我国东部地区养殖北极狐弓形虫、微孢子虫、隐孢子虫流行病学调查和基因型研究

发布时间：2018-04-19 18:47

本文选题：弓形虫 + 微孢子虫　；参考：《吉林农业大学》2016年硕士论文

【摘要】：弓形虫(Toxoplasma gondii)是一种重要的专性细胞内原虫,可以感染包括狐狸在内的所有恒温动物。据估计全世界有三分之一的人呈慢性感染弓形虫,其中部分人表现出了症状。人类通过食用未煮熟的含有卵囊的组织肉类和饮用被终末宿主污染的含有卵囊的水而感染。弓形虫感染能导致先天性感染弓形虫病的胎儿严重的疾病,并且对免疫缺陷的病人有致命的伤害,例如艾滋病病人和器官移植癌症患者。隐孢子虫(Cryptosporidium)作为一种重要的人与动物的肠道寄生虫,在世界范围内呈多样性分布,截至目前至少发现有26种虫种以及73种有宿主特异性的未确定基因型。犬隐孢子虫是寄生于犬体内的一种常见寄生虫,并且已经被证实发现于狐狸体内。从全世界各国感染的病人的症状来看,犬隐孢子虫已经确定为一种潜在的人兽共患的隐孢子虫虫种。微孢子虫病是一种在动物和人群中常见的疾病。微孢子虫(Enterocytozoon bieneusi)是人群中常见的孢子虫属的虫种,许多动物物种都可以成为微孢子虫感染的潜在感染对象。然而,关于养殖狐狸的微孢子虫感染率和基因型信息几乎还没有报道。因此,本研究使用基因检测方法对我国东部地区养殖狐狸的微孢子虫阳性率,风险因素以及基因型进行了调查。本研究第一部分对我国东部地区的养殖狐狸进行了弓形虫血清学调查,使用改良凝集试验法对1346份养殖狐狸样品进行了检测,其中滴度在1:25判定为阳性,阳性样品数为113个(8.39%),其中滴度在1:25的为66个,1:50的为26个,1:100的为9个,1:200的为7个,1:400的有3个,1:800的有2个。雄狐的弓形虫阳性率为8.68%,高于母狐的7.95%,在白狐中的阳性率为7.07%,低于蓝狐的感染率(9.75%)。在黑龙江,吉林,辽宁,山东的弓形虫感染率分别为:85%,9.21%,7.37%和8.68%。经过二元逻辑回归分析后,亚种、性别、地区因素与血清阳性率之间均没有显著的差异性。这项调查结果表明,养殖狐狸的弓形虫感染情况是比较严重的,这是我国关于北极狐的弓形虫血清学研究方面的首篇报道。本研究第二部分检测了北极狐的微孢子虫的感染率、风险因素以及基因型。在302份养殖狐狸粪便样品中,经过巢式PCR的检测之后37份为微孢子虫阳性(12.25%,95%CI 8.55-15.95),这个结果只与北极狐的养殖模式有显著相关性:处于户外养殖模式的北极狐微孢子虫阳性率(26.03%positive,95%CI=18.91-33.15)明显高于室内的(5.77%,95%CI=2.11-9.43),共鉴定出11种ITS基因型,其中4种为常见的基因型:Peru8,Types IV,CHN-DC1 and D;7种新的基因型:NCF1-NCF7。NCF2基因型是阳性个数中占比最多的,存在于三个省份的五个养殖场中。所有的基因型属于系统发育第一种群。本研究第三部分采用巢式PCR方法检测北极狐的隐孢子虫阳性率为15.9%(48/302),其中雄性感染率为12.9%(18/139),雌性感染率为18.4(30/163),来自于各个不同养殖场的北极狐隐孢子虫感染率为0到31.4%不等。不同的年龄段的北极狐隐孢子虫的感染率也在14.1%到19.0%变化。此外,河北养殖场的北极狐隐孢子虫感染率(7.8%,11/141)明显的低于黑龙江省(22.9%,16/70)的和吉林省(23.1%,21/91)(P=0.0015)的感染率。SSU r RNA序列分析结果表明所有的分离株均为犬隐孢子虫。本研究还首次对我国养殖狐狸的弓形虫基因型进行了鉴定。264脑组织样品分别采集于吉林、黑龙江以及山东。样品经过组织DNA提取后采用半巢式PCR方法检测弓形虫B1基因,然后再以11个遗传标记为基础(SAG1,alternative SAG2,5’-and3’-SAG2,SAG3,L358,BTUB,c22-8,GRA6,c29-2,PK1 and Apico),采用PCR-RFLP方法对阳性样品做进一步的基因分型,264份组织样品中有21份组织DNA的弓形虫B1基因检测呈阳性(7.96%)。一共鉴定两种基因型(Toxo DB#9和Toxo DB#10)。本研究使用MAT方法检测了我国东部地区北极狐弓形虫感染的血清阳性率。使用巢式PCR方法对微孢子虫和隐孢子虫进行了流行病学调查和基因型研究。本研究使用PCR-RFLP从基因层面对弓形虫分离株进行了基因型鉴定,发现了一种主要流行弓形虫基因型Toxo DB#9型和另一种基因型Toxo DB#10型。这些研究结果为我国控制和预防北极狐的弓形虫病、微孢子虫病和隐孢子虫病提供了基本数据,填补了相关学科空白。
[Abstract]:Toxoplasma gondii is an important specific intracellular protozoa that can infect all the thermostat animals, including foxes. It is estimated that 1/3 of the world are infected with chronic infection of Toxoplasma gondii, some of which show symptoms. Human beings eat uncooked tissue meat containing oocysts and drink the end host. Infected with the water of the oocyst. Toxoplasma infection can cause serious diseases of the fetus with congenital infection of toxoplasmosis, and can be fatal to immunodeficient patients, such as AIDS patients and organ transplant cancer patients. Cryptosporidium is an important intestinal parasite of human and animal, in the world. At least 26 species and 73 host specific unidentified genotypes have been found at least. Cryptosporidium canis is a common parasite parasitic in dogs, and has been found in foxes. From the symptoms of infected patients all over the world, Cryptosporidium canis has been confirmed. A latent zoonozoic species of Cryptosporidium. Microsporia is a common disease in animals and populations. Microspore (Enterocytozoon bieneusi) is a common species of Sporozoa in the population. Many animal species can become a potential target for infection of microspore. However, foxes are raised. The infection rate and genotype information of microspore have hardly been reported. Therefore, this study used gene detection methods to investigate the positive rate, risk factors and genotypes of Microspore in cultured foxes in eastern China. The first part of this study conducted a serological survey on the cultured foxes in eastern China. The modified agglutination test was used to detect 1346 cultured foxes, of which the titer was judged positive at 1:25, and the number of positive samples was 113 (8.39%), of which the titer was 66 at 1:25, 26 for 1:100, 7 for 1:200, 3 for 1:400 and 2 for 1:800. The positive rate of Toxoplasma gondii in male fox was 8.68%, higher than 7.95% of the female fox. The positive rate in white fox was 7.07%, lower than the infection rate of blue fox (9.75%). The infection rates of Toxoplasma gondii in Heilongjiang, Jilin, Liaoning and Shandong were respectively 85%, 9.21%, 7.37% and 8.68%. after two yuan logical regression analysis. There were no significant differences between subspecies, sex, regional factors and serum positive rates. The infection of Toxoplasma gondii is very serious in fox. This is the first report on the serological study of the Toxoplasma gondii in the Arctic fox. The second part of this study examined the infection rate, risk factors and genotypes of the microspore worm in the Arctic fox. In 302 foxes feces samples, 37 were detected by nested PCR. Sporozoite positive (12.25%, 95%CI 8.55-15.95), this result is only related to the Arctic Fox culture model: the positive rate of the Arctic Fox microspore (26.03%positive, 95%CI=18.91-33.15) in the outdoor culture model is significantly higher than that in the indoor (5.77%, 95%CI= 2.11-9.43), and 11 kinds of ITS genotypes are identified, of which 4 are common genotypes. Peru8, Types IV, CHN-DC1 and D, 7 new genotypes: the NCF1-NCF7.NCF2 genotype is the most positive in the positive number, and exists in five farms in three provinces. All genotypes belong to the first phylogeny population. The third part of this study uses the nested PCR method to detect the positive rate of Cryptosporidium in arctic fox 15.9% (48/302). The male infection rate was 12.9% (18/139) and the female infection rate was 18.4 (30/163). The infection rate of Cryptosporidium from arctic fox was 0 to 31.4% from different farms. The infection rate of Cryptosporidium of arctic fox was 14.1% to 19% in different age groups. In addition, the infection rate of Cryptosporidium of arctic fox in Hebei farm was 7.8%, 11/141). Significantly lower than Heilongjiang (22.9%, 16/70) and Jilin province (23.1%, 21/91) (P=0.0015) infection rate.SSU R RNA sequence analysis results showed that all the isolates were canine Cryptosporidium. This study also identified the Toxoplasma gondii genotypes of cultured foxes in China for the first time,.264 brain tissue samples were collected in Jilin, Heilongjiang and mountains, respectively. The B1 gene of Toxoplasma gondii was detected by the semi nested PCR method after the tissue DNA extraction. Then 11 genetic markers were used as the basis (SAG1, alternative SAG2,5 '-and3' -SAG2, SAG3, L358, BTUB, c22-8). The positive samples were further genotyping, and 21 of the 264 tissue samples were used. The B1 gene of Toxoplasma gondii in DNA was positive (7.96%). A total of two genotypes (Toxo DB#9 and Toxo DB#10) were identified. The serum positive rate of Toxoplasma gondii infection in the eastern region of China was detected by MAT method. The epidemiological investigation and genotyping of microspore and cryptospora were investigated by nested PCR method. This study used PCR-RFLP to identify the genotypes of Toxoplasma gondii isolates from the gene layer, and found a major genotype Toxo DB#9 and another genotype Toxo DB#10. These results provide basic data for the control and prevention of toxoplasmosis, sporozoon and cryptosporidiosis in the Arctic fox in China. It fills the gap in the related disciplines.

【学位授予单位】：吉林农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S858.92

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