Isolation And Identificationof Bovine Mastitis Streptococcus Agalactiae And Its Establishment Of PCR Detection Method

Dairy bovine mastitis is one of the most harmful infectious diseases to the global dairy farming industry.Its etiology is many and complex.Pathogenic bacteria are the most common and main cause of dairy bovine mastitis.In the diagnosis of mastitis,although accurate diagnosis can be made through the morphological and biochemical characteristics of bacteria,it is difficult to meet the needs of rapid clinical detection due to the long identification process and cumbersome steps.In this thesis,a strain of Streptococcus lactis was isolated from clinical dairy cow mastitis milk samples,and the bacterial morphology,biochemistry and bacterial 16 S r RNA gene were identified;The pathogenic characteristics were analyzed by screening the main virulence genes;The drug resistance to 15 antibiotics was detected;The PCR detection method of Streptococcus agalactiae was established and its sensitivity,specificity and clinical applicability were preliminarily verified.The results show that:(1)The strains isolated from clinical dairy cow mastitis milk samples were spherical,arranged in chains and positive for Gram staining,arranged in chains,forming small needle tip colonies with gray white and smooth surface on THB medium,and forming on sheep defibrillation blood agar plate β Hemolysis;The contact enzyme test was negative,decomposition of lysine,lactose and salicin,no decomposition of aescin,mannitol and sorbitol,positive VP test,consistent with the morphological and biochemical characteristics of Streptococcus agalactiae;The sequencing result of 16 S r RNA gene fragment of the isolated strain had 99.76 % homology with Streptococcus agalactiae,and the isolated strain was identified as Streptococcus agalactiae;The isolates contained five major virulence genes: Sip,hyl B,cps E,rib and bib A;The results of drug sensitivity test showed that the isolate was highly sensitive to clindamycin,azithromycin,ciprofloxacin,gentamicin,ofloxacin,cefazolin,vancomycin,enrofloxacin,penicillin,amoxicillin and ceftriaxone,and resistant to polymyxin B;(2)The minimum concentration of DNA detected by the PCR method of Streptococcus agalactiae established in this thesis is 1×103copies/μL;It has good specificity and can identify common pathogens of dairy bovine mastitis such as Escherichia coli and Streptococcus lactis;The amplified product is about 401 b P,and its size is consistent with the expected size.The sequencing results show that its nucleic acid sequence has 98.20 %homology with the published Streptococcus agalactiae.It is determined that the method established in this thesis can accurately detect Streptococcus agalactiae;The PCR detection method established in this thesis was used to detect clinical samples.The results showed that 6 positive samples were detected in 92 normal milk samples,and the detection rate was 7 %;9 positive samples were detected in 78 clinical mastitis milk samples,and the detection rate was 12 %.The results of this study provide a material basis for the in-depth study of Streptococcus agalactiae and a reliable method for the diagnosis of dairy cow mastitis caused by Streptococcus agalactiae.