Chamchoy Tapakorn, Williams Deneice R, Adaska John M, Anderson Randall J, Aly Sharif S
Veterinary Medicine Teaching and Research Center, School of Veterinary Medicine, University of California, Davis, Tulare, CA, USA.
California Animal Health and Food Safety Laboratory, Tulare Branch, Tulare, CA, USA.
PeerJ. 2019 Nov 19;7:e8081. doi: 10.7717/peerj.8081. eCollection 2019.
subspecies (MAP) is a bacterium that can cause substantial economic losses in infected dairy herds due to reduced milk production and increased cow-replacement costs. In order to control MAP in dairies with drylot pens, a standardized environmental sampling protocol to quantify MAP in fecal slurry was developed based on an existing protocol for freestall pens. Specifically, following a 24 h hold of the flush, a grab sample of approximately 10 ml of fecal slurry was collected every 1 m along the flush lane of the drylot pens, avoiding individual cow fecal pats. To determine the reliability and repatability of the new environmental sampling protocol for estimation of MAP bioburden at the pen level, two collectors simultaneously collected fecal slurry samples every day for 3 days from six drylot cow pens on two Central California dairies. During the study period no cow movement between pens was allowed with the exception of sick cows. The study herds had MAP seroprevalence of 5.8% and 3.2%, respectively, based on whole pen serum ELISA results. Variance components models for quantitative real-time PCR (qPCR) results showed samples collected from different pens on different dairies accounted for greater variablitiy in MAP concentration (65%), while samples collected by different collectors had the least variability (0.1%). In contrast, variability in MAP concentration in environmental samples collected on different days had 25% variability. The intraclass correlation coefficient showed high reliability (93%) of environmental sampling simultaneously by different collectors. In contrast, the reliability of environmental sampling at different days was 65%, which was similar to the reliability for sampling by different collectors on different days. Investigators can expect high reliability when employing the new environmental sampling protocol along with qPCR testing of environmental samples from drylot pens.
副结核分枝杆菌（MAP）是一种细菌，由于牛奶产量下降和奶牛更换成本增加，它会给受感染的奶牛群造成巨大的经济损失。为了控制采用干栏式牛舍的奶牛场中的MAP，在现有的散栏式牛舍协议基础上，制定了一种标准化的环境采样方案，用于量化粪便泥浆中的MAP。具体而言，在冲洗后保持24小时，沿着干栏式牛舍的冲洗通道每隔1米采集一份约10毫升的粪便泥浆抓取样本，避开单个奶牛的粪便堆。为了确定新的环境采样方案在牛栏水平上估计MAP生物负荷的可靠性和可重复性，两名采集人员每天同时从加利福尼亚州中部两个奶牛场的六个干栏式奶牛栏中采集粪便泥浆样本，持续3天。在研究期间，除了生病的奶牛外，不允许牛在不同牛栏之间移动。根据整栏血清ELISA结果，研究牛群的MAP血清阳性率分别为5.8%和3.2%。定量实时PCR（qPCR）结果的方差成分模型显示，从不同奶牛场的不同牛栏采集的样本在MAP浓度方面的变异性更大（65%），而由不同采集人员采集的样本变异性最小（0.1%）。相比之下，在不同日期采集的环境样本中MAP浓度的变异性为25%。组内相关系数显示不同采集人员同时进行环境采样具有较高的可靠性（93%）。相比之下，不同日期环境采样的可靠性为65%，这与不同采集人员在不同日期采样的可靠性相似。研究人员在采用新的环境采样方案并对来自干栏式牛舍的环境样本进行qPCR检测时，可以预期具有较高的可靠性。
