| Campylobacter spp are the most commonly reported bacterial causes of gastroenteritis worldwide. C. jejuni and C.coli are responsible for the most of human Campylobacteriosis, and Campylobacter spp may cause human sporadic and local epidemic of gastroenteritis outbreaks. The cases caused by Campylobacter spp are rising all over the world. As the normal asymptomatic bacteria colonizing intestinal tract of animals, Campylobacter spp are widespread in intestines of livestock and poultry. The waterfowl act as major reservoirs, without signs of pathology, shedding C. jejuni in their stools to environment in long term, which form a source of infection, causing other animals and human infections. This is a potential threat to society and human. Therefore, control and clear Campylobacter in intestinal tract of waterfowl is an important prerequisite to control human Campylobacteriosis. The objectives of this study were to: (1) investigate the prevalence of C. jejuni and C. coli among waterfowl flocks including geese and ducks and detect the antimicrobial susceptibility of isolates with K-B method recommended by Clinal and Laboratory Standard Institute in 2007; (2) Recombinant labeling C. jejuni SOC0612-2-pUOA18-dsRed was constructed successfully and was used to research the major means of transmission of C. jejuni among chickens and waterfowl. (3) Track the source and define molecular epidemiology and population structure of C. jejuni isolates from different origins by multilocus sequence typing.1 Epidemiological investigation and antibiotic resistance of C. jejuni and C. coli isolates from waterfowl and chicken flocksIn this study we attempted to investigate the prevance of C. jejuni and C. coli in waterfowl flocks in parts of Jiangsu and chicken flocks in Jiangsu, Shandong and Anhui from December 2007 to December 2009. Among 15 waterfowl flocks, 12 were positive (80.0%) for C. jejuni and 7 were positive (53.3%) for C. coli. 2182 of waterfowl cloaca were collected. The results showed that there were 380 strains of C. jejuni and 16 strains of C. coli, the positive rate was 17.42% and 0.73% respectively. Among all the waterfowl samples, 850 of goose cloaca were collected. The results showed that the positive rate was 34.94% of C. jejuni and 0.82% of C. coli respectively. The positive rates of C. jejuni varied from 0% to 65.52% and C.coli from 0 to 1.85%. The results showed that the C. jejuni incidence of geese from Market was significantly higher than that of geese from farm (p<0.05). And 1332 of duck cloaca were collected. The results concluded that the positive rate was 6.23% of C. jejuni and 0.15% C. coli respectively. The positive rates of C. jejuni varied from 0% to 18.87% and C.coli from 0 to 1.57%. The results showed that the C. jejuni incidence of ducks from Market was significantly higher than that of ducks from farm (p<0.01). Also, 960 of chicken cloaca from 5 chicken flocks were collected. The results showed that there were 251 strains of C. jejuni and 36 strains of C. coli, the positive rate was 26.15% and 3.75% respectively.Epidemiological investigation of C. jejuni and C. coli from different poultry flocks showed that C. jejuni incidence of goose and chicken mixed farming were significantly higher than that of them from sigle farm cultivation respectively (p<0.01). The results indicated that waterfowl, especially geese are the important reservoir of Campylobacter spp.The 101 C. jejuni strains including 54 goose strains,36 duck strains and 11 chicken strains were analyzed for susceptibility to 20 kinds of antibiotics. The results showed that these strains isolated from poultry were highly sensitive to seven kinds of antibiotics such as Azithromycin 96.04%, Erythromycin 91.09%, Gentamicin 82.18%, and were resistant to some frequently used medicines such as Cefoperazone 100.00%, Penicillin 93.07%, Cefradine 98.02%, Enrofloxacin 80.2%, Ciprofloxacin 82.18%, Co-trimoxazole 97.03%, Tetracycline 83.17%, Doxycycline 82.18%. The range of multidrug resistant of 101 C. jejuni from poultry was from 4 drugs to 18 drugs, most of which were resistant 11 drugs and 12 drugs, sharing 47.52% percentage of all isolates.The results of this study showed that the C. jejuni strains isolated from poultry were sensitive to some antibiotics, such as Amoxicillin, Cefotaxime, Cefaclor, Aminoglycosides, Macrolides and Clindamycin, but meanwhile, they were resistant to a number of antibiotics used in clinic, for instance, Quinolones, Tetracyclines, Cefradine and Cefoperazone, and so on. In a word, antimicrobial resistance especially multiresistance of C. jejuni became more and more diverse and complicated. It should pay more attention to the surveillance of antimicrobial resistance in C. jejuni, 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 The major transmission routes of C. jejuni between waterfowl and chickenTo study the major transmission routes of C. jejuni between waterfowl and chicken, recombinant plasmid pUOA18-dsRed was constructeded and electrotransformated into C. jejuni isolated strain SOC0612-2, and a recombinant labeling strain SOC0612-2-pUOA18-dsRed was constructeded successfully. The results of stability experiment in vitro showed that recombinant plasmid pUOA18-dsRed still existed in SOC0612-2 after it was passed 25 times on CCDA medium in the absence of antibiotics. The results indicated that it was feasible when recombinant bacteria SOC0612-2-pUOA18-dsRed was used as medium for researching the major transmission routes of C. jejuni between chicken and waterfowl.The results of animal experiments showed that before challenged with lable bacteria, all of the poultry were none C. jejuni infections. After challenged with lable strain, cloacal awab of each duck of 5 groups were detected at 3rd, 6th, 9th, 12th, 15th and 18th days after infections. The results indicated that the ducks in group of mixed farming with chickens first infected with lable bacteria, and the number of infected ducks had been rising with days increase. Meanwhile, one or two ducks of other groups expence control group were infected with lable bacteria, and the number of infected ducks also had been rising with days increase. On 18th days, more than half of ducks from group of transmission with stool and group of mixed farming were infected and half of ducks from other two groups were infected by recombinant strain.The ducks of group mixed farming were first infected with lable bacteria, and the number of C. jejuni of cloaca were 1250 CFU per duck. On the 6th day, the CFU number reached the largest amount and the average number were 4000CFU per duck, then with the increase in the number of days, the average number of C. jejuni of each duck showed constancy. In the ducks of other groups expect control group, the CFU number reached the largest amount, and then with the increase in the number of days, the average number of C. jejuni of each duck showed constancy. In a word, the results showed that the the major transmission routes of C. jejuni between chickens and ducks were diverse and complicated. The transmission route by digestive tract was regard as the most common route, but the feces, food, water and aerosol nearby the poultry had an important influence on C. jejuni infected by poultry flocks. Separation of different flocks of poultry farming and maintain a clean sanitation will help to reduce the spread of Campylobacter spp infection.3 Genotyping of C. jejuni isolates from different origins using MLST methodAccording to the previously studies published, 7 housekeeping genes were selected and their primers for amplification and sequencing were synthesized. 167 C. jejuni strains from different sources were analyzed using MLST method. The genotyping results of MLST showed that there were 94 STs in these isolates and all 94 STs were grouped to 18 clonal complex and unassigned. Among all of these isolated strains, 60 strains belong to 45 novel STs. Among these 60 strains, 20 strains had 22 new allele sequences: aspA (n=3), glnA (n=5), gltA (n=5), glyA (n=2), pgm (n=1), tkt (n=6), and the uncA had no new allele sequence.The largest number of allele sequence was glnA (n=26) and the least was uncA (n=11). Among 94 STs, 9 STs belonged to ST-353 clonal complex, accounting for 9.57% of all STs, meanwhile, ST-21, ST-45, ST-52 and ST-692 complex comprised 6 STs respectively. Moreover, 27 STs belonged to singletons, accounting for 28.72% of all STs. Among all 18 complex, ST-353 had the largest number of isolated strains, and it contained 20 strains, accounting 12.0% of all strains. ST-354 complex and ST-45 complex had the second largest and the third largest number of strains. 76.6% of all isolates had complex, and 23.4% of all isolates belonged singletons. 102 isolates, accounting for 61.1% of all isolates, belonged to eight complex that contained the largest number of strains. 51.70% of human isolates, 60.40% of chicken isolates, 65.00% of cattle isolates, 80.00% of food strains, 80.00% of pet strains and 68.40% of waterfowl isolates belonged these 8 complex.Among all 94 STs, ST-354 comprised the largest number of isolates and it contained 11 isolates, accounting for 6.59%. 35 different STs contained 2 or more isolates, and the rest 59 different STs contained only one isolates respectively. Among all typed 60 human isolates, 18 isolates belonged to singletons and 12 isolates belonged to ST-353 complex, accounting for 30.0% and 20.0% respectively. Among 48 chicken isolates, 13 isolates belonged to singletons, meanwhile, 10 isolates and 9 isolates belonged to ST-354 complex and ST-574 complex, accounting for 20.83% and 18.75% of chicken strains respectively. Among 20 cattle isolates, the ST-22 complex had the largest number isolates and it contained 9 isolates, accounting for 45.0%. Among 10 isolates from food, ST-353, ST-354, and ST-45 complex contained 8 strains, accounting for 80.0%. 10 strains isolated from pet were separated to 6 different complex, and ST-45 complex contained 3 isolates, accounting for 30.0%. Among all isolated 19 strains, 7 isolates belonged to ST-692 complex, accounting for 36.84%.In this study, among all 94 different STs, 21 different STs contained 2 or more different sources isolates. Two STs, ST-354 and ST-137 contained 4 different sources isolates, and the rest 19 STs contained 2 different sources isolates. Among these 21 different STs, 15 STs contained human isolates and isolates from other sources. 167 isolates from different origins and 2 standard C. jejuni strains that belonged to 96 different STs, were classificated into 9 clonal groups and singletons by UPGMA dendrogram of the mean normalized pairwise differences between alleles for seven genes fragments. Group 5 had the largest number of STs and it contained 55 different STs.The results showed that MLST could be a powerful for the investigation of molecular epidemiological pattern, tracing the source of C. jejuni and researching population structure of C. jejuni. It was concluded that many complex and STs had the isolates from different source by genotyping of C. jejuni isolates from human, chicken, cattle, waterfowl, food and pet. These findings manifested that these animals and poultry food products was closely related with human C. jejuni infections, and they may had the same infection sources. In a word, human Campylobacteriosis was intimate relevant to animal and food, especially chicken. |
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