我国部分地区种鸡场沙门菌流行现状调查、耐药性分析及分子分型

发布时间：2018-05-26 00:51

本文选题：沙门菌 + 种鸡场　；参考：《扬州大学》2017年硕士论文

【摘要】：沙门菌病(Salmonellosis)是指由沙门菌属(Salmonella)细菌所引起的各种动物及人的疾病的总称,是世界动物卫生组织法定报告的动物疾病,是我国政府规定的二类动物疫病,是《国家中长期动物疫病防治规划(2012-2020年)》确定的优先防治的病种。沙门菌感染的畜禽及其被污染的产品是食源性沙门菌的重要来源之一,这不仅阻碍了畜禽相关产业的发展,而且也对人类健康构成了巨大威胁。种鸡养殖是养鸡业上游的生产环节,减少这一环节鸡群中沙门菌的感染能够有效降低其后代次鸡群及其相关产品的沙门菌检出比例。许多发达国家在种鸡群中采取的检疫淘汰制度有效控制甚至消灭了鸡白痢、鸡伤寒沙门菌在鸡群中的传播;近年来这些国家的沙门菌流行病学调查结果显示鸡群中副伤寒沙门菌如肠炎沙门菌、鼠伤寒沙门菌、肯塔基沙门菌等开始成为优势血清型。目前针对我国种鸡场鸡群中沙门菌感染情况的研究还不是很多,种鸡养殖过程中不同阶段的沙门菌流行规律、防控关键点还不是完全清晰。本研究以国内不同地区的7家种鸡场为研究对象,调查了种鸡养殖场中沙门菌的流行状况、血清型分布及药物敏感性特点,并以其中一个规模化种鸡场作为场内流行规律调查分析的样板,分阶段分析流行状况,综合应用CRISPR、PFGE分子分型技术分析不同沙门菌分离株之间的内在联系,期望探明国内种鸡场沙门菌的流行现状及传播特点,摸清种鸡养殖过程中的防控关键点,为种鸡场沙门菌的科学有效防控提供依据。一、种鸡场沙门菌的分离鉴定及耐药性分析在2015年6月至2017年3月期间,对A、B、C、D、E、F、G共7家种鸡场进沙门菌的分离鉴定,共采集样品2,185份,分离沙门菌326株,总体分离率为14.92%。统计学分析表明,不同种鸡场沙门菌分离率存在差异(P0.05),分离率最高可达18.37%,最低的一家种鸡场则尚未分离到沙门菌。针对F种鸡场不同养殖阶段沙门菌分离率的调查研究表明,产蛋期和孵化期的沙门菌分离率较高,分别达到24.66%和24.52%;而引种阶段的分离率则相对较低,为6.67%。统计分析表明,在种鸡养殖的各个阶段中,引种阶段、育雏期、产蛋期沙门菌分离率逐渐上升;产蛋期和孵化期的沙门菌分离率维持在较高水平;孵化期到孵化后期沙门菌分离率有所下降。7家种鸡场分离的沙门菌血清型鉴定结果显示,肠炎沙门菌是优势血清型,其次是鸡-雏沙门菌,而其他血清型分布比例则相对较小。研究对A、B、F、G四家种鸡场分离的223株沙门菌进行了 16种抗菌素药物敏感性试验,整体情况显示,萘啶酸耐药比例最高,达94.62%;其次是氨苄西林,耐药率为56.05%;链霉素的耐药率也相对较高,为39.91%。多重耐药统计结果显示,细菌耐药种类在1-3种的比例最高,达76.23%;耐药种类在4-6种之间的比例为17.94%;7种及以上的比例相对较低。分析F种鸡场中沙门菌的耐药情况,细菌同样对萘啶酸表现出很高的耐药率,达91.30%;分析不同养殖阶段沙门菌的多重耐药分布情况,产蛋期分离细菌的多重耐药种类明显增多,并且16种测定药物中有11种药物都在产蛋期时耐药率最高。二、不同分子分型技术在种鸡场沙门菌传播规律及溯源研究上的应用本研究首先对F种鸡场不同养殖阶段分离的115株肠炎沙门菌进行了 CRISPR分型,结果显示所有的肠炎沙门菌被分类成了 3种CRISPR型,这表明F种鸡场内存在有基因型不同的多种肠炎沙门菌克隆亚群。此外,研究还发现同种CRISPR型的肠炎沙门菌往往来自于多个分离阶段,这表明种鸡场中可能存在沙门菌的交叉传播。引种阶段分离的SET1型和SET2型肠炎沙门菌,同样能在其后的养殖阶段中被发现,说明引种阶段的沙门菌传播至其后的养殖阶段,提示引种阶段可能是沙门菌防控的一个关键环节。CRISPR型和耐药表型的联合分析显示,同一 CRISPR型的细菌耐药谱往往完全或部分相似;耐药种类和CRISPR型的对应统计结果还显示SET2和SET3型的细菌耐药谱数目都较为相似。这些结果提示,CRISPR分型技术可能在单一血清型分子亚分型中提供了较好的亚类区分。对存在供货关系的G和F两家种鸡场的60株肠炎沙门菌进行PFGE分型,PFGE聚类分析共分成了 4个聚类簇,其中PFGE聚类簇A既包含17株G种鸡场来源的肠炎沙门菌,还有32株则来源于F种鸡场。这一结果提示,供需链间种苗鸡的引入是F种鸡场沙门菌传播来源的途径之一。同时,研究还发现除PFGE聚类簇A外,细菌还分成了几类细菌数目相对较少的PFGE聚类簇B、C和D,分析每个聚类簇中细菌的来源,发现PFGE聚类簇C的细菌都来源于F种鸡场,PFGE聚类簇B和D则都属于G种鸡场,这些结果说明供需链间沙门菌的传播感染还存在其他途径,不同沙门菌亚型在鸡场中的传播能力可能存在差异。
[Abstract]:Salmonellosis (Salmonellosis) refers to the general name of all kinds of animals and human diseases caused by Salmonella bacteria. It is an animal disease reported by the world animal health organization. It is the two kind of animal disease stipulated by the government of our country. It is the priority prevention and control disease in the plan of prevention and control of the middle and long term animal epidemic disease (2012-2020 years). Salmonella infected livestock and poultry and their contaminated products are one of the important sources of food borne Salmonella, which not only hinders the development of livestock and poultry industry, but also poses a great threat to human health. The breeding of chicken is a production link in the upstream of the chicken breeding industry. Reducing the infection of Salmonella in this ring chicken group can effectively reduce its infection. The detection ratio of Salmonella in the offspring of descendants and their related products. The quarantine and elimination system adopted by many developed countries effectively control and even eliminate the spread of chicken white dysentery and Salmonella typhimurium in the chickens. In recent years, the epidemiological investigation of Salmonella in these countries shows that the paratyphoid salmonella in the chicken group, such as enteritis sand, has been shown. Bacteria, Salmonella typhimurium, Salmonella, Kentucky, and Salmonella became dominant serotypes. At present, there are not many studies on the infection of Salmonella in chicken farms in China. The epidemic law of Salmonella in different stages of chicken breeding and the key point of prevention and control are not completely clear. This study is based on 7 domestic chicken farms in different regions of China. In this study, the epidemic situation of Salmonella, serotype distribution and drug sensitivity in the breeding farm were investigated, and one of the large-scale chicken farms was used as a sample for the investigation and analysis of the field epidemic. The epidemic situation was analyzed in stages, and the CRISPR and PFGE molecular typing techniques were used to analyze the different Salmonella isolates. In order to find out the epidemic situation and transmission characteristics of Salmonella in domestic chicken farm, we can find out the key points of prevention and control in the breeding process of the chicken, and provide the basis for the scientific and effective prevention and control of Salmonella in the chicken farm. First, the isolation and identification of Salmonella in the chicken farm and the analysis of drug resistance from June 2015 to March 2017, 7 species of A, B, C, D, E, F, G The isolation and identification of Salmonella in the chicken farm, 2185 samples were collected and 326 strains of Salmonella were isolated. The total separation rate was 14.92%. statistical analysis showed that the isolation rate of Salmonella in different kinds of chicken farms was different (P0.05), the highest separation rate was up to 18.37%, and the lowest one was still not isolated from Salmonella. The isolation rate of Salmonella strains showed that the isolation rate of Salmonella was higher in laying and hatching period, reaching 24.66% and 24.52% respectively, while the separation rate of the introduction stage was relatively low. The 6.67%. statistical analysis showed that the separation rate of Salmonella in the introduction stage, brooding stage and laying period was gradually rising in each stage of breeding, laying period and incubation period. The isolation rate of Salmonella in the period of incubation was maintained at a high level, and the isolation rate of Salmonella from the incubation period to the later stage of the hatchery decreased. The serotype identification of Salmonella isolated from the.7 poultry farm showed that Salmonella enteritis was the dominant serotype, followed by the chicken chick Salmonella, and the other serotype distribution ratio was relatively small. The study on four chickens with A, B, F and G was a study. 223 strains of field isolated Salmonella were tested with 16 antimicrobial susceptibility tests. The overall situation showed that the drug resistance rate was the highest, up to 94.62%, followed by ampicillin, the drug resistance rate was 56.05%, and the drug resistance rate of streptomycin was relatively high. The statistical results of 39.91%. multiple resistance showed that the rate of bacterial resistance in the 1-3 species was the highest, up to 76.2. 3%, the proportion of the resistant species in the 4-6 species was 17.94%, and the proportion of the 7 species was relatively low. The resistance of Salmonella in the F chicken farms was analyzed, and the bacteria also showed a high drug resistance rate to nnidium acid, up to 91.30%. The multi drug resistance distribution of Salmonella in different breeding stages was analyzed, and the multidrug resistance of the isolated bacteria in the laying period was obvious. In addition, the drug resistance rate of 11 of the 16 kinds of drugs at the laying period was the highest. Two, the application of different molecular typing techniques to the propagation and traceability of Salmonella in the chicken farm was applied to the CRISPR classification of Salmonella enteritis isolated at different breeding stages in F chicken farms. The bacteria were classified into 3 CRISPR types, which indicated that there were different genotypes of the clone subgroups of Salmonella enteritis in F chicken farms. In addition, the study also found that the same CRISPR type of Salmonella enteritis was often derived from multiple separation stages, which indicated that the cross transmission of Salmonella may exist in the chicken farm. SET1 and SE in the introduction stage were isolated. The Salmonella enteritis type T2 can also be found in the subsequent breeding stage, indicating the spread of Salmonella in the introduction stage to the subsequent breeding stage, suggesting that the introduction stage may be a key link in the prevention and control of Salmonella. The joint analysis of the.CRISPR type and drug resistant phenotype shows that the same CRISPR type of bacterial resistance spectrum is often completely or partially similar. The corresponding statistical results of resistance species and type CRISPR showed that the number of SET2 and SET3 type bacterial resistance profiles were similar. These results suggest that CRISPR typing may provide better subclass differentiation in single serotype subtypes. PFGE points for 60 strains of Salmonella enteritis in G and F two chicken farms with supply relations. The PFGE cluster analysis was divided into 4 cluster clusters, in which the PFGE cluster A contained not only 17 strains of Salmonella enteritis from G chicken farms, but also 32 from F chicken farms. The results suggested that the introduction of the interseed and supply chain of the supply chain is one of the ways for the transmission of Salmonella in F chicken farms. Meanwhile, the study also found that the bacteria except the PFGE cluster A, the bacteria were also found to be bacteria. PFGE cluster B, C and D were also divided into several kinds of cluster clusters, C and D were used to analyze the origin of bacteria in each cluster. It was found that all the bacteria of PFGE cluster C were derived from F chicken farms, PFGE cluster B and D were all G seed farms. These results showed that the transmission infection of Salmonella among the supply chains still existed other ways, different Salmonella subtypes were in the field. There may be a difference in the ability of the chicken farm to spread.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S858.31

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