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.

Allvar Gullstrand and the slit lamp 1911.

The Swedish ophthalmologist and self-taught mathematician Allvar Gullstrand (1862-1930) invented the slit lamp to illuminate the anterior of the eye. With its rectangular beam of very bright light, he studied the structure of the cornea and the function of the lens. His dioptric investigations showed that, as well as the extracapsular mechanism described by Helmholtz, changes in the substance of the lens, that he termed intracapsular, also contribute to accommodation. However, his invention has been appropriated by clinical ophthalmologists and is now routinely used in examination of the eye.

Infection of embryos following insemination of donor mares with equine arteritis virus infective semen.

The objective was to evaluate the potential risks associated with embryo transfer from mares bred with equine arteritis virus (EAV) infective semen. Twenty-six mares were embryo donors, whereas 18 unvaccinated and EAV antibody seronegative mares were embryo recipients. Of the 26 donor mares, 15 were unvaccinated and seronegative for antibodies to EAV and 11 were vaccinated for the first time with a commercially available modified live virus vaccine against EVA before breeding and subsequent embryo transfer. All donor mares were bred with EAV-infective semen from a stallion persistently infected with the virus. Twenty-four embryos were recovered 7 d post-ovulation; all were subjected in sequential order to five washes in embryo flush medium, two trypsin treatments, and five additional washes in embryo flush medium (prior to transfer). Twelve and seven embryos (Grades 1 or 2) were transferred from the non-vaccinated and vaccinated donors, respectively, and pregnancy was established in 3 of 12 and 2 of 7. Perhaps trypsin reduced embryo viability and pregnancy rate. The uterine flush fluid of 11 mares (9 of 15 and 2 of 11 from non-vaccinated and vaccinated donor groups, respectively) was positive for EAV by VI (confirmed by real-time RT-PCR); the wash fluid from the embryos of nine of these mares was negative following 10 washes and two trypsin treatments. However, the embryo wash fluid from two mares was still positive for EAV after all 10 washes and the two trypsin treatments, and one embryo was positive for EAV. Two of 18 recipient mares had seroconverted to EAV 28 d after embryo transfer. Virus was not detected in any fetal tissues or fluids harvested after pregnancies were terminated (60 d). In conclusion, we inferred that the washing protocol of 10 washes and two trypsin treatments did not eliminate EAV from all embryos; due to limitations in experimental design, this requires confirmation. Furthermore, there may be a risk of EAV transmission associated with in vivo embryo transfer from a donor mare inseminated with EAV infective semen.

Horse species symposium: contagious equine metritis: an insidious threat to the horse breeding industry in the United States.

Contagious equine metritis (CEM) has given rise to international concern since it was first recognized as a novel venereal disease of equids in 1977 and the etiologic agent was identified as a previously undescribed bacterium, Taylorella equigenitalis. Horse industry concerns over CEM centered on the ease with which this bacterium could be disseminated, the significance of T. equigenitalis as a cause of short-term infertility in the mare, and the existence of the carrier state in the stallion and the mare. The first known outbreak of CEM in the United States was in Kentucky in 1978. The economic impact on the Thoroughbred industry in the state was substantial. Before 2008, additional small-scale outbreaks occurred in Missouri in 1979, Kentucky in 1982, and Wisconsin in 2006, nearly all attributed to the importation of carrier animals. On each occasion, appropriate measures were taken to eliminate the infection, resulting in the United States regaining its CEM-free status. With the exception of the 1978 occurrence in Kentucky, none of the subsequent outbreaks significantly affected the horse industry. That changed dramatically in 2008, however, after the discovery of a Quarter horse stallion in Kentucky that cultured positive. Subsequent investigations turned up 23 carrier stallions and 5 carrier mares belonging to 11 breeds and located in 8 states. Shipment of infective semen and indirect venereal contact in stallion collection centers through the use of contaminated fomites were major factors in the spread of T. equigenitalis. Trace-back investigations of some 1,005 exposed and carrier stallions and mares in 48 states have failed to identify the origin of this latest CEM event. Neither clinical evidence of CEM nor decreased pregnancy rates were reportedly a feature in infected or exposed mares. In light of these findings, there was some question of whether or not the considerable expense incurred in investigating the latest CEM occurrence was warranted. Regaining CEM-free status for the United States will present considerable challenges.

Initial occurrence of Taylorella asinigenitalis and its detection in nurse mares, a stallion and donkeys in Kentucky.

In 1998, a newly identified bacterium Taylorella asinigenitalis was isolated from the external genitalia and reproductive tracts of nurse mares, a stallion and donkey jacks in Kentucky. An extensive regulatory effort was implemented to contain the outbreak including the tracing and testing of 232 horses and donkeys on 58 premises. T. asinigenitalis was isolated from the reproductive tract of 10 adult equids, including two donkey jacks, one Paint Quarter-horse stallion and seven draft-type breeding mares. None of the infected horses had clinical signs of reproductive tract disease. The odds of being culture positive were 20 times greater for a mare bred to a donkey than for a mare bred to a stallion. Approximately 18% of mares bred to either a carrier stallion or donkey jack were confirmed culture positive. Seventy-one percent of infected mares required more than one course of treatment to clear the organism from their reproductive tracts and one mare harbored the organism for more than 300 days.

Douglas Argyll Robertson (1837-1909) and his pupil.

Douglas Argyll Robertson's (1837-1909) experimental work with physostigmine in 1863 sharpened his knowledge of the innervation of the internal muscles of the eye. So he was ideally prepared in 1869 to analyse the conundrum when he saw patients with spinal cord disease who had lost the response to light even though accommodation to near objects was normal. By translating his knowledge of basic science to a clinical problem he drew attention to this phenomenon, known subsequently as the Argyll Robertson pupil that came to be considered pathognomonic of tabes dorsalis, general paresis and neurovascular syphilis.

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.

Genetic variation and phylogenetic analysis of 22 French isolates of equine arteritis virus.

Genetic variation and phylogenetic relationships among 22 French isolates of equine arteritis virus (EAV) obtained over four breeding seasons (2001-2004) were determined by sequencing open reading frames (ORFs) 2a-7. The ORFs 2a-7 of 22 isolates differed from the prototype virulent Bucyrus strain of EAV by between 14 (99.5% identity) and 328 (88.7% identity) nucleotides, and differed from each other by between 0 (100% identity) and 346 (88.1% identity) nucleotides, confirming genetic diversity among EAV strains circulating in France. Phylogenetic analysis based on the partial ORF5 sequences (nucleotides 11296-11813) of 22 French isolates and 216 additional EAV strains available in GenBank clustered the global isolates of EAV into two distinct groups: North American and European. The latter could be further divided into two large subgroups: European subgroup 1 (EU-1) and European subgroup 2 (EU-2). Phylogenetic analysis based on 100 EAV ORF3 sequences yielded similar results. Of the 22 French EAV isolates, the 11 isolates obtained before January 28, 2003 clustered with either the EU-1 (9 isolates) or EU-2 (2 isolates) subgroup. In contrast, by the criteria used in this study, the 11 isolates obtained after January 30, 2003 belong to the North American group, strongly suggesting that these strains were recently introduced into France.

Lateral transmission of equine arteritis virus among Lipizzaner stallions in South Africa.

REASONS FOR PERFORMING STUDY: A serological study conducted in 1995 revealed that 7 stallions at the Lipizzaner Centre, Gauteng, South Africa, were seropositive for antibody to equine arteritis virus (EAV). A Lipizzaner stallion imported into South Africa from Yugoslavia in 1981 had previously (1988) been confirmed to be an EAV carrier. Despite being placed under life-long breeding quarantine, EAV had been transmitted between stallions at the Lipizzaner Centre. OBJECTIVES: To investigate the phylogenetic relationships between the strain of EAV shed in the semen of the original carrier stallion and strains recovered from the semen of 5 other stallions; and to investigate the means whereby lateral transmission of EAV occurred among 7 in-contact, nonbreeding stallions at the Centre. METHODS: EAV was isolated from semen collected from the seropositive stallions using RK-13 cells. Viral RNA was reverse transcribed and amplified by polymerase chain reaction using ORF 5-specific primers, subjected to sequence and phylogenetic analysis. RESULTS: Phylogenetic analysis of strains of EAV recovered from the semen of 6 persistently infected stallions confirmed that all viruses were closely related and probably derived from a common ancestor, i.e. the stallion imported from Yugoslavia. Lateral transmission subsequently occurred among 7 in-contact, nonbreeding stallions at the Centre. It is speculated that these stallions may have been exposed to virus from bedding or fomites contaminated with semen. CONCLUSIONS: These data confirm that lateral transmission of EAV can occur from shedding stallions to susceptible, in-contact horses, including other stallions, which may become persistently infected with the virus. POTENTIAL RELEVANCE: The findings are consistent with lateral spread of a single, unique strain of EAV among a group; and suggest that transmission of EAV may be initiated by infection of one or more stallions with virus on bedding or other fomites contaminated with EAV- infected semen.

Prevalência de anticorpos antivírus da arterite dos eqüinos em cavalos criados no Estado de Säo Paulo / Prevalence of equine arteritis virus specific antibodies in horses of Säo Paulo State, Brazil

Utilizou-se a prova de soroneutralização em microplacas para detecção de anticorpos antivírus da arterite dos eqüinos em 659 amostras de soro sangüíneo de animais criados no Estado de São Paulo. A prevalência de anticorpos na população estudada foi igual a 18,2 por cento. A raça Mangalarga foi a que apresentou maior taxa de prevalência, 33,3 por cento. Animais na faixa etária de 6 a 24 meses de idade apresentaram a maior taxa de prevalência, 30,4 por cento, e as fêmeas apresentaram prevalência de 22, 9 por cento, mais alta do que nos machos

With the purpose of studying the prevalence of equine viral arteritis in horses raised in São Paulo State, Brazil, by the standard microtiter serum neutralization test, 659 serum samples were investigated. The prevalence of antibodies in the horse population was 18.2 per cent, which was significantly higher in Mangalarga horses (33.3 per cent) than in any other breed (Thoroughbred, Arab, Quarter Horse, mixed breeds and others). The distribuition of horses by age showed that horses between 6 to 24 months of age (30.4 per cent) had a higher prevalence (30.4 per cent) rate than others. The female horses prevalence rate of 22.9 per cent was significantly higher than in male horses.

Taylorella asinigenitalis sp. nov., a bacterium isolated from the genital tract of male donkeys (Equus asinus).

Three bacterial isolates that were phenotypically indistinguishable from Taylorella equigenitalis were obtained from the urethral fossae of three male donkeys (Equus asinus), one located in the state of California and the other two in the state of Kentucky, USA. Based on results of pulsed-field gel electrophoresis, the isolate from California differed from the two Kentucky isolates, which were the same. Mares bred artificially (California) or naturally (Kentucky) did not show signs of disease, even though infection with the organism was established in those bred naturally. Mares and, uncharacteristically, all three jacks produced antibodies that reacted in the complement fixation test utilized to identify mares recently infected with T. equigenitalis. Sequence analysis of DNA encoding the 16S rRNA revealed that the gene sequences of these isolates were virtually identical to each other (>99.8% similarity), but different (97.6% similarity) from those of several confirmed isolates of T. equigenitalis. The 16S rDNA sequences of the latter were 100% identical. DNA-DNA hybridization studies revealed a mean hybridization level of 89% between the donkey isolate from California and the donkey isolate from Kentucky. On the other hand, the mean DNA-DNA hybridization level from the donkey isolates with DNA from a strain of T. equigenitalis was 23%. The DNA G+C composition was 37.8 mol% for the two donkey isolates, as well as the strain of T. equigenitalis used in the hybridization studies. These data support our opinion that micro-organisms isolated from the male donkeys are different from T. equigenitalis and it is proposed that they be considered a new species within the genus Taylorella and named Taylorella asinigenitalis sp. nov. The type strain is strain UCD-1T (= ATCC 700933T = LMG 19572T).

Fatal nonneurological EHV-1 infection in a yearling filly.

A case of fatal nonneurological equine herpesvirus 1 (EHV-1) infection in a yearling filly is described. Gross lesions included extensive pulmonary edema, prominent laryngeal lymphoid follicles, and congestion and edema of the dorsal third ventricle choroid plexus. Histologically, there was vasculitis, hemorrhage, and edema in the lungs and dorsal third ventricle choroid plexus as well as mild intestinal crypt necrosis with occasional intranuclear inclusion bodies. The perivascular and vascular inflammatory infiltrates were comprised mainly of T lymphocytes and macrophages. EHV-1 antigen was identified within the nucleus and cytoplasm of endothelial cells, dendritic-like cells of the pharyngeal lymphoid follicles, pharyngeal glandular epithelium, crypt enterocytes, and monocytes. Attempted virus isolation was negative. Weak seroconversion for EHV-1 was observed. Herpesvirus-like particles were identified within pharyngeal endothelial cells by transmission electron microscopy. Polymerase chain reaction amplified 369 and 188 base-pair fragments specific for EHV-1. The scarcity of pathognomonic viral inclusions and lesions in this case suggests that this disease may not be recognized, particularly in situations when ancillary laboratory procedures are limited.

Ataxia and paresis with equine herpesvirus type 1 infection in a herd of riding school horses.

An outbreak of neurologic disease associated with serologic evidence of equine herpesvirus type 1 (EHV-1) infection occurred in a herd of 46 riding school horses. Ataxia and paresis were observed in 14 geldings and 5 barren mares. Eight affected horses had distal limb edema, 1 horse had a head tilt, and 3 others had urinary incontinence. Other clinical signs included fever, depression, and inappetance in 30 horses. Seven horses with neurologic signs were treated with acyclovir. Serum neutralizing antibody titers against EHV-1 increased 4-fold between acute and convalescent samples or exceeded 1:256 in 19 of 44 horses, confirming recent infection. A significantly greater proportion of horses that seroconverted were mares (P = .014). Of the 19 horses exhibiting ataxia and paresis, 17 made a complete recovery, 1 made a partial recovery, and 1 was euthanized.

Detection of antibodies to equine arteritis virus by a monoclonal antibody-based blocking ELISA.

A potent ELISA antigen was prepared from equine arteritis virus (EAV) by differential centrifugation of EAV-infected cell culture fluid, followed by solubilization of the preparation by Triton X-100 treatment. Using this antigen and a mouse monoclonal antibody against the G(L) protein of EAV, a reliable blocking ELISA (bELISA) was developed for the detection of EAV antibodies in equine sera. The bELISA was evaluated using a total of 837 test serum samples. The relative sensitivity (n = 320) of the bELISA compared to the serum neutralization (SN) test was 99.4%. The bELISA appears to be a highly specific test, the specificity of which did not appear to be adversely affected by previous exposure of horses to non-EAV-containing biologicals. Of 119 serum samples, 21 from horses without any history of exposure to EAV and 98 from racetrack Thoroughbreds, 118 were negative in the SN test and bELISA. One sample was SN-negative but suspicious with the bELISA. Based on testing 465 SN-negative field samples and 52 SN-negative samples from experimental horses, and excluding any sera giving a suspicious reaction, the relative specificity of the bELISA was 97.7%. Samples should be examined undiluted and diluted 1/10 in the bELISA because the testing of sera of high neutralizing antibody titer may be affected by a prozone-like phenomenon. The bELISA is a more rapid and cost-efficient test than the SN test for the detection of EAV antibodies in equine sera.

Factors influencing the international spread of equine diseases.

In an era of increasing globalization, the risk of spread of infectious diseases in humans and animals, including equids, has never been greater. International movement of equids and trade in semen are the most important factors responsible for the dissemination of various equine pathogens. Other factors that can or do have the potential to influence the global distribution of equine infectious diseases include: multinational trade agreements, emergent diseases, mutation of pathogens, climate related phenomena, migration of amplifying/reservoir hosts or vectors, availability of new vectors, vaccine contamination and agroterrorism. The relative importance of each of these factors is considered in relation to the spread of equine diseases.

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.