyperglycaemia and hyperinsulinaemia. An electron microscopic study using the in vitro hoof explant laminitis model has commenced to determine what ultrastructural events in the lamellar basement membrane occur as lamellar tissues separate.

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We have discovered the supernatant of cultured Streptococcus bovis isolated from equine faeces induces in vitro laminitis by activating MMP-2. This bacterium proliferates in the large bowel during the development of grain induced laminitis and may produce a pyrogenic exotoxin that reaches hoof lamellae via the circulation to activate MMP-2 and trigger laminitis.

Project Title

Respiratory ventilation and tracheal sounds in horses with airway disease

RIRDC Project No.:

US-53A

Start Date: 1/10/97 Finish Date: 1/12/99 Researcher: Dr. David Evans Organisation: The University of Sydney

Dept of Animal Science SYDNEY NSW 2006


• To investigate the use of two techniques for field studies of airway function during

and/or after exercise of moderate intensity.

Current Progress:

A new method of measuring breath by breath air flow has had to be developed over the last six months to enable completion of the experiments. This new technology is now being used to study instantaneous velocities of respiratory gas flow during inspiration and expiration in resting and exercising horses, and during recovery after exercise. Measurements of respiratory air flow at each nostril has also been successfully combined with measurements of tracheal sounds in Thoroughbred horses exercising on a treadmill. Studies of the tracheal sounds during and after field exercise in Thoroughbred horses at Randwick racecourse have been completed. Current treadmill experiments are investigating respiratory sounds, and respiratory gas flow rates and volumes in normal horses and horses with reversible, induced laryngeal hemiplegia. These experiments are using techniques not previously conducted in Australian horses, such as recordings of breath by breath air flow, and the generation of tidal breathing flow volume loops during exercise. Tidal breathing flow volume loops relate the air flow rate to the phases of breathing during a respiratory cycle, and enable calculation of peak inspiratory and expiratory flow rates, and flow rates at intermediate proportions of the tidal volume.

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Project Title

Training and Fitness in Athletic Horses

RIRDC Project No.:

US-77A

Start Date: 1/12/98 Finish Date: 31/08/99 Researcher: Dr. David Evans Organisation: The University of Sydney

Dept of Animal Science SYDNEY NSW 2006


a. The objective of this project is technology transfer, or the transfer of information

to industry participants. b. A book will be written which will serve as a reference for those in the horse

training industry. The book will collate the results of research on horse training and fitness assessment, and present the information in a format suitable for industry participants.

Current Progress:

A literature review has been completed. Consultations with several industry participants have been conducted concerning suitable topics for inclusion in the book.

Project Title

Diagnosis of and protection against infections caused by Rhodococcus equi

RIRDC Project No.:

USA-6A

Start Date: 3/02/98 Finish Date: 30/03/00 Researcher: Dr. Mary Barton Organisation: School of Pharmacy and Medical Sciences

University of South Australia GPO Box 2471 ADELAIDE SA 5001


• To develop an accurate, reproducible and easily performed serological test for early

diagnosis of R.equi infections in foals. • To identify antigenic epitopes that may be further investigated as potential vaccines

to protect foals from infection.

Current Progress:

An ELISA has been developed for the detection of IgG antibodies to R. equi outer membrane proteins. PCR has also been developed for the detection of the vapA (virulence-associated protein) gene, as well as for the detection of the 16S rRNA gene of R. equi.

A biotinylated peptide bank based on the VapA protein sequence has been prepared by Chiron diagnostics, Victoria and this peptide bank will be used to screen sera from R .equi infected horses using ELISA techniques. The ELISA technique developed has the potential to be useful as a relatively quick and easy diagnostic test for R .equi infections

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in horses but must first be evaluated in the field.

Electron microscopy studies were carried out on various clinical isolates of R. equi (both equine and human) some of these isolates were found to possess pili-like appendages and phage –like particles. More studies will be carried out to determine the clinical significance of the pili-like appendages and phage-like particles. These results support the view that human and equine isolates are different ie R equi is not a zoonotic organism as is sometimes suggested.

The groEL gene of R. equi ATCC 6939 was successfully cloned in E. coli DH5α and is currently being sequenced. This gene will be used in the development of a DNA vaccine which initially be trialed in a mouse model and will hopefully be useful in the prevention of R. equi disease in horses.

HORSES - AgriFutures

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