Equine viral arteritis: current status and prevention.

Recently, there has been increased interest in equine viral arteritis (EVA) among veterinarians and horse owners. Outbreaks of the disease were identified initially in New Mexico, USA in 2006, and in the Normandy region of France in the summer of 2007. Both occurrences were associated with AI of cool-shipped semen. Each was linked to respiratory illness, neonatal death, abortion, development of carrier stallions, and cancellation of equestrian events. In light of the increased interest, this paper will present a brief case history, followed by a review addressing common concerns regarding EVA, current status, and control and prevention strategies, including vaccination, and recommended bio-security measures.

Further evaluation and validation of a commercially available competitive ELISA (cELISA) for the detection of antibodies specific to equine arteritis virus (EAV).

The purpose of this study was to further evaluate and validate two commercially available equine arteritis virus (EAV) competitive ELISAs (original and enhanced cELISAs) using archived equine sera from experimentally inoculated animals and field sera submitted for laboratory diagnosis. First, the original and subsequently enhanced cELISAs were compared with the virus neutralisation test (VNT) using a panel of archived serum samples from experimentally inoculated animals. Then, the enhanced cELISA was compared with the VNT using a large panel of archived serum samples. The total number of equine sera tested was 3255, which included sera against 25 different EAV strains. The study confirmed that the enhanced cELISA was more sensitive than the original cELISA. Based on testing sera from experimentally inoculated animals and field sera, the enhanced cELISA had an estimated sensitivity (98.9 percent and 99.6 percent, respectively) and specificity (98.3 percent and 98.7 percent, respectively). The currently marketed enhanced VMRD EAV antibody cELISA test kit (VMRD Inc., Pullman, Washington, USA) has high sensitivity and specificity relative to the VNT. Based on the findings of this study, the authors would propose that the enhanced cELISA should be considered as an alternative approved method to the VNT for the detection of antibodies to EAV.

Franciscus Cornelis Donders (1818-1889).

Franciscus Cornelis Donders was educated at Duizel and Boxmeer before entering the Military Medical School and the medical faculty at Utrecht University in 1835. In 1840, he received his MD from Leiden and spent 2 years in practice at Vlissingen before returning to Utrecht, where he was appointed as an extraordinary professor to lecture on forensic medicine, anthropology, general biology and ophthalmology. Refraction by the eye is complex, since the ray of light passes through many changes of refractive index in its path, and Donders simplified the account of the process by establishing an equivalent refractive system: the reduced eye. When Donders opened an Eye Hospital in 1858, he devoted himself to clinical ophthalmology, making fundamental advances in providing spectacles to correct errors of refraction-which he separated from errors of accommodation. In 1862, Donders was promoted as an ordinary professor at Utrecht and he handed over the greater part of his practice to his pupil Hermann Snellen. From narrow specialisation, Donders was freed to return to the broader physiology; subatmospheric pressure in the pleura was for a while referred to as 'Donders' pressure'; he also devised a method of measuring the mental reaction time taken in making discrimination, rather than the simple reaction time in which no choice is involved. He was widely honoured, presiding at international congresses, and elected as a foreign member of the Royal Society. He died suddenly on 14 March 1889, but his work lives on.

Genetic variation in open reading frame 2 of field isolates and laboratory strains of equine arteritis virus.

The open reading frame 2 (ORF2) of three laboratory strains, the live attenuated vaccine virus, and 18 field isolates of equine arteritis virus (EAV) from Europe and North America was sequenced. The ORF2 of EAV encodes the Gs protein that is abundantly expressed in infected cells but constitutes less than 2% of the virion protein mass. Variation of ORF2 among the isolates facilitated phylogenetic analysis that largely confirmed results of an earlier study based on sequence divergence of ORF5 of the same isolates of EAV, despite exposure of the proteins encoded by ORF2 (Gs) and ORF5 (GL) to potentially different selective pressures in vivo. The data indicate that the Gs protein is highly conserved between isolates, considerably more so than the GL protein, consistent with an important role of the Gs protein in virus replication.

The increasing significance of international trade in equids and its influence on the spread of infectious diseases.

Expansion in international trade in equids and equine semen has been especially notable over the past 10-15 years among those countries historically identified as having significant breeding and performance horse industries. The continuing trend towards globalization of the horse industry received additional impetus in January, 1995, following establishment of the World Trade Organization (WTO), whose primary goal is to promote freer economic exchange between member countries through the reduction or elimination of protectionist barriers to trade. Continued growth in international trade, closely related to changing trends in the horse industry, has greatly increased the risk of spread of a wide range of equine infectious diseases between countries. In consequence, the global distribution of certain of these diseases is likely to change in the future. Within the past 30-40 years, there have been numerous confirmed instances of the spread of specific diseases through the international movement of equids or shipment of semen, some of which have resulted in epidemics of major economic importance. Under the Sanitary-Phytosanitary Agreement of the WTO, national agencies have had to rethink their traditional "zero-risk" approach in regulating the importation of equids or equine semen from other countries. Mindful of the risks of disease spread inherent in such transactions, authorities must now accept that primary emphasis in today's global economic climate must be on greater facilitation of trade, rather than attempting to provide absolute disease preventive safeguards.

Detection of antibodies to equine arteritis virus by enzyme linked immunosorbant assays utilizing G(L), M and N proteins expressed from recombinant baculoviruses.

Indirect enzyme linked immunosorbant assays (ELISAs) utilizing the three major structural proteins (M, N, and G(L)) of equine arteritis virus (EAV) expressed from recombinant baculoviruses were developed. A large panel of sera collected from uninfected horses, and from animals experimentally and naturally infected with EAV or vaccinated with the modified live virus vaccine against equine viral arteritis, were used to characterize the humoral immune response of horses to the three major EAV structural proteins. The data suggest that the M protein was the major target of the equine antibody response to EAV. The responses of individual animals varied and ELISAs that utilized individual EAV structural proteins were not reliable for detecting antibodies in all sera that contained neutralizing antibodies to EAV. An ELISA based on a cocktail of all three EAV structural proteins, however, was used successfully to detect antibodies in most equine sera that were positive in the standard serum neutralization assay following natural or experimental EAV infection (100% specificity, 92.3% sensitivity). In contrast, this ELISA did not reliably detect antibodies in the sera of vaccinated horses. EAV frequently causes a persistent infection in stallions and all sera from carrier stallions evaluated in this study had obvious reactivity with the N protein, whereas seropositive non-carrier stallions, mares and geldings did not respond consistently to the N protein.

Contagious equine metritis: experimental infection in the donkey.

Contagious equine metritis (CEM) was reproduced in 3 of 4 donkey mares with an Irish streptomycin-resistant strain of Haemophilus equigenitalis isolated from an experimental case of the disease in a pony mare. Although some variability in clinical response occurred, there was no evidence that semen enhanced the clinical severity of the infection. Variable amounts of vaginal discharge and associated inflammatory changes of the vagina and/or cervix, similar to those seen in the horse, were observed. All the affected donkeys made spontaneous clinical recoveries and so far as could be detected, subsequent persistence of H. equigenitalis in the genital tract was of limited duration. Recovery of the bacterium was not associated with oestrus and there was no evidence that it persisted in the clitoral area after it could no longer be cultured from the anterior genital tract. Cytological examination of smears of intra-uterine or cervical swabs was of diagnostic value only during the clinical phase of the infection. Serological responses demonstrated in 3 of the 4 donkey mares by the agglutination, complement-fixation and passive haemagglutination tests, were of low magnitude and short duration. The diagnostic value of the agglutination and complement-fixation tests was limited by the presence of low levels of non-specific reactivity and pronounced anti-complementary reactivity, respectively, in many of the donkey sera. The passive haemagglutination test proved superior for demonstrating elevation in antibody and for confirming infection. The overall results indicate that the donkey has the potential to act as a source of CEM infection and under certain circumstances, could have a role to play in the epidemiology of this disease.

Genomic variability among globally distributed isolates of equine arteritis virus.

Equine arteritis virus (EAV), a non-arthropod borne togavirus, has been shown to have a global distribution. To date, no major antigenic variation has been demonstrated between EAV isolates from different geographic origins. In this study, the genomic RNA of EAV isolates obtained from horses of different breeds in various countries around the world was oligonucleotide fingerprinted. Comparisons of these fingerprints were used to determine the extent of genomic variation among such isolates. Comparisons among isolates from North American horses revealed, for the most part, oligonucleotide homologies of less than 60%. Only 29 of the 98 comparisons revealed greater than 60% oligonucleotide homology. Nonetheless, several comparisons indicated a close epidemiologic relationship between isolates from horses of different breeds located in different states. Though all European isolates were of Standardbred origin and were from horses located in northern European countries, the majority had oligonucleotide homologies of less than 60%. Where oligonucleotide homology was apparent, it was, with one exception, greater than 70%. The two isolates from New Zealand had 93.2% oligonucleotide homology. This is indicative of an extremely close epidemiologic relationship. Comparisons between EAV isolates from around the world revealed oligonucleotide homologies between viruses from North America, Europe and New Zealand. In several instances, this homology was greater than 70% and in one case greater than 80%. No oligonucleotide homology was evident in comparisons involving the virus from South Africa. The high level of genomic conservation between certain EAV isolates of disparate geographic origins may reflect dissemination of the virus associated with the international movement of horses. The extent of genomic variation demonstrated between most of the EAV isolates used in this study confirms the need for further investigation of genomic heterogeneity among strains of this virus before techniques that rely upon nucleic acid hybridization can be effectively applied as diagnostic procedures.

Contagious equine metritis.

Contagious equine metritis (CEM) is a highly contagious venereal infection of equids caused by Taylorella equigenitalis, a bacterium with fastidious growth requirements. A disease of major international concern, CEM can be the cause of short-term infertility and, very rarely, abortion in mares. Unlike the mare, stallions exposed to T. equigenitalis do not develop clinical signs of disease. CEM is transmitted by direct or indirect venereal contact. The carrier state occurs in the mare and the stallion and carrier animals are frequently the source of infection for new outbreaks of the disease. There are streptomycin-sensitive and -resistant biotypes of T. equigenitalis, and diagnosis is based primarily on culture of the bacterium from its predilection sites in the reproductive tract of the mare and the stallion. Treatment modalities are available for elimination of the carrier state. Prevention and control of CEM is achievable through a comprehensive programme of breeding farm management that includes early detection and treatment of carrier mares and stallions.

Equine influenza.

A highly contagious virus infection of horses, influenza is the single most important equine respiratory disease in many countries. Two subtypes of equine influenza virus have been identified, A/equine-1 and A/equine-2, neither of which immunologically cross-reacts. In the case of A/equine-2 virus, two lineages exist, American and European, which appear to have evolved independently of one another. The acute febrile respiratory disease characteristic of influenza is frequently complicated by secondary bacterial infection, especially in unvaccinated horses. Primarily a respiratory-borne infection, influenza has been spread to a significant number of countries through the international movement of horses. Strains of A/equine-2 virus have been responsible for all known outbreaks of the disease since 1980. Simple rapid procedures are now available for the diagnosis of equine influenza. Prevention and control of influenza is based on frequent use of inactivated, adjuvanted vaccines, which confer only incomplete and short-term protection against this disease. To be maximally effective, vaccines need to be periodically updated and include influenza virus strains closely related to those in current circulation.

Transmission studies with the contagious equine metritis bacterium in albino Swiss mice.

Aspects of experimental transmission of the causal bacterium of contagious equine metritis (CEM) to albino Swiss mice were investigated. Whereas infection was established in the majority of female mice, the organism was recovered from only a limited number of male mice after challenge. No clinical evidence of infection was observed in the experimental mice. There was only one instance of presumptive venereal transmission of the CEM bacterium. One third of infected females conceived and had normal litters.

Transmissibility of the contagious equine metritis organism for the cat.

A group of SPF cats were moderately susceptible to the causal organism of contagious equine metritis (CEM) following intra-uterine or intrapreputial challenge with an Irish streptomycin resistant strain isolated from a clinically infected mare. Subclinical infections were established in only 50% of the cats, none of which became long-term carriers of the organism. Cytological examination of vaginal smears was of no diagnostic value in confirming infection in inapparently infected cats. Bacteriological responses after primary or secondary challenge with the CEM organism were essentially similar, with one exception, a female cat in which there was possible evidence of local immunity persisting after the primary infection. Efforts to reactivate shedding subsequent to the immediate post-challenge period were unsuccessful. Throughout the experimental period, the cats remained sero-negative to the complement-fixation test, and they failed to develop any significant increase in the levels of antibody activity as measured by the kinetics-based ELISA or KELA system. On day 89 after primary challenge, the cats were euthanized and various sites in the genitourinary tract and the internal iliac lymphatic glands subjected to bacteriological and pathological examination for evidence of CEM infection with negative results. The findings of this study, although establishing the transmissibility of the CEM organism for the cat, demonstrate the limited value of this species as an experimental model system for the disease in the horse.

Serologic response of horses to the structural proteins of equine arteritis virus.

Equine arteritis virus (EAV) is the causative agent of equine viral arteritis, an apparently emerging disease of equids. In this study, the antibody response of horses to the structural proteins of EAV was evaluated using gradient-purified EAV virions and baculovirus-expressed recombinant EAV structural proteins (G(L), G(S), M, N) as antigens in a Western immunoblotting assay. Thirty-three sera from horses that previously had been naturally or experimentally infected with EAV were evaluated, including samples from mares, geldings, and both persistently and nonpersistently infected stallions. Sera also were evaluated from 4 horses that had been vaccinated with the commercial modified live EAV vaccine. The data suggest that the serologic response of individual horses to EAV may vary with the infecting virus strain and duration of infection. The M protein was most consistently recognized by the various serum samples, whereas the response to the N and G(L) proteins was variable and the G(S) protein was bound by only 1 serum sample. The immunoblotting assay definitively established the protein specificity of the humoral response of horses to EAV; however, it clearly is less sensitive than the standard serum neutralization (SN) test--2 of the 37 sera that were seropositive by the SN test failed to react in the immunoblot assay with any EAV structural protein. Furthermore, the G(L) protein expresses the known neutralization determinants of EAV, yet only 22 of the 37 sera that had SN antibodies bound the G(L) protein in the immunoblotting assay. Information from this study will assist ongoing efforts to develop improved methods for the serologic diagnosis of EAV infection of horses.

Variation in susceptibility of ten mouse strains to infection with a strain of Ehrlichia risticii.

Eight inbred strains of mice (A/J, AKR/NCrlBR, BALB/cAnNHsd, C3H/HeJ, C3H/HeNHsd, C57BL/6J, C57BL/10SnJ and CBA/J) and two outbred strains (CF1 and ICR) were inoculated with a strain of Ehrlichia risticii and their relative susceptibility to disease was determined. The strains varied widely in susceptibility, some showing profound illness, with illness being barely detectable in others. Severity of illness was uniform within strains. This study confirmed that the A/J and BALB/cAnNHsd strains were very susceptible to illness, but the C3H/HeJ, CBA/J and the CF1 strains were slightly less susceptible. The C57BL/6J, C57BL/10SnJ, C3H/HeNHsd, AKR/NCrlBR and ICR strains were resistant.

Clinical, virological and serological responses of donkeys to intranasal inoculation with the KY-84 strain of equine arteritis virus.

The clinical, virological and serological responses of seven female donkeys (Equus asinus) to inoculation with the KY-84 strain of equine arteritis virus (EAV), a strain that causes moderate to severe clinical signs in horses, was investigated. In the donkeys, the only clinical signs observed were fever (mainly 3-9 days after inoculation), mild depression in four animals, and a slight nasal or ocular discharge in three. All of the donkeys became infected with EAV as shown by recovery of the virus for periods of up to 14 days from the nasopharynx and buffy coat and, in three out of four donkeys sampled, from the cervix or vagina. Virus replication in the donkey appeared to mirror that previously described for the horse. The donkeys had "sero-converted" to EAV by the 10th day after inoculation. Additional studies are needed to obtain a better understanding of the pathogenesis of EAV in donkeys.

Variable persistence of the contagious equine metritis organism in the genital tract of CBA/J, CBA/N, LAF1/J, BALB/c and congenitally thymus-deficient (nude) mice.

Five inbred strains of mice, CBA/J, CBA/N, LAF1, BALB/c and congenitally thymus-deficient nude mice of BALB/c background, varied considerably in their susceptibility to the contagious equine metritis organism (CEMO). Whereas all the strains were virtually refractive to vaginal challenge, LAF1 and CBA/N mice were readily infected by intra-uterine inoculation. Based on infection rate and nature of the bacteriological response, CBA/N mice appeared the more susceptible of the 2 strains. Attempts to transmit CEMO to thymus-deficient nude mice were unsuccessful by both of these routes of challenge and by intraperitoneal inoculation, indicating that host resistance to the causal agent is independent of thymus-mediated immune phenomena. No clinical evidence of infection was observed in any of the experimentally infected mice. Although persistence of CEMO in the female reproductive tract varied widely, it could be isolated from some of the CBA/N mice for as long as 19 weeks after challenge by the intra-uterine route. The organism was cultured from the ovaries and/or oviducts of a high percentage of one group of CBA/N mice after 50 days, when it could no longer be recovered from the remainder of the genital tract. Limited attempts to achieve venereal transmission of CEMO between culture-positive female and companion male CBA/N mice were unsuccessful. The relative susceptibility of the CBA/N strain of mice to CEMO would suggest that host resistance to this infection is at least partly dependent on the presence of a fully functional B lymphocyte system. Further studies in this experimental model may elucidate some of the immunological mechanisms underlying development of resistance in the horse, more specifically as they relate to the occurrence of the carrier state in this disease.

Relationship between onset of puberty and establishment of persistent infection with equine arteritis virus in the experimentally infected colt.

The relationship between stage of reproductive tract maturity and susceptibility to the experimental establishment of persistent infection with equine arteritis virus (EAV) was investigated in 21 prepubertal and 15 peripubertal colts. Five of six prepubertal colts inoculated intranasally remained infected in the reproductive tract from post-challenge day 28 to 93 and two of six from post-challenge day 120 to 180. No virus was detected in five of these animals killed on post-challenge day 210. Each of two peripubertal colts remained infected in the reproductive tract at post-challenge day 60 and one of nine was found to be persistently infected with EAV 15 months after challenge. These findings confirm that the virus can replicate in the reproductive tract of a significant proportion of colts for a variable period of time after clinical recovery in the absence of circulating concentrations of testosterone equivalent to those found in sexually mature stallions. Long-term persistent infection with EAV does not appear to occur in colts exposed to the virus before the onset of peripubertal development. We suggest that colts should be vaccinated at approximately 6 months of age, before peripubertal development but after the disappearance of maternally acquired antibodies.

Pathological changes associated with equine arteritis virus infection of the reproductive tract in prepubertal and peripubertal colts.

The nature and extent of changes associated with equine arteritis virus (EAV) infection of the reproductive tract was documented in 21 prepubertal and 15 peripubertal colts. This study was part of an investigation into the relationship between stage of reproductive tract maturity and susceptibility to the experimental establishment of persistent infection with EAV. After intranasal challenge with a field isolate of EAV, all colts developed clinical signs of equine viral arteritis (EVA) from which they recovered rapidly. Clinical signs during the acute phase consisted of fever, serous to mucopurulent ocular and nasal discharge, oedema of the limbs, scrotum or prepuce, scleral injection, conjunctivitis, icterus, cough, diarrhoea, stiff gait, lethargy, inappetence and depression. At necropsy, the most significant macroscopic lesions included excessive accumulation of fluid within the thoracic and abdominal cavities, lymph node enlargement and oedema of the reproductive tract. Colts killed 7 to 14 days after challenge had acute necrotizing vasculitis involving the testes, epididymides, vasa deferentia, ampullae, prostatic lobes, vesicular glands and bulbourethral glands. Vasculitis was characterized by striking fibrinoid necrosis of small muscular arteries with extravasation of erythrocytes and proteinaceous material into the media, adventitia and perivascular tissues. Colts examined on days 28-180 had lymphocytic and plasmacytic inflammatory cell infiltrates in the lamina propria and muscularis of the epididymides and accessory sex glands. The vascular lesions found during the acute phase of EAV infection contrasted with the multifocal lympho-plasmacytic infiltrates found within the parenchyma of the reproductive tract during the chronic phase. One peripubertal colt was found to be persistently infected with EAV 15 months after challenge. This colt had marked lympho-plasmacytic infiltrates in the ampullae at necropsy.

Resistance of castrated male horses to attempted establishment of the carrier state with equine arteritis virus.

Twelve geldings all became infected when inoculated intranasally with the KY-84 strain of equine arteritis virus (EAV), a strain previously shown to be capable of establishing the carrier state in the stallion. With the exception of one animal that showed no effects other than pyrexia, all of the geldings developed clinical signs characteristic of equine viral arteritis (EVA). The geldings were febrile for varying periods within the range of 2-10 days after inoculation. Viraemia occurred from day 2 onwards, for periods varying from 9 to at least 19 days. Nasal shedding of virus began 2-4 days after inoculation and persisted for periods ranging from 7-14 days. All geldings "seroconverted" to EAV by day 11, with serum neutralization titres ranging from 8 to 64. The titres ranged from 8 to 32 after 4 weeks. Low concentrations of EAV were detected in the kidney and blood of one gelding killed 30 days after inoculation and in the blood of another killed after 57 days. Virus was not isolated from any tissue or fluid sample collected from the remaining 10 geldings, all of which were killed between days 30 and 148. The findings confirm that persistent EAV infection is unlikely to occur in geldings and support the results of previous studies, which demonstrated that testosterone plays an essential role in the establishment and maintenance of the carrier state.