A six-year study on respiratory viral infections in a bull testing facility

Viral infection dynamics and bovine respiratory disease (BRD) treatment rates were studied over six years at a Swedish bull testing station with an 'all in, all out' management system. In August of each of the years 1998-2003, between 149 and 185 4-8-month-old calves arrived at the station from 99 to 124 different beef-breeding herds, and remained until March the following year. Only calves that tested free from bovine viral diarrhoea virus (BVDV) were allowed to enter the station and original animal groups were kept isolated from new cattle in their original herds for three weeks before admission. Although neither prophylactic antibiotics, nor BRD vaccines were used, less than 0.7-13.2% (mean 5%) of the calves (n=970) required treatment for BRD during the first five weeks following entry. This was probably due, at least in part, to the season (the summer months) when the animals were commingled. In the six-month period August-February, 38% of the animals were treated one or more times for BRD and mortality was 0.7%. Hereford and Aberdeen Angus calves had significantly higher treatment rates than Charolais, Simmental and Blonde d'Aquitaine. Serological testing on samples obtained in August, November and January indicated that bovine parainfluenza virus 3 (PIV-3) infections occurred each year before November after entry. Bovine coronavirus (BCoV) infections also occurred every year, but in 3/6 years this was not until after November. Bovine respiratory syncytial virus (BRSV) infections occurred only every second year and were associated with a treatment peak and one death on one occasion (December). The herd remained BVDV free during the entire study period. The infection patterns for PIV-3 and BCoV indicated a high level of infectivity amongst bovine calves, whereas the incidence for BRSV was observed at a lower level. Although the rearing of the animals differed from conventional beef production, the study has shown that commingling animals from many sources is not necessarily associated with high morbidity within the first few weeks after arrival. By preventing BRD soon after commingling the prerequisites for protective vaccination at entry might be improved. Applied management routines are discussed.     

牛冠状病毒S基因的序列分析及原核表达

为了解牛冠状病毒（bovine coronavirus,BCoV）的S基因变异情况并建立ELISA检测方法,本研究对采自不同牛场的新生犊牛腹泻（CD）和成年牛冬痢（WD）腹泻样本提取总RNA,反转录合成cDNA,利用PCR扩增S全基因和S1基因。将S1基因目的片段连接表达载体pET-32a （+）,并转化大肠杆菌BL21（DE3）感受态细胞,经PCR、双酶切及测序验证正确后,进行IPTG诱导表达。结果显示,CD与WD分离株S基因核苷酸为98.4%,CD和WD分离株与参考毒株BCoV-ENT株核苷酸同源性最高,分别为98.4%和98.5%,CD分离株与参考毒株SUN5株的同源性最低,为97.5%,WD分离株与FRA/EPI/Caen/2003/13同源性最低,为97.3%。通过比对可知,分离株与已知毒株之间存在较大差异,为疫苗候选毒株筛选提供依据。本试验同时构建了pET-32a-S1表达载体,在0.2 mmol/L IPTG诱导5 h时,重组菌能在大肠杆菌BL21感受态细胞中产生大量的S1融合蛋白,获得约58 ku表达产物。本试验成功表达了S1蛋白,并对BCoV进行了核苷酸进化分析,为疫苗免疫效果评价方法的建立奠定了基础。     

BVDV、BRV和BCoV TaqMan三重RT-qPCR检测方法的建立

为建立牛病毒性腹泻病毒(BVDV)、牛轮状病毒(BRV)和牛冠状病毒(BCoV)的快速检测方法,根据GenBank中登录的BVDV 5'-UTR、BRV NSP5和BCoV N基因序列设计特异性引物和探针,通过优化反应体系和条件,建立了同时检测上述3种病毒的TaqMan三重RT-qPCR方法。该方法仅对BVDV 5'-UTR、BRV NSP5和BCoV N基因扩增呈阳性,而对牛传染性鼻气管炎病毒、牛副流感病毒、魏氏梭菌(A型、B型和D型)、多杀性巴氏杆菌(A型和B型)等犊牛腹泻相关病原扩增均呈阴性;最低检出限为10拷贝/μL;组内和组间变异系数均小于2%。利用本研究建立的TaqMan三重RT-qPCR方法对29份临床样品进行检测,获得BVDV、BRV、BCoV的4种混合感染型,其中BVDV与BCoV的混合感染率最高(27.6%);与已有单重RT-qPCR方法比较,发现两种方法检测BVDV、BRV和BCoV的符合率分别为100%、96.5%、100%。结果表明,本研究建立的TaqMan三重RT-qPCR方法具有特异性强、敏感性高、重复性好、可行性高等优点,可为今后BVDV、BRV和BCoV共感染引起的牛腹泻性疾病的鉴别诊断和流行病学调查提供新技术手段。