Establishment Of An Infection Model Of Pathogenic Escherichia Coli From The Lungs Of Forest Musk In BALB/c Mice And Sequencing And Analysis Of The Whole Genome

Pneumonia is one of the most severe diseases with strong pathogenicity in forest musk deer that affects the survival of musk deer breeding farms,the diseases seriously hindered the development of the musk deer farming.In the previous study,Lung pathogenic Escherichia coli?LPEC?strains were found to be one of the principal bacterial pathogens,and 078 was found to be the dominant serotype,thus it was selected as further study.In this study,the method of artificial infection was used to establish the BALB/c mice model of the LPEC 078 infection.The technology platform of Illumina Miseq and the bioinformatics methods were used to whole genome sequencing and comparative genome analysis,respectively.The study results were as follows:1 Establishment of the model of the LPEC 078 infection in BALB/c miceThe results of LD50 tests showed that LPEC 078 was pathogenic to BALB/c mice,the LDso to mice was about 3.6 × 108 CFU/pice.All the infected mice had clinical symptoms including reduced activity,reduced food intake,lethargy and ruffled fur.The mice of the control group had no clinical symptoms and were survived.The significant changes of blood biochemical index of infected mice were detected.The levels of ALT and AST were increased in different degrees.Paraffin sections from the liver,spleen,lungs,kidney,duodenum,jejunum,ileum,cecum,stomach,trachea,pancreas,rectum and colon of infected group had different degree of histopathological changes.2 Complete genome sequence,annotation and analysis of LPEC 078The genome size of LPEC 078 is 5,376,541 bp,gene average sequencing depth is 105×,GC content is 50.58%,203 contigs,88 tRNAs and 2 rRNA.Other components were predicted using bioinformatics softwares and databases.The major virulence genes of urinary tract infection?UTI?,neonatal meningitis-causing E.coli?NMEC?and avian pathogenic E.coli?APEC?were detected in the genome of LPEC 078,including type I fimbriae,hemin uptake proteins,hemolysin,iron/manganese transport proteins,type VI secretion protein and AslA,OmpA,TraJ,iss?traT?iroN?hly capsule,respectively.The results showed that an example of a genome of chimeric pathogenic properties was found.Furthermore,in the genome of LPEC 078,30 ISs and 8 intact phages were detected.3 Genetic evolution analysis and comparative genomics of LPEC 078The phylogenomic study indicated that LPEC 078 belonged to the D group of E.coli and clustered with human UPEC UMN026?GenBank:NC₀11751?,and had close relationship with EAEC 042?GenBank:NC₀17626?and UPEC IAI39?GenBank:NC₀11750?.The results were in favor of the possibility that strain of animal origin LPEC 078 was dangerous for public health.Orthologous analysis were completed by using genome sequences of the strains LPEC 078,UMN026,042 and IAI39.The total number of orthologous of 4 strains was 6362,in which there are 3579 orthologous were determined as the core gene clusters that shared by all 4 strains,accounting for 56.26%of the total orthologous.Significantly,strains LPEC 078 and UMN026 shared the largest gene clusters?165?more than other combinations.Results indicated that LPEC 078 was close to UMN026 in evolutionary relationships,the results were also consistent with phylogenetic analysis.Furthermore,orthologous analysis were performed by using genome sequences of the strains LPEC 078,APEC 078,ACN001 and ACN002.The total number of orthologous of 4 strains was 6208,in which there are 3664 orthologous were determined as the core gene clusters that shared by all 4 strains,accounting for 59.02%of the total orthologous.Significantly,strains APEC 078,ACN001 and ACN002 shared the largest gene clusters?388?more than other combinations.Results indicated that APEC 078 was close to ACN001 and ACN002 in evolutionary relationships,but far away with LPEC 078,the above results were also consistent with phylogenetic analysis.The specific genes were annotated with functional categories using the COG and GO databases.This study investigated found that most proteins involved in the amino acid,inorganic ions and carbohydrate metabolism.In the COG's classification and annotation features,the major function of specific genes is replication,recombination and repair,which might be related to the different habitats of the strains and also thought to be essential for growth and survival in LPEC 078.The experiment results also approved that the relatively large genome of strains usually had the strong metabolic capacity and stress tolerance,so that it could be adapted to different ecological environment.Furthermore,the core genes were annotated with functional categories using the COG and GO databases,the core genes of strains displayed an obvious enrichment in cell growth and metabolism,it also reflects the importance of the existence of the core gene for cell survival.