【作者】 董和平；

【导师】 张龙现； 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】 本研究对河南省部分地区犬肠道寄生虫进行了流行病学调查,并对检查到的对隐孢子虫从形态学、动物感染试验、寄生部位和寄生特点、虫体超微结构和核糖体小亚基(18S rRNA)基因、肌动蛋白(actin)基因和热休克蛋白(HSP70)基因进行扩增和生物学信息学分析等方面进行了种类鉴定及其生物学特性研究。用饱和蔗糖溶液漂浮法对郑州地区的犬养殖场、宠物市场、河南农业大学实验动物房、宠物医院临床病例以及郑州郊县农村的不同品种、不同年龄的309只犬进行了肠道寄生虫的感染情况调查。结果,共检出虫卵和卵囊6类,其中原虫2种(球虫、犬隐孢子虫);线虫3种(犬弓首蛔虫、类圆线虫、犬鞭虫);绦虫1种(未定种)。本次调查中共发现96份粪便样品为寄生虫阳性,寄生虫总感染率为31.07%(96/309),其中等孢球虫感染率为28.16%(87/309),隐孢子虫感染率2.59% (8/309),蛔虫感染率为11.33%(35/305),狐毛尾线虫感染率为1.94%(6/309),粪类圆线虫感染率为4.21%(13/309),绦虫感染率为0.65%(2/309)。调查发现,郑州地区犬肠道寄生虫超过66%是人兽共患种类;等孢球虫和弓首蛔虫感染在6月龄以下犬只中较为常见。用饱和盐水漂浮法检查了郑州部分地区的105份犬粪便样品。结果,33份粪便样品为球虫阳性,球虫总感染率为31.4%(33/105)。球虫阳性犬均为6月龄以下幼犬,本次调查中共发现犬球虫2种,为俄亥俄等孢球虫(I.ohioenais)和伯氏等孢球虫( I.burrowsi),其平均感染强度(OPG)为1.28x103。在调查中,对患病犬进行了口服增效联磺片药物治疗,结果表明该药对治疗犬球虫病具有良好的治疗效果。用饱和蔗糖溶液漂浮法和改良抗酸染色法对犬养殖场、宠物市场、实验动物房、宠物医院以及郑州郊县农村的309只犬进行了隐孢子虫感染情况调查。结果显示,隐孢子虫感染率为2.59% (8/309)。其中,犬养殖场、郑州郊县农村和实验动物房犬只隐孢子虫感染率分别为0.56%(1/179)、10.53%(2/19)、16.67%(5/30),而宠物市场、宠物医院被调查犬只未发现隐孢子虫感染。从8份阳性样品中分离到的卵囊大小和形态结构基本一致,卵囊平均大小为4.81μm (4.210～5.263μm)×4.39μm(4.210μm～5.263μm),卵囊形状指数平均为1.09。动物感染试验表明,犬隐孢子虫不感染35日龄SCID小鼠和正常幼犬,但感染免疫抑制幼犬。隐孢子虫感染幼犬潜隐期为3～5d;感染后出现2个排卵囊高峰期,第10d出现第一个排卵囊高峰,高峰期持续2d,第17d又出现一个高峰;试验中排卵囊持续期为22d。通过石蜡切片技术、扫描电镜技术研究,在幼犬小肠上发现虫体寄生,主要在十二指肠中段、后段,空肠前段、中段肠绒毛上发现有较多隐孢子虫寄生。十二指肠前段发现有较少虫体寄生,其它部位未发现虫体寄生。根据发表的隐孢子虫18S rRNA、actin基因和HSP70基因序列,建立PCR方法。扩增18S rRNA、actin基因和HSP70基因序列并进行测序,测序后的序列用Blast在NCBI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、actin基因位点、HSP70基因位点种系进化关系分析,河南郑州犬感染隐孢子虫种类为犬隐孢子虫(C.canis)。犬隐孢子虫病是人兽共患隐孢子虫病之一,对人类的健康是一种潜在威胁。因此,研究犬隐孢子虫具有非常重要的公共卫生意义。犬隐孢子虫研究国内尚未见文献报道,研究结果为进一步研究犬隐孢子虫的生物学特性、分子流行病学、虫株之间的种系进化关系等提供了重要的参考依据。更多还原

【Abstract】 In the present study, the epidemiology investigation on intestinal parasites from dogs were conducted in Henan province, then the Cryptosporidium isolates obtained were identified from the following aspects, i.e., the morphology of oocysts, cross-transmission study, PCR amplification of 18S rRNA, actin and 70kDa heat shock protein (HSP70) genes and bioinformation analyses using phylogenetic softwares, moreover, the biology character studies of parasitic site, parasite characterization and ultrastruction were also involved.A total of 309 dogs with differnet age and different species, in dog breeding farms, the pet market in the urban area of Zhengzhou, the experimental animal house of Henan Agricultural University, pet hospitals and the rural areas of Zhengzhou, were surveyed for the prevalence of intestinal parasites using the Sheather’s sugar flotation technique. As a result, 6 species parasites were found, namely, 2 species of protozoon (Coccidian, Cryptosporidium), 3 species of nematode (Toxocara canis, Strongyloides stercoralis, Trichuris vulpis) and 1 specie of Cestode (unnamed). In present investigation, 96 fecal samples were positive and therefore the average infection rate was 31.07%(96/309). Thereinto, the infectioin rate of Isospora sp. was 28.16%(87/309), Cryptosporidium sp. was 2.59% (8/309), Toxocara canis was 11.33%(35/305), Trichuris vulpis was 1.94%(6/309), Strongyloides stercoralis was 4.21%(13/309), and Cestode was 0.65%(2/309). The results showed that more than 66 % parasite species found were zoonotic species. Isospora sp. and T. canis were commonly found in lower than 6-month-old dogs.105 fecal samples of dog from partial area in Zhengzhou were examined by Sheather’s sugar flotation technique. 33 samples were positive and the general infection rate was 31.4%(33/105). The positive dogs detected were all less than 6-month-old. Two species were discovered, namely, Isospora ohioenais and Isospora burrowsi. The average infection intensity (OPG) was 1.28×10~3. At the same time, SMZ-SD-TMP was used to treat coccidiosis and shown a better effect. 309 fecal samples, collected from dog farms, the pet market in the urban area of Zhengzhou, the experimental animal house of Henan Agricultural University, pet hospitals and the rural areas of Zhengzhou, were examined using the Sheather’s sugar flotation technique and the modified acid-fast stain technique to study the prevalence of Cryptosporidium in dogs. The results showed that the average infection rate was 2.59% (8/309). Thereinto, The infection rate of dog farms, the suburban areas, the experimental animal house were 0.56%(1/179), 10.53%(2/19), 16.67%(5/30), respectively. No positive case was found in the pet market and pet hospitals. The mean size of oocysts was 4.81μm(4.210μm～5.263μm)×4.39μm(4.210μm～5.263μm), and the ratio of length to width was 1.09.The results of cross-transmission showed that C.canis can infect immunosuppressed dogs, however, can not infect 35-day-old SCID mice and immunocompetent dogs. The latent period of Cryptosporidium infection in dog was 3～5 days, two peak periods were found, the first peak was at DPI 10 days and lasts 2 days, and DPI 17 was the second peak. The duration of oocyst excretion was 22 days.By the routine histotomy and the H.E staining technique and the scanning electron microscope technique, Cryptosporidium was only found in the small intestine of dogs, including duodenum and jejunum. There was a large amount of Cryptosporidium that mainly located in the middle section and the last section of the duodenum, and in the front section and the middle section of the jejunum. There was little Cryptosporidium in the front section of the duodenum, and no Cryptosporidium was found in the other parts.The PCR methods were developmented according to the sequences of 18S rRNA, actin and 70-kDa heat shock protein (HSP70) genes published previously. The partial sequence of 18S rRNA, HSP70 and actin genes were amplified and sequenced. Sequence data were analyzed with BLAST online. Then, sequences alignment, phylogenetic relationships and homological analysis were performed using Clustal X 1.81, Phylip3.64 and DNAstar 4.0 respectively. Based on the phylogenetic analyses of the 18S rRNA, HSP70 and actin genes, the Cryptosporidium isolates from Henan were identified as Cryptosporidium canis.Cryptosporidium canis is one of the zoonotic Cryptosporidium species. It is a potential threat to humans’public health. To study Cryptosporidium canis is of important public health significance. No report on cryptosporidiosis in dogs in China, therefore, the present study provides an important data for further studying the biological characteristics, molecular epidemiology and the evolution relationship of different isolates, etc. in the future.更多还原