| Swine Pasteurellosis is a sporadic or endemic infectious disease caused by different serotypes of Pasteurella multocida.Part of the pigs infected with toxin-producing P.multocida showed progressive atrophic rhinitis.This makes swine pasteurellosis a bacterial disease that has a huge impact on the development of the pig industry in China.P.multocida can be divided into multiple serotypes according to different antigen components.Moreover,in the prevention and control of swine pasteurellosis,regular vaccination is still the main effective means.At present,the inactivated vaccines used clinically for the prevention and control of swine pasteurellosis are mainly multiple vaccine,the vaccine strain has a single serotype and cannot provide cross-immunity protection.there is a need to develop an inactivated vaccine that can provide immune protection against different epidemic serotypes of P.multocida.Therefore,this study carried out the isolation and identification of P.multocida from the lung and nasal swab samples of pigs with suspected respiratory diseases on large-scale pig farms in some regions of my country,and the isolated strains were analyzed by molecular epidemiological investigation methods.On the basis of molecular epidemiological investigations,strains of epidemic serotypes are screened as seed strains to prepare inactivated P.multocida vaccines,and the safety,immune efficacy of the vaccines were evaluated.Establish a foundation for the prevention and control of Pasteurellosis.The main findings are as follows:1.Isolation and identification of P.multocida from pigDuring the period 2019-2020,from large-scale pig farms in parts of China（16provinces and cities including Hubei,Hebei,Henan,Guangdong,Zhejiang）,A total of 158Pm strains were isolated from nasal swab samples and lung tissue samples from pigs with suspected respiratory diseases and without typical clinical symptoms,with an average separation rate of 11.5%.The 158 isolated P.multocida strains were classified by capsular PCR.Among them,95 strains（60%）of type A strains,56 strains（36%）of type D strains,and 7 strains（4%）of type F strains.The isolated strains were subjected to lipopolysaccharide PCR typing,and a total of 2 different lipopolysaccharide genotypes and"undetermined"（the lipopolysaccharide genotype does not belong to any of the lipopolysaccharide L1-L8 types）were identified.There are 105 strains of L6 type P.multocida,accounting for 66%;45 strains of lipopolysaccharide L3 type P.multocida,accounting for 29%;a total of 8 untyped strains,accounting for 5%.Excluding 8 strains of untyped lipopolysaccharide,the remaining 150 Pm strains include 4 types of capsular lipopolysaccharide combinations,of which 38 strains（25.3%）of type A:L3 and 50 strains（33.3%）of type A:L6,55 strains（36.7%）of D:L6 strain,7 strains（4.7%）of F:L3 strain.2.Distribution of P.multocida virulence genes from pigsThe 158 strains of P.multocida isolated were detected by PCR for 23 virulence-related genes,including nan H,hgb B,hsf-2,plp B,omp A,oma87,ptf A,fim A,sod A,sod C,Fur,omp H and other virulence-related genes The detection rate exceeded 80%;the detection rates of tox A,tbp A,hgb A,and nan B were less than 40%.In addition,there are significant differences in the detection rate of some virulence genes in different capsule genotypes,lipopolysaccharide genotypes,and capsular lipopolysaccharide combination types.The detection rates of different virulence-related genes in different capsular P.multocida The detection rate of hsf-1 in capsular D and F type P.multocida is significantly higher than that in capsular A type P.multocida;while pm HAS and nan B are detected in capsular A type P.multocida,the detection rate is significantly higher than the detection rate in capsule D and F P.multocida.3.Analysis of P.multocida resistance in pigsA drug susceptibility test was conducted on the 211 isolated P.multocida strains,and the results showed that most P.multocida strains were sensitive to the antibiotics used in this test,and only a few strains were resistant to ciprofloxacin（6 strains,accounting for2.8%）,tigecycline（19 strains,accounted for 9.0%）,ampicillin（16 strains,accounted for7.6%）,cefepime（1 strain,accounted for 0.5%）,chloramphenicol（6 strains,accounted for2.8%）However,more than half of the strains are resistant to tetracycline（108 strains,accounting for 51.2%）,polymyxin E（135 strains,accounting for 64.0%）,and none of the strains showed resistance to iminium.South（0 strains,accounting for 0%）of drug resistance.The 158 isolated P.multocida strains were tested for drug resistance genes by PCR using specific identification primers.Only two drug resistance genes（tet B,flo R）were detected,and the detection rates were 38.3%and 39.0%,respectively;The remaining resistance genes include the resistance genes bla SHV and bla TEM encodingβ-lactamase,the resistance genes cat A1 and cat A2 for chloramphenicol and the detection of the resistance genes qnr S,oqx A and oqx B for fluoroquinolones The rates are all 0%.4.Evaluation of the immune effect of the bivalent inactivated vaccine against swine pasteurosisOn the basis of epidemiological investigations,A:L3 strain HB03 and D:L6 strain HN05 were selected as vaccine seedling strains.After expanding and cultivating the seed bacteria liquid,it was inactivated,concentrated by centrifugation,and mixed with hydrogen in a certain proportion.After mixing alumina adjuvant,a bivalent inactivated vaccine for swine pasteurellosis was prepared.The Kunming mouse was used as an animal model to evaluate the safety and immune effect of the vaccine.The results showed that the vaccine is safe for mice and can provide more than 80%of the immune protection rate against HB03and HN05.The ELISA antibody test results show that The mice produced higher levels of ELISA antibodies 7d and 14d after the second immunization. |
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