【作者】 党海亮；

【导师】 张龙现； 何国声； 宁长申；

【作者基本信息】 河南农业大学 ， 预防兽医学， 2008， 硕士

【摘要】 野生动物是指生存于自然状态下,非人工驯养的各种哺乳动物、鸟类、爬行动物、两栖动物、鱼类、软体动物、昆虫及其他动物。野生动物是多数自然界病原体的天然宿主,野生动物重大疫病与人类公共卫生问题息息相关。寄生虫病是野生动物的常见疾病之一,由于寄生虫病病原种类繁和分布广泛,以及野生动物感染数量大等因素,寄生虫病常造成野生动物机体消瘦,机体防御能力下降,甚至造成动物的死亡。因此,寄生虫病是造成野生动物种群数量下降的重要因素之一。分别采集17种鸟类,18种草食动物和23种肉、杂食动物的粪便样品162,140,162份,应用饱和蔗糖溶液漂浮法对其寄生虫感染情况进行调查。结果显示:鸟类寄生虫总感染率为19.75%(32/162),主要感染球虫、线虫。草食动物的寄生虫平均感染率为25.71%(36/140)。其中,球虫感染率为14.29%(20/140);线虫总感染率为9.29%(13/140),其中鞭虫和蛔虫感染率分别为7.86%(11/140),1.43%(2/140)。11份云豹的粪便样品中,毛首线虫感染率高达36.4%(4/11);黑豹球虫感染率为100%(1/1),金钱豹球虫感染率为50%(1/2);虎球虫感染率高达100%(4/4),而在三色犬粪便样品中未发现寄生虫虫卵。90份猕猴的粪便样品中,寄生虫感染率高达37.8%(34/90),其中球虫感染5份,鞭虫感染27份,蛔虫感染1份;在采集的其他品种的猴子粪便样品中,寄生虫感染情况也不相同,金丝猴感染鞭虫2份,感染率40%(2/5):食蟹猴球虫感染1份,感染率20%(1/5)。应用饱和蔗糖溶液漂浮法和改良抗酸染色法,对上海地区野生和圈养的部分爬行类及两栖类动物的隐孢子虫流行情况进行调查。结果显示:改良抗酸染色法检测31份野生蛇胃内容物样品,隐孢子虫感染率为45.2%(14/31);改良抗酸染色法检测44份圈养蛇粪便样品,总感染率为13.6%(6/44),蜥蜴感染率为30%(3/9),乌龟感染率为100%(1/1),巨龟感染率为100%(1/1),蟒蛇感染率为25%(1/4);而饱和蔗糖溶液漂浮法检测,仅发现2份蜥蜴样品感染,感染率为22.2%(2/9)。本调查表明,来自野外蛇的隐孢子虫感染率要显著高于动物园驯养的爬行动物,对喜食野生蛇类的陋习提出了严重的警示。利用巢式PCR扩增1个蛇源隐孢子虫分离株18S rRNA基因部分片段,PCR产物经过连接转化,挑选阳性克隆进行测序,产物序列长度均为845bp;用限制性内切酶SspI对PCR产物进行消化酶切确定种类和基因型。SspI酶切结果显示:该隐孢子虫分离株酶切后产生383bp、414bp二个片段。根据酶切片段,初步确定这个隐孢子虫分离株为蛇隐孢子虫C.serpentis。为从分子种系发育关系确定蛇源隐孢子分离株的种类或基因型,利用巢式PCR对这个隐孢子虫分离株进行18S rRNA基因特定片段扩增,并对扩增片段进行测序,获得的序列用Blast和Fasta在NCBI、EMBL和DDBJ三大核酸序列数据库搜索同源序列,然后利用Clustal X1.81、Phylip3.64和DNAstar4.0等生物学软件对序列进行多重比对、构建分子进化树以及同源性分析。根据在18S rRNA基因位点种系进化关系分析,表明该蛇源隐孢子虫分离株为蛇隐孢子虫C. serpentis。采集郑州地区4个鹿场499份鹿粪便样品,应用饱和蔗糖溶液漂浮法和改良抗酸染色法,对其进行隐孢子虫病的检测,获得2个鹿源隐孢子虫分离株。对其中一个分离株进行巢式PCR检测,基于核糖体小亚基(18S rRNA)基因、70kDa热休克蛋白(HSP70)基因、肌动蛋白(actin)基因和隐孢子虫卵囊壁蛋白(COWP)基因的种类与基因型分析,鉴定郑州地区鹿源隐孢子虫分离株种类、基因型,确定本地区鹿源隐孢子虫分离株和其它种类隐孢子虫之间的分子种系进化关系,为预防和控制鹿隐孢子虫病提供理论依据。对河南省郑州市动物园,野生动物保护站,邙山鹿场等鹿养殖区进行了的隐孢子虫病感染情况调查。在2份1岁的梅花鹿粪便样品中发现有隐孢子虫感染,总感染率为0.40%(2/499)。低感染率初步揭示了鹿群不是隐孢子虫的重要储藏宿主。更重要的是,这些养殖场远离城市的饮水供应系统。因此,鹿群可能不是郑州地区人隐孢子虫病的重要感染来源。测序获得的序列用邻接法构建进化树进行种系发育分析。结果显示,四个基因位点有相似的拓扑结构,梅花鹿分离株的序列与其他的隐孢子虫鹿基因型序列属于一个群中,该分离株和隐孢子虫鹿基因型在18S rRNA , HSP70 , actin ,COWP基因位点上的相似性分别为99.1%-99.8%, 99.8%, 99.7%和100%。尽管在三个位点上有微小的差异,该分离株属于隐孢子虫鹿基因型。更多还原

【Abstract】 Wild animals are non-domesticated animals and survive in natural environment, which include various mammals, birds, reptiles, amphibians, fish, mollusks, insects and some other animals. Wildlives are the natural host of most pathogens in nature, The great loemia of wild animals are closely related to the public health of humans. Parasitosis is one of the most common diseases in wildlives. Due to various pathogens of parasitosis, with a wide distribution, and large number of infected wild animans, etc. It ofen causes the loss of body weight, the decline of body defense capability, and even leads to death. So, parasitosis is one of the most reasons for causing the number decreasing of wildlife populations.162, 140 and 162 fecal samples were respectively collected from 17 species birds, 18 species herbivores, 23 zoophagous, and examined for understanding the prevalence of parasites infection by Sheather’s sugar flotation technique.The overall infection rate of parasites in birds was 19.75%(32/162), the common parasites found were Coccidia and Nematode. And the average infection rate of herbivore was 25.71%(36/140), thereinto, the infection rates of Coccidia, Nematode, Trichuris trichiura, and Lumbricoide were 14.29(20/140), 9.29% (13/140), 7.86%(11/140) and 1.43%(2/140), respectively. The infection rate of Neofelis nebulosa in whipworm was 36.4%(4/11). The infection rates of Coccidia in black panther, Panthera pardusl and tiger were 100%(1/1), 50%(1/2)and 100%(4/4), respectively. However, no eggs or oocysts were discovered in Grand Anglo-Francais Tricolore. 37.8% (34/90) of infection rate was found in the 99 fecal samples from macaque, thereinto, the positive numbers of Coccidia, Trichuris trichiura and lumbricoide were respectively five, 27 and one. Moreove, there were various infection rate in other monkeys, namely, 40%(2/5)of Trichuris trichiura infection in Rhinopithecus roxellanae, and 20%(1/5)of Coccidia infection in Cynomolgi.Survey on the prevalence of Cryptosporidium in partial wild and captive reptiles and amphibians was conducted in Shanghai area. The methods of Sheather’s sugar flotation and modified acid-fast staining were used to check the oocysts of Cryptosporidium. The results showed that 45.2%(14/31) and 13.6%(6/44) of infection rates were respectively found in 31 wild snakes and 44 captive snakes by modified acid-fast staining method. Similarly, the infection rates of 30%(3/9), 100%(1/1), 100%(1/1) and 25%(1/4) were also discovered in lizard, tortoise, huge tortoise and python. However, only two positive samples were found in lizards by Sheather’s sugar flotation technique, with a average infection rate of 22.2%(2/9). This survey showed that the Cryptosporidium infection rate in wild snakes was significantly higher than that of infection rate in captive snakes in Zoo. Therefore, the results propose a essential warning to persons who have the bad habits that like eating wild snakes.The specific fragment of 18S rRNA gene was amplified by nested-PCR from a Cryptosporidium isolate obtained from snake. PCR product was cloned and the positive clones were selected and sequenced, and the results showed that the length of fragment was 854bp. Moreover, the PCR product was digested by SspI restriction enzyme to determine the Cryptosporidium species and genotype. After digestion, two fragments of 383bp and 414bp were discovered, and based on the profiles, the Cryptosporidium isolate was primarily identified as C. serpentis. In order to determine the Cryptosporidium species/genotype from the aspect of molecular phylogenetic relationship, sequence obtained was searched by Blast and Fasta provided in three nucleotide databases, i.e., NCBI, DDBJ and EMBL. Then, sequence alignment, constructing phylogenetic trees and similarities were respectively performed by using the softwares of Clustal X1.81,Phylip3.64, DNAstar4.0, and etc. According to the results of phylogenetic analyses based on 18S rRNA gene locus, the isolate from snake was identified as C. serpentis.A total of 499 fecal samples were collected from four deer farms in Zhengzhou City and examined for Cryptosporidium by Sheather’s sugar flotation technique and modified fast acid staining method. Two Cryptosporidium isolates were obtained. The partial sequences of 18S rRNA, HSP70, actin and COWP genes were amplified from one of the two isolates. Based on the four gene loci, the phylogenetic analyses were conducted to identify the Cryptosporidum species and genotype. Thus to determine the molecular evalution relationship between the cervine isolate and other Cryptosporidium species, and to provide a theory foundation for the prevention and treatment of cryptosporidiosis in deer.Investigation on cryptosporidiosis in deer was conducted in Zhengzhou Zoo, wildlife rescure center, deer farm in Mangshan, etc. Tow positive samples were found from two one-year-old deer and the average infection rate was 0.40%(2/499). The low infection rate suggests that deer studied here are not an important reservior of cryptosporidiosis. Moreover, the farms are all far from the drinking water supply system. Therefore, the deer populations may be not a major source of cryptosporidiosis in humans in Zhengzhou region. The sequences of 18SrRNA,Actin,COWP and HSP70 genes were used to conduct phylogenetic analyses based on neighbour-joining method. The analyses of four genes produced similar topology structure, i.e., the Cryptosporidium isolate shared a identical group with other Cryptosporidium cervine genotypes. The similarities in 18S rRNA, HSP70, actin and COWP genes between this isolate and other Cryptosporidium cervine genotype isolates were respectively 99.1%-99.8%, 99.8%, 99.7% and 100%. All in all, the isolate from sika deer was identified as Cryptosporidium cervine genotype although minor difference was noticed in three of four gene loci studied.更多还原