Yang R, Abraham S, Gardner G E, Ryan U, Jacobson C
School of Veterinary and Life Sciences, Murdoch University, Murdoch, Western Australia 6150, Australia.
Aust Vet J. 2017 May;95(5):143-148. doi: 10.1111/avj.12572.
Develop a multiplex quantitative PCR assay to investigate the prevalence and shedding of Escherichia coli O157/O145, Salmonella spp. and Campylobacter spp. in sheep at sale yards and abattoirs.
A qPCR for E. coli O157/O145 was developed, validated and multiplexed with an existing qPCR for Campylobacter and Salmonella enterica. The absolute numbers of E. coli O157/O145, Campylobacter and Salmonella in control samples was determined using droplet digital PCR. These were then used as the controls in the multiplex qPCR on a total of 474 sheep faecal samples collected from two saleyards over a 4-month period (April-July 2014) and 96 effluent samples from an abattoir.
The mutiplex qPCR was specific with a sensitivity of 5 organisms/μL faecal DNA extract for Campylobacter, S. enterica and E. coli O157/O145. The overall prevalence of Campylobacter, S. enterica and E. coli O157/O145 in faecal samples was 5.7%, 3.6% and 8.4% and in effluent samples was 18.8%, 6.3% and 5.2%, respectively. The pathogen loads of Campylobacter, S. enterica and E. coli O157/O145 in faecal and effluent samples was also determined via mutiplex qPCR.
The overall prevalences of Campylobacter, S. enterica and E. coli O157/O145 were generally low (<6%), but point prevalences ranged considerably in healthy sheep (up to 26% for E. coli O157/O145). Further work to determine risk factors for shedding of bacterial organisms in meat sheep in the pre-slaughter period (on-farm, sale yards and lairage at abattoirs) could further reduce the risk of contamination of meat products.
开发一种多重定量PCR检测方法，以调查在交易市场和屠宰场的绵羊中大肠杆菌O157/O145、沙门氏菌属和弯曲杆菌属的流行情况及排毒情况。
开发并验证了一种针对大肠杆菌O157/O145的qPCR，并将其与现有的针对弯曲杆菌和肠炎沙门氏菌的qPCR进行多重检测。使用滴液数字PCR测定对照样品中大肠杆菌O157/O145、弯曲杆菌和沙门氏菌的绝对数量。然后将这些用作多重qPCR的对照，对在4个月期间（2014年4月至7月）从两个交易市场收集的总共474份绵羊粪便样本和来自一个屠宰场的96份废水样本进行检测。
多重qPCR具有特异性，对弯曲杆菌、肠炎沙门氏菌和大肠杆菌O157/O145的粪便DNA提取物的检测灵敏度为每微升5个菌。粪便样本中弯曲杆菌、肠炎沙门氏菌和大肠杆菌O157/O145的总体流行率分别为5.7%、3.6%和8.4%，废水样本中的总体流行率分别为18.8%、6.3%和5.2%。还通过多重qPCR测定了粪便和废水样本中弯曲杆菌、肠炎沙门氏菌和大肠杆菌O157/O145的病原体载量。
弯曲杆菌、肠炎沙门氏菌和大肠杆菌O157/O145的总体流行率通常较低（<6%），但健康绵羊中的点流行率差异很大（大肠杆菌O157/O145高达26%）。进一步开展工作以确定肉羊在屠宰前期（农场、交易市场和屠宰场的圈舍）排出细菌的风险因素，可进一步降低肉类产品受污染的风险。
