Sanderson M W, Sargeant J M, Nagaraja T G
Department of Clinical Sciences, Kansas State University, Veterinary Medical Teaching Hospital, 103J Mosier Hall, Manhattan, KS, USA.
Vet Microbiol. 2005 Sep 30;110(1-2):125-30. doi: 10.1016/j.vetmic.2005.08.003.
To assess the effect of pooling fecal samples on the sensitivity of detection of E. coli O157:H7, 12 calves, inoculated orally with 10(8)cfu per calf of nalidixic acid resistant E. coli O157:H7, were used to provide positive fecal samples. After inoculation, calves were sampled twice weekly. Negative fecal samples were from calves at a local dairy. Samples from inoculated calves were incubated without pooling or were mixed with known negative fecal samples in a 1:4 ratio or a 2:3 ratio (positive:negative) for detection of E. coli O157:H7. Samples were enriched 6h in Gram negative broth with vancomycin, cefixime, and cefsoludin, underwent immunomagnetic separation with Dynabeads, and were plated onto sorbitol MacConkey agar with cefixime, and tellurite (SMACct). Morphologically typical colonies were plated onto blood agar, incubated overnight at 37 degrees C and an indole test was performed on each colony. Indole positives colonies were plated on SMAC agar with 20 microg/ml nalidixic acid (SMACnal). Colonies that grew on SMACnal were confirmed by O157 agglutination. Sensitivity of detection in non-pooled samples was 77%. Samples pooled 1:4 and 2:3 with negative samples were 55 and 52% sensitive, respectively. Pooling decreased sensitivity of detection for E. coli O157:H7 in bovine fecal samples (P<0.01). A deterministic binomial probability model was developed to assess the probability of detecting pens of cattle shedding E. coli O157 using a pooling protocol or individual samples. Pooling decreased sensitivity of detection at low pen prevalence compared to individual samples but was similar at high prevalence.
为评估粪便样本混合对大肠杆菌O157:H7检测灵敏度的影响，选用12头犊牛，每头犊牛经口接种10(8)cfu耐萘啶酸的大肠杆菌O157:H7，以提供阳性粪便样本。接种后，每周对犊牛进行两次采样。阴性粪便样本取自当地一家奶牛场的犊牛。对接种犊牛的样本不进行混合培养，或将其与已知阴性粪便样本按1:4或2:3（阳性:阴性）的比例混合，用于检测大肠杆菌O157:H7。样本在含有万古霉素、头孢克肟和头孢磺啶的革兰氏阴性肉汤中富集6小时，用磁珠进行免疫磁珠分离，然后接种到含头孢克肟和亚碲酸盐的山梨醇麦康凯琼脂（SMACct）上。将形态典型的菌落接种到血琼脂上，于37℃过夜培养，并对每个菌落进行吲哚试验。吲哚阳性菌落接种到含20μg/ml萘啶酸的SMAC琼脂（SMACnal）上。在SMACnal上生长的菌落通过O157凝集试验进行确认。未混合样本的检测灵敏度为77%。与阴性样本按1:4和2:3比例混合的样本，检测灵敏度分别为55%和52%。样本混合降低了牛粪便样本中大肠杆菌O157:H7的检测灵敏度（P<0.01）。建立了一个确定性二项概率模型，以评估使用混合方案或单个样本检测感染大肠杆菌O157的牛栏的概率。与单个样本相比，在低牛栏患病率时，样本混合降低了检测灵敏度，但在高患病率时相似。
