Equine herpesvirus-1 myeloencephalopathy: a review of recent developments

Equine herpes myeloencephalopathy (EHM), although a relatively uncommon manifestation of equine herpesvirus-1 (EHV-1) infection, can cause devastating losses on individual farms or boarding stables. Although outbreaks of EHM have been recognized for centuries in domestic horse populations, many aspects of this disease remained poorly characterized. In recent years, an improved understanding of EHM has emerged from experimental studies and from data collected during field outbreaks at riding schools, racetracks and veterinary hospitals throughout North America and Europe. These outbreaks have highlighted the contagious nature of EHV-1 and have prompted a re-evaluation of diagnostic procedures, treatment modalities, preventative measures and biosecurity protocols for the disease. This review concentrates on these and other selected, clinically relevant aspects of EHM.     

The first reported outbreak of equine herpesvirus myeloencephalopathy in New Zealand

CASE HISTORY AND CLINICAL FINDINGS: On 9 January 2014 (Day 0) a mare from a stud farm in the Waikato region presented with urinary incontinence without pyrexia. Over the following 33 days 15 mares were clinically affected with neurological signs. All but one mare had a foal at foot. The most commonly observed clinical signs were hind limb paresis and ataxia. In some cases recumbency occurred very early in the course of disease and seven mares were subject to euthanasia for humane reasons.

LABORATORY FINDINGS: Equid herpesvirus (EHV) type 1 was detected using PCR in various tissues collected post mortem from two mares with neurological signs. DNA sequencing data from the DNA polymerase gene of the virus showed a nucleotide transition at position 2254, a mutation encoding amino acid D₇₅₂ that is highly associated with the neuropathogenic genotype of EHV-1. In total 12/15 mares were confirmed positive for EHV-1 on PCR. Results from a virus neutralisation test and ELISA on paired serum samples, and PCR on whole blood and nasal swabs, indicated that of four paddocks in a high-risk area where a cluster of cases had occurred, 20/21 (95%) horses were likely to have been exposed or were confirmed infected with EHV-1. Subsequent to the outbreak two mares aborted, one at 9 months and one at 10 months of gestation. The cause of abortion was confirmed as EHV-1 with the same genotype as that involved in the outbreak.

DIAGNOSIS: Equine herpesvirus myeloencephalopathy.

CLINICAL RELEVANCE: The outbreak described shows the considerable impact that can occur in outbreaks of equine herpesvirus myeloencephalopathy in New Zealand. Early biosecurity controls not only reduced the effect on the farm but mitigated the potential for the virus to spread to other horse enterprises.     

马冠状病毒检测试剂盒的研制

参考GenBank发表的马冠状病毒的基因序列,设计合成一对引物,对马冠状病毒进行RT-PCR扩增,产物经琼脂糖电泳分析,呈现一条约427bp的条带,并进行序列测定,与GenBank中发表的基因序列比较发现,与马冠状病毒核苷酸的同源性为98.7%,证实为马冠状病毒基因。通过对RT-PCR反应条件进行优化,研制了用RT-PCR方法检测马冠状病毒的试剂盒,该试剂盒的敏感性和特异性都较好。     

Equine Protozoal Myeloencephalitis: An Updated Consensus Statement with a Focus on Parasite Biology,Diagnosis, Treatment,and Prevention

Equine protozoal myeloencephalitis (EPM) remains an important neurologic disease of horses. There are no pathognomonic clinical signs for the disease. Affected horses can have focal or multifocal central nervous system (CNS) disease. EPM can be difficult to diagnose antemortem. It is caused by either of 2 parasites, Sarcocystis neurona and Neospora hughesi, with much less known about N. hughesi. Although risk factors such as transport stress and breed and age correlations have been identified, biologic factors such as genetic predispositions of individual animals, and parasite‐specific factors such as strain differences in virulence, remain largely undetermined. This consensus statement update presents current published knowledge of the parasite biology, host immune response, disease pathogenesis, epidemiology, and risk factors. Importantly, the statement provides recommendations for EPM diagnosis, treatment, and prevention.     

Frequency of latent equine herpesvirus type-1 infection among a sample of horses in the central North Island of New Zealand

ABSTRACT

Aim: To estimate the frequency of infection with equine herpesvirus type-1 (EHV-1) among horses from the central North Island of New Zealand, including the frequency of detection of the D₇₅₂ genotype.

Methods: Samples of retropharyngeal lymph nodes (RLN) and submandibular lymph nodes (SLN) were dissected from the heads of 63 horses that were humanely killed for various unrelated reasons between March and November 2015. DNA extracted from these tissues was subjected to enrichment for EHV-1 sequences by hybridisation with biotin-labelled EHV-1 specific probe, followed by recovery of EHV-1 sequences on streptavidin-coated magnetic beads. Enriched samples were tested for the presence of EHV-1 using nested quantitative real-time PCR. The EHV-1 amplicons were sequenced to determine the genotype of the virus.

Results: The median age of the horses was 6 (min 2, max 30) years, and 47/63 (75%) were Thoroughbreds. EHV-1 DNA was detected in RLN samples from 6/63 (10%) horses, and three of these horses were also positive for EHV-1 DNA in SLN. The remaining horses were negative for EHV-1 DNA in both RLN and SLN samples. The N₇₅₂ genotype was detected in all positive samples and the D₇₅₂ genotype was not detected in any of the samples.

Conclusions: EHV-1 continues to circulate among horses in New Zealand. The frequency of latent EHV-1 infection among sampled horses may have been underestimated due to the sensitivity limit of the assay or because of the limited anatomical sites sampled in the study. Lack of detection of the D₇₅₂ genotype suggests that infection with this genotype is not common in horses in New Zealand.

Clinical Relevance: If live animals are tested for EHV-1 using SLN biopsy it should be kept in mind that negative results do not rule out the presence of latent EHV-1 infection at other sites inaccessible for testing. The RLN appear to be the preferred sample for detection of EHV-1 DNA in horses following recent euthanasia.     

爱滋病毒抗原(P_(24))检测马传染性贫血病毒的研究

本研究通过基因工程技术生产了爱滋病毒的Ｐ２４抗原,并且运用于马传贫病毒的血清学检查,与传统方法所制备的马传贫抗原对照检测军马２５２４匹以及本实验室保留的各种马传贫阳性血清２２４份。结果显示马传贫血清能够免疫沉淀爱滋病毒（ＨＩＶ）的Ｐ２４,表明它们之间有着共同的抗原决定簇,且Ｐ２４检测法检测率高,检测周期仅３～４ｈ,明显优于传统法,可用于马传贫的快迅检测。     

Platelets in equine recurrent airway obstruction

Platelets contribute to the pathogenesis of human allergic airway disease. The aim of this study was to compare platelet activating factor (PAF)-induced platelet aggregation and thromboxane (Tx) production, plasma Tx and 5-hydroxytryptamine (5-HT) in ponies with recurrent airway obstruction (RAO), an hypersensitivity to inhaled antigens, and normal ponies, before and after antigen exposure. Plasma 5-HT was significantly higher in ponies with RAO but was not further increased by antigen challenge. There was no difference between PAF-induced platelet aggregation or Tx production, or in plasma Tx before or after challenge. These data suggest there may be a difference between platelet 5-HT uptake in RAO and normal ponies but do not provide evidence of platelet activation following antigen exposure.     

Abnormal synaptic protein expression in two Arabian horses with equine degenerative myeloencephalopathy

Numerous swollen neurons and multiple dystrophic axons were observed in the gracillis and cuneatus nuclei of two male Arabian horses, aged six and 12 months of age, with equine degenerative myeloencephalopathy. Swollen neurons and dystrophic axons showed synaptophysin, synaptosomal-associated protein of 25 kDa, syntaxin-1 and alpha-synuclein immunoreactivity. Moreover, dystrophic axons were strongly immunopositive against the ubiquitin protein and against the anti-phosphorylated 200 kDa neurofilament protein. Abnormal expression of integral synaptic vesicle, synaptic vesicle-associated presynaptic plasma membrane and cytosolic proteins, which participate in the trafficking, docking and fusion of the synaptic vesicle to the plasma membrane, suggest that severe disruption of axonal transport plays a crucial role in the pathogenesis of dystrophic axons in equine degenerative myeloencephalopathy (EDM).     

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.     

马疱疹病毒1型感染马疾病模型的建立及评价

试验旨在建立马疱疹病毒1型（Equine herpesvirus type1,EHV-1）人工发病模型,确定EHV-1感染马的半数感染量（ID₅₀）及感染发病的判定标准,为该病的预防与治疗药物的研发奠定基础。以新疆伊犁地区某发病马场流产胎儿中分离的EHV-1 XJ2015株为研究对象,设立4组不同病毒剂量感染组及对照组,经鼻内喷雾感染马,5 mL/匹,每天观察试验马的临床症状和发病情况,14 d后进行剖检,观察各组织脏器病理变化并应用实时荧光定量PCR方法检测鼻腔排毒及病毒分布情况。结果显示,EHV-1 XJ2015株感染马的ID₅₀为10^-6.67/5 mL,其病毒含量为10^4.33 TCID₅₀/mL。与对照组相比,1×10⁶和1×10⁵ TCID₅₀/mL感染组马临床评分显著升高,主要表现为体温升高（高达39.5 ℃,一般持续2~6 d）、食欲不振、流浆液性鼻液和下颌淋巴结肿大;且1×10⁶和1×10⁵ TCID₅₀/mL感染组试验马均表现出不同程度的排毒,肺脏及脑组织中可检测出大量病毒,与对照组相比极显著或显著升高（P<0.01;P<0.05）;病理学检查发现,患马脑组织出现非化脓性脑炎及神经元水肿,肺脏组织出现间质性肺炎、嗜中性粒细胞、炎性细胞浸润、出血和肺泡间隔增厚。以上结果表明,EHV-1 XJ2015株对马具有较强的致病性,患病马临床症状典型,病毒主要随鼻液排出,并富集在肺脏及脑组织,通过上述指标确定EHV-1感染马发病的判定标准,本试验成功建立EHV-1感染本体动物疾病模型。     

Results of Quantitative Cultures of Urine by Free Catch and Catheterization from Healthy Adult Horses

Quantitative urine cultures were performed on 11 male and 11 female healthy adult horses. Urine was collected by free catch and catheterization using standard methods. Results showed that all samples collected by free catch contained less than 20,000 colony-forming units (CFU)/mL. All samples collected by catheterization contained 500 CFU/mL or less. A significant difference was found between collection methods ( P < .005), with catheterization having less contamination. In samples collected by free catch, females had significantly greater contamination than did males ( P < .03). Predominant bacterial species isolated included Streptococcus spp., Escherichia coli, Enterohacter sp., Bacillus sp., Staphylococcus spp., Diptheroids sp., Proteus spp., and Enterococcus sp. Many samples contained multiple bacterial species. Bacterial isolates were representative of the normal bacterial flora of the equine urogenital tract. This paper establishes reference values for quantitative urine culture results in healthy adult horses to aid in the diagnosis of urinary tract infections.     

Cytotoxic Effects of Serum from Equine Grass Sickness Cases on Neuro-2a and PC12 Tet-Off Cell Lines: Implication for Using In Vitro Methods as Antemortem Diagnostic Tools

A wide variety of clinical and paraclinical methods have been used for diagnosis of equine grass sickness (EGS), but none of them could absolutely confirm the diagnosis, and postmortem pathologic examination is still considered the final step in precise diagnosis of EGS. Use of in vitro cell toxicity caused by EGS serum on neuronal cell lines was investigated. Three well-known cytotoxic methods were used to investigate the cytotoxicity of EGS serum on neuro-2a and genetically engineered PC12 Tet-Off P53 cells. The results of alamar blue reduction as an index for mitochondrial activities and intracellular adenosine triphosphate content assays, but not neutral red uptake, indicate that the EGS serum may affect the mitochondrial function and cellular metabolism at up to 60% of the cases. The results of present study might be used for diagnosis of EGS cases. Further studies with high sample size may lead us to uncover the pathogenesis of EGS and to increase the sensitivity and applicability of in vitro techniques as diagnostic tools.