| Pasteurella multocida(P.multocida)is classified into five capsular serogroups(A,B,D,E and F),different capsular serogroups of P.multocida are associated with host adaptation.Serogroup A strains are causative agent of fowl cholera in avian and shipping fever in cattles.The main site of infection of fowl cholera is respiratory tract,with a wide variety of manifestations ranging from acute/acute systemic diseases characterized by high mortality to relatively mild chronic localized infections.The shipping fever is mainly caused by the stress reaction during cattles transportation,which is associated with fever,dyspnea,exudative and necrotizing pneumonia.Previous research found that,among serotype A strains,avian strain PmQ can simultaneously kill mice and chickens,while bovine strain PmCQ2 can kill mammalians and mice,but seems to not effect on chickens;but PmCQ7 isolated from bovine pneumonia,can simultaneously kill mice and chickens,and was found to have a large difference with other bovine A strains by RIRDC MLST.This experiment aims to determine classified status and the host-origined of PmCQ7 by comparing the biological characteristics and genetic evolution relationship between PmCQ7 and bovine and avian P.multocida of serotype A;on this basis,the adaptive mutation of PmCQ7 was studied by outer membrane proteins,and the common specific genes of PmCQ7 and avian strains and pseudogenesdistributionwerediscovered.So that,lay the foundation for the research of P.multocida pathogenic mechanism and cross-host cross infection.Main contents are as follows:1 Comparison of biological characteristicsThe results showed that RIRDC MLST genotypes and LPS genotypes of 27 P.multocida strains.The LPS genotype of bovine strains of serotype A is L3,bovine strain of B serotype is L2,and the avian strains are L1.All bovine strains wereassigned into one categorie of MLST genotypes(ST79),excepting PmCQ7(ST7)and PmCQ3(only a single base mutation of pgi gene compared to ST79).PmB and PmF are ST122 and ST9,respectively.Two avian strains sPL665 and PmQ are ST342 and ST129,respectively.Growth curve based on electrical conductivity showed that electrical conductivity change is similar between PmCQ7 and PmQ at 37 ? and 41.5 ?,while the conductivity of PmCQ2 is significantly greater at 37 °C.Growth curve based on bacterial count showed that the growth rate and stress resistance of PmCQ7 and PmQ are faster or stronger than PmCQ2 in the medium.The results suggested that PmCQ7 and PmQ have faster growth rate and stronger stress resistance at 41.5 °C(bird temperature),but the growth of PmCQ2 was inhibited at this temperature.Pathogenicity tests in KM mice and chicks revealed differences in pathogenicity of P.multocida isolated from different host.The pathogenicity of avian strains sPL665 and PmQ and PmCQ7 were significantly stronger than that of bovine strains,and the bovine strain PmCQ2 did not kill chicks.Colonization experiment showed that the colonization of PmCQ7 and PmQ were higher than PmCQ2 in the heart,liver,spleen and kidney of KM mice.The result suggested that the high colonization of PmCQ7 and PmQ may be one of the reasons why pathogenicity is stronger than PmCQ2.2.Whole genome sequencing of PmCQ7 strainWhole genome sequencing of PmCQ7 was performed using the Whole Genome Shotgun(WGS).Genome of PmCQ7 is composed of 2,243,276 base pairs(bps)forming a single circular chromosome with a GC content of approximately 40.41%,does not carry plasmid.88.6% genome of PmCQ7 is made up of 2112 functional genes,including 2036 CDSs and 76 RNA.Genomic analysis revealed that the PmCQ7 genome contains 2 CRISPRs sequences,2 incomplete phages,3 gene islands(GIs),60 carbohydrate active enzyme-related genes,at least 44 virulence-related genes,and 43antibiotic-related genes.Subcellular localization analysis revealed that PmCQ7 strain encoded 172 proteins containing signal peptide and 512 membrane proteins containing transmembrane structures.3.Evolutionary relationship between PmCQ7 and other Pm strainsAverage nucleotide identity(ANI)and cluster analysis based on Multi-host MLST,RIRDC MLST,core genome SNPs and 54 outer membrane proteins were found.The results showed that PmCQ7 has no significantly cluster relationship with other bovine strains,but has a closer phylogenetic relationship with avian strains.Further analysis ofthe adaptive mutation of OmpA and OmpH1 revealed that PmCQ7 is clustered with bovine strains.The results suggested that PmCQ7 were derived from avian,and OmpA and OmpH1 had undergone adaptive mutation after trans-host transfer.4.Compositionanalysis and collinearity analysis of genomeAnalysis of the genomic composition by binary abscent/present value of accessory genome revealed that PmCQ7 is similar to avian strain.However,genomic collinearity analysis was found that the collinearity of PmCQ7 and bovine strains was almost identical at the genes level.Analysis of sequences in syntenic block revealed that sequences encoding tRNA and rRNA often were found between two syntenic blocks.The results suggested that sequences encoding RNA may play an important role in genomic rearrangement.5.Host-specific genes and pseudogenes analysisProtein sequence similarity of 80% as a threshold,no host-specific gene was found.Further pseudogenes analysis revealed that four proteins were broken in the core genome of bovine or partial strains,which are pyrimidine/purine permease,nutA homologous protein(bifunctional UDP-sugar hydrolase/5'-nucleotidase),TonB-dependent receptor and O-antigen ligase.The inactivation of the former three may reduce the growth and proliferation rate of the bovine strains by reducing metabolic efficiency or nutrient uptake.Since P.multocida lipopolysaccharide does not has O-antigen,it is speculated that the fragmentation of the O-antigen ligase may not affect on the synthesis of the lipopolysaccharide of the bovine strains. |
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