EQUINE ARTERITIS VIRUS

Equine arteritis virus (EAV) is the prototype member of the family Arteriviridae, which includes porcine reproductive and respiratory syndrome virus (PRRSV), lactate dehydrogenase elevating virus of mice (LDV), and simian hemorrhagic fever virus (SHFV). EAV is a small, enveloped, single-stranded RNA virus that is inactivated by most disinfectants. The endothelium of the small blood vessels and macrophages are the primary targets of EAV, but the virus can also replicate in different cell types in vivo and in vitro. It is transmitted by venereal and respiratory routes.^3,⁴ EAV can infect horses, donkeys, and mules, but natural infection in zebras has not been demonstrated.⁵

Clinical Signs

Symptoms of EAV vary widely in severity and range and depend on the virus strain and other factors such as age, breed, and immune status of the host.⁶ The range of clinical signs varies between individuals within and between outbreaks, with breed appearing to be an important determinant of symptoms. In particular, susceptibility to infection appears to be the same for Standardbreds and Throroughbreds, but clinical disease is less frequently reported in the former, despite their high seroprevalence.

Infected horses may or may not present, after an incubation period of 3 to 14 day,⁷ any combination of the possible clinical signs, the most common of which are fever (lasting up to 7 days and reaching up to 41°C); depression; lethargy; anorexia; conjunctivitis; rhinitis with serous nasal discharge; palpebral, periorbital, and/or supraorbital edema; edema of mammary glands, prepuce, ventral abdomen, and/or legs; and urticarial skin rash. Other signs include weakness, ataxia, congestion or petechia of mucosal membranes, respiratory distress, diarrhea, and abortion in the pregnant mare.

The two features of the disease that make its control important are its ability to cause both abortion in pregnant mares and persistent infections in stallions. Detailed study of the disease indicates that the venereal route, particularly stallion to mare, plays a critical role in the maintenance of the infection in the horse population.⁸^–¹⁰

Infection of pregnant mares can result in abortion, which has a serious economic impact for the horse breeding industry. It usually occurs 10 to 33 days post infection and can occur without the mare displaying any clinical signs.⁷ Abortions have been reported in mares between 3 and 10 months of gestation. Mares do not appear to suffer fertility problems after EAV infection, but stallions experience a reduction in the quality and quantity of the sperm during the acute phase of the disease. In contrast, no fertility problems have been observed in persistent carrier stallions.¹¹

A proportion of stallions become chronically infected with EAV after the acute phase and can continue to excrete virus in the semen for months or years without displaying any signs of disease. EAV localizes in cells of the entire male urogenital tract, and it has been recovered from the epididymis, vas deferens, ampulla, seminal vesicle, bulbourethral gland, prostate, urinary bladder, and proximal urethra in persistently infected stallions.¹² Such persistent infection is testosterone dependent; castration results in cessation of virus excretion in semen after castration, although it can be maintained through administration of testosterone.¹³ Persistent infections do not occur in colts infected before puberty.¹⁴ Results from recently reported attempts to stop shedding by temporary chemical castration have been interesting,¹⁵ but more work is required before this can be recommended as an effective procedure.

EAV is well preserved in both frozen and chilled equine semen and so can be transmitted most effectively through the use of natural covering and artificial insemination (AI),^3,⁴ whether the semen has been preserved by chilling or freezing.

Immune Responses

EAV infection in the horse induces virus-neutralizing antibody (VNAb) and cytotoxic T-lymphocyte responses.¹⁶ Experimental infections indicate that VNAb in serum appears within the first week after infection¹⁷^–²² and remains at high levels for months or years. The appearance of VNAb in serum coincides with clinical recovery in experimentally infected animals.¹⁸ Passively acquired maternal antibodies, which last between 2 and 8 months in foals,^23,²⁴ prevented or moderated EAV infection, and horses vaccinated with formalin-inactivated vaccine showed various degrees of protection, which correlated to the VNAb at the time of challenge.^25,²⁶ Although infection with EAV results in a long-lasting, if not lifelong, immunity and recovered horses do not show signs of disease, they are rarely protected from reinfection, and sterile immunity may not be achieved reliably with either inactivated or live vaccines.^21,^27,²⁸

Diagnosis of Equine Viral Arteritis (EVA) and Detection of EAV in Carrier Stallions

Due to the asymptomatic nature of many natural EAV infections and the fact that several of the clinical signs of EVA are nonspecific, it is always necessary to confirm suspected EAV cases by laboratory testing.

Retrospective serologic diagnosis of EAV infection is made by detection of virus-specific antibodies in serum from convalescent horses. The most widely used serologic test and the one prescribed for international trade by the Office International des Epizooties (OIE) is the virus neutralization test (VNT) performed on microtiter plates as described by Senne et al.²⁹ The test, despite being widely used and suitable for large sample screening, can present problems in its performance, and discrepant results have been obtained by different laboratories.³⁰

In specialist laboratories conversant with the use of the VNT, VNAb has been demonstrated in all persistently infected stallions. Thus the VNT is a useful screening assay for all stallions to help determine infection status. Nonspecific cytotoxicity of equine sera obtained from horses vaccinated with tissue culture–derived vaccines can, however, complicate the use of the VNT.³¹ Use of enzyme-linked immunosorbent assay (ELISA) tests to replace the VNT has been investigated for some time.³²^–³⁴ The diagnostic ELISA test developed by Nugent et al³⁵ appears useful but has not yet become available commercially.

Virus isolation from clinical specimens appears most sensitive when RK-13 cells are used.³⁶ Isolation of EAV from clinical material is difficult and time consuming, for both semen and samples collected during the acute phase of the disease.³⁷

All unvaccinated stallions with VNAb may be persistently infected semen shedders, and their semen should be examined for the presence of EAV. Detection of infected stallions can also be made indirectly by test mating. The suspected stallion is mated to two seronegative mares on two consecutive days, and detection of a rise in VNAb titer of the mares is taken as an indication of the shedding status of the stallion. Although slow and expensive, this is a safe method for determining the shedding status of a stallion.

Due to these inconveniences, molecular diagnostic methods have become increasingly popular in the last 10 years for the detection of EAV from clinical samples. The potential of these techniques for EAV diagnosis was demonstrated by Chirnside and Spaan,³⁸ and their methods provide the basis for molecular diagnostic protocols.^39,⁴⁰ Modern real-time polymerase chain reaction (PCR) methods have facilitated high sample throughput, while minimizing cross contamination.^11,⁴¹

Vaccination Against EAV

Currently there are two commercially available vaccines for the prevention of EVA, a live attenuated vaccine and a killed virus adjuvanted vaccine. The live vaccine, used extensively in some parts of the United States,^42,⁴³ prevents clinical disease and significantly reduces viremia and virus excretion. However, its use is not recommended in pregnant mares. Recently a virus with an identical sequence to the vaccine strain was derived from an aborted fetus whose dam was vaccinated a few days earlier with the live vaccine.⁴⁴

Inactivated vaccines were first developed in Japan and consisted of concentrated preparations of formalin-inactivated whole virus formulated without adjuvant. These vaccines stimulate neutralizing antibodies after an initial two-dose course. The antibody levels decline over a period of 6 months, and revaccination is recommended to sustain protective immunity. This immunity correlates with circulating neutralizing antibody levels.^25,^26,^28,⁴⁵ The commercial inactivated vaccine (Artervac, Southampton, UK) is currently licensed in Germany and available in the United Kingdom (UK) and other European states under test certificates, although only limited data on efficacy and duration of immunity exist. The vaccine claims to stimulate VNAb after two doses, but some field studies and experimental observations showed that detectable titers may not be achieved even after three or more inoculations,⁴⁶ although higher titers were usually achieved after multiple doses.

The available vaccines do not allow discrimination of infected from vaccinated animals, which is necessary to allow serologic surveillance in nonendemic areas. The lack of serologic discrimination also hugely complicates international trade in vaccinated stallions and their semen. All stallions being vaccinated should be shown to be seronegative in an accredited laboratory on the day of vaccination, and thereafter serologic titers should be monitored. The induction of protective immune responses that can be differentiated from natural infections by using “marker” vaccines would overcome the problems mentioned above.^47,⁴⁸

Prevention of EAV Transmission at Covering or Insemination

Control relies on combining serologic surveillance, vaccination, and sound animal management practices. Special emphasis is put on detection of persistent shedding in stallions and prevention of the carrier state developing. However, the policies vary widely between different countries, being motivated in part by the differences in disease prevalence. There are various codes of practice that exist to prevent transmission of EAV on stud farms in Europe, including a Common Code of Practice used in many European states ⁴⁹ and the British Equine Veterinary Association’s Code of Practice for artificial insemination.⁵⁰ European Community directives cover the intracommunity trade in semen.⁵¹

In contrast to these European protocols, the American Horse Council Protocol on EAV bases its recommendations for the control of EAV on the extensive use of the live vaccine in stallions, permitting the breeding of immune or immunized mares to shedding stallions. The control measures implemented in the UK and Ireland, where prevalence is low, contrast to these; recommendations are based on serologic screening of mares and stallions before covering to ensure that infectious animals are restricted from the breeding population. Stallions vaccinated with the whole virus killed vaccine should also be serologically monitored annually. EVA is notifiable in stallions in several European states, including the UK and Ireland.

European legislation requires that stallions used for frozen semen collection should be demonstrated to be seronegative for EAV 14 days after collection of semen and that horses used for chilled semen production should be seronegative before entry into a licensed collection center.⁵¹

The use of AI is probably the best means of protecting stallions from becoming infected as a result of covering acutely infected, visiting mares. If stallions, such as Thoroughbreds, need to cover mares naturally, then they should be vaccinated and care exercised to ensure that mares are not acutely infected before covering. Serosurveillance of mare bands immediately before covering is useful in achieving this, but is not foolproof.⁵²

CONTAGIOUS EQUINE METRITIS

Contagious equine metritis (CEM) is caused by infection with Taylorella equigenitalis, a microaerophilic bacterium also referred to as contagious equine metritis organism (CEMO). Two biotypes of the organism, distinguished by streptomycin sensitivity, have been characterized.⁵³ Infection in naïve animals causes an acute endometritis in most naïve mares, following a latent period of 2 to 12 days.⁵³ Primary infection in the mare usually results in short-term infertility, and this is the major impact of the disease. Infection is usually asymptomatic in stallions. It is spread venereally between mares and stallions and between infected mares and their offspring at parturition. It can also be spread mechanically.⁵⁴^–⁵⁷

CEM is endemic in non-Thoroughbreds in several European countries but is largely absent from most Thoroughbred populations, including in Ireland, Britain, and France. Horses in the United States and Canada are thought to be free from infection, although there is some evidence of infection of donkeys with a related organism, Taylorella asinigenitalis.^54,^58,⁵⁹

There is a wide range of opinions as to the clinical significance of the infection, in particular in Europe. Although some countries, including Ireland, Britain, the United States, and Canada, regard the infection as highly significant, others such as Belgium and the Netherlands regard the organism as an occasionally pathogenic commensal. It is clear that the major impact of the infection is economic, both through reduction of high levels of fertility now expected in intensive breeding operations and through stringent international controls on the movement of infected animals and germplasm.⁵¹

Outbreaks in the UK and Ireland have been controlled highly effectively by implementation of the recommendations of the Horserace Betting Levy Board’s Codes of Practice.⁴⁹ A small number of cases of CEM have been diagnosed recently in the UK, but these have occurred in recently imported non-Thoroughbred animals and their contacts.⁶⁰ Recent cases have not been diagnosed in Ireland.

Long-term male and female carriers of the infection play a key role in the dissemination and maintenance of infection,⁵³ and the vast majority of persistently infected mares harbor T. equigenitalis on the mucous membrane surfaces of the clitoral sinuses and fossa.⁵³ It was noted around 25 years ago that available methods for the detection of CEMO in mares were not capable of detecting all infected mares.⁶¹ The methods in common use today have not altered in substance since that time, even though more sensitive methods have become available.⁶²^–⁶⁶