【作者】 李静；

【导师】 菅复春；

【作者基本信息】 河南农业大学 ， 预防兽医学（专业学位）， 2015， 硕士

【摘要】 羊腹泻是由多种原因引起羊的以拉稀为主要症状的一类疾病的总称。近些年来一直困扰着养羊业的发展,已经成为羊养殖中影响羊的种群繁育速度和经济效益的主要疾病之一,应引起高度重视。引起羊腹泻的因素多而复杂,但以寄生虫性病原和细菌性病原为主导因素。隐孢子虫、贾第虫和球虫是引起羊腹泻较为常见的寄生虫性病原,这三种病原在世界范围内均有分布,羊均易感染。羊细菌性腹泻病是一种以腹泻为特征性症状的羊消化道疾病,严重影响羊早期的生长发育,然而引起羊腹泻的细菌性病原主要以羊魏氏梭菌和沙门氏菌为主,这二种细菌性病原危害最为严重。为了解致羊腹泻的三种病原的流行情况,为羊腹泻病的防治提供科学的参考和依据。根据不同地区和饲养方式,选取河南省的郑州市、登封市、中牟县、开封市、新乡市、新密市、长葛市、安阳市、南阳市、平顶山市以及安徽省合肥市、陕西省等地12个县市的13个羊场,共采集羊粪便样品553份。经显微镜检查后发现,球虫感染率为88.61%,其中感染率最高为100%,最低为67.31%,感染强度最大为4200000(OPG),隐孢子虫感染率为1.27%,贾第虫感染率为3.25%。对收集的粪便DNA样品同时采用PCR法对隐孢子虫、贾第虫、魏氏梭菌进行检测。结果,基于SSUrRNA的nested-PCR共检出50份隐孢子虫阳性样品,基于16SrRNA的nested-PCR共检出39份贾第虫阳性样品,隐孢子虫和贾第虫感染率分别为9.04%(50/553)、7.05%(39/553);基于ɑ毒素基因的常规PCR共检出13份魏氏梭菌阳性样品,感染率为2.35%(13/553)。幼龄羊的隐孢子虫、贾第虫和魏氏梭菌感染率明显高于成年羊,其中幼龄羊的感染率分别为13.02%(41/315)、8.25%(26/315)、2.86%(9/315),成年羊的感染率分别为4.04%(8/198)、6.57%(13/198)、2.02%(4/198);放牧羊的隐孢子虫、贾第虫和魏氏梭菌的感染率分别为31.34%(21/67)、7.46%(5/67)、5.97%(4/67),舍饲羊的感染率分别为5.97%(29/486)、7.00%(34/486)、1.85%(9/486);绵羊隐孢子虫、贾第虫、魏氏梭菌的感染率分别为9.28%(31/334)、8.08%(27/334)、3.59%(12/334),山羊的感染率分别为8.68%(19/219)、5.48%(12/219)、0.46%(1/219),绵羊感染率均高于山羊。为了确定引起羊腹泻的各个病原的种类,将扩增出的阳性样品序列扩增后分别在NCBI中进行Blast比对,下载相关参考序列,应用Clustalⅹ2.11软件分别进行序列比对,鉴定出致病种。在本次调查的羊粪便样品中共鉴出3个隐孢子虫虫种,分别是C.ubiquitum(N=27)、C.xiaoi(N=19)、C.parvum(N=4);贾第虫经鉴定分别为集聚体A(N=1)和集聚体E(N=38);魏氏梭菌经鉴定分属于A型(N=9)、C型(N=2)和D型(N=2)。其中C.parvum、C.ubiquitum、贾第虫集聚体A和魏氏梭菌A、C型均属人畜共患的病原种类,应引起高度重视。本研究表明羊腹泻病原在各种年龄和饲养方式中普遍存在,实践中也发现该病已经严重影响我国养羊业的经济效益和健康发展,其中的人畜共患性寄生虫和细菌性病原是人类健康的潜在威胁。所以,对引起羊腹泻的重要病原应尽早做出准确诊断,采取有效的综合预防措施。本研究为羊腹泻的诊断和防控提供技术手段和参考依据。更多还原

【Abstract】 Diarrhea of sheep and goats is the disease which caused by a variety of pathogens, and lead the animals to appeare the main symptom of diarrhea. It has already become a strong factor of hindering development of sheep industryon affecting the population of sheep breeding speed and economic lose, so it should be highly attentioned.The factors causing diarrhea in sheep and goats is complex, parasitic and bacterial pathogensare the key factors. Cryptosporidium, Giardia and Coccidiosis are common parasitic pathogens distributed worldwide caused diarrhea in sheep and goats, however, diarrhea caused by the bacterial pathogen, Clostridium welchii and Salmonella, play central role in the most cases. Sheep and goats are susceptible to pathogens mentioned above, and gastrointestinal diarrhea symptoms impact on lamp early growth and development seriously.In order to understand the epidemic status of three diarrheal pathogens in sheep and goats, and provide scientific basis for the prevention and control of diarrheal disease, a total of 553 sheep fecal samples were collected according to different areas and feeding mode from total 13 sheep farms, there localed in Henan, Anhui, Shaanxi Province respectively, total 11 farms of Henan were selected, they are Zhengzhou, Dengfeng, Zhongmu, Kaifeng, Xinxiang, Xinmi, Changge, Anyang, Nanyang, Pingdingshan, one farm was selected from Hefei and Shaanxi Province respectively. These samples were treated by microscopic examination firstly, Results show that the total infection rate was 88.61% among the Eimeria, in which the highest one was 100%, and the lowest is 67.31%, the maximum intensity of infection was 4200000(OPG), the infection rate of Cryptosporidium, Giardia infection rate was 1.27%, 3.25% respectively. Three pathogens, Cryptosporidium, Giardia, Clostridium welchii, were detected using PCR method from feces DNA,50 of 553 samples of Cryptosporidium positive were detected by nested-PCR based on SSUr RNA locus were detected 39 and 13 isolates of Giardia and Clostridium welchii were found based on the nested-PCR of 16 Sr RNA and PCR of alpha toxin gene respectively. Cryptosporidium,Giardia,Clostridium welchii infection rates were 9.04%(50/553),7.05%(39/553) and 2.35%(13/553) respectively; The kids infection rate of Cryptosporidium, Giardia, and Clostridium welchii was significantly higher than that of adult, the sinfection rates of three pathogens were 13.02%(41/315), 8.25%(26/315), 2.86%(9/315), however, adult infection rates were 4.04%(8/198), 6.57%(13/198), 2.02%(4/198) respectively; free-range sheep and goat infection rates of Cryptosporidium, Giardia and Clostridium perfringens were 31.34%(21/67), 7.46%(5/67), 5.97%(4/67), while, that of stall-feeding were 5.97%(29/486), 7%(34/486), 1.85%(9/486); Sheep infection rate of Cryptosporidium, Giardia and Clostridium perfringens of sheep were 9.28%(31/334), 8.08%(27/334), 3.59%(12/334), while that ofgoat were 8.68%(19/219), 5.48%(12/219), 0.46%(1/219) respectively, these data means that the infection rates of sheep were higher than that of goats in different diarrleal pathogens.In order to determine the species, type of different diarrheal pathogens, the positive samples which detected by PCR on different loci and methods were sequenced and blasted in NCBI to find sequences similar to objective one, then the reference sequences were download and compared using Clustal X 2.11 software. In this study, 3 Cryptosporidium species were identified, they are C. ubiquitum(N = 27), C.xiaoi(N = 19), C. parvum(N = 4) respectively; Two Giardia lamblia assemblage were fonud, they are Assemblage A(N = 1) and E(N = 38); Clostridium welchii was identified and belonged to type A(N = 9), type C(N = 2) and type D(N = 2) respectively. There are more attention should be paid to these zoonotic pathogens, C. ubiquitum C. parvum, Giardia Assemblage A and Clostridium perfringens type A, C.This study shows that sheep and goats diarrheal pathogens exists in all ages and feeding mode generally, diseases these pathogens caused has affected the sheep industry’s economic and healthily development seriously in our country, and the zoonotic parasites and bacterial pathogens exist as a potential threat to human health. Therefore, an accurate method of diagnosis on is should be used for important diarrheal pathogen detection as soon as possible, effective and comprehendsive prevention measures should be takenbased on detection data too. This study provides technology method and reference for the diarrheal disease diagnosis and prevention of sheep and goats.更多还原