Selection Of Candidate Strains Of Bovine Pasteurella Multocida Avaccine And Preliminary Evaluation Of Immune Effect

Object:.In this study,9 strains of bovine Pasteurella multocida?Pm?isolated in some areas of northern Xinjiang were used as research objects.On the basis of determining the prevalent Pm serotype and ST type isolated from suspected cases in some areas of northern Xinjiang,1-2 strains of bovine type A Pm vaccine candidates with strong toxicity,good immunogenicity and high vaccine protection rate were screened out for later study of Pm vaccine.Methods:?1?Bacterial samples were collected to isolate and culture bacteria according to conventional methods,and the isolated strains were identified by biochemical identification,PCR identification of 16S rRNA and pm-specific identification gene Kmt,and the Pm isolates were classified by capsular,lipopolysaccharide multiple PCR and MLST typing methods.The main Pm serotypes and ST types isolated from suspected cases in some areas of northern Xinjiang were preliminarily determined.?2?PCR amplification was used to detect the distribution of 25 virulence genes in 7 classes and 22 resistance genes in 12 classes of 9 bovine Pm isolates;Sensitivity determination to 20 drugs of 9 Pm isolates were tested by K-B;The virulent strains were screened by artificially infecting mice with the same dose,and the half-lethal lethal dose(LD₅₀)of the virulent isolates was determined by improved Karber method;Pathological sections were made to observe the pathological changes of lung,liver and spleen of mice infected with Pm.?3?The selected 4 strains of Pm were cultured and concentrated to the required concentration,bacterial fluid was inactivated with 0.4%formaldehyde for 10 hours and emulsified to prepare inactivated vaccine;Indirect ELISA method was used to detect antibody levels produced within 5weeks after vaccination of mice;Immune protection rate of 4 groups of Pm vaccines in mice was determined by challenge-protection test;Isolation and identification of pathogenic bacteria in mice infected with the immune challenge protection test by conventional bacterial isolation method and PCR amplification of specific genes.Result:?1?9 strains of Pm were identified by biochemical identification and PCR identification?Named Pm8,Pm9,Pm12,Pm13,Pm14,Pm17,Pm18,Pm122,PmM6?,8 strains of Pm the capsular:lipopolysaccharide?LPS?:multilocus sequence typing genotypes are A:L3:ST1,other one Pm the capsular:lipopolysaccharide?LPS?:multilocus sequence typing genotypes are B:L2:ST44.?2?The carrying rate 9 Pm of 17 virulence-associated genes were 100%?exbD,fimA,fur,hgbA,hsf2,nanB,oma87,ompA,ompH,plpB,psl,ptfA,sodA,sodC,tonB,tbpA?and the toxA was 0%;All 9 strains of Pm were detected?-lactam bla_TEM gene,and some strains carried strA,sul2,strB resistance genes;The isolates showed different degrees pathogenicity;The LD₅₀ values of the 4 strains with strong virulence were10^0.614 CFU for Pm8,10^0.629 CFU for Pm12,10⁰.547.547 CFU for Pm13,and 10^0.505 CFU for Pm122.?3?Antibody level results:The antibody levels of the 4 Pm vaccines increased slowly in the first week and the second week after the first immunization of mice,and the antibody level was relatively low in the two weeks,the antibody titer was mostly 1:80,the antibody titer reached a high level in the week after the second immunization,and the titers were 1:320-1:640,Pm8 and Pm12 have a high antibody antibody titer of 1:640 after the second immunization;The protective rate of the immune challenge test in mice was 80%for Pm12,56%for Pm122,66%for Pm8,and 40%for Pm13;The corresponding Pm was isolated from the liver of the immune challenge protection test.Conclusion:The main serotype of Pm isolated from suspected cases of large-scale cattle farms in northern Xinjiang was type A:L3:ST1,and a candidate strain with strong virulence and good antigenicity was preliminarily screened out,laying a foundation for further to research and development of Pm vaccine.