Isolation And Identification Of Clostridium Perfringens From The Process Of Dairy Cattle Breeding And Milking And Risk Factors Analysis Of Raw Milk Contamination

As an important zoonosis pathogen,Clostridium perfringens(C.perfringens)causes a varity of animals diseases and mass death of livestock,and it is also directly harm to human health.At present,study on risk factors analysis of C.perfringens contamination is rare from the process of dairy cattle breeding and milking.In this study,the multiple fluorescent quantitative PCR method was developed and used to detect and type the C.perfringens of samples from dairy cattle breeding and milking processes in order to understand the contamination and the transmission pattern.At the same time,the contamination quantity of each sample was detected,and the growth prediction model of the bacteria in milk was established,using@Risk and Excel software to confirm the critical control points of risk factors.Results are shown as follow:1.The limit of detection for cpa-p MD18-T,cpb-p GEM-T,etx-p GEM-T and ia-p GEM-T plasmids was 1×10~2 copies/μL by the developed multiplex fluorescence quantitative PCR method.The limits of detection of bacterial solution and DNA were 10~2 CFU/m L and 100fg/μL,respectively,which showed good sensitivity,specificity and reproducibility.2.A total of 91 strains of C.perfringens type A were isolated and identified from 244samples collected from two large-scale dairy cattle farm in Shaanxi Province,it was consistent with results detected by multiple fluorescence quantitative PCR,indicating that C.perfringens type A was the prevalent serotype in the process of dairy cattle breeding and milking.3.The Logistic growth prediction model of C.perfringens in milk was established,and the contamination quantity of samples in the process of dairy farming and milking was detected.Using@Risk and Excel software,the initial milk contamination quantity and workers hands as well as the feces were confirmed as the critical control point of risk factors,while the contamination from air,nipples and milking cups is not negligable.In conclusion,this study preliminarily understood the contamination level and transmission pattern of C.perfringens in dairy breeding and milking process,the initial milk contamination quantity and workers hands as well as the feces were confirmed as the critical control point of risk factors.It can provide scientific basis for effective prevention and control of C.perfringens contamination in dairy farming enterprises.