【作者】 朵红；

【导师】 吴润； 李伟；

【作者基本信息】 甘肃农业大学 ， 预防兽医学， 2012， 博士

【摘要】 【目的】本研究拟以青海省东南部棘球蚴病和棘球绦虫为研究对象，通过中间宿主人、羊、牦牛和高原属兔的感染调查，终末宿主藏犬和野生狐狸的感染调查，掌握青海省棘球蚴病和棘球绦虫感染的基线数据。对收集的青海省藏犬和野生狐狸感染的棘球绦虫进行基因多态性研究，掌握青海省流行的棘球绦虫种类及基因多态性，为该病的预防控制提供理论依据。夏季在青海省河卡地区进行野生狐狸调查并收集粪便进行食性分析，冬季收集青海省贵南、刚察、海晏和称多野生狐狸胃内容物进行食性分析，了解野生狐狸的分布状况以及冬、夏季野生狐狸的食物组成，为棘球蚴病的野生终末宿主的防治提供数据。选择适合藏犬驱虫的药品，在青海省称多和河卡种羊场，进行不同模式的藏犬防治研究，以期探讨一种适合青海省牧区切实可行的防治方法。【方法】本论文中主要研究方法如下：（1）应用血清学方法对青海省部分地区的易感人群进行包虫病感染调查，血清学检查阳性的患者用B超腹部脏器扫描，综合判断是否感染包虫病，分析不同性别人群感染包虫病的差别；结合秋、冬季牧区大量屠宰菜畜的机会，对屠宰畜的肝、肺脏用肉眼观察和仔细触摸的方法检查棘球蚴囊肿，记录数量、部位及性质，统计感染率，分析不同脏器的感染率；捕捉高原鼠兔，剖检，用肉眼观察和仔细触摸的方法检查棘球蚴囊肿，记录囊肿的数量、部位及性质，统计感染率，分析不同脏器的感染率。（2）用氢溴酸槟榔碱泻下法和犬粪便用金标试剂条法，对青海省东南部的藏犬进行感染调查，收集虫体，统计感染率和感染强度。在青海省贵南、刚察、海晏和称多县收集野生狐狸，剖检，收集虫体，统计感染率和感染强度。（3）对收集的感染藏犬和野生狐狸的棘球绦虫，选择mtDNA的CO基因、ND基因，设计特异性引物，PCR扩增，测序，通过分子生物学软件，分析青海省不同地区、不同宿主来源的棘球绦虫基因序列的差异，研究棘球绦虫的遗传多样性和遗传进化特征。（4）利用探照灯计数，放置红外线自动感应相机，调查青海省河卡地区野生狐狸。野外收集狐狸粪便，用犬粪便用金标试剂条。检测野生狐狸的棘球绦虫感染率。对夏季收集的狐狸粪便，冬季收集狐狸的胃内容物，进行食性分析。（5）对防治藏犬棘球绦虫的药物进行筛选，在青海省兴海县河卡羊场和称多县进行棘球绦虫防治研究，青海省河卡种羊场每45天左右，藏犬药物驱虫一次，称多县每年4月和10月，藏犬药物驱虫一次，连续防治2年，考核防治效果。【结果】本论文得到的研究结果如下：（1）对青海省玉树县、兴海县河卡种羊场、海晏县和刚察县主要从事畜牧业生产的牧民，进行包虫病血清学调查，在受检的752人中（男性387人，女性365人）共检出50份血清呈阳性，阳性率为6.6%，经B超扫描确诊21人（男性10人，女性11人）为棘球蚴病患者，总患病率2.8%，其中男性患病率2.6%，女性患病率3.0%，女性稍大于男性（P﹥0.05）；在海晏、刚察、共和、泽库和贵南5县及湖东种羊场检查993只屠宰羊，检出570只感染棘球蚴,感染率为57.4%，其中肝脏感染331只，肺脏感染239只羊棘球蚴，分别占感染总数的58.1%和41.9%；在海晏、刚察、共和、祁连、达日和门源6县的屠宰场检查594头屠宰牦牛，检出205头牦牛感染棘球蚴，感染率为34.5%，其中肝脏感染128头、肺脏感染91头，分别占感染总数的58.5%和41.6%；在兴海县河卡羊场及湟源县和称多县草原剖检高原鼠兔347只，检出17只感染棘球蚴，感染率4.9%，其中肝脏感染13只、肺脏感染4只，分别占感染总数的76.5%和23.5%；仅在称多县的高原鼠兔肺脏上发现包囊。（2）用氢溴酸槟榔碱泻下法调查青海省玉树、称多、贵南、刚察、兴海、海晏和天峻7县等地藏犬175只，检出16只感染棘球绦虫、感染率为9.1%，感染强度1～10195条，其中称多和兴海2县藏犬棘球绦虫感染率较高，分别为13.8%和9.1%；用犬粪便用金标试剂条调查青海省称多、刚察、海晏3县和玉树县国有牧场及河卡羊场藏犬粪便611份，检出128份粪便反应阳性、阳性率21.0%，其中称多县、河卡羊场和玉树县国有牧场藏犬棘球绦虫感染率较高，分别为34.8%、25.0%和21.2%；在青海省称多、贵南、刚察和海晏4县捕获或收集藏犬咬死野生狐狸28只，检出5只狐狸感染棘球绦虫、感染率17.9%，感染强度116～1670条。（3）经PCR扩增和DNA测序，从21个棘球绦虫分离株中均扩增获得285bp的mtDNACOI基因片段。该基因片段的同源性分析表明，在21个棘球绦虫分离株中，11株分离株为E.g1型，占52.4%，6株为E.m，占28.6%，4株为E.s，占19.0%；所有核苷酸序列未见有插入和缺失，碱基置换中颠换数明显高于转换数。E.g1型分离株间同源性为98.9%～100%，E.m型分离株间同源性为97.9%～99.6%，E.s型分离株间同源性为98.9%～100%，E.g1型与E.m型分离株间同源性为90.2%～92.6%， E.g1型与E.s型分离株间同源性为89.1%～90.2%，E.s型与E.m型分离株间同源性为88.8%～90.2%。E.g1型存在7个不同的基因序列，涉及7个位点核苷酸异义码变异，占全部分析位点数的2.5%，基因序列间差异值在0～1.1%之间；E.m型存在6个不同的基因序列，涉及到13个位点核苷酸异义码变异，占全部分析位点数的4.6%，基因序列间差异值在0.4%～2.1%之间。E.s型存在3个不同的基因序列，涉及到7个位点核苷酸异义码变异，占全部分析位点数的2.5%，基因序列间差异值在0～1.1%之间。在得到的21个基因序列中，有15个基因序列在GenBank上未找到相同序列，是在青海省新发现的棘球绦虫COI基因序列。经PCR扩增和DNA测序，从21个棘球绦虫分离株中均扩增获得507bp或510bp的mtDNAND基因片段。该基因片段序列的同源性分析表明，在21个棘球绦虫分离株中，11株为E.g1型，占52.4%，6株为E.m型，占28.6%，4株为E.s型，占19.0%。所有核苷酸序列未见有插入和缺失，碱基置换中颠换数明显高于转换数。E.g1型分离株间同源性为99.0%～100%，E.m型分离株间同源性为99.2%～100%，E.s型分离株间同源性为99.2%～100%，E.g1型与E.m型分离株间同源性为83.4%～84.2%， E.g1型与E.s型分离株间同源性为74.8%～75.3%，E.s型与E.m型分离株间同源性为75.9%～76.5%。 E.g1型存在6个不同的基因序列，涉及到12个位点核苷酸异义码变异，占全部分析位点数的2.4%，基因序列间差异值在0～1.0%之间；E.m型存在5个不同的基因序列，涉及到9个位点核苷酸异义码变异，占全部分析位点数的1.8%，基因序列间差异值在0%～0.8%之间；E.s型存在3个不同的基因序列，涉及到10个位点核苷酸异义码变异，占全部分析位点数的2.0%，基因序列间差异值在0%～0.8%之间。在得到的21个ND基因序列中，有14个基因序列在GenBank上未找到相同序列，是在青海省新发现的棘球绦虫ND基因序列。（4）用探照灯计数调查5次共发现野生狐狸5只、野兔3只，按此计算，青海省河卡地区约10Km2分布有野生狐狸1只左右；红外线自动感应照相机拍到野生狐狸1次、鹰1次、旱獭1次、高原鼠兔若干次。用犬粪便用金标试剂条检测，在野外调查地点的1.76hm2内，收集野生狐狸粪便6份，试剂条检测阴性，1.76hm2外，收集粪便14份，13份试剂条检测阴性、1份阳性。野生狐狸食性分析，夏季收集的17份野生狐狸粪便分析显示，夏季食物中哺乳类动物占19.5%、蝗亚目昆虫占58.0%、鞘翅目昆虫占11.7%、幼虫占0.05%、鸟类占4.7%、植物占6.1%。冬季收集的18份野生狐狸胃内容物食性分析显示，冬季食物中牛羊占36.3%、高原鼠兔占53.7%、鸟类占3.5%、植物占6.5%。（5）筛选出吡喹酮片剂为犬的棘球绦虫首选驱虫药物，用糌粑包裹投喂，藏犬采食率高，驱虫效果好。在青海省的河卡羊场和称多县进行为期2年的藏犬棘球绦虫防治，藏犬的感染率从防治前的25%和38.6%下降到了4.9%和6.8%，防治效果明显。更多还原

【Abstract】 【Objective】To understand the situations of echinococcosis and echinococcus in southeast ofQinghai province, we checked human, yaks, sheep, pikas, Tibetan dogs and wild foxes. First, mainspecies of echinocossus which have found to be infectious in human and animals were confirmedby analyzing mitochondria gene polymorphism of echinocossus collected from Tibetan dogs andwild foxes of Qinghai province. Second, distribution, behavior and feeding habits of wild foxeswere investigated by analyzing feces at Heka district in summer and gastric content at somecounties such as Guinan,Gangcha, Haiyan and Chenduo in winter which provide importantdatas about how to prevent wild animals to transmit echinococcosis. Third, to establishingtreatments by drugs treatment and then using praziquantal into Chengduo county and Heka sheepfarm of Qinghai province to find an effective way to prevent and cure echinococcsis of Tibetan dogsin pastoral areas of Qinghai province.【Methods】The main research methods used in this paper were as follows:(1) A rapid sero-diagnosis kit for detecting specific antibodies against echinococcus was usedto screen villagers as the primary survey, then B ultrasound and X-ray were used furtherconfirmation check among these sero-positive objectives; Yaks and sheep were inspected by visualand palpation at slaughterhouses and those cysts which confirmed were taken to laboratory forfurther study like numbers， size,location and statistics of infection rates of yaks and sheep; Pikaswere trapped and dissected for checking lungs and livers and other abdominal organs to recordingnumbers, size, location and statistics of infection rates.(2) Detect dohemg feces gold standard reagent strips and arecoline purge were used to detectEchinococcus in Tibetan dogs in southeast of Qinghai province. Wild foxes were trapped and theintestines were checked by necropsy.(3) The genetic diversity of Echinococcus were assessed by two mitochondrial target genes, i.e.coxI and nadI, in which the genetic polymorphism of Echinococcus of isolated strain of differentdistricts and hosts was analyzed.(4)Searchlight and infrared camera were used to count numbers of wild foxes and indentify thespecies of wild foxes in Heka district of Qinghai province. Further more, we used detect dog fecesgold standard reagent strips to detect the infection rate of Echinococcus in wild foxes. And feedinghabits of wild foxes was carried out by detecting feces in summer and gastric content in winter.(5)To screen medicine for prevention and cue echinococcosis, we choose two sites(Hekadistrict and Chengduo county)to do research. In addition, we gave medicine to Tibetan dogs every45days in Heka district and twice a year (April and October) in Chengduo county for two years. 【Results】The results was obtained in the paper as follows:(1)752residents(male:387,female:365) were serologically screened and of them50arepositive,thus the positive of rate is6.6%(50/752). And21sero-positive villagers(male:10,female:11) were confirmed positive by physical image techniques, so the positive rate of allresidents were2.8%（21∕752）the male is2.6%and the female is3.0%.57.4%(570/993) of sheepwere infected with cystic echinococcosis(liver,331;lung,239) and34.5%(205/594）of yaks wereinfected with cystic echinococcosis(liver,128;lung,91).Besides,4.9%(17/374)of Ochotonacurzoniaes were found hydatid cysts (liver:13, lung:4), but we found hydatid cysts in lungs only atChengduo county.(2)Arecoline purge was used to detect the prevalence of Echioncoccus at Tibetan dogs in manyarea in Qinghai Province like Yushu county, Chengduo county, Guinan county, Xinghai county, etc.And175of Tibetan dogs were investigated,9.1%(16/175) of them were infected Echinococcus, theinfection intensities are different from1to10195. The infection rate of Tibetan dogs of Chengduocounty（13.8%） and Xinghai county（9.1%）were much higher. With the detect dog feces goldstandard reagent strips, we surveyed611Tibetan dogs in Chengduo county, Gangca county, etc. inQinghai Province. And21.0%（128/611）of Tibetan dogs were positive. In addition,28foxes werearrested and collected in Chengduo county, Guinan county,Haiyan county, etc., and5of foxes(Chengduo conty:4,Haiyan county:1)were infected, the infection rate reach to17.9%, the infectionintensities were different from116to1670.(3)As a result of the amplification of partial sequence of mtDNA, we obtained fragments of285bp long CO1gene from21isolates. The results of alignment of our21mtDNA CO1genesequences show that the genotype of11isolates were E.g1(52.4%), of6isolates (28.6%) were E.m,and of4isolates (19.0%)were E.s. All the nucleotide sequences were found no insert mutation anddelete mutation, only base transition and transversion can be found and base transversion wasobviously more than base transition. The homology analysis showed that the similarity of E.g1strains were from98.9%to100%, while of the E.m strains’ were from97.9%to99.6%and of theE.s strains’were from98.9%to100%. It was shown90.2%～92.6%similarity with E.g1and E.m,and89.1%~90.2%similarity with E.g1and E. s strains, the homology similarity between E.s and E.m strains was88.8%~90.2%. There were7different sequences for E.g1genotype, which involved7different Nucleotide mutation points and made up2.5%of total gene. Haploid sequencedifference value are from0to1.1%. And there were6different sequences for E.m, which involved13different Nucleotide mutation points and made up4.6%of the total gene, and the haploidsequence are different from0.4to2.1%.And there were3different sequences for E.s, which waschecked involved7different Nucleotide mutation points and made up2.5%of the total gene, andthe haploid sequence are different from0to1.1%.By the alignment of sequences imported prior inGenBank,15of21isolates were can’t find the same variation strains, they are new genotype sequences never been found before in Qinghai Province.As a result of the amplification of partial sequence of mtDNA, we obtained fragments of507bp and510bp long ND1gene from21isolates. The results of alignment of our21mtDNA ND1gene sequences show that the genotype of11isolates were E.g1, reach to52.4%,6of isolates(28.6%) were E.m, and4of isolates (19.0%)were E.s. All the nucleotide sequences were found noinsert mutation and delete mutation, only base transition and transversion can be found and basetransversion was obviously more than base transition. The homology analysis showed that thesimilarity of E.g1strains were from99.0%to100%, while of the E.m strains’ were from99.2%to100%and of the E.s strains’ were from99.2%to100%. It was shown83.4%～84.2%similaritywith E.g1and E.m, and74.8%~75.3%similarity with E.g1and E. s strains, the homology similaritybetween E.s and E.m strains was75.9%~76.5%. There were6different sequences for E.g1genotype, and the Nucleotide mutation points were made up2.4%of the total gene, which involved12different Nucleotide mutation points, haploid sequence differences are from0to1.0%. E.m werechecked5different sequences, and the mutation points were made up1.8%of the total gene, whichinvolved9different Nucleotide mutation points, the Haploid sequences are different from0%to0.8%. While there were3different sequences for E.s, which involved10different Nucleotidemutation points were made up2.0%of the total gene involving the mutation of9amino acids, andthe difference value of Haploid sequence are from0%to0.8%. By the alignment of sequencesimported prior in GenBank,14of21isolates were can’t find the same variation strains, they arenew genotype sequences never been found before in Qinghai Province.(4)During our experiment at night, we surveyed five times and finally found five wild foxesand three rabbits. And in the whole Heka district if calculated in this way, there will be one wild foxeach10km2. And by our Infrared Automatic Induction Camera we shot wild fox, eagle and marmoteach for one time, and pikas for several times. Use detect dog feces gold standard reagent strips，Inthe area within1.76hm2of our experimental site, we selected6pieces of fox feces and all wereexamined negative, meanwhile,14pieces were collected beyond400m2and of13were examinednegative besides the positive one. We do trophic analysis for our17pieces collected in summerdays, it showed that in summer,19.5%food for fox were from mammals,58%were fromOrthoptera Acridoidea, and11.7%were from Coleoptera,0.05%were from larval worms,4.7%were from birds,6.1%were from plants. Also trophic analysis was conducted to our18piecescollected in winter days, and we finally found that in cold winter,36.3%food for fox were fromdomestic yak and sheep,53.7%were plateau pikas,3.5%were birds, and6.5%were plants.(5)We have selected Praziquantel tablet as the prior deworming pills for the canine, which wasused with excellent results. The drug should better be fed to the Tibetan dogs together with tsamba.For that Tibetan dogs usually like to eat with food other than to take it directly. Prevention andtreatment work of Echinococcosis for Tibetan dogs had conducted in Heka County Sheep Stud and Chengduo County area in Qinghai Province since two years’ prevention. The infection ratedecreased from25%and38.6%counted before prevention, down to4.9%and6.8%at present, sothe prevention effect is obvious.更多还原