Analysis Of Virulence-associated Genes Of Isolated Campylobacter Strains In Parts Of China And Establishment And Application Of A Campylobacter Jejuni Infection Model On HT-29 Cells

Campylobacter jejuni is currently recognized as one of the leading causes of bacterial-induced gastroenteritis in human worldwide. It is also the most frequent infectious agent associated with the development of immunoreactive complications such as Guillain-Barre and Miller-Fisher Syndrome. Genetic characteristics and pathogenic mechanisms of Campylobacter jejuni are rarely known due to the lack of appropriate infection models that mimic the disease seen in human bodies. The objectives of this study are to: (i) investigate the prevalence of virulence-associated genes in Campylobacter jejuni and Campylobacter coli isolates in parts of China; (ii) determine the adhesion, invasion and multiplication properties of Campylobacter jejuni by using HT-29 cells in vitro and preliminarily analyze the relationships between invasive ability and virulence-saaociated genes of Campylobacter jejuni.1. Analysis of virulence-associated genes in C. jejuni and C. coli isolates in parts of ChinaIn this study we attempted to investigate the prevalence of virulence-associated genes in Campylobacter isolates in parts of China. A total of 453 C. jejuni isolates and 40 C. coli isolates from diarrheal patients, fowl, cattle, food products and market sewages were detected their virulence-associated genes. The results showed that the average rates of all kinds of virulence-associated genes in C. coli isolates were lower than those of C. jejuni isolates. Of the 453 C. jejuni isolates tested, the average positive rates of the adhesion related gene cadF, peb1A, racR were 91.39%, 95.36% and 97.79% respectively, the average rates of the chemotaxis gene cheY and docA were 95.81% and 92.27% respectively, invasion-associated gene iamA was 85.87%, the flagellar gene flaA was 83.66%, but the average rates of the virulence-associated genes cdt, wlaN and vir B11 were 72.19%, 48.12%, 6.57% respectively. Comparing of the positive rate of same gene in C. jejuni isolates from various sources, it was shown that the positive rates of gene flaA and virB11 were higher in the isolated diarrheal patients strains, cadF, docA, iamA and cheY were higher in the isolated cattle strains, and peb1A, racR, cdt and wlaN were higher in the isolated food product and market sewage strains, the average positive rates of genes in fowl isolates were at the medium level. From above results, it was concluded that 393 of 453 strains (86.75%) carried over 6 virulence-associated genes. There were remarkable differences in the presence of virulence-associated genes carried by C. jejuni isolates from various sources. Presumably the pathogenesis of C. jejuni in various hosts may be different.2. Establishment and application of a C. jejuni infection model on HT-29 cellsIn an in vitro cell culture model using HT-29 cells, the adhesion, invasion and multiplication properties of C. jejuni isolates from various sources were studied. Additionally, the relationships between invasive ability and virulence-associated genes of C. jejuni were preliminarily analysed. 42 isolates were tested for their ability to adhere to, invade and multiply in HT-29 cells in vitro. Of the 42 strains tested, 11 did not adhere to the HT-29 cells, 13 did not invade the HT-29 cells, 3 did not multiply in the HT-29 cells. The average adhesion rate, invasion rate of the remaining 15 C. jejuni isolates were 0.2270% and 0.0778% respectively and their average multiplication times was 12.25.212 C. jejuni isolates were tested for their ability to invade HT-29 cells in vitro. Of the 212 strains tested, 116 (54.72%) did not invade the HT-29 cells, 32 out of 212 strains (15.09%) invaded the cells at a very low level (<0.01%), 27 strains (12.74%) invaded the cells at a very high level (>0.1%) and the remaining 37 strains (17.45%) invaded the cells at a medium level (0.01%-0.1%). C. jejuni isolates from diarrheal patients were more invasive than the isolates from fowl.The analysis of the relationships between invasive ability and virulence-saaociated genes of C. jejuni showed the positive correlation between the prevalence of cdt, virB11, wlaN genes and C.jejuni invasive ability, the negative correlation between the prevalence of cadF, peb1A, docA, flaA genes and C. jejuni invasive ability. Comparing with the positive rate of same gene in C. jejuni isolates from fowl, the positive rates of gene virB11, cdt were much higher in C. jejuni isolates from diarrheal patients. Possibly it was one of the reasons why C. jejuni isolates from diarrheal patients were more invasive than the isolates from fowl.