Investigation,Isolation And Identification Of Four Pathogenic Bacteria In Carcass And Viscera Of Minks From Shandong Province

Mink is one of the important fur-bearing animals depended mostly on artificial breeding at present.China has more minks than any other country,of which Shandong province accounts for more than 70% of the country's mink farmers.There is a growing national awareness of the increasing number of carcass and viscera of minks,lacking for an effective way for by-product.Due to the lack of scientific knowledge and processing technology of carcasses and viscera of minks at home and abroad,the utilization or treatment of those is certainly confusing.They are processed by farmers into raw food for the feeding of other fur animals,and also considered as game.It has also been buried or discarded processing,which results in a very large biosafety hazard to the ecological environment and fur animal breeding.Then,to provide the basis for the biological assessment of safety and scientific utilization of the carcasses and visceral tissues of minks,this study was to carry out the research of investigation of the main pathogens Escherichia coli,Salmonella,Klebsiella pneumoniae and Staphylococcus aureus in those.Firstly,four pairs of specific primers were designed according to the conservative regions of phoA gene for E.coli,invA gene for Salmonella,khe gene for K.pneumoniae,nuc gene for S.aureus.The quadruple PCR system was established through optimization of multiplex PCR and detection of its specificity,sensitivity and stability.Secondly,the samples of carcass and viscera of minks in the main producting areas,namely Zhucheng,Wendeng,Liaocheng,Rizhao,and Haiyang cities in Shandong province,were collected.The four pathogens were tested;then the pathogens in the positive samples were identified by physical and chemical tests and other methods.The results showed that the method can identify E.coli(622?bp),Salmonella(801?bp),K.pneumoniae(303?bp)and S.aureus(464?bp)specifically and simultaneously from the same sample containing the four pathogens,with a detection sensitivity of Salmonella and K.pneumoniae of 10?pg,and that of E.coli and S.aureus of 1?pg by this newly developed multiplex PCR assay,and non-specifically amplify for C.welchii,P.aeruginosa and Streptococcus.268 samples were detected by the multiplex PCR and it was proved that themethod could identify the four pathogens rapidly and accurately,While,there is no significant difference compared to the single PCR.Although there was significantly different for the contamination of different pathogens in carcasses of minks,the positive detection rates of E.coli was the highest(33.96%),then,K.pneumoniae(2.61%),Salmonella(0.75%)and S.aureus(0.37%)and the rate of E.coli/K.pneumoniae superinfection is 2.61%.83 strains of E.coli,2 strains of Salmonella,7 strains of K.pneumoniae and 1 strains of S.aureus were isolated from the positive samples.The above results indicated that we had succeeded to establish the quadruple PCR assay for the specific detection of E.coli,Salmonella,K.pneumoniae and S.aureus;the different pathogen had different degrees of infection in the carcass and viscera of minks.Among them,E.coli drew the highest rate of contamination.