运用重组隐孢子虫特异性抗原对中国社区人群隐孢子虫感染的血清流行病学研究

发布时间：2018-07-21 09:47

【摘要】： 隐孢子虫(Cryptosporidium)为一种主要寄生于宿主消化道上皮细胞，引起以腹泻为主要临床症状的人兽共患寄生原虫。免疫功能正常者，腹泻可自限，但免疫功能低下者，，尤其是婴幼儿和艾滋病患者，一旦感染则病情严重，迁延不愈，甚至危及生命。近年来，在美国和加拿大等国家因水源污染引起了大规模的隐孢子虫病爆发流行，造成了严重的经济损失，发达国家政府已经意识到监测隐孢子虫对水源的污染以及在环境中分布情况的重要性。特别是近年来艾滋病蔓延，各种环境和职业性有害因素导致机体免疫抑制的情况广泛存在，使得隐孢子虫感染和传播成为可能。因此，隐孢子虫病的研究受到广泛关注。以往，隐孢子虫病的诊断和流行病学调查依赖于通过镜检发现粪便中的隐孢子虫卵囊，其中改良抗酸染色法因其特异性较好而被广泛使用。但是，作为“金标准”的粪便检查不仅工作量大，现场应用时人群的依从性差，而且缺乏足够的敏感性。免疫学诊断方法因其快捷、简便易行等优点，愈来愈受到重视。特异性抗体检测是一种间接诊断方法，并不能肯定地对个案作出有或无的回答。但相关研究表明，血清学群体特征可以在一定程度上反映相应流行区的流行状态，并为疾病的防控提供重要资料。本研究选择了两个社区经济条件、日用水源及健康卫生状况存在显著差异的中国农村社区，收集其全人群的户口信息，同时采集血清，运用CP23重组蛋白为抗原的间接ELISA法，定量检测人群隐孢子虫CP23抗原特异的抗体水平，并根据阳性判断值，估计隐孢子虫在上述两个社区的流行状态及其影响因素。本研究获得结果如下： 1．成功制备了纯化的CP23蛋白，并在本室建立了以此蛋白为抗原的间接ELISA法用于检测隐孢子虫特异性抗体。 2．首次对中国农村人群的隐孢子虫感染状态进行血清流行病学调查。浙江麻车村人群隐孢子虫感染的血清抗体阳性率为32.16％(173／538)，江西驾湖村为70.20％(688／980)，两社区血清抗体阳性率差异显著(P=0.000)。 3．对浙江及江西两地隐孢子虫感染人群血清抗体阳性率与相关变量的广义估计模型分析表明在社区内部，教育程度、家庭收入和有无血吸虫感染对隐孢子虫抗体阳性率没有影响，而性别和年龄是影响隐孢子虫抗体阳性率的重要因素。人群隐孢子虫感染血清抗体阳性率存在性别差异。浙江麻车村人群，在6-10岁年龄组女性低于男性，21-30岁年龄组男性与女性的血清抗体阳性率接近，而其余各年龄组女性隐孢子虫感染血清抗体阳性率高于男性。男性在各年龄组血清抗体阳性率相当，而女性的隐孢子虫血清抗体阳性率随着年龄增长而呈上升的趋势，在41-50岁年龄组达到最高值48.05％。江西驾湖村人群各年龄组女性隐孢子虫感染血清抗体阳性率均高于男性。且男性的隐孢子虫感染血清抗体阳性率有随着年龄增长而上升的趋势，在41-50岁年龄组达到最高值(73.86％)。而女性各年龄组血清阳性率相当，无此趋势。 4．两个地区相比，江西人群的家庭收入显著低于浙江人群(P=0.000)，江西人群的经济收入多在7000-10000元／年，而95％的浙江人群家庭年收入在16000-20000元及以上。通常，经济水平的差异会造成卫生条件的不同。隐孢子虫病是一种经口传播的肠道寄生虫病，它的传播途径与大部分肠道蠕虫相似，该寄生虫的感染与卫生条件有关，这些结果提示了家庭收入可能与两地隐孢子虫血清阳性率的差异相关。 5．利用网络数据库，包括最新的ABCpred服务器对微小隐孢子虫SA35和SA40蛋白的B细胞抗原表位进行预测。隐孢子虫SA35蛋白的103～115和129～146以及SA40蛋白的77～89、127～136、156～174和200～209等区段为B细胞表位。所得表位为这两种蛋白今后应用于合成肽检测血清中抗体、制备相应的抗体、联合CP23抗原发展高特异性和敏感性的诊断系统等提供了理论基础。
[Abstract]:Cryptosporidium (Cryptosporidium) is a kind of parasitic protozoa which mainly parasitic on the epithelial cells of the digestive tract of the host, causing the parasitic protozoa with diarrhea as the main clinical symptom. The immune function is normal, the diarrhea can be self limiting, but the low immune function, especially in infants and AIDS patients, is serious, not more prolonged and even endangered once the infection is infected. Life.
In recent years, a large scale of Cryptosporidium disease caused by water pollution in the United States and Canada has caused a massive outbreak of Cryptosporidium disease, causing serious economic losses. The government of developed countries has realized the importance of monitoring the pollution of Cryptosporidium to the water source and the importance of its distribution in the environment. It is possible that the infection and transmission of Cryptosporidium is possible because of the widespread harmful factors that cause the immune suppression of the body. Therefore, the study of Cryptosporidium is widely concerned.
In the past, the diagnosis and epidemiological investigation of cryptosporidiosis depended on the discovery of Cryptosporidium oocyst in feces by microscopic examination. The improved anti acid staining method was widely used. However, the fecal examination, as the "gold standard", was not only a large amount of work, but poor adherence to the population in the field, and it was not enough. Sensitivity.
Immunological diagnosis has been paid more and more attention because of its advantages, such as quick, easy and easy to do. Specific antibody detection is an indirect diagnostic method, and it is not sure to make any answer to the case. But the related research shows that the serological characteristics can reflect the epidemic state of the corresponding epidemic area to a certain extent and for the disease. Prevention and control provide important information.
In this study, we selected two community economic conditions, Chinese rural communities with significant differences in daily use of water and health and health conditions, collecting their population information, collecting serum and using the indirect ELISA method of CP23 recombinant protein as antigen to detect the specific antibody level of CP23 antigen of Cryptosporidium, and the positive judgment. The prevalence and influencing factors of Cryptosporidium in these two communities were estimated.
The results of this study are as follows:
1. the purified CP23 protein was successfully prepared, and an indirect ELISA method was established in this laboratory to detect Cryptosporidium specific antibodies.
2. the first seroepidemiological survey of Cryptosporidium infection in Chinese rural population was carried out. The positive rate of serum antibody of Cryptosporidium infection in Zhejiang mache village was 32.16% (173 / 538), 70.20% (688 / 980) in Jiangxi driving Lake Village, and significant difference in the positive rate of serum antibody in the two community (P=0.000).
3. a generalized estimation model of the positive rate of serum antibody and related variables of Cryptosporidium infective people in Zhejiang and Jiangxi showed that the degree of education, family income and infection without schistosomiasis had no effect on the positive rate of Cryptosporidium antibody in the community, and sex and age were important factors affecting the positive rate of Cryptosporidium. The positive rate of serum antibody of Cryptosporidium infection in the population of the population of Zhejiang Ma Che village was lower than that of male in the age group of 6-10 years old. The positive rate of serum antibody was close to the male and female in 21-30 years old age group, but the positive rate of serum antibody in the other age groups was higher than that of the male. The positive rate was equal, but the positive rate of serum antibody of Cryptosporidium in women increased with age, and the positive rate of serum antibody positive rate of Cryptosporidium infecting in the age group of 41-50 years old was higher than that of men in the age group of 41-50 years old, and the positive rate of serum antibody of Cryptosporidium infection in male was higher than that of men. The rising trend of age was the highest in the 41-50 year old group (73.86%).
4. compared with the two regions, the family income of the Jiangxi population was significantly lower than that of the Zhejiang population (P=0.000), and the economic income of the Jiangxi population was 7000-10000 yuan per year, while the annual income of the 95% Zhejiang population was 16000-20000 yuan and above. Generally, the difference in the economic level would cause the difference of health conditions. The transmission pathway of intestinal parasitosis is similar to that of most intestinal worms. The infection of the parasite is related to the hygienic conditions. These results suggest that the family income may be related to the difference in the positive rate of the sera of Cryptosporidium.
5. using the network database, including the latest ABCpred servers, the B cell epitopes of the SA35 and SA40 proteins of Cryptosporidium parvum are predicted. The 103~115 and 129~146 of Cryptosporidium SA35 protein and 77~89127 to 136156~174 and 200~209 of the protein are B cell epitopes. The epitopes are the future of these two proteins It provides a theoretical basis for the detection of antibodies in serum by synthetic peptides, the preparation of corresponding antibodies, and the development of highly specific and sensitive diagnostic systems combined with CP23 antigen.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392;R531.5

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