水禽空肠弯曲菌流行病学及分离株的多位点序列分型分析
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摘要
弯曲菌(Campylobacter)是全球范围内最常见的细菌性肠道病原菌,对人类致病的主要是空肠弯曲菌和结肠弯曲菌,能引起散发性和地方流行性的胃肠炎暴发,由其引起的弯曲菌病病例在世界各地普遍呈上升趋势。弯曲菌广泛存在于家禽家畜的肠道和自然环境中,水禽是导致人弯曲菌感染的潜在宿主,感染的动物通常无明显病症,但可长期向外界排菌,通过排泄物污染食物和饮水,引起人类及其它动物感染。因此,控制或清除水禽弯曲菌是预防和控制人弯曲菌感染的重要前提之一。本文研究的主要目的是:(1)较为系统地调查与分析水禽中弯曲菌的流行现状与特点,对分离株进行耐药性分析。(2)通过构建重组标签空肠弯曲菌分析弯曲菌在水禽和家禽之间可能的传播途径。(3)应用多位点序列分型技术(Multilocus sequence typing, MLST)对不同源空肠弯曲菌分离株进行分子分型研究,探究菌株间的分子流行病学特征及种群结构。
一、水禽弯曲菌流行现状和耐药性调查与分析
2007年12月至2009年12月,采集江苏部分地区的鹅源、鸭源和江苏、山东、安徽鸡群样品进行空肠弯曲菌和结肠弯曲菌流行病学调查。15个水禽群体中,空肠弯曲菌阳性的有12个、阳性率为80.0%,结肠弯曲菌阳性的有7个、阳性率为53.33%。在2182份水禽泄殖腔棉拭样品中,共检测出380份空肠弯曲菌阳性、阳性率为17.42%,16份结肠弯曲菌阳性、阳性率为0.73%,其中空肠弯曲菌和结肠弯曲菌混合感染的样品为6份,阳性率为0.27%。其中,在850份鹅泄殖腔棉拭样品中,空肠弯曲菌平均阳性率为34.94%、结肠弯曲菌平均阳性率为0.82%;在1332份鸭群泄殖腔棉拭样品中,空肠弯曲菌阳性率为6.23%、结肠弯曲菌阳性率为0.68%。不同水禽群感染率悬殊较大,空肠弯曲菌阳性率从0到65.52%、结肠弯曲菌阳性率从0到1.85%。数据分析显示,鹅群空肠弯曲菌感染率极显著高于鸭群感染率( p<0.01),活禽市场鹅群感染率显著高于养殖场中鹅群感染率( p<0.05);活禽市场鸭群空肠弯曲菌感染率极显著大于养殖场中鸭群感染率( p<0.01)。
在960份鸡泄殖腔棉拭样品中,共检测出251份空肠弯曲菌阳性、阳性率为26.15%,36份结肠弯曲菌阳性、阳性率为3.75%,其中空肠弯曲菌和结肠弯曲菌混合感染的样品为15份、阳性率为1.56%;不同鸡群感染率悬殊较大,空肠弯曲菌阳性率从7.58%到73.08%、结肠弯曲菌阳性率从0到5.77%。5个鸡群均为空肠弯曲菌阳性,群体阳性率为100%、结肠弯曲菌群体阳性率为80%,数据分析显示,规模化圈养条件下鸡群的空肠弯曲菌携带率显著高于散养鸡(p<0.05)。进一步的流行病学调查表明,混合养殖方式中鹅群和鸡群的空肠弯曲菌感染率极显著高于单一养殖方式的感染率(p<0.01)。
水禽弯曲菌流行病学调查表明,水禽特别是鹅群是弯曲菌的重要宿主,是弯曲菌感染流行和环境污染的重要因素。
54株鹅源、36株鸭源和11株鸡源分离株的耐药性分析显示,对8大类20种抗生素高度敏感的是:阿齐红霉素(AZM) 96.04%、红霉素(E) 91.09%、庆大霉素(CN) 82.18%,高度耐药的是:头孢哌酮(CFP)100.00%、青霉素G (P)93.07%、头孢拉定(CE)98.02%、恩诺沙星(ENR)80.2%、环丙沙星(CIP)82.18%、复方新诺明(SXT)97.03%、四环素(TE)83.17%、强力霉素(DO)82.18%。耐药菌株的多重耐药分布于4耐到18耐不等,其中11耐至12耐最多,占47.52%。
不同禽源空肠弯曲菌分离株药敏试验结果比较表明,分离株均对头孢菌素类的头孢哌酮和头孢拉定、青霉素G及磺胺类药物产生了很强的耐药性,水禽源分离株对头孢克罗产生的耐药性显著低于鸡源(p<0.01),而对喹诺酮类和四环素类抗生素,虽然水禽源分离株也产生了较强的耐药性,但低于鸡源分离株的耐药性。对其它种类抗生素,各不同源分离株之间的耐药性差别较小。
二、鸡空肠弯曲菌对水禽传播途径分析
鸡群是全球公认的弯曲菌的主要宿主之一,为研究鸡群体内空肠弯曲菌能否传播给水禽,本研究通过构建重组质粒pUOA18-dsRed,并将其用电转化的方法转化至空肠弯曲菌分离株SOC0612-2,成功获得重组菌SOC0612-2-pUOA18-dsRed,体外稳定性实验表明质粒pUOA18-dsRed在无抗性CCDA固体培养基上经过连续传代25次后仍能稳定存在。将该重组菌作为标签细菌研究空肠弯曲菌在水禽和鸡之间可能的传播途径。动物实验中,动物分成5个试验组,每只动物从1日龄养至4周龄。第一组放置8只笼养鸭子,接种方式为肌肉注射;第二组平行放置两个笼子,分别各养殖8只鸡和鸭子,接种方式为对鸡进行口服;第三组放置8只笼养鸭子,每日将第二组鸡的粪便喂食于改组鸭子;第四组放置笼养的鸡鸭混合饲养,鸡鸭各8只,接种方式为对鸡进行口服;第五组放置两个笼子,分别各养殖8只鸡和鸭子,作为空白对照。
空肠弯曲菌在水禽与鸡之间的传播实验表明,在第3、6、9、12、15、18天各组鸭子泄殖腔的检测结果显示,鸡鸭混合养殖组中的鸭子首先被重组标签空肠弯曲菌感染,鸭子感染的数量一直呈现上升的趋势,其它组鸭子(除空白组之外)均在第6天被重组空肠弯曲菌感染,感染的数量也呈现一种上升的趋势。在第18天的检测结果中,经粪便传播和鸡鸭混合养殖的两组均有超过一半的鸭子被感染,其它两组也有一半的鸭子被感染。
鸡、鸭混合养殖组中,鸭子首先被空肠弯曲菌感染,泄殖腔排菌量约为1250CFU/只,并在第6天达到最高排菌量,约为4000CFU/只,其它三组鸭子也均在第6天达到最高排菌量。之后随着时间的推移,各组鸭子排菌量保持水平趋势。
研究表明,水禽与鸡之间的空肠弯曲菌以多种途径进行传播,呈现多样化和复杂化,但主要以消化道传播为主。同时,鸡群周围的粪便、饲料和水以及环境中的空气粉尘均对水禽的感染具有重要影响。不同禽群的分开养殖以及保持良好的养殖环境卫生将有助于降低弯曲菌感染传播。
三、不同源空肠弯曲菌分离株多位点序列分型研究
选取空肠弯曲菌7个管家基因aspA、glnA、gltA、glyA、pgm、tkt、uncA,对不同来源的167株空肠弯曲菌分离株和2株标准株进行多位点序列分型研究。结果表明,167株分离株共分成94种不同的序列型,分属18种同源复合体和独特型。有60株细菌分属45个新序列型,其中,20株细菌存在新等位基因序列,新等位基因序列的数量分别为aspA(n=3)、glnA(n=5)、gltA(n=5)、glyA(n=2)、pgm(n=1)、tkt(n=6) ,uncA中没有新等位基因序列。
等位基因数量最多的是glnA(n=26),最少的是uncA(n=11)。94种不同的序列型中,属于ST-353同源复合体的序列型有9种,占总序列型的9.57%,ST-21、ST-45、ST-52和ST-692同源复合体各包含6种序列型,分别占总序列型的6.38%。另外,属于独特型的序列型共27种,占总序列型的28.72%。包含菌株数最多的同源复合体是ST-353,共有20株,占总分离株的12.0%,其次为ST-354同源复合体和ST-45同源复合体。具有同源复合体的菌株占总数的76.6%,属于独特型的细菌占总分离株的23.4%。含菌株数排前8位的同源复合体涵盖了102株,占总数的61.1%。人源、鸡源、牛源、食品源、宠物源和水禽源分离株中这8种主要同源复合体所包含的分离株分别占该来源分离株的51.70%、60.40%、65.00%、80.00%、80.00%和68.40%,为优势同源复合体。
包含菌株数最多的序列型ST-354,共有11株,占总数的6.59%,含有2株或2株以上分离株的序列型共有35种,其余59种序列型只含有1株细菌。60株人源分离株中,属于独特型的分离株共18株,除此之外,ST-353同源复合体包含的人源菌株最多,共有12株细菌,占人源总分离株的20.0%;48株鸡源分离株中,属于独特型的分离株共13株,除此之外,ST-354和ST-574同源复合体包含的鸡源菌株最多,分别含有10株和9株细菌,分别占鸡源总分离株的20.83%和18.75%;20株牛源分离株中,ST-22同源复合体含有9株细菌,占牛源总分离株的45.0%;10株食品源分离株中,8株细菌分属ST-353、ST-354和ST-45同源复合体;10株宠物源分离株分属6种同源复合体,其中含菌株最多的是ST-45同源复合体,共3株;19株水禽源分离株中,7株细菌属于ST-692同源复合体,占36.84%。
在所获得的94种序列型中,共有21种序列型含有两种或两种以上不同来源的空肠弯曲菌分离株,其中ST-354和ST-137两种序列型分别各含有4种不同来源的分离株,另有19种序列型含有2种不同来源的空肠弯曲菌分离株。21种序列型所包含的细菌中,含有人源的序列型有15种,该15种序列型中除了含有人源分离株之外,同时还含有其它来源细菌。
通过UPGMA作图对空肠弯曲菌进行遗传聚类分析,选择相邻序列型之间有4个或4个以上的管家基因位点序列是相同的,可将167株分离株和两株标准株共96种序列型分为9个克隆组群和1个单独型,其中组群5占有的序列型最多,达55种序列型。
分子分型研究表明,MLST可作为研究空肠弯曲菌传播来源、分子流行病学和种群结构研究的有力工具。人源、鸡源、牛源、水禽源、食品源和宠物源空肠弯曲菌分离株之间的遗传关系分析显示,同一种同源复合体和同一种序列型包含不同来源空肠弯曲菌分离株,表明动物和禽肉食品与人类感染空肠弯曲菌密切相关,可能存在相同的传染源,而且动物和禽肉食品源弯曲菌是人类弯曲菌病的主要传染源之一。
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 flocks
In 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 chicken
To 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 method
According 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.
一、水禽弯曲菌流行现状和耐药性调查与分析
2007年12月至2009年12月,采集江苏部分地区的鹅源、鸭源和江苏、山东、安徽鸡群样品进行空肠弯曲菌和结肠弯曲菌流行病学调查。15个水禽群体中,空肠弯曲菌阳性的有12个、阳性率为80.0%,结肠弯曲菌阳性的有7个、阳性率为53.33%。在2182份水禽泄殖腔棉拭样品中,共检测出380份空肠弯曲菌阳性、阳性率为17.42%,16份结肠弯曲菌阳性、阳性率为0.73%,其中空肠弯曲菌和结肠弯曲菌混合感染的样品为6份,阳性率为0.27%。其中,在850份鹅泄殖腔棉拭样品中,空肠弯曲菌平均阳性率为34.94%、结肠弯曲菌平均阳性率为0.82%;在1332份鸭群泄殖腔棉拭样品中,空肠弯曲菌阳性率为6.23%、结肠弯曲菌阳性率为0.68%。不同水禽群感染率悬殊较大,空肠弯曲菌阳性率从0到65.52%、结肠弯曲菌阳性率从0到1.85%。数据分析显示,鹅群空肠弯曲菌感染率极显著高于鸭群感染率( p<0.01),活禽市场鹅群感染率显著高于养殖场中鹅群感染率( p<0.05);活禽市场鸭群空肠弯曲菌感染率极显著大于养殖场中鸭群感染率( p<0.01)。
在960份鸡泄殖腔棉拭样品中,共检测出251份空肠弯曲菌阳性、阳性率为26.15%,36份结肠弯曲菌阳性、阳性率为3.75%,其中空肠弯曲菌和结肠弯曲菌混合感染的样品为15份、阳性率为1.56%;不同鸡群感染率悬殊较大,空肠弯曲菌阳性率从7.58%到73.08%、结肠弯曲菌阳性率从0到5.77%。5个鸡群均为空肠弯曲菌阳性,群体阳性率为100%、结肠弯曲菌群体阳性率为80%,数据分析显示,规模化圈养条件下鸡群的空肠弯曲菌携带率显著高于散养鸡(p<0.05)。进一步的流行病学调查表明,混合养殖方式中鹅群和鸡群的空肠弯曲菌感染率极显著高于单一养殖方式的感染率(p<0.01)。
水禽弯曲菌流行病学调查表明,水禽特别是鹅群是弯曲菌的重要宿主,是弯曲菌感染流行和环境污染的重要因素。
54株鹅源、36株鸭源和11株鸡源分离株的耐药性分析显示,对8大类20种抗生素高度敏感的是:阿齐红霉素(AZM) 96.04%、红霉素(E) 91.09%、庆大霉素(CN) 82.18%,高度耐药的是:头孢哌酮(CFP)100.00%、青霉素G (P)93.07%、头孢拉定(CE)98.02%、恩诺沙星(ENR)80.2%、环丙沙星(CIP)82.18%、复方新诺明(SXT)97.03%、四环素(TE)83.17%、强力霉素(DO)82.18%。耐药菌株的多重耐药分布于4耐到18耐不等,其中11耐至12耐最多,占47.52%。
不同禽源空肠弯曲菌分离株药敏试验结果比较表明,分离株均对头孢菌素类的头孢哌酮和头孢拉定、青霉素G及磺胺类药物产生了很强的耐药性,水禽源分离株对头孢克罗产生的耐药性显著低于鸡源(p<0.01),而对喹诺酮类和四环素类抗生素,虽然水禽源分离株也产生了较强的耐药性,但低于鸡源分离株的耐药性。对其它种类抗生素,各不同源分离株之间的耐药性差别较小。
二、鸡空肠弯曲菌对水禽传播途径分析
鸡群是全球公认的弯曲菌的主要宿主之一,为研究鸡群体内空肠弯曲菌能否传播给水禽,本研究通过构建重组质粒pUOA18-dsRed,并将其用电转化的方法转化至空肠弯曲菌分离株SOC0612-2,成功获得重组菌SOC0612-2-pUOA18-dsRed,体外稳定性实验表明质粒pUOA18-dsRed在无抗性CCDA固体培养基上经过连续传代25次后仍能稳定存在。将该重组菌作为标签细菌研究空肠弯曲菌在水禽和鸡之间可能的传播途径。动物实验中,动物分成5个试验组,每只动物从1日龄养至4周龄。第一组放置8只笼养鸭子,接种方式为肌肉注射;第二组平行放置两个笼子,分别各养殖8只鸡和鸭子,接种方式为对鸡进行口服;第三组放置8只笼养鸭子,每日将第二组鸡的粪便喂食于改组鸭子;第四组放置笼养的鸡鸭混合饲养,鸡鸭各8只,接种方式为对鸡进行口服;第五组放置两个笼子,分别各养殖8只鸡和鸭子,作为空白对照。
空肠弯曲菌在水禽与鸡之间的传播实验表明,在第3、6、9、12、15、18天各组鸭子泄殖腔的检测结果显示,鸡鸭混合养殖组中的鸭子首先被重组标签空肠弯曲菌感染,鸭子感染的数量一直呈现上升的趋势,其它组鸭子(除空白组之外)均在第6天被重组空肠弯曲菌感染,感染的数量也呈现一种上升的趋势。在第18天的检测结果中,经粪便传播和鸡鸭混合养殖的两组均有超过一半的鸭子被感染,其它两组也有一半的鸭子被感染。
鸡、鸭混合养殖组中,鸭子首先被空肠弯曲菌感染,泄殖腔排菌量约为1250CFU/只,并在第6天达到最高排菌量,约为4000CFU/只,其它三组鸭子也均在第6天达到最高排菌量。之后随着时间的推移,各组鸭子排菌量保持水平趋势。
研究表明,水禽与鸡之间的空肠弯曲菌以多种途径进行传播,呈现多样化和复杂化,但主要以消化道传播为主。同时,鸡群周围的粪便、饲料和水以及环境中的空气粉尘均对水禽的感染具有重要影响。不同禽群的分开养殖以及保持良好的养殖环境卫生将有助于降低弯曲菌感染传播。
三、不同源空肠弯曲菌分离株多位点序列分型研究
选取空肠弯曲菌7个管家基因aspA、glnA、gltA、glyA、pgm、tkt、uncA,对不同来源的167株空肠弯曲菌分离株和2株标准株进行多位点序列分型研究。结果表明,167株分离株共分成94种不同的序列型,分属18种同源复合体和独特型。有60株细菌分属45个新序列型,其中,20株细菌存在新等位基因序列,新等位基因序列的数量分别为aspA(n=3)、glnA(n=5)、gltA(n=5)、glyA(n=2)、pgm(n=1)、tkt(n=6) ,uncA中没有新等位基因序列。
等位基因数量最多的是glnA(n=26),最少的是uncA(n=11)。94种不同的序列型中,属于ST-353同源复合体的序列型有9种,占总序列型的9.57%,ST-21、ST-45、ST-52和ST-692同源复合体各包含6种序列型,分别占总序列型的6.38%。另外,属于独特型的序列型共27种,占总序列型的28.72%。包含菌株数最多的同源复合体是ST-353,共有20株,占总分离株的12.0%,其次为ST-354同源复合体和ST-45同源复合体。具有同源复合体的菌株占总数的76.6%,属于独特型的细菌占总分离株的23.4%。含菌株数排前8位的同源复合体涵盖了102株,占总数的61.1%。人源、鸡源、牛源、食品源、宠物源和水禽源分离株中这8种主要同源复合体所包含的分离株分别占该来源分离株的51.70%、60.40%、65.00%、80.00%、80.00%和68.40%,为优势同源复合体。
包含菌株数最多的序列型ST-354,共有11株,占总数的6.59%,含有2株或2株以上分离株的序列型共有35种,其余59种序列型只含有1株细菌。60株人源分离株中,属于独特型的分离株共18株,除此之外,ST-353同源复合体包含的人源菌株最多,共有12株细菌,占人源总分离株的20.0%;48株鸡源分离株中,属于独特型的分离株共13株,除此之外,ST-354和ST-574同源复合体包含的鸡源菌株最多,分别含有10株和9株细菌,分别占鸡源总分离株的20.83%和18.75%;20株牛源分离株中,ST-22同源复合体含有9株细菌,占牛源总分离株的45.0%;10株食品源分离株中,8株细菌分属ST-353、ST-354和ST-45同源复合体;10株宠物源分离株分属6种同源复合体,其中含菌株最多的是ST-45同源复合体,共3株;19株水禽源分离株中,7株细菌属于ST-692同源复合体,占36.84%。
在所获得的94种序列型中,共有21种序列型含有两种或两种以上不同来源的空肠弯曲菌分离株,其中ST-354和ST-137两种序列型分别各含有4种不同来源的分离株,另有19种序列型含有2种不同来源的空肠弯曲菌分离株。21种序列型所包含的细菌中,含有人源的序列型有15种,该15种序列型中除了含有人源分离株之外,同时还含有其它来源细菌。
通过UPGMA作图对空肠弯曲菌进行遗传聚类分析,选择相邻序列型之间有4个或4个以上的管家基因位点序列是相同的,可将167株分离株和两株标准株共96种序列型分为9个克隆组群和1个单独型,其中组群5占有的序列型最多,达55种序列型。
分子分型研究表明,MLST可作为研究空肠弯曲菌传播来源、分子流行病学和种群结构研究的有力工具。人源、鸡源、牛源、水禽源、食品源和宠物源空肠弯曲菌分离株之间的遗传关系分析显示,同一种同源复合体和同一种序列型包含不同来源空肠弯曲菌分离株,表明动物和禽肉食品与人类感染空肠弯曲菌密切相关,可能存在相同的传染源,而且动物和禽肉食品源弯曲菌是人类弯曲菌病的主要传染源之一。
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 flocks
In 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 chicken
To 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 method
According 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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