乳腺炎主要病原体的早期发现对于乳牛群的乳房健康管理至关重要。混合牛奶样品的测试,单独的测试日奶牛样本(TDCSs)或无菌收集的奶前季度样本(PMQSs)可以提供易于使用且具有成本效益的组水平筛选工具。因此,这项研究的目的是(1)使用贝叶斯潜在类别分析评估2种商业多重实时PCR检测试剂盒应用于合并牛奶样品的敏感性(Se)和特异性(Sp),以及(2)估计与池大小和池内BC阳性检测母牛数量相关的检测概率。从2个商业奶牛场的2,045头母牛中收集的1,912个测试日样本和7,336个PMQSs集合了10头、20头和50头母牛。两种商业定量实时PCR试剂盒用于检测葡萄球菌(葡萄球菌。)金黄色葡萄球菌,链球菌(链球菌。)无乳和链球菌。在汇集的样本中,并将细菌学培养物(BC)应用于PMQSs,产生累积池结果。如果池包含至少一个BC阳性PMQS,则其被认为是BC阳性。在PMQS池中比在TDCS池中更频繁地检测到病原体。与较大的池大小相比,无论样品类型或PCR试剂盒类型如何,10头母牛的池显示出最高的检测概率。使用贝叶斯潜伏类别分析进行估算,导致PCR试剂盒I的10头母牛的PMQS池中的硒中位数为63.3%,PCR试剂盒II为78.1%,BC为95.5%;Sp值分别为97.0、97.6%和89.1%,分别。链球菌的估计中值硒。PCR试剂盒的种类介于77.5和85.6%之间,BC介于73.7和79.2%之间;PCR试剂盒的Sp中值介于93.6和99.2%之间,BC在96.9%至97.4%之间。此外,检测的可能性随着每个池BC阳性母牛数量的增加而增加。要实现90%的检测概率,试剂盒I的10头PMQS池中阳性母牛的估计数量为葡萄球菌的4.1头。金黄色葡萄球菌,1.5为Strep。无乳和链球菌1.3。中毒;对于等效的TDCS池和病原体,需要6.9、1.9和2.0头阳性奶牛,分别。对于套件II和PMQS池,葡萄球菌所需的阳性母牛数量为2.8。金黄色葡萄球菌,1.4为Strep。无乳和链球菌1.2。中毒;对于等效的TDCS池和病原体,需要5.3、1.8和2.0阳性母牛,分别。总之,用于汇集的样本类型,池的大小和每个池的感染母牛的数量决定了通过PCR检测检测池内主要乳腺炎病原体感染的可能性。
The early detection of major mastitis pathogens is crucial for the udder health management of dairy herds. Testing of pooled milk samples, either individual test-day cow samples (TDCS) or aseptically collected pre-milk quarter samples (PMQS) may provide an easy to use and cost-effective group level screening tool. Therefore, the aim of this study was (1) to evaluate the sensitivity (Se) and specificity (Sp) of 2 commercial multiplex real-time PCR test kits applied to pooled milk samples using a Bayesian latent class analysis and (2) to estimate the probability of detection in relation to the pool size and the number of cows positively tested by bacteriological culture (BC) within a pool. Pools of 10, 20 and 50 cows were assembled from 1,912 test-day samples and 7,336 PMQS collected from a total of 2,045 cows from 2 commercial dairy farms. Two commercial quantitative real-time PCR kits were applied to detect Staphylococcus aureus, Streptococcus agalactiae, and Streptococcus dysgalactiae in the pooled samples, and a BC was applied to PMQS yielding a cumulative pool result. A pool was considered BC-positive if it contained at least one BC-positive PMQS. Pathogens were more frequently detected in the PMQS pools than in the TDCS pools. Pools of 10 cows showed the highest probability of detection irrespective of sample type or type of PCR kit compared with larger pool sizes. Estimation with a Bayesian latent class analysis resulted in a median Se in PMQS pools of 10 cows for Staph. aureus of 63.3% for PCR kit I, 78.1% for PCR kit II, and 95.5% for BC; the Sp values were 97.0%, 97.6%, and 89.1%, respectively. The estimated median Se for Strep. species for PCR kits ranged between 77.5 and 85.6% and for BC between 73.7% and 79.2%; the median Sp values ranged between 93.6 and 99.2% for PCR kits, and between 96.9% and 97.4% for BC. In addition, the probability of detection increased with an increasing number of BC-positive cows per pool. To achieve a probability of detection of 90%, the estimated number of positive cows in PMQS pools of 10 cows for kit I was 4.1 for Staph. aureus, 1.5 for Strep. agalactiae, and 1.3 for Strep. dysgalactiae; for the equivalent TDCS pools and pathogens, 6.9, 1.9, and 2.0 positive cows were required, respectively. For Kit II and PMQS pools, the number of positive cows required was 2.8 for Staph. aureus, 1.4 for Strep. agalactiae, and 1.2 for Strep. dysgalactiae; for the equivalent TDCS pools and pathogens, 5.3, 1.8, and 2.0 positive cows were required, respectively. In conclusion, the type of samples used for pooling, the pool size and the number of infected cows per pool determine the probability of detecting an infection with major mastitis pathogens within a pool by PCR testing.