Isolation,Identification And Pathogenicity Of Pasteurella Multocida From Ducks

Pasteurella multocida often causes avain cholera in poultry,which often causes acute death in poultry with an extremely high mortality rate.Even several bacteria of a Highly pathogenic strains make young ducks infected and sick.Our laboratory identified 17Pasteurella multocida strains from four duck farms in 2021.4 were selected for pathogenicity tests and preparation of inactivated vaccines,and then the immune efficiency were evaluated.Finally,the strain with the most pathogenic and the best immune effects were selected for whole genome sequencing analysis,providing theoretical basis and technical support for the prevention and control of the disease.First,20 suspected strains were cultured and then extracted DNA by boiling at 100℃for5 min.The DNA was used as a template for species specificity identification by PCR.The results showed that 17 of the 20 strains were positive for Pasteurella multocida species-specific KMT1,and then common biochemical reagents in biochemical spectrum of Pasteurella multocida were used to identify the 17 positive strains.The results found that all17 strains showed consistent biochemical reaction characteristics with Pasteurella multocida.The drug sensitivity assay showed that 17 isolates showed different degrees of resistance to a variety of antibiotics,among which 14 isolates were sensitive to polymyxin,13 isolates were resistant to cefotaxime,butamicana,neomycin and furazolidone.Identification of capsular serotype showed that all the 17 isolates were capsule type A.The results of animal regression assay showed that all 17 strains could kill ducklings.The dead ducks were dissected for bacteria isolation and pathological changes observation.PCR identification of the isolated bacteria was carried out,the results showed that all the 17 strains were successfully returned.Subsequently,four strains from different sources（PM-3,PM-7,PM-11 and PM-17）were selected for median lethal dose(LD₅₀)determination by animal pathogenicity tests.The concentration of bacterial solution was adjusted to 2×10⁷-2×10²CFU/m L,and 0.5m L was injected into the legs of 20-day-old ducklings.The results showed that PM-3 strain was the most pathogenic with an LD₅₀ of 10^2.1CFU,while PM-11 strain the least,with LD₅₀ of 10^4.5CFU.The inactivated vaccine of the four strains(containing 4×10¹⁰CFU/m L)was prepared for vaccine immunization and protection evaluation.The results showed that the inactivated PM-3 vaccine had the best protective effect,and it also had good protective effect against other three strains.The results of the growth curve showed that there was no significant difference in the growth curve of the four strains.Whole genome sequencing analysis of PM-3 showed that the whole-genome chromosome length of PM-3 was 2,345,687 bp,and PM-3contains 2,181 open reading frames（ORFs）and one plasmid.The length of the plasmid was5,259 bp,containing 8 ORFs.A total of 97 non-coding RNAs,3 CRISPR structures,4prophages,61 virulence factors and 41 antibiotic-associated genes were predicted from the whole genome.In summary,this study identified 17 strains of Pasteurella multocida capsule type A,which were resistant to a variety of antibiotics.Animal regression of 17 isolates was successful.The pathogenicity test identified the differences in the virulence of different strains,Inactivated vaccine with good immune effect was prepared,and sequencing test of the strain with the strongest virulence and best immune effect was taken,which provided the theoretical reference for the prevention and control of Pasteurella multocida.