| Nasopharyngeal microorganism is an important part of animal microecosystem and plays a role in respiratory tract health.Among them,Pasteurella multocida,Mannheimia haemolytica and Mycoplasma bovis etc.are opportunistic pathogens.When cattle are stressed from long-distance transportation,mixing flocks,overcrowding,climate variations etc.,animal immunity decreases and bacteria will proliferate,which lead respiratory tract infection.Bovine respiratory disease complex(BRDC)is a general term for a variety of respiratory diseases,including bovine pneumonia and bronchitis,which results in huge economic losses to the global cattle industry.Many pathogens are associated with BRDC,and Pasteurella multocida is one of the most important pathogens.At present,Pasteurella multocida serogroup A is the most common in BRDC in China.Comparative genomics has sprung up rapidly in recent years.It is a powerful tool to study the genomes of Pasteurella multocida isolates.Many virulence-associated factors of P.multocida have been identified,such as adhesin,lipopolysaccharide,capsule and outer membrane proteins,but the pathogenic mechanism is still unclear.In this work,16S rRNA high-throughput sequencing technology was used to analyze the composition of cattle nasopharyngeal microflora with respiratory diseases in bovine nasal mucus.Then the whole genome of P.multocida virulent isolate PMPAN001 and avirulent isolate PMPAN007 were sequenced by the next generation sequencing technology.The comparative genomics was carried out to investigate the difference of gene function and structure between PMPAN001 and PMPAN007.The bacterial load of organs,transcription level of virulence-associated gene and lung tissue damage in mice between virulent and avirulent strains were compared.In order to explore the function of virulence-associated genes flpC?LH0147?fbpA ? flpD,recombinant plasmids were accomplished for contructing the gene-deleted strains of P.multocida.The purpose of the present study was to provide evidence and data for the pathogenesis of P.multocida infection.1.Detection of microbial community structure and diversity in lactating cows by 16S rRNA high-throughput sequencingThe 16S rRNA high-throughput sequencing technology was used to evaluate the differences on microbial flora of the upper respiratory tract between the lactating cows and replacement heifers with symptoms of respiratory diseases.This work collected nasopharyngeal deep swabs from 14 replacement heifers and 37 lactating cows.The results showed that Proteobacteria,Firmicutes and Bacteroidetes were the dominant bacteria.Alpha diversity analysis showed that the species diversity of nasal mucus in replacement heifers was significantly higher than that in lactating cows.At the phylum level,Proteobacteria in nasal pharynx microflora from replacement heifers was significantly higher than lactating cows.At the genus level,the bacteria of Moraxella and Acetobacterium in lactating cows were significantly higher than replacement heifers.This study providing references for the prevention and controlling of respiratory diseases in cattle farms.2.Comparative genomic analysis of virulent and avirulent strains of P.multocidaComparative genomic analysis was carried out between P.multocida high-virulence isolate PMPAN001 and low-virulence isolate PMPAN007.Although the genomes of PMPAN001 and PMPAN007 were highly similar,obviously differences also were found.Compared with PMPAN007,PMPAN001 had large deletions in 1640kb?1800kb.Based on homologous analysis,gene islands,insertion sequences,secreted proteins and virulence-associated genes were screened.The number of gene islands(GIs)in PMPAN001 was twice more than GIs in PMPAN007.There were many unique genes in PMPAN001,including ISAac3,ISKpn25,IS1016C2 and ISRso21,as well as virulence-associated genes flpC,fles,narH and so on.These unique genes may play important roles in virulence.The results showed the differences in genomic structure and function between virulent and avirulent strains.3.Differences of virulence-associated genes expression between virulent and avirulent P.multocida isolates in vivo and in vitro(fimA,tbpA,exbD,fur,oma87,pmHAS,nanH and tonB)This study compared the differences of PMPAN001&PMPAN007 isolates in organ bacterial loads,transcription level of virulence-associated genes in lung tissues and in vitro,and lung tissue injury.The proliferation rate of PMPAN001 was much higher than PMPAN007.In addition,the transcription levels of the same virulence-associated genes were different between PMPAN001&007.The transcription level of virulence-associated genes in vitro can not reflect the true situation in vivo.Genes fimA,exbD,fur,oma87,pmHAS and tonB which were highly expressed in high-virulent strains can be used as markers for evaluating the virulence of P.multocida.Among the 29 unique virulence-associated genes in PMPAN001,eight genes(flpD,fbpA,LH0147,flpC,HD1156,HIBPF19140,HD1505 and neuA)were expressed in lung tissue.Most of them were related to adhesion.It is possible that these genes play important roles in the pathogenicity progress.4.Construction of gene mutation vector of multocidaThe upstream and downstream homologous arms of PMPAN001 isolate genes flpC,LH0147,fbpA and flpD were amplified by PCR,and then the kanamycin resistance sequence from pUC-4K was inserted between the upstream and downstream homologous arms by fusion PCR.Finally,the fusion fragments were inserted into plasmid pBC-SK for constructing four recombinant suicide plasmids:pBC-SK-?flpC,pBC-SK-?LH0147,pBC-SK-?fbpA,pBC-SK-?flpD.Electrotransformation was used to transfer four recombinant plasmids into competent PMPAN001 cells.The colonies were all negative growing on the selective plates after many attempts.The correct gene knockout strains could not be screened.In this study,the conditions for constructing P.multocida mutants were explored,which laid a foundation for the further construction of P.multocida deletion strains. |
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