Epidemiological Survey Of Campylobacter Jejuni In Chickens In Parts Of China And The Immunobiological Properties Of Chitosan Harboring Recombinant Plasmid Expressing FlaA

Campylobacter are the most common zoonotic bacteria associated with human diarrhea in both developing and developed countries. Two thermophilic species, C. jejuni and C. coli are responsible for the vast majority of human Campylobacteriosis (～99% and～1%, respectively). A considerable number of Guillain-Barre syndrome(GBS) patients present with a prior history of Campylobacteriosis, and GBS is considered a sequela of infections caused specifically by C. jejuni. GBS is the most common acute flaccid paralysis due to an autoimmune disorder in nature. Animals (such as chicken, cattle, pigs, sheep, dogs and so on) may act as asymptomatic reservoirs, shedding C. jejuni in their stools resulting in the contamination of animal food products and the surface water nearby during slaughter and carcass dressing. Chickens, which are often heavily colonized with Campylobacter spp. without signs of pathology, are considered the most important source for human infection. It is generally assumed that Campylobacter spp. contaminate poultry constitute a major risk to human health.The objectives of this study were to: (i) investigate the prevalence of C. jejuni and C. coli among poultry flocks in parts of China; identify the isolates by phenotypic tests and detect the antimicrobial susceptibility with K-B method recommended by Clinical and Laboratory Standard Institute in 2005; (ii) establish and apply a C. jejuni infection model on HT-29 cells (iii) track the source of C. jejuni isolates form different origins by RAPD and PFGE; (ⅳ) develop and charaterize novel DNA vaccine candidates against C. jejuni.1 Epidemiological investigation and antibiotic resistance of C. jejuni and C. coli among chicken flocks in parts of ChinaIn this study we attempted to investigate the prevalence of C. jejuni and C. coli in chicken flocks in parts of China from June 2005 to December 2007. Among 40 flocks, 34 of them (85.0%) were detected positively for C. jejuni, and 16 were positive (40.0%) for C. coli. 4891 of chicken cloaca samples from grandparent,parent and commercial flocks of different breeds chickens were collected for the isolation and identification of C. jejuni or C. coli. The results showed that there were 773 strains of C. jejuni and 55 strains of C. coli isolated, the average positive rate was 15.80% and 1.12% respectively. The positive rates of C. jejuni varied from 0 to 73.3%, and C. jejuni from 0 to 7.4% on different chicken farms. The results showed that the C. jejuni incidence of poultry raised in large scales was significantly higher than that of chicken raised in a free-range system (P<0.01). Statistical analysis indicated that the C. jejuni positive rate of grandparent flocks was significantly higher than that of parent and commercial flocks (P<0.01).The 240 isolated C. jejuni from patients with sporadic diarrhea were analyzed for susceptibility to 21 kinds of antibiotics, and the results showed that these isolated strains were highly sensitive to seven kinds of antibiotics such as Amoxicillin 96.67%, Gentamicin 90.00%, Azithromycin 91.25%, Streptomycin 87.92%, Erythromycin 85.42%, and were resistant to some frequently used medicines such as Cefradine 98.33%, Cefoperazone 100%, Cefaclor 92.50%, Norfloxacin 92.08%, Enrofloxacin 95.68%, Ciprofloxacin 92.08%, Levofloxacin 91.25%, Co-trimoxazole 99.58%, Tetracycline 89.58%, Deoxycycline 88.33%. The percentage of isolates were 90.00% with resistance to 8 to 14 antimicrobials. Antibiotic resistance of 34 isolated C. coli strains were all evaluated by 21 kinds of antibiotics, and the results showed that these isolates were only high sensitive to Amoxicillin 91.18%, compared with the isolates of C. jejuni, the multidrug resistance of isolated C. coli was more severe.The results of this study showed that the chicken isolated strains were resistant to a number of common antibiotics used in clinic including Quinlones, Sulfonamides, Tetracyclines, and most cephalosporins. Compared with the isolates of C. jejuni, the resistance of isolated C. coli was more severe. It should pay more attention to the surveillance of antimicrobial resistance in Campylobacter spp., and it is important for inspecting the trends of antimicrobial susceptibility and supporting development of public policies for better prevention and treatment of Campylobacteriosis.2 Analysis of virulence-associated genes of chicken C. jejuni isolates and their infection capacity on HT-29 cell lineOf the 282 C. jejuni isolates tested, the average positive rates of the adhesion related gene cadF, peb1A, racR were 90.07%, 95.04% and 97.87% respectively, the average rates of the chemotaxis gene cheY and docA were 95.39% and 92.20% respectively, invasion-associated gene iamA was 87.94%, the flagellar gene flaA was 86.17%, but the average rates of the virulence-associated genes cdt, wlaN and vir B11 were 71.28%, 48.94%, 7.08%, respectively. 86.75% strains carried over 6 virulence-associated genes. From above results, these data did not show remarkable differences in the presence of pathogenic genes carried by C. jejuni from various sources in chicken.Chicken C. jejuni isolates were tested for their ability to invade HT-29 cells in vitro. Of the 119 strains tested, 65 (54.62%) did not invade the HT-29 cells, 44 out of 119 strains (36.97%) invaded the cells at a very low level (<0.1%), 10 strains (8.40%) invaded the cells at a very high level(>0.1%). The analysis of the relationships between invasive ability and virulence-associated genes of C. jejuni showed the positive correlation between the prevalence of cdt, wlaN genes and C. jejuni invasive ability, the negative correlation between the prevalence of flaA, cadF, docA, racR, iamA, peb1A and cheY genes and C. jejuni invasive ability. There was no one to one association between virulence-associated genes and invasion or colonization ability in the chick gut.In vitro studies showed adhesion and invasion ability to HT-29 cells of strains of lineageⅣwere higher than those of lineageⅠ,ⅡandⅢ. The analysis of the relationships between invasive ability and virulence-associated genes of Chicken C. jejuni isolates showed that cadF,peb1A,racR,cheY,docA,iamA were the major factors to infect HT-29 cells3 Genotyping of C. jejuni isolates from different origins using PCR-RAPD and PFGE subtyping methodsC. jejuni isolated from different sources (n=337) were subtyped by PCR-RAPD. The PCR products showed 27 different banding profiles, 3 lineages and 10 clonal groups. Profile number 5, 6, 7, 9, 12, 14, 15, 22 were the most frequently observed among different sources, accounting for 83.09%. In this study, profile number 5, 9 14 and 15 were predominant in the isolates, which were 14.84%,17.51%,11.28% and 12.76%, respectively. In addition, profile number 5, 7, 9, 14 and 15 were found in human, bovine and poultry isolates, accounting for 63.51%. PFGE analyses showed that the Chinese isolates could be separated into 4 genetic lineages and 46 clonal groups, respectively. The PFGE lineage classification for chicken isolates possessed dominance among lineageⅠ,Ⅱ,ⅢandⅣ, which were 50%, 65%, 70.59%, 64.10%, respectively. The lineage of isolate from human patient, cattle and food were distributed all lineages. These findings indicated that isolated strains from different source overlaped each other in all clonal groups, presenting the possibility that bovine and food, as well as chickens, may serve as a source of human Campylobacteriosis.The results showed that PCR-RAPD and PFGE could be powerful tool for the investigation of molecular epidemiological pattern of C. jejuni and for tracing the source of C. jejuni in clinical patients, and PFGE allowed much higher discrimination among the isolated strains than RAPD. These findings indicated that C. jejuni isolates had predominant lineage, genotype distribution of the difference resource isolates overlaped. Human Campylobacteriosis was intimate relevant to animal and food, especially chicken.4 Immunobiological properties of the nanoparticle of chitosan harboring DNA Vaccine pCAGGS-flaA against C. jejuniAccording to the sequences published in GenBank(GenBank Accession NO. gi30407139), a pair of primers were designed to amplify gene flaA of C. jejuni by PCR. Then the PCR product was cloned into pGEM-T easy vector to give rise to pT-flaA. The Eco R I-Xba I-restricted insert of pT-flaA was cloned into eukaryotic expression vector pCAGGS, the recombinant vector pCAGGS-flaA was constructed. The plasmid pCAGGS-flaA was transfected into COS-7 in order to dectect the expression of inserted gene by immunofluorescence staining. The results showed flaA was expressed in COS-7. To develop the oral immunological DNA vaccicnes with chitosan(CS), the recombinant plasmid was encapsulated with chitosan to nanoparticles. The imaging by transmission electron microscope (TEM) demonstrated that the diameter of the particles was around 100 nm and the shape was spherical or eliptical. The result of encapsulated efficiency of chitosan-plasmid nanoparticles showed that more than 90% plasmid DNA was encapsulated with chitosan.In order to evaluate the novel DNA vaccine, 1-day-old commercial White Leghorn chickens were immunized orally with chitosan-plasmid nanoparticles at two weeks interval. Negative control groups, including blank control and CS-pCAGGS control, were Low titers of specific antibodies were observed in all immunized groups and negative control groups two weeks at second postinoculation (PI). But the titers of specific antibodies in positive control group were elevated significantly at third PI two weeks (p<0.01). Ratio of CD4+/CD8+ T cell in cecal tonsil and spleen of pCAGGS-flaA group was step-up at third PI two weeks. RT-PCR showed that the expression of IL-4 in spleen was enhanced at third PI two weeks, but the level of IFN-γhad no significant difference between two and third PI.After challenged orally with 5×107 isolated strain ALM-80, the percentage of C. jejuni -positive chickens in cloaca showed downtrend in vaccine group and no detection after 21d, but the negative control groups holded metastable high positive ratio. Among vaccine groups, the number of strain ALM-80 in small intestine, large intestine, cecum and blood acquired peak from 9d to 15d, and then downtrend. But otherwise, there was significant difference compared with the negative control groups, which were given to uptrend. Among CS-pCAGGS-flaA group, ALM-80 could be cleaned in small intestine at 18d, compared with the negative control groups, there was 2lg-3 lg, 2lg and 2lg-3lg reduction of quantity in large intestine, caecal and blood respectively when chicken were challenged with the homologous strain.