Second O.I.E. International Workshop on Equine Viral Arteritis:
October 13th – 15th, 2008
Lexington, Kentucky, USA
Introduction
The workshop was convened by The Dorothy Russell Havemeyer Foundation Inc., represented by Mr.
Gene Pranzo (President and Foundation Director) and Mr. David Schaengold (Foundation Director), and
organized by Dr Peter Timoney with the support of colleagues and staff at the Maxwell H. Gluck Equine
Research Center, Department of Veterinary Science, College of Agriculture, University of Kentucky.
The workshop was endorsed by the World Organization for Animal Health (OIE) and included
contributions from scientists from Asia, Australia, the European Union, the Middle East, North and South
America.
Goals of the Symposium
1. To share information on new and existing laboratory tests for the diagnosis of equine arteritis
virus (EAV) infection; the prescribed tests for international trade described in the current edition
[6th] of the OIE Standards Manual are the virus neutralization (serology) and virus isolation (virus
detection) assays.
2. To share information on the epidemiology of EAV infection of horses for the purpose of
optimizing prevention and control strategies that mitigate the risk of virus transmission but which
are not excessively restrictive nor economically overly burdensome to equine industries
worldwide.
Detection and Identification of Equine Arteritis Virus
The sensitive and specific detection of EAV is central to the diagnosis of this infection, and the accurate
identification of carrier stallions is especially critical to effective control of equine viral arteritis (EVA).
Although virus isolation (VI) in RK-13 cells is the prescribed test for detection of EAV, it has certain
disadvantages in terms of cost, assay duration, and labor requirement. Several participants presented data
on rapid and more convenient molecular diagnostic assays for the detection of EAV, including:
conventional reverse transcription – polymerase chain reaction (RT-PCR), nested and hemi-nested PCR,
real time PCR, and new generation technologies such as the loop-mediated isothermal amplification
(LAMP) assay. However, although these assays often can rapidly and accurately identify EAV in samples
collected during outbreaks of EVA, it has been shown repeatedly that none of these assays, as described,
provides equal sensitivity to virus isolation for the detection of EAV in semen samples from acute or
chronically infected (carrier) stallions or other clinical or necropsy specimens that contain low levels of
virus infectivity.
Conclusions:
1. VI is the current “gold standard” and the OIE prescribed method for international trade for EAV
detection based on its proven ability to determine infectivity in a wide range of clinical and
necropsy material from horses experimentally or naturally infected with this virus. Test mating of
stallions is a financially costly alternative method for detection of the carrier state. OIE
acceptance of a molecular method for the detection of EAV will require development of an assay
1
with equivalent sensitivity and specificity to the VI assay; false negatives are unacceptable for
regulatory purposes.
2. Current efforts to develop an improved molecular virus detection test have been largely
uncoordinated at an international level with little concerted effort to promote harmonization and
standardization among laboratories.
3. The relatively lower sensitivity of current molecular assays (as compared to VI), likely reflects
factors such as:
a. lack of template (small sample volume for example)
b. sequence mismatches in primers and/or probes, reflective of the genetic diversity among
global strains of the virus as evidenced by more recent sequence analysis of field strains
of EAV
c. inhibitors of PCR
Recommendations:
1. Establish a working group to determine the optimal test platform(s) – real time PCR for example
with thought to:
a. assays that can be run by various laboratories and provide comparable results, and which
would facilitate standardization and harmonization
b. increase assay sensitivity to equivalency with VI
i. Template concentration from equivalent starting volumes of sample for instance
ii. Re-evaluate strategies for statistical validation of assays to ensure maximal
accuracy
c. Second generation platforms, especially those like LAMP that might be used in countries
where expensive laboratory equipment is not available.
2. Collate all (including future) sequence data into a public repository, then revisit assay design
a. evaluate the full genome of as many global strains of EAV as possible to determine the
optimal region(s) to target
b. optimize the design of primers and probes to minimize mismatches
3. Coordinate the efforts of different laboratories to standardize, harmonize and trouble shoot
respective assays – development of an improved molecular virus detection assay is feasible and
should be considered a high priority!
Assays for Antibody Determination to Equine Arteritis Virus
Serologic tests are vital to confirm prior exposure of horses to EAV (including vaccination), recent
infections (for diagnostic purposes), and for assessing risk – specifically, seronegative animals are
susceptible to infection, whereas seropositive horses (mares, stallions, geldings, foals) are considered
protected. Importantly, however, a variable percentage of seropositive stallions can be persistently
infected virus carriers. Serological tests should have high sensitivity and specificity, to minimize false
positive and false negative results. The serum virus neutralization (VN) assay is the OIE prescribed test
for international trade; it has certain disadvantages, however, including cost, assay duration, and labor
requirement. Problems with VN assay include cytotoxicity that can be encountered with a variable
percentage of sera and which can make assay interpretation more difficult and “cut-off” values that could
2
potentially result in false positive results. While more convenient and rapid ELISA tests have been
described, however, none currently offer equivalent sensitivity and specificity to the VN test.
Conclusions:
1. There are marked differences in the “expectations/requirements” of EAV serology, expectations
that vary between countries/laboratories and even sectors of the equine industry. Basic goals of
serology include:
a. Retrospective surveys to monitor the overall seroprevalence of infection amongst horses
in a country or region, to monitor prior infection status etc – clearly, there is some
latitude for lower test sensitivity in the case of assays used for such purposes
b. Diagnostic serology to confirm recent infection using paired sera
c. Prospective assessment of risk in respect of individual animals, which requires optimal
assay sensitivity and specificity
i. serological tests and vaccines are not yet available that differentiate vaccinated
from infected animals (DIVA vaccines and companion diagnostic tests)
2. Performance of the current VN test can be complicated by the problem of cytotoxicity
encountered with certain serum samples and which can interfere with assay interpretation.
However, as currently configured, ELISAs and their derivatives lack equivalent or superior
sensitivity compared to the VN and therefore, they cannot realistically be applied to determine
risk on an individual animal basis.
Recommendations:
1. Continue to use the VN assay for regulatory purposes until such time as an ELISA with
equivalent sensitivity and specificity to the VI test becomes available. However, methods for
minimizing or eliminating the problem of serum toxicity in the VN test should be standardized
and harmonized.
2. Where accepted at a national and industry level, utilize the best ELISAs for surveillance so that
large numbers of samples can quickly be screened in a more cost effective manner.
3. Future development of vaccines and serological tests that facilitate DIVA
Epidemiology of Equine Arteritis Virus Infection
Equine arteritis virus infection of horses most commonly is subclinical or asymptomatic, however
outbreaks of EVA appear to be occurring on a more frequent basis in different areas of the world.
Molecular typing, sequencing and phylogeny of EAV isolates is increasingly being done and is a useful
tool in furthering our understanding of the global epidemiology of EAV infection.
Conclusions and Recommendations:
1. Continue to monitor the global occurrences of EVA to establish the mechanisms of virus spread
in such instances, so that appropriate control strategies can be implemented.
2. Permanent systems aimed at the unique identification of horses and premises would greatly assist
epidemiologic studies and control programs.
3. Ongoing genetic and phenotypic assessment of viruses contained in the semen of carrier stallions
is critical. Not only are these animals the essential natural reservoir of EAV, it would appear that
3
genetic diversity among strains of EAV is generated during the course of persistent infection in
the carrier stallion. Ongoing documentation of this genetic diversity is critical to:
a. Molecular investigations to trace the source of individual outbreaks of EVA, and to
monitor the global situation through molecular topotyping
b. Continued improvement of molecular diagnostics
c. To the rational design of new generation vaccines
Control of Equine Viral Arteritis
Equine viral arteritis is a preventable and controllable disease provided that available strategies and
diagnostic technologies are appropriately and consistently implemented. The primary focus of current
national programs is to prevent the introduction and spread of EAV in breeding populations and the risk
of virus-related outbreaks of abortion and establishment of the carrier state in stallions. The single most
important factor in reducing the risk of spread of EAV in breeding populations of unprotected equids is to
vaccinate all breeding stallions against natural infection with the virus and thereby minimize the potential
for establishment of the carrier state. This should be expanded also to include vaccination of prepubertal
colts between 6 and 12 months of age. This would interrupt the natural cycle of infection, and over time,
eliminate the essential virus reservoir. More thought needs to be given globally to this approach even
though different sections of the equine industry are likely to have different perspectives – even in the
same country. The treatment of carrier stallions with a GnRH antagonist in order to “cure” or eliminate
persistent infection is gaining favour in some parts of the world. However, currently it is not clear
whether such treatment can effect a permanent cure or not, nor whether it can interfere with the accuracy
of diagnostic tests. Finally, assessment of vaccine efficacy in preventing persistent infection should be an
ongoing priority in countries that adopt the vaccination of prepubertal colts.
Many of the persisting issues regarding the national/international control of EAV infection are political,
not scientific.
Conclusions and Recommendations:
1. Recommend the monitoring of breeding stallion populations for the presence of carrier animals.
2. Continue to strongly recommend vaccination of all seronegative breeding stallions to prevent the
risk of establishment of the carrier state.
3. Continue to monitor the situation concerning the use of GnRH antagonists for the treatment of the
carrier state in the stallion.
4. In the event of any emerging evidence, consider whether there are international trade or
movement implications and advise OIE accordingly.
4