Study On Some Biological Characteristics Of Streptococcus Agalactiae From Dairy Cows And Preliminary Preparation Of Subunit Vaccine

Mastitis is a common disease on dairy farms.There are more than 150 species of pathogenic bacteria that cause bovine mastitis.Streptococcus agalactiae is one of the main pathogenic bacteria.In addition,the bacteria is also potentially harmful to humans and aquatic animals.In order to understand the situation of S.agalactiae infections in dairy cow mastitis cases in some areas of Sichuan province,the sensitivity of isolates to antibacterial drugs,virulence factor carrying status,and preliminary exploration of subunit vaccines.In this study,clinical mastitis samples from large-scale dairy farms in 5 different regions of Sichuan were collected for the isolation and culture of S.agalactiae,and the isolated bacteria were identified by routine microbial identification methods and 16 S rRNA.The serotype,drug resistance and virulence genes of isolates were analyzed.At the same time,using the S.agalactiae enolase protein as the antigen protein,a subunit vaccine against the bacteria was initially prepared,and the serum antibody levels of the immunized mice were detected by indirect ELISA to verify the immunogenicity of the antigen protein.It is expected to provide basic data for the prevention and control of dairy cow mastitis and the research and development of subunit vaccine.1.Isolation and identification of S.agalactiaeA total of 313 clinical mastitis milk samples were collected from pastures in 5different regions of Sichuan.The milk samples were inoculated on Columbia blood agar plates containing 5% defibrillated sheep blood.The biochemical reaction and16 S rRNA test were used to identify pathogenic bacteria in milk samples,and the serotypes of isolated bacteria were identified by multiplex PCR.The biochemical reaction and 16 S rRNA test were used to identify pathogenic bacteria in milk samples,and the serotypes of isolated bacteria were identified by multiplex PCR.The results showed that 105 strains of S.agalactiae were isolated by isolation culture,biochemical test and 16 S rRNA identification,and the separation rate was 33.55%.Serotype multiplex PCR results showed that all isolates were type Ia.2.Study on drug resistance and virulence genes of S.agalactiaeA total of 10 antibiotic-resistant phenotypes of β-lactams(piperacillin,ceftriaxone,penicillin,amoxicillin,ceftazidime)and aminoglycosides(Kanamycin,gentamicin,neomycin,streptomycin,tobramycin)were determined on 105 isolates.Detection of β-lactams(TEM,IMP,DHA,OXA)and aminoglycosides(aph(3 ’)Ia,ant(3’)I,aac(6 ’)Ib,aac(3’)Ib))8 drug-resistant genes and isolates bac,cfb,cylE,fbsA,fbsB,hylB,α-enolase,lmb 8 virulence factors by PCR.The results showed that the isolates were up to 100% sensitive to aminoglycosides(kanamycin,gentamicin,neomycin,tobramycin),and to β-lactams(penicillin,amoxicillin,Ceftazidime,piperacillin)resistance rate is as high as 98.10%.Only β-lactam-resistant genes TEM were detected in all isolates,which basically accorded with drug resistant phenotype.Virulence gene test results showed that all isolates carried cfb,cylE,fbsA,fbsB,hylB,α-enolase virulence factors,bac and lmb were not detected.3.Preliminary preparation of enolase protein subunit vaccineThe recombinant expression plasmid pET-32a-enolase was constructed by cloning the isolate’s enolase gene.It was identified by PCR and double enzyme digestion,sequenced and transferred into BL21 E.coli.In vitro induction of IPTG,the expression products were analyzed by SDS-PAGE and Western-blot,and then the recombinant protein was purified by Ni-NTA column.At last,the mice were immunized with purified enolase recombinant protein and adjuvant,and the immunity was enhanced again on the 14 th day after 1 immunization.Serum antibody levels of mice on day 14,21 and 28 were detected by indirect ELISA to evaluate the immune effect of enolase protein subunit vaccine on mice.To evaluate the immune effect of enolase protein subunit vaccine on mice.The results showed that the sequencing and double enzyme digestion identification results showed that the enolase gene amplified from S.agalactiae(MN686586)had more than 97% sequence homology with the enolase gene(KF607046.1)published by Gen Bank.The pET-32a-enolas recombinant plasmid was successfully constructed,transferred into E.coli BL21 and induced by IPTG in vitro.After SDS-PAGE electrophoresis analysis,the recombinant protein with molecular weight of 67 k Da was obtained.Western-blot analysis showed that the recombinant protein had good immunogenicity.Mice were immunized with the enolase subunit vaccine,and the serum antibody levels of the mice were measured by indirect ELISA.The titer of serum antibody levels at the 14 th day after the second immunization reached 1: 25600.It shows that the enolase subunit vaccine could stimulate mice to produce a certain immune response and produce specific antibodies.A subunit vaccine against S.agalactiae of bovine origin has been initially prepared.