Analysis of ORFs 2b, 3, 4, and partial ORF5 of sequential isolates of equine arteritis virus shows genetic variation following experimental infection of horses

Samples from horses experimentally infected with the “large plaque variant (LP3A+)” of equine arteritis virus were analysed. These included 182 nasal swabs collected from day 1 to 14 post-infection (p.i.), and 21 virus isolates obtained from white blood cells of animals that showed a prolonged viraemia between days 30 to 72 p.i. In order to determine the genetic stability of the virus and particularly to characterise the genetic variants found during the prolonged viraemia, partial sequences of open reading frame 5 (ORF5) encoding glycoprotein 5 (GP5) were generated. Viruses with amino acid substitutions in GP5 were used for further amplification and sequencing of a fragment encompassing ORFs 2b, 3, and 4. The ORF5 nucleotide sequences of the virus present in 65 out of 66 nasal swabs were identical to that of the inoculated virus, suggesting that the ORF5 gene of LP3A+ was genetically stable during the first 2 weeks p.i. Contrary to this, a number of mutations were found in the ORF5 of virus isolates obtained from day 30 p.i. The mutations mainly clustered in antigenic neutralization site C within variable region 1 of the GP5 ectodomain. Sequence variability was also identified in ORFs 2b, 3 and 4, with ORF 4 having the highest proportion of non-synonymous changes (4/6).     

马病毒性动脉炎诊断技术研究进展

马病毒性动脉炎(EVA)是马属动物之间通过呼吸道或生殖器官传播的传染病,是危害养马业及赛马比赛的重要疾病之一,应用各种诊断技术对该病做出准确的诊断是预防和控制该病的前提.EVA的诊断技术包括检测病毒,检测血清抗体,涉及的技术和方法包括病毒的分离及中和试验、血凝及血凝抑制试验、免疫荧光试验以及酶联免疫吸附试验等.每种诊断技术都有其各自的优势和局限性,应根据目的及工作条件等综合情况予以选择,论文就各种诊断技术的原理、方法及特点进行了概述和比较,并对今后的应用前景和发展趋势进行了展望.     

Evidence for absence of equine arteritis virus in the horse population of New Zealand

AIM: To summarise investigation and laboratory data collected between 2001 and 2011 to provide evidence that equine arteritis virus is not present in the horse population of New Zealand.

METHODS: Analysis was carried out on results from laboratory tests carried out at the Ministry for Primary Industries Animal Health Laboratory (AHL) for equine arteritis virus from horses tested prior to being imported or exported, testing of stallions as part of the New Zealand equine viral arteritis (EVA) control scheme and testing as part of transboundary animal disease (TAD) investigations for exclusion of EVA. Horse breeds were categorised as Thoroughbred, Standardbred or other.

RESULTS: A total of 7,157 EVA serological test records (from import and export testing, EVA control scheme testing and TAD investigations) were available for analysis between 2005 and 2011. For the three breed categories a seroprevalence of ≤1.6% at the 95% confidence level was determined for each category. Between 2001 and 2011, as part of the EVA control scheme, the EVA status of 465 stallions was determined to be negative. During 2005–2011 EVA was excluded from 84 TAD investigations.

CONCLUSIONS: There was no evidence of equine arteritis virus being present in the general horse population outside of carrier stallions managed under the EVA control scheme.

CLINICAL RELEVANCE: Equine arteritis virus is absent from the general horse population of New Zealand.     

马动脉炎病毒实时RT-PCR检测方法的建立

选取马动脉炎病毒基因组中高度保守的开放阅读框7序列,设计了特异性引物和TaqMan探针,利用一步法的实时定量RT-PCR来定量检测马动脉炎病毒.分别使用马动脉炎病毒的总RNA和含有选定检测序列的克隆标准品做扩增曲线,两曲线斜率之差小于0.1,证明两者的扩增效率相同,可用选定检测序列的克隆标准品对马动脉炎病毒进行定量检测.该方法敏感,快速,准确,且无交叉污染,能满足海关及检疫部门对马动脉炎病毒快速、准确的检测要求.     

马动脉炎病毒N蛋白基因GST融合表达载体的构建及表达

采用RT-PCR方法扩增出马动脉炎病毒核衣壳蛋白基因0RF7,并将其克隆到pMDl8-T载体,构建成重组质粒pMDl8-N,在上海生物工程公司测序。结果表明,所克隆的核衣壳蛋白基因序列与EAVNC-002532株的同源性为99％。表明0RF7是EAV基因组内的保守序列,将0RF7亚克隆到原核表达载体pGEX-6P-1中,构建成重组质粒pGEX-6P-N,用pGEX-6P-N转化表达菌株BL21（DE3）,诱导表达后SDS-PAGE和Westernblotting分析表明,克隆在谷胱苷肽硫转移酶（GST）下游的核衣壳蛋白基因与GST获得了高效融合表达,表达的融合蛋白GST-N分子质量约为40ku,为马动脉炎病毒病血清学诊断方法的建立奠定了基础。     

Interstate Equine Semen and Embryo Shipment Regulations in the United States and Their Implications on Control of Disease Transmission

Outbreaks of both equine viral arteritis and contagious equine metritis (CEM) in the United States have occurred in recent years. Shipped semen has been implicated in disease transmission and rapid spread. A survey was performed of state regulations regarding testing for equine arteritis virus (EAV) and CEM before interstate semen or embryo shipment. Results demonstrated lack of any requirements in 31 states. Four states had regulations regarding EAV; 17 required a Certificate of Veterinary Inspection (CVI) to accompany a semen or embryo shipment. Ten states required a negative equine infectious anemia test, primarily as a requirement of a CVI versus because of risk of dissemination of disease. No states required CEM testing. A comparison of state department of agriculture or veterinary medical association Web sites and direct communication with state veterinarians or their offices demonstrated contradictory information in six states. The lack of uniform regulations concerning CEM and EAV testing for shipped semen and embryos, and the threat they pose to the equine breeding industry and horse health, should be alarming to veterinarians and horse owners. Routine testing of animals before shipment of semen or embryos and veterinary involvement in collection and breeding activities are appropriate and necessary to help prevent future outbreaks and protect equine health.     

A study of the effects of Marek's disease in a broiler flock

In a flock of 17000 broilers, characteristic Marek's disease (MD) paralysis, accompanied by increased mortality occurred during the fourth week of the flock's life. Cytolytic lesions and atrophy in the bursae of Fabricius, and the severity of lymphocytic infiltration of nerves, livers, proventriculus and brain are suggestive of the involvement of MD virus of greater pathogenicity than has been encountered previously in New Zealand.     

Autosomic 27 Trisomy in a Standardbred Colt

A 19-month-old Standardbred colt was donated to the University of Pennsylvania School of Veterinary Medicine with a suspicion of intersexuality. The anal−genital distance and penis were normal, and there was no evidence of intersexuality, but the colt was bilaterally cryptorchid. Several aspects of the colt's behavior appeared unusual, including general temperament and behavior described as sympathetically dull and affable. With herd mates, the colt appeared slow to perceive or to learn the usual intraspecies social cues. An atypical gait characterized by intermittent unnatural shuffle of the hind limbs, sliding them along in short rhythmic strides for 3 to 10 seconds at a time was noted at times when a horse might normally transition from a slow walk to a fast walk or a slow trot. Occasionally the colt exhibited slight protrusion of the tongue through the teeth and lips with jaw movements and smacking of the tongue against the teeth as if struggling to retract the tongue to the normal position. Evaluation of the karyotype combined with fluorescent in situ hybridization (FISH) revealed an abnormal male karyotype showing trisomy of chromosome 27 (65, XY + 27). The colt was euthanized at 24 months of age, and a necropsy revealed no significant abnormalities. This case of trisomy was not associated with developmental abnormalities described in other rare reports of trisomy in horses; however, some features were strikingly similar to that of humans with trisomy 21. FISH was demonstrated to be an excellent method for correct identification of equine chromosomes.     

Equine Performance and Autonomic Nervous System Improvement After Joint Manipulation: A Case Study

A nine-year-old gelding quarter horse, whose discipline is barrel racing, was experiencing difficulty performing tight turns around the barrels for 8 months prior to treatment. He demonstrated tail swishing as if aggravated when under the saddle, which would escalate to bucking for 3 weeks prior to treatment. This gelding had no previous history of bucking under the saddle. Static and motion palpation findings indicated multiple segmental joint fixations located throughout the spine and extremities. High-velocity low-amplitude adjustments were performed to address the joint fixations found during examination. A comparison of pre and post-treatment thermographic images showed a temperature change indicative of autonomic nervous system improvement caused by joint manipulation. A follow-up at two weeks revealed subjective long term improvements. Subjective, objective, and thermographic evidence indicated that segmental joint dysfunction was causing increased nociception and autonomic dysregulation, most notably over the sacroiliac joints, lateral front left cannon bone and right carpus. Previous research has indicated causative effects of joint manipulation on the autonomic nervous system and nociceptive processes. This case shows the positive thermographic effects post-adjustment on the nervous system, and a two week follow-up indicated that the gelding no longer showed signs or symptoms of pain. This case demonstrates how joint manipulation can affect the autonomic and nociceptive nervous systems in the equine patient.     

Sarcocystis neurona-specific immunoglobulin G in the serum and cerebrospinal fluid of horses administered S neurona vaccine

A vaccine against Sarcocystis neurona, which induces equine protozoal myeloencephalitis (EPM), has received conditional licensure in the United States. A major concern is whether the immunoglobulin G (IgG) response elicited by the vaccine will compromise the use of Western blotting (WB) as a diagnostic tool in vaccinated horses with neurologic disease. Our goals were to determine if vaccination (1) causes seroconversion: (2) causes at least a transient increase in S neurona-specific IgG in the cerebrospinal fluid (CSF); and (3) induces an IgG response that can be differentiated from that induced by natural exposure. Horses included in the study (n = 29) were older than 6 months with no evidence of neurologic disease. The presence or absence of anti-S neurona antibodies in the serum of each horse was determined by WB analysis. Seropositive horses had CSF collected and submitted for cytology, CSF index, and WB analysis. The vaccine was administered to all the horses and boostered 3-4 weeks later. On day 14 after the 2nd administration, serum and CSF were collected and analyzed. Eighty-nine percent (8 of 9) of the initial seronegative horses seroconverted after vaccination, of which 57% (4 of 7) had anti-S neurona IgG in their CSE Eighty percent (16 of 20) of the seropositive horses had an increase in serum S neurona IgG after vaccination. Of the 6 of 20 horses that were initially seropositive/CSF negative, 2 were borderline positive for anti-S neurona IgG in the CSF, 2 tested positive, and 2 were excluded because the CSF sample had been contaminated by blood. There were no WB banding patterns that distinguished samples from horses that seroconverted due to vaccination versus natural exposure. Caution must be used in interpreting WB analysis from neurologic horses that have been recently vaccinated for EPM.     

Equine Viral Respiratory Pathogen Surveillance at Horse Shows and Sales

Equine respiratory viral infections cause significant worldwide disease and economic loss. Common causes include equine influenza virus (EIV) and equine herpesviruses-1 and -4 (EHV-1 and -4), and risk of exposure to these agents may be highest in young horses commingling at sales and competitive events. A surveillance study was conducted at two horse shows and two Thoroughbred sales to determine whether horses shed EHV-1, EHV-4, or EIV on arrival, or 2-4 days later, and whether shedding was associated with identifiable risk factors. Real-time polymerase chain reaction assays were used to detect EHV-1, EHV-4, and EIV nucleic acid in nasal swabs obtained from 369 horses at the four events. In response to evidence of clinical disease, 82 additional horses were sampled at two farms providing horses for one of the sales. On arrival at the events, shedding of EHV-1 was detected in 3.3%, EHV-4 in 1.1%, and EIV in 0.8% of horses. EHV-1 was detected at low levels, and EHV-1 and EHV-4 detection was not associated with clinical disease. EIV was detected only in horses at a Thoroughbred sale, in association with an outbreak of respiratory disease traced back to regional farms. On arrival at events, horses younger than 2 years had a significantly greater risk of shedding EHV-1 compared with older horses; no other significant risk factors associated with viral shedding were identified. Thus, there is a risk of exposure to EIV, EHV-1, and EHV-4 at equine events, and horses and events should be managed to mitigate this risk.     

Retrospective Review of Carriage Horse and Mule Welfare in Charleston,South Carolina (2009–2012)

Equine Welfare and Carriage Horse Companies have been recently highlighted as matters of public concern, although little data exists to describe working conditions, municipal regulations, and veterinary attention. In light of the current pandemic of unwanted horses in this country, it is prudent for the veterinary community to identify objective measurements of satisfactory equine welfare in the carriage horse industry to regulate and maintain working horses in sound conditions. Retrospective data from a carriage company in South Carolina was collected from 2009 to 2012. Variables included days in work, hours and/or tours worked, veterinary charges, management practices, and environmental conditions. City veterinary reports were also reviewed for all five local carriage companies. The average number of days in work per animal ranged from 163 to 188 per year. On average, each animal performed 4.6 hour long tours per working day, resulting in an average 865 tours per year. Each animal cost on average $2.00 per day in veterinary costs, and $0.84 of each animal's tour went toward veterinary bills. Welfare among carriage horses is a subject on which equine veterinarians should be well versed; not only to answer questions from clientele but also to promote good working and living conditions for these animals in an effort to stem the number of unwanted horses in America. The carriage company reported in detail here provides acceptable welfare for their animals. Prospective studies are needed to collect objective, evidence-based data regarding stress levels in the animals.