Isolation,Identification,Molecular Typing And Virulence Related Gene Detection Of Campylobacter Jejuni Isolated From Poultry In Some Areas Of Sichuan Province

The incidence and prevalence of campylobacteriosis caused by Campylobacter jejuni,one of the most widespread infectious diseases in the last century,have increased over the last ten years.Many animal may act as the asymptomatic reservoirs,and poultry is the main carriers.Animals do not have obvious symptoms,but they can be excreted long to the outside,polluting the environment.Ingestion of polluted food and water is the main cause of infection.Therefore,the prerequisite for controlling infection is to strengthen its monitoring.In order to provide a basis for monitoring epidemiology,this study isolated and identified C.jejuni from avian cecum and rectal swabs collected in Chengdu,Maishan and Yaan city of Sichuan Province,using ERIC-PCR,MLST typing and investigating seventeen kinds of virulence related genes to understand the distribution of pollution and virulence genes.A total of 469 chicken,duck and quail cecum and anal swab samples collected from Chengdu,Meishan and Yaan,were examined for the presence of C.jejuni.There are forty-eight strains of C.jejuni isolated,the positive rate is 10.23%.The result shows that the C.jejuni isolated from poultry market is 17.05%,which is extremely higher than that from farm(7.65%)(P<0.01).The C.jejuni incidence ofpoultry(12.30%)is higher than that of quail(5.41%)(P<0.05).The C.jejuni isolated from Jintang(22.50%)is extremely higher than that from Meishan(5.41%)(P<0.01).The positive rate in Wenjiang is 11.38%,which is higher than that from Meishan(P<0.05).For the different ways of breeding,C.jejuni isolated in scatter ways is higher than that in captivity(P<0.05),and C.jejuni isolated from cecum is higher than that from anal swab,but there is no significant difference between them(P>0.05).This study choosing the primers which are widely used,use ERIC-PCR method to classify all of the forty-eight C.jejuni.Cluster analysis showes that there are thirty-one genotypes,which are classified into sixteen groups and six clusters The genotype of each field shows a rich genetic diversity,and the isolates from different field have a unique genotype,while a few of genotypes show cross distribution on different regions and field except farm one on Wenjiang.Then all of the isolates are classified by MLST,producing twenty-four sequence types.Among all of the strains,twenty-two strains are assigned to a total of eleven new sequence types,which account for 45.83%of the total strains and sequence types.The genotype of strains showe diversity from other field except farm one in Wenjiang and farm five in Meishang.The strains belonging to the same sequence type are always from the same field,and different field isolates have a unique sequence type,while a few of strains isolated from different regions and different field show cross distribution.The isolates of C.jejuni in this study along with strains at home and abroad are did genetic evolution analysis,and the results show that isolates in this study have closely relationship with the international strains,and five isolates have closely genetic distance with the isolates from domestic patients with diarrhea.Twenty-one strains can be divided into the same group,accounting for 43.75%of the total number of isolates,and the results indicate that both two typing of methods have good synchronization.This study use PCR to investigate seventeen kinds of virulence related genes.The positive rates of virulence related genes including flaA,peb1A and cadF,which are related with adhesion,are 91.7%,81.25%and 93.75%.Colonization related gene cheY and racR infection rate are 95.83%.The carrying rate of invasion related genes pldA,iamA,ciaB and che W are 89.58%,68.75%,87.5%and 100%.There are 79.17%,97.92%and 83.33%strains carrying toxin regulatory gene cdtA,cdtB and cdtC.C.jejuni lipopolysaccharide core oligosaccharide(LOS)synthesis related genes wlaN and neuBl carrying rate are 52.05%and 35.42%.Heat shock protein grpE and dnaJ carrying rate are 95.83%and 89.58%.The plasmid vir B? gene carrying rate is 2.08%.Virulence related genes carrying rate of farm isolates is generally higher than the market sources of isolates,and quail strains carrying rate is generally higher than that from chicken and duck.The virulence related genes carrying rate of different types of aquaculture isolates are complex,and isolates that carrying more than 13 kinds of virulence related genes account for 72.92%of the total strains.