Characterisation Of Type D Pasteurella Multocida Strains From Goats And Screening Of Vaccine Candidate Strains

Pasteurella multocida,a member of the genus Pasteurella,is a gram-negative short bacillus that infects a variety of animals,causing fowl cholera,bovine pneumonia,and hemorrhagic septicemia,as well as porcine infectious atrophic rhinitis and rabbit nasal fistulas.Similarly,this pathogen is also an important pathogen of goats and sheep,leading to the occurrence of sheep pneumonia and hemorrhagic septicemia,therefore,posing a serious threat to the sheep industry.According to different capsular antigens,P.multocida can be divided into five serotypes:A,B,D,E,and F,and different serotypes show host specificity to some extent.However,there is a lack of in-depth understanding of the serotypes and other important biological characteristics of P.multocida in goats in China,which is not conducive to the development of a vaccine for P.multocida in goats and the comprehensive prevention and control of the disease.At the same time,because there is no good cross-protection between different serotypes of Pasteurella multocida,in the development of vaccines,it is necessary to develop vaccines specifically for the dominant serotypes circulating in specific hosts and regions in order to get a good immune protection effect of vaccines.Accordingly,in this study,samples were collected from multiple large-scale goat farms in Sichuan province for molecular detection and isolation and identification of P.multocida,and the biological characteristics of the isolates such as serotypes,antimicrobial resistance,and virulence factor were analyzed.Subsequently,four strains isolated from the lungs of dead goats were selected,and a candidate strain of Pasteurella multocida was screened by evaluating its pathogenicity and immunoprotective effect,thus providing a basis for further development of commercial vaccines.The main contents and results of this study are as follows:1.Isolation identification and characterisation of P.multocida from goatsIn order to investigate the infection of P.multocida in goats and its main biological characteristics.in this study,respiratory swabs as well as lung samples were collected from five large-scale goat farms with respiratory diseases in Sichuan province,and their total DNA was extracted and detected using P.multocida species-specific PCR,Isolation and identification of P.multocida from the samples were also performed Subsequently,the serotype,antimicrobial resistance,virulence factor distribution and omp A gene sequence of clinical isolates of P.multocida were analyzed.The results showed that the infection rate of Pasteurella multocida in goats was 11.53%（34/295）,and a total of 27 strains of Pasteurella multocida were isolated;The serotypes of the isolates were capsular type D,which was the dominant serotype in this area;the virulence gene detection results revealed that the isolates carried abundant virulence factors;antimicrobial resistance analysis showed that the isolated strains were highly sensitive to ampicillin and penicillin,of which the multi-drug resistance of the strains from the two sheep farms in Shuangliu and Huidong counties was serious;omp A gene sequence analysis showed that there was a correlation between sequence types and the infected hosts.The results strongly enriched the biological characteristics of P.multocida derived from goats in China and provide a basis for the development and comprehensive control of P.multocida vaccines.2.Screening of candidate strains for type D P multocida vaccineIn order to screen candidate strains of P.multocida type D for vaccine development and control of Pasteurellosis in goats,four strains of Pasteurella multocida（Pm1,Pm2,Pm3,Pm4）isolated from the lungs of disease-dead animals were used in this study to determine the LD₅₀ of each strain to mice,as well as the immune response level and cross-challenge protection ability after immunization of mice.The results showed that the LD₅₀ of Pm1,Pm2,Pm3 and Pm4 strains to mice were 2.70×10⁵CFU/0.5m L and 2.57×10⁴CFU/0.5m L,3.09×10⁵CFU/0.5m L,9.29×10⁵CFU/0.5m L,respectively.All of which have pathogenicity and can cause pathological damage of liver,spleen,lung,kidney,brain and other organs in mice.The inactivated vaccine prepared by each strain could induce good antibody level after immunization of mice,of which the antibody level produced by Pm2 strain was the highest;the results of cross challenge protection test also showed that the inactivated vaccine prepared by this strain had the best immune protection effect against artificial infection of each strain after immunization of mice,and the inactivated vaccine prepared by this strain could induce good humoral immune response in goats.Therefore,Pm2 has the potential to be further developed to develop inactivated P.multocida type D vaccines.In conclusion,in this study,we performed molecular detection of P.multocida infection in goats,isolated 27 P.multocida strains,and analyzed their biological characteristics.On this basis,a strain with strong virulence,good immunogenicity,and high immune protection rate was screened as a vaccine candidate.The results provided a useful basis for the comprehensive control of P.multocida infection in goats.