| Campylobacter is the most commonly identified bacteria causing human gastroenteritis worldwide, only after Salmonella and Shigella. Poultry are considered the natural resevoir of Campylobacter. Many chicken flocks are infected with Campylobacter but showed no signs of illness during their rearing period. And Campylobacter are shedding sustainable to the outside world, which is easily to cause contamination of chicken meat and its products during slaughtering, processing and retail. The public health benefits of controlling Campylobacter in primary broiler production are expected to be greater than control later in the food chain, and which would reduce the risk of human exposure to this pathogen.It is a necessary prerequisite to prevent and control Campylobacter contamination that we should fully recognize the ecological transmission rule of Campylobacter infection during rearing period. Although many countries had carried out systematic studies of Campylobacter on different host and food, but the quantitative epidemiological studies on Campylobacter during poultry rearing period are relatively scarce. This study aims to survey the qualitative and quantitative epidemiological characteristics of Campylobacter on fixed point chickens during rearing period. We then used pulsed-field gel electrophoresis (PFGE) for genotyping research, and detected the antimicrobial resistance and the virulence genes of Campylobacter isolates, in order to explore the ecological rule of colonization and dissemination during rearing period, providing valuable guidance for daily management and the control of Campylobacter.1. Quantitative tracking survey of Campylobacter in broilers during rearing periodTo assess the risks of Campylobacter in poultry rearing period, qualitative and quantitative surveys were performed in succession. In this study, cloacal swabs from 30 fixed point chickens of each flock, and environmental samples were collected from poultry rearing period. The level of Campylobacter contamination was tested using the direct plating method and molecular biology method.The results showed that Campylobacter were detected after three weeks of age in chicken flocks. The infection rate and enumeration were quickly reached a peak after infection, then declined and tended to be stable. The highest infection rate was 100%, and the Campylobacter enumeration were 103 to 107 CFU/g. Most broiler chickens had been carried Campylobacter throughout their rearing period, and the separation of C. jejuni was higher than C. coli; the mixed contamination of C. jejuni and C coli was also higher (14.4%). In environmental samples, the positive rate of Campylobacter isolated from sewage was the highest (15.8%), and the isolates were usually C. coli. Quantitative results of different age broiler’s tissues showed that cecum was the predominant site for Campylobacter colonization, and the enumeration reached up to 2.55×108 CFU/g. Survey results indicated that once Campylobacter colonized in a chicken, it would quickly spread to the whole flock. And accompanied with high infection rate, the enumeration was also higher, posed a serious threat to slaughtering, processing and retail.2. Analysis of ecological transmission rule of Campylobacter during rearing period by using PFGEIn total 365 Campylobacter isolates from different broiler flocks during rearing period were typed by PFGE. The PFGE results were analyzed, and PFGE patterns with Dice coefficients (F value) greater than 0.75 were classified as related.The results showed that 7 flocks of Campylobacter isolated from non fixed point chickens had 7 to 25 PFGE genotypes.164 Campylobacter isolated from fixed point chickens were subdivided into 79 PFGE genotypes, and each chicken had 6 to 10 PFGE genotypes during their rearing period. For some chickens of native chicken flock, Campylobacter which were obtained at the same time point had different PFGE genotypes (PF1, PF8 and PF15 had isolates recovered from 8 weeks), and Campylobacter which were obtained at different time point had the same PFGE genotypes (PF4, PF8 and PF9). All these results showed that the genetic diversity of Campylobacter isolates recovered from broiler rearing periods was both diverse and conservative. The PFGE genotypes of Campylobacter isolated from the same weeks of the fixed point chickens ranged from 6 to 21, which had a correlation with the age of flocks and the number of isolates. The PFGE genotypes of Campylobacter isolates from environmental samples were related genotypes with the isolates from chickens, and Campylobacter isolates from environmental samples had multiple PFGE genotypes, which was one of reasons for the prevalence and genetic diversity of Campylobacter in chicken flocks.These findings manifested that the PFGE patterns were closely related with strains isolated from chicken and environmental samples, which indicated that Campylobacter in the environment was a main source for the infection of chicken flocks. The results showed that PFGE could be a powerful tool for the investigation of infection, spread and population structure of Campylobacter.3. Drug sensitivity and virulence genes analysis of Campylobacter isolated from poultry rearing periodThe 165 Campylobacter strains from different weeks of age were analyzed for susceptibility to 21 kinds of antibiotics. The results showed that these strains were highly resistant to penicillin G, cefaclor, norfloxacin, ciprofloxacin, levofloxacin, nalidixic acid, sulfamethoxazole, tetracycline, and were highly sensitive to amoxicillin/clavulanate, imipenem. The range of multidrug resistant of 165 Campylobacter from poultry was from 8 to 18 drugs, most of which were resistant 8 and 15 drugs, sharing 86.05% percentage of all isolates. Compared with the isolates of C. jejuni, the drug resistance and multidrug resistance of isolated C. coli were more severe. The resistant characteristics analysis of Campylobacter isolated from different weeks of age showed that penicillin G, quinolones, sulfonamides and tetracyclines antibiotic resistance did not change with the increase of age. Cephalosporin antibiotic resistance rates increased with the increase of age. And the antibiotic resistance rates of penicillins, aminoglycosides, macrolides, lincomycin in 7 weeks,9 weeks,11 weeks of age was significantly higher than other weeks (p <0.05). In addition, the multidrug resistance of Campylobacter isolates from different weeks was changed. The range of multidrug resistant of Campylobacter from 7 weeks,9 weeks,11 weeks of age was from 12 to 18 drugs, while other weeks were resistant 8 to 12 drugs.Of the 95 Campylobacter isolates tested, the positive rates of flaA (91.58%) and cadF (95.79%) were highest in the adhesion-associated genes. For colonization- associated genes, positive rate of racR (90.53%) was highest. The positive rate of invasion-associated genes was less than 60%. For LOS-associated genes, positive rate of neuBl (80%) was highest. For the 3 toxin-associated genes, positive rate of cdtB was highest. The positive rate of 27 virulence genes changed with the increase of age. The positive rate of peblA, wlaN and neuBl increased with the increase of age. The positive rate of 19 genes (flaA, cadF,peb4, galE, waaF, racR,fliG, cheY, rpoN,fliA, doc A, flgK, imaA, pldA,flaC, ciaB, cdtB, cdtC and hipO) decreased at first and then increased. The analysis of drug susceptibility and distribution of virulence genes provided important information for the prevention and control of Campylobacter. |
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