Selection Of Candidate Strains Of Bovine Pasteurella Multocida A Vaccine

Object:To screen out candidate strains of inactivated vaccine against Pasteurella multocida?Pm?that are fast-growing,highly virulent,highly immunogenic,and have little side effects,6 strains were selected for this experiment.Isolation and identification of isolates from cattle decidual A type Pasteurella multocida isolates from diseased cattle farms in different parts of Xinjiang that cause yak cow pneumonia and their growth curve determination,drug susceptibility testing,and mouse toxicity The determination of the force,the determination of bacterial lipopolysaccharide?LPS?content,the inactivated vaccine of each strain immunized mice,rabbits antibody determination,challenge protection and calves immune effect determination.Test 1:Growth characteristics of different Pm isolates,LPS content of bacteria and determination of drug susceptibility.Pure cultures of 6 isolates were subjected to gram staining and microscopic examination to observe staining and morphological characteristics to ensure that each strain was a pure culture.The growth curve of 6 strains was detected by the method of viable count?CFU?and absorbance?OD?of the bacteria solution and compared;6 strains of LPS were extracted with LPS extraction kit and 6 strains were separated.The nucleic acid content,polysaccharide content,and protein content of strain LPS;Determination of drug susceptibility of six strains by disk methodTest 2:Virulence genes of different Pm isolates and determination of pathogenicity in miceExtract DNA from different Pm isolates,and design specific primers by referring to genes related to decidual formation-related gene ceaA,bacterial adhesion-associated gene fimA,type-four pilin-related gene ptfA,and iron-obtaining factor-related gene tbpA.PCR amplification was carried out separately;The mouse half-lethal dose?LD50?of six Pm strains was determined by intraperitoneal injection in mice.Test 3:Preparation of inactivated bacterins of different Pm isolates and determination of their immune effectsPreparation of inactivated bacterins:Six strains of the isolates were each subjected to expansion culture,viable counts,and after inactivation by formaldehyde,a Pm inactivated oil adjuvant vaccine was prepared with an ISA 206 biphasic adjuvant.Seedlings for sterility testing,physical property testing,vaccine stability testing,vaccine storage time testing,animal safety testing,etc;Mice,rabbits,animal immune efficacy determination:6 inactivated vaccines to be prepared After subcutaneous immunization of mice and rabbits,the appetite and mental status of rabbits and rabbits,as well as changes in the body temperature of rabbits,were measured.The serum of immunized mice and rabbits was determined by the established IHA method 14 days after the second immunization.Determination of immune effect of yak:select the inactivated vaccine of mouse and rabbit serum with high antibody titer to conduct yak immunity test;observe and record the health status of immune calf and collect it on the second exemption 14 days.Bovine serum was tested for antibodies.Results:1.The growth curves of six Pm isolates are similar,among which the Pm2,Pm3,and Pm5 isolates grow faster;the Pm1,Pm4,and Pm6 isolates grow more slowly.The Pm3 isolate LPS had the highest nucleic acid content,polysaccharide content and protein content,and the Pm5 and Pm2 strains took the second place.The other strains contained less.Drug susceptibility tests showed that the six strains were similar in sensitivity to the same drug,and all had higher sensitivities to gentamicin and ofloxacin,and were less sensitive to neomycin and ciprofloxacin.It is almost insensitive to ampicillin sodium.2.The virulence factor test showed that the six isolates all contained the type IV fimbriae-related gene ptfA and the adhesion-associated gene fimA;the mouse LD50 assay showed that the isolates Pm1,Pm2,Pm3,Pm4,Pm5,Pm6 The LD50 were 10^3.17,10^1.97,10^1.72,10^2.30,10¹.89.89 and 10².52.52 CFU/mL,respectively.Among them,Pm3 strain had the strongest virulence;3.The inactivated bacterin produced by the system was free from contamination by bacteria,the viscosity was in line with requirements,there was no delamination after centrifugation,and the stability was good;the immunoprotective test results of mice and rabbits showed:Pm2,Pm3 The antibody titers of immunized mice and rabbits with inactivated vaccines prepared from Pm5isolates were high,reaching 1:64 to 1:128,and all of them survived virulent challenge.All of the PBS control groups died.The immunized rabbits produced fewer side effects,while the Pm1,Pm4,and Pm6antibodies had lower titer and side effects on rabbits;the inactivated vaccines prepared from the three isolates of Pm2,Pm3,and Pm5 were paralyzed.Bovine immunization trials showed that the calf's feed intake was normal after immunization,and its mental state was good.Body temperature rose slightly and returned to normal within 1 to 3 days after immunization.The side reactions to calves were small,and the second exemption was 14 days later.The antibody titer of yak serum was measured.As a result,the antibody titer of inactivated vaccine prepared from Pm3 strain was higher than that of Pm2 and Pm5.Six of the three immune calves achieved serum antibody 1:32 and one reached 1:64.Conclusion:The results showed that the purified Pm3 and Pm5 strains of six different Pm isolates had stable morphological characteristics,rapid growth rate,high LPS content,and carrying two major virulence genes,and had strong pathogenicity to experimental animals;After immunization,the rabbits immunized with bacterin vaccine achieved a protection rate of 100%;after immune yak,the side reactions were small and the immune antibody level was high,which can be used as a candidate strain of inactivated vaccine against type A multi-kill Pasteurella multocida type A.Among them,Pm3 strain can be used as the first strain.The results provide strain support for the further development of this vaccine.