马动脉炎病毒Eva Green荧光定量RT-PCR检测方法的建立及初步应用

为建立马动脉炎病毒(EAV)的快速检测方法,本研究设计了一对针对EAV Bucyrus株ORF7基因序列的特异性引物,建立了检测EAV的Eva Green荧光定量RT-PCR (qRT-PCR)方法.结果表明,该方法在102拷贝/μL～107拷贝/μL范围内具有良好的线性关系;最低检出量为1015 TCID50/mL病毒,敏感性为常规RT-PCR的100倍,与其他马常见病毒无交叉反应;组内及组间重复性试验的变异系数均小于2％,具有较好的重复性.此外,采用该方法测定了EAV Bucyrus株及其拯救病毒icEAV感染RK-13细胞后的复制动态,并与TCID50方法进行了比较.结果显示icEAV与其亲本病毒接种RK-13细胞后,0h测定病毒TCID50及拷贝数分别为103.33TCID50/mL(1 04 65拷贝/2μL)和10350 TCID50/mL(104 70拷贝/2μL);60 h时,细胞病变达到100％,icEAV及其亲本病毒的TCID50及拷贝数分别为105.67TCID50/mL(107 0拷贝/2μL)和105.57 TCID50/mL(106 98拷贝/2μL),两株病毒在RK-13细胞内的复制效率相似.本研究建立的Eva Green qRT-PCR方法可以为细胞培养物中EAV的检测提供有效的技术手段.     

Management,health, and abnormal behaviors of horses: A survey in small equestrian centers in Brazil

Care conditions of horses housed in small equestrian establishments and the occurrence of health and behavioral problems were assessed in 197 animals. Based on questionnaires and direct observations of the animals, it was noted that 8% of all animals were fed exclusively on pasture, whereas 92% received commercial concentrate or grain with limited or no grass. Sixty percent of the horses spent 20-24 hours/day in the stalls, 30% spent 12 hours/day, and 10% were kept outdoors. Eighty five percent exercised regularly, whereas 15% did not exercise at all. Wounds (18%), colic (6%), and strangles (3%) were the most cited diseases in the interviews. Fifty-six percent of the horses showed behavioral problems; the frequency and type of abnormal behavior varied according to horse use (P < 0.001). The management of the horses presented numerous concerns regarding feeding and housing, probably leading to increased behavioral problems. The results presented here would be a useful tool for the education of owners and caretakers.     

Study on Inducing Equine Bone Marrow Mesenchymal Stem Cells Differentiated into Chondrocytes

This study was aimed to establish equine bone marrow mesenchymal stem cells(BMSCs) line and induce it to differentiate into chondrocytes. The BMSCs were collected by cutting the bone and flushing the cutting surface by PBS, and then the bone marrow was washed with PBS,the collected cells were cultured after centrifugation. The cells were purified by passaging,the stem cell properties were tested before the induction. And the cells were also appraised by the expression of the special gene of chondrocytes as well as staining the differentiated cells with Alcian blue to insure the induction was effective. The obtained BMSCs expressed Sox2 and Nanog genes, which were the stem cell special genes, and also expressed CD44, CD90 and CD105 genes, but absent of CD34 and CD45 genes. The shapes of the 3rd passage BMSCs were changed after cultured in inducing medium for a few days. Furthermore, the cells were positive to Alcian blue staining, increased expression of the Col special gene of chondrocyte day by day as well. According to this study, the BMSC line was established, and the BMSCs were induced and differentiate into chondrocytes successfully.     

马A3Z3V1抑制马传染性贫血病毒早晚期反转录的鉴定

马A3Z3V1是宿主细胞内的一种APOBEC3免疫因子,它可以有效抑制马传染性贫血病毒(EIAV)在马宿主细胞内的复制.为鉴定马A3Z3V1分子对EIAV早晚期反转录的抑制作用,本实验根据EIAV的R/U5和gag基因序列分别设计了检测EIAV早晚期反转录产物的引物并建立了SYBR Green Ⅰ荧光定量PCR方法,利用该方法评价免疫因子A3Z3V1对EIAV逆转录过程的影响.结果显示,在早期反转录过程中,A3Z3V1可以使EIAV的反转录产物最高减少7倍;在晚期反转录过程中,A3Z3V1可以使EIAV的反转录产物最高减少3倍.本实验结果将有助于进一步研究APOBEC3分子的抗病毒作用机制.     

马传染性贫血病毒在慢性感染体内的抗原变异

应用在日本发离的马传染性贫血病毒（ＥＩＡＶ）强毒株Ｐ３３７－Ｖ７０实验感染了１匹健康马，经２次感染后，这匹实验感染并未呈现临床症状。从这匹马的外周和肝组织获得了覆盖马传染性贫血病毒ｇｐ９０基因主要中和抗原功能区（ＰＮＤ）的前病毒序列。通过对这些序列的比较分析发现，一些序列在ＰＮＤ功能区含有核苷酸的插入变异。     

Viruses associated with outbreaks of equine respiratory disease in New Zealand

AIM: To identify viruses associated with respiratory disease in young horses in New Zealand.

METHODS: Nasal swabs and blood samples were collected from 45 foals or horses from five separate outbreaks of respiratory disease that occurred in New Zealand in 1996, and from 37 yearlings at the time of the annual yearling sales in January that same year. Virus isolation from nasal swabs and peripheral blood leukocytes (PBL) was undertaken and serum samples were tested for antibodies against equine herpesviruses (EHV-1, EHV-2, EHV-4 and EHV-5), equine rhinitis-A virus (ERAV), equine rhinitis-B virus (ERBV), equine adenovirus 1 (EAdV-1), equine arteritis virus (EAV), reovirus 3 and parainfluenza virus type 3 (PIV3).

RESULTS: Viruses were isolated from 24/94 (26%) nasal swab samples and from 77/80 (96%) PBL samples collected from both healthy horses and horses showing clinical signs of respiratory disease. All isolates were identified as EHV-2, EHV-4, EHV-5 or untyped EHV. Of the horses and foals tested, 59/82 (72%) were positive for EHV-1 and/or EHV-4 serum neutralising (SN) antibody on at least one sampling occasion, 52/82 (63%) for EHV-1-specific antibody tested by enzyme-linked immunosorbent assay (ELISA), 10/80 (13%) for ERAV SN antibody, 60/80 (75%) for ERBV SN antibody, and 42/80 (53%) for haemagglutination inhibition (HI) antibody to EAdV-1. None of the 64 serum samples tested were positive for antibodies to EAV, reovirus 3 or PIV3. Evidence of infection with all viruses tested was detected in both healthy horses and in horses showing clinical signs of respiratory disease. Recent EHV-2 infection was associated with the development of signs of respiratory disease among yearlings [relative risk (RR)=2.67, 95% CI=1.59-4.47, p=0.017].

CONCLUSIONS: Of the equine respiratory viruses detected in horses in New Zealand during this study, EHV-2 was most likely to be associated with respiratory disease. However, factors other than viral infection are probably important in the development of clinical signs of disease.     

Effect of training location and time period on racehorse performance in New Zealand. 1. Descriptive analysis

AIM: To describe characteristics of Thoroughbred training stables in Matamata and in all other locations in New Zealand combined, over two 19-month time periods in 1996–1997 and 1998–1999, representing equal length periods immediately prior to and after the construction of a new training surface at the Matamata Racing Club.

METHODS: Retrospective records covering all horses training and racing in New Zealand during two 19-month time periods (1996–1997 and 1998–1999), covering 161 locations, were obtained from New Zealand Thoroughbred Racing (NZTR). Outcome variables included whether a horse was raced again in the 6 months following any start in the first 13 months of either time period, number of race starts for every horse, and finishing position. Summary measures with confidence intervals (CI) and unadjusted odds ratios (OR), measuring strength of associations for various factors, were computed.

RESULTS: The datasets contained information on 45,446 horses, 11,336 races, 5,110 trials and a total of 110,643 race starts. Horses trained at Matamata represented 8% (3,715) of the total horse datasets, and accounted for 11,977 race starts (10.8%). They were more likely to start in a race or trial in either time period and were 1.4 and 1.3 times as likely to finish first, second or third compared with horses trained at other locations in 1996–1997 and 1998–1999, respectively. A 6-month no-race period occurred for 9,306/12,584 (74%) horses that started at least once in the first 13 months of either time period. Horses trained at Matamata were less likely to have a 6-month no-race period than horses trained at other locations in both time periods. There was no effect of time period within each location on the probability of either a horse having a 6-month no-race period or of a race start being followed by a 6-month no-race period, but there was an overall effect of time and more 6-month no-race periods were observed in 1998–1999 relative to 1996–1997.

CONCLUSION: Summary statistics are presented for Thoroughbred racing in New Zealand over two 19-month time periods. Differences between the populations of horses trained in Matamata compared with those trained at other locations were attributed, in part, to the fact that many of the more successful racehorse trainers in the country have stables at Matamata. As a result, the population of horses in Matamata may not be representative of the racehorse population in New Zealand. Although more likely to win or place in both time periods, the magnitude of the advantage to horses in Matamata was reduced in 1998–1999 relative to 1996–1997, and this could be due, in part, to effects of the new track surface at Matamata. There was no evidence of a rise in risk of a 6-month no-race period following any race start in those horses trained in Matamata in 1998–1999 relative to either horses trained at other locations or to horses trained in Matamata during the earlier time period.     

Risk factors for injury to the superficial digital flexor tendon and suspensory apparatus in Thoroughbred racehorses in New Zealand

AIM: To investigate risk factors for injury to the superficial digital flexor tendon (SDFT) and suspensory apparatus (SA) of the forelimbs in Thoroughbred racehorses in New Zealand.

METHODS: Poisson and negative binomial regression, with exposure time represented by cumulative training days for each horse, were used to relate explanatory variables to the incidence rate (IR) of cases of inflammation of the SDFT (n=51), and injuries involving the SA (n=48) in a population of 1,571 commercially-trained racehorses over 554,745 study days. Only the first occurrence of an injury for any one horse was eligible for inclusion. Separate analyses were run for data from horses in training regardless of whether they had started in a trial or race, and using a subset of these data restricted to those preparations associated with at least one start in a trial or race. Results were reported as incidence rate ratios (IRR) and 95% confidence intervals (CI).

RESULTS: Male horses had a higher risk of injury to the SA (IRR 2.57; p=0.005) and tended to have a higher risk of injury to the SDFT (IRR 1.74; p=0.09) than female horses. Increasing age was associated with increased risk of injury. Horses aged 4 and ≥5 years were 6.76 (p<0.001) and 15.26 (p<0.001) times more likely to incur injury to the SDFT, and 2.91 (p=0.02) and 3.54 (p=0.005) times more likely to incur injury to the SA, respectively, than 2-year-olds. Horses were more likely to suffer an injury to the SDFT or SA in a training preparation that was not associated with any starts in official trials or races compared with those preparations that were associated with more than one start (p<0.001), and more likely to injure the SA compared with preparations containing one start (p=0.03). The IR of injury to the SDFT tended to be lower between November–January (IRR 0.78; p=0.08) and February-April (IRR 0.75; p=0.08) compared with August–October. Incidence of injury to the SDFT or SA was not associated with the cumulative distance raced in the last 30 days of a training preparation.

CONCLUSION: This study identified risk factors for injury to the SDFT and SA in Thoroughbred racehorses in New Zealand. Injuries were more likely in males, older horses and in horses in training preparations without any starts. There was no evidence of association between injury and cumulative high-speed exercise.     

The seasonal incidence of coccidia infections in trade cattle,sheep, and goats in Nigeria

Summary

A survey of coccidia infections in trade cattle, sheep, and goats was undertaken in Nigeria between April 1978 and March 1979.

Faecal examinations showed coccidia oocysts in 1,456 (56 per cent) of 2,600 calves, 832 (80 per cent) out of 1,040 sheep, and 468 (45 per cent) out of 1,040 goats. There were relatively few coccidia oocysts between October and March and peaks occurred in August and September. Nine species of Eimeria were identified in bovine faeces, and seven species in both sheep and goats.

Eimeria bovis and E. zurnii are predominant in cattle, whereas E. faurei and E. ninakohlyakimovae have the highest percentage occurrence in sheep and goats in Nigeria. The effects of seasonal influences on the abundance of coccidia oocysts among ruminants in Nigeria are stressed.