Isolation Of Corynebacterium Pseudotuberculosis And Its Specific Bacteriophage

Corynebacterium pseudotuberculosis(CP)is the pathogen of caseous Lymphadenitis(CLA)in goats and sheep,which can cause the decrease of meat,milk and wool production in diseased sheep.CP is a chronic infectious disease with no obvious symptoms at the initial stage of infection.Moreover,CP can survive in the natural environment for a long time,easily causing repeated infection of sheep and huge economic losses.In clinical practice,the therapeutic effect of antibiotics on CLA is not ideal.Therefore,it is of great significance to find an effective method to control and remove CP.Phages,as natural antimicrobials,are promising as a powerful tool for the prevention and control of CLA.The purpose of this study was to investigate the prevalence of CLA in Qinghai Province,isolate CP phages,and explore the possibility of using CP phages for the treatment of pseudotuberculosis.The research results are as follows:(1)A total of 66 serum samples were collected from three regions of Qinghai Province,and the ELISA method established in our laboratory was used to detect the serum.The positive rates of CP antibody in sheep farms in three different regions were 68.75%,61.29% and31.58%,respectively.(2)A strain of CP was isolated from the pus sample.This strain grew slowly on the blood plate.needle tip-sized white dry colonies began to appear at 48 h.The strain was identified by 16 S rRNA and pld gene with the the identity of 100% and 98%,respectively.(3)A strain of bacteriophage that could lyse CP was isolated from the environmental sewage,and the plaque was named M2 with the shape of needle.Transmission electron microscopy revealed that phage M2 was tailless with a head about 50 nm in diameter.One-step growth curve showed that the incubation period of phage was 20 min.The cracking period was 60 min.The average outbreak volume is 690 PFU/cell.(4)The phage injection into the pus found that the phage could make the pus burst in advance,and the number of viable bacteria in the pus decreased greatly.The prevalence of CLA in Qinghai province was investigated by indirect ELISA.One strain of CP and one strain of phage capable of cracking CP were successfully isolated.Bacteriophages can make pustules burst in advance and reduce the number of viable bacteria in pus,which provides theoretical support for the prevention and control of CLA.