Wisselink Henk J, Cornelissen Jan B W J, van der Wal Fimme J, Kooi Engbert A, Koene Miriam G, Bossers Alex, Smid Bregtje, de Bree Freddy M, Antonis Adriaan F G
Wageningen Bioveterinary Research, P.O. Box 65, 8200, AB, Lelystad, The Netherlands.
BMC Vet Res. 2017 Jul 13;13(1):221. doi: 10.1186/s12917-017-1141-1.
Pasteurella multocida, Mannheimia haemolytica, Histophilus somni and Trueperella pyogenes are four bacterial agents commonly associated with bovine respiratory disease (BRD). In this study a bacterial multiplex real-time PCR (the RespoCheck PCR) was evaluated for the detection in bronchoalveolar lavage fluid (BALF) of these four bacterial agents.
The analytical sensitivity of the multiplex real-time PCR assay determined on purified DNA and on bacterial cells of the four target pathogens was one to ten fg DNA/assay and 4 × 10 to 2 × 10 CFU/assay. The analytical specificity of the test was, as evaluated on a collection of 118 bacterial isolates, 98.3% for M. haemolytica and 100% for the other three target bacteria. A set of 160 BALF samples of calves originating from ten different herds with health problems related to BRD was examined with bacteriological methods and with the RespoCheck PCR. Using bacteriological examination as the gold standard, the diagnostic sensitivities and specificities of the four bacterial agents were respectively between 0.72 and 1.00 and between 0.70 and 0.99. Kappa values for agreement between results of bacteriological examination and PCRs were low for H. somni (0.17), moderate for P. multocida (0.52) and M. haemolytica (0.57), and good for T. pyogenes (0.79). The low and moderate kappa values seemed to be related to limitations of the bacteriological examination, this was especially the case for H. somni.
It was concluded that the RespoCheck PCR assay is a valuable diagnostic tool for the simultaneous detection of the four bacterial agents in BALF of calves.
多杀性巴氏杆菌、溶血曼氏杆菌、睡眠嗜组织菌和化脓隐秘杆菌是四种通常与牛呼吸道疾病（BRD）相关的细菌病原体。在本研究中，评估了一种细菌多重实时PCR（RespoCheck PCR）用于检测支气管肺泡灌洗液（BALF）中这四种细菌病原体的能力。
在纯化DNA和四种目标病原体的细菌细胞上测定的多重实时PCR检测的分析灵敏度分别为每检测1至10 fg DNA和每检测4×10至2×10 CFU。在对118株细菌分离株的集合进行评估时，该检测的分析特异性对于溶血曼氏杆菌为98.3%，对于其他三种目标细菌为100%。使用细菌学方法和RespoCheck PCR对来自十个不同牛群且有与BRD相关健康问题的160份犊牛BALF样本进行了检测。以细菌学检查作为金标准，四种细菌病原体的诊断敏感性和特异性分别在0.72至1.00之间和0.70至0.99之间。细菌学检查结果与PCR结果之间一致性的Kappa值，对于睡眠嗜组织菌较低（0.17），对于多杀性巴氏杆菌（0.52）和溶血曼氏杆菌（0.57）中等，对于化脓隐秘杆菌较好（0.79）。低和中等的Kappa值似乎与细菌学检查的局限性有关，睡眠嗜组织菌尤其如此。
得出结论，RespoCheck PCR检测是同时检测犊牛BALF中四种细菌病原体的有价值的诊断工具。
