Analysis Of Epidemiological Characteristics Of Escherichia Coli And Its Virulence Factors In Diarrhea Rabbits From Parts Of Sichuan

E.coli?Escherichia coli?can cause diarrhea in a wide range of hosts,such as humans,mammals,and so on.Some strains with high virulence,propagation and drug resistance pose a great threat to public health and livestock production.In order to master the epidemic situation of diarrhea-related virulence genes and clarify the molecular epidemiologic characteristic of E.coli in major large scale breeding areas of rabbits in Sichuan,the distribution of virulence genes and molecular subtyping of the E.coli was studied.Gathering anal swab samples of diarrhoeal rabbits from parts of Sichuan?Chengdu,Deyang,Yaan,Zigong,Mianyang?during 2015²017,then the rabbit E.coli was isolated by selective mediums of Mac conkey?MAC?and Eosin-methylene blue?EMB?,identification of morphology and specific PCR.After that,17 virulence genes about enterotoxin,Stx?Shiga-toxin?,fimbrial adhesins,invasive factors,EAEC?Enteroaggreg-ative Escherichia coli?and PAI?Pathogenicity island?were detected by PCR,and the virulence gene distribution of the isolates was compared by Fisher's exact test.After using MLST?Multilocus sequencing typing?method to do PCR amplification and sequencing of 7 housekeeping genes,it would get the sequence type in the Warwick database.Gathering anal swab samples of diarrheal rabbits from parts of Sichuan?Chengdu,Deyang,Yaan,Zigong,Mianyang?during 2015²017,then the rabbit E.coli was isolated by selective mediums of Mac conkey?MAC?and Eosin-methylene blue?EMB?,identification of morphology and specific PCR.After that,17 virulence genes about enterotoxin,Stx?Shiga-toxin?,fimbrial adhesins,invasive factors,EAEC?Enteroaggreg-ative Escherichia coli?and PAI?Pathogenicity island?were detected by PCR,and the virulence gene distribution of the isolates was compared by Fisher's exact test.After using MLST?Multilocus sequence typing?method to do PCR amplification and sequencing of 7 housekeeping genes,it would get the sequence type in the Warwick database.Then,constructing phylogenetic trees and analyzing phylogenetic relationships among different sequence types based on housekeeping gene sequence by using MAGA6.0 and SplitsTree4 software.In addition,DnaSP6 software was used to analyze the nucleotide diversity of housekeeping genes.The PFGE?Pulsed field gel electrophoresis?scheme of CDC?Centers for Disease Control and Prevention?'s PulseNet was used for genotyping with Quantity One software.Finally,the correlation among the typing results of MLST,PFGE and the detection of virulence genes was analyzed by synthesis.The result,39 strains of E.coli were isolated and identified from diarrheal rabbits.Strains carried 6 virulence genes among 17 virulence genes which were detected by PCR.LEE?Locus of enterocyte effacement?pathogenicity island,eae?41.0%??ler?41.0%?;Rabbit fimbrial adhesins,ral?33.3%??afr2?10.3%?;HPI?Yersinia high-pathogenicity island?,irp2?15.4%?;Enterotoxin,astA?7.7%?,respectively.The other 11 virulence genes?aggR?aap?aaf?aggA?IpaH?ST_a?ST_b?LT?stx₁?stx₂?afr1?were not detected.24 of 39 E.coli carried at least one or more virulence genes related to diarrhea.6virulence gene combinations are eae-ler-ral?50%??eae-ler-afr2?13%??eae-ler-afr2-ral?4%??irp2?21%??astA?8%?and astA-irp2?4%?,respectively.The results of statistical analysis showed that there was a distinguished difference in the virulence distribution in Chengdu and Deyang between 2015²016 and 2017,and in the virulence distribution among Chengdu,Deyang,Yaan,Zigong and Mianyang in 2015²016.MLST analysis of 39 strains of E.coli identified 17 sequence types.The main sequence types were U328?8 strains?,ST162?6 strains?,ST328?4 strains?,ST380?3 strains?and ST20?3strains?.Genetic evolution analysis of housekeeping gene sequences showed that U328expressed a close relationship to ST328.Nucleotide diversity?Pi?of 7 housekeeping gene sequences was 0.00342?purA?⁰.01374?fumC/icd?.39 strains of E.coli were subtyped into 21 types by PFGE.The strains showed high diversity in general,but some regions also have a single genotype.The research showed that 39 strains of diarrhea rabbit E.coli had high diversity in 5areas of Sichuan,but there was a single genotype of E.coli in some breeding areas.The study of virulence genes showed that there were space-time differences in the virulence gene profile of rabbit E.coli in the investigated area,and the virulence genes of eae,ler,ral and afr2 were closely related to MLST sequence type.