水中隐孢子虫卵囊的研究进展

隐孢子虫病（Cryptospotidiosis）是新近发现的,由隐孢子虫卵囊（Cryptospotidium Oocyst）引起的一种可介水传播的人畜共患急性腹泻病。近年来,由于隐孢子虫病的介水暴发流行不断发生,使之对水中隐孢子虫卵囊的研究日益增多。现综述如下。     

混凝沉淀法富集分离水中隐孢子虫卵囊的实验研究

目的 建立混凝沉淀富集回收水中隐孢子虫卵囊的方法.方法 以4 μm荧光微球为研究对象,采用混凝沉淀试验,通过正交设计筛选出最佳混凝剂[聚合氯化铝(PAC)、水溶性壳聚糖(WSC)、1 mol/L的NaOH溶液用量]的配方,并观察不同pH值(6～9)、浑浊度(0～15 NTU)和水温(5～15℃)对回收率的影响.结果 确定了最佳混凝配方,聚合氯化铝浓度为80 mg/L,壳聚糖浓度为30 mg/L,1 mol/L氢氧化钠溶液用量为0.6 ml.当pH值在6～9范围内,回收率均在86％以上;当水温在5～25 c℃范围内,回收率在55％以上;当浑浊度为0～15 NTU范围内,回收率均在90％以上.且回收率均随着pH值、水温和浑浊度的升高而上升.向实际水样投加隐孢子虫,采用混凝沉淀法与EPA1623法进行富集比较,差异无统计学意义.结论 该方法能够高效富集回收水中的隐孢子虫卵囊,并对实验条件要求较低,适用于一般实验室对水中隐孢子虫卵囊的检测.     

臭氧灭活水中微小隐孢子虫卵囊的效果

目的评价臭氧灭活水中微小隐孢子虫卵囊的效果,为研制饮用水中隐孢子虫的灭活措施提供理论和技术依据。方法利用纯化后的微小隐孢子虫卵囊制成悬液,采用动态通入臭氧方式进行消毒试验。分别观察臭氧浓度、作用时间、水温、pH和色度对卵囊灭活效果的影响。利用DAPI和PI荧光活体染色方法进行活性评价,计算灭活率。结果提高臭氧浓度和作用时间均有利于隐孢子虫卵囊的灭活,0.3 mg/L臭氧消毒60 min,灭活率达到99.81%,3 mg/L臭氧消毒10 min,灭活率达到99.99%。水温、pH和色度均对臭氧灭活隐孢子虫卵囊有一定的影响。结论臭氧灭活微小隐孢子虫卵囊速度快、效果好,是灭活水中隐孢子虫卵囊较理想的消毒剂。     

介水性隐孢子虫病及其预防控制

介水性隐孢子虫病是由于饮用了含隐孢子虫卵囊的水引起的以腹泻为主要症状的消化道传染病。隐孢子虫为一类寄生在哺乳类、鸟类和人类体内的肠道原虫,感染此病的动物和人粪便中有大量隐孢子虫卵囊。卵囊体积小(直径4~6μm),人感染130个左右的卵囊即可致病。卵囊能抵抗饮用水常用消毒剂,在水和土壤中可存活数月。因此,如水源附近,或水源上游地表中存在高浓度的卵囊,而水厂处理工艺不良或处理失误时,就极易发生隐孢子虫病的介水性爆发流行。当前,随着城市规模发展演进到城市群带来人口的高度聚集和城乡集约化畜、禽养殖业的发展,城乡生活垃圾、生活污水和养殖业污水的排放量迅速增加,使得部分人、畜、禽粪尿污染环境和水,故对隐孢子虫病介水传播的危险性必须予以重视。     

隐孢子虫卵囊基因组的提取及以保守基因为靶标的PCR检测

目的探讨提高PCR检测微小隐孢子虫卵囊方法的特异性和敏感性,为防止隐孢子虫病流行提供检测依据。方法应用BALB／c小鼠感染扩增卵囊,不连续蔗糖密度梯度离心法从粪便中纯化卵囊,通过冻融和超声法破碎卵囊,然后以酚／氯仿／异戊醇法提取基因组DNA作为PCR扩增模板,根据隐孢子虫保守基因18S rRNA,设计一对特异性的引物,扩增约450bp的目的片段。结果超声处理法对隐孢子虫卵囊的破囊率为92％,明显高于冻融法的86％。2个组抽提的DNA作为模板均扩增出目的片段,但超声处理组敏感性高于冻融处理组,分别能检测到卵囊2个／mL和20个／mL。结论超声法对微小隐孢子虫卵囊的破碎效果优于冻融法,经改进的PCR检测微小隐孢子虫卵囊方法具有特异性好和敏感性强的特点。     

不同剂量隐孢子虫感染正常和免疫抑制小鼠的效应研究

目的探讨昆明小鼠在灌服地塞米松、地塞米松+隐孢子虫卵囊(150万)、地塞米松+隐孢子虫卵囊(300万)的免疫功能的变化。方法采用MTT法检测40只昆明小鼠的免疫功能。随机分组:空白对照组(n=10),免疫抑制组(n=10),免疫抑制+隐孢子虫卵囊150万(n=10),免疫抑制+隐孢子虫卵囊300万(n=10)。结果实验组小鼠与对照组小鼠比较,巨噬细胞吞噬功能明显降低,T、B淋巴细胞的增殖明显降低,NK细胞活性均明显降低(p<0.01)。结论40只昆明小鼠吞噬功能、脾细胞增殖、NK细胞活性检测结果见表(1-3),从表中可以看出,实验组小鼠与对照组小鼠三项检测指标比较均有明显降低,差异显著(P<0.01),实验组小鼠各组间比较,差异无显著性(P>0.05)。     

中试装置去除饮水中贾第虫与隐孢子虫的简介

为了从饮水中有效地去除贾第虫孢囊和卵囊,需要采取多种处理方法,通常包括进行有效的水源防护,优化处理及配备完好的配水系统。本研究主要针对3种处理装置开展了研究,分析了原水和净化后的水样,测定贾第虫孢囊与隐孢子虫卵囊的浓度和去除率,以及孢囊与卵囊浓度和水的浑浊度及微粒物的粒度之间的关系。     

关于人源隐孢子虫排卵规律的探讨

目的探讨昆明小鼠在免疫抑制和非免疫抑制条件下,感染相同剂量3株人源隐孢子虫分离株的排卵规律。方法饮水中加地塞米松(DEX)抑制小鼠免疫功能,人工灌胃隐孢子虫卵囊(CSO)感染小鼠,观察小鼠粪便中CSO排出规律和小鼠临床表现。结果1号和2号分离株感染小白鼠后的排隐孢子虫卵囊规律几乎一致,二者与3号分离株感染小白鼠后的排隐孢子虫卵囊规律差别较大。结论可见能够感染人的隐孢子虫的虫种具有广泛性,这些研究结果,为进一步了解隐孢子虫的人畜互传途径和隐孢子虫病的防治提供了重要的理论依据,并为人源隐孢子虫病的分子流行病学研究打下了良好基础。     

水中隐孢子虫和贾第虫检测方法的比较

目的分析比较水中隐孢子虫(Cryptosporidium)和贾第虫(Giardia)的检测方法。方法采用FiltaMax xpress快速法、美国环保局(EPA)标准检测方法1623中的两种方法(即IDEXX的Filta-Max系统和Pall Gelman的EnvirochekTM HV浓缩器)对不同环境水样加标样品的回收率和精密度进行比较。结果Filta-Max xpress快速法对隐孢子虫和贾第鞭毛虫的回收率符合美国环保局IPR/OPR样品检测标准。就环境水样加标样品来说,Filta-Max xpress快速法的检测性能等同于目前认可的标准方法。     

隐孢子虫病研究进展

隐孢子虫是一类寄生于人和动物体内的肠道原虫，从粪便以卵囊形式排出体外。隐孢子虫存在于各种水体中，如地表水、地下水、过滤水、污染湖水、游泳池水等。隐孢子虫有多种，引起人类感染的主要是伯温隐孢子虫。介水性隐孢子虫病是由饮用含隐孢子虫卵囊的水所引起的以腹泻为主要症状的消化道传染病。目前，隐孢子虫病已被视为全球腹泻病中最常见的病因之一，且因其为艾滋病的常见并发症而引起重视。世界卫生组织和美国疾病预防控制中心均将该病列为新发传染病。现对其研究进展作一综述。     

Detection of Cryptosporidium oocysts and Giardia cysts in swimming pool filter backwash water concentrates by flocculation and immunomagnetic separation

The purpose of the present study was to evaluate techniques for detection of Giardia cysts and Cryptosporidium oocysts in swimming pool filter backwash water. Calcium carbonate flocculation was used for water samples concentration of 1 l filter backwash water samples. Immunomagnetic separation (IMS) was used for separation of cysts and oocysts from detrital material. Cysts and oocysts were then detected using direct immunofluorescence. ColorSeed C&G was used as an internal standard. Recoveries were examined at several processing points. Highest recoveries (23% Cryptosporidium, 18% Giardia) were obtained using lower filter backwash volumes, greater IMS volumes, and addition of Tween20 to backwash samples prior to processing. The combination of CaCO3 flocculation, IMS and direct immunofluorescence was found to be an effective tool for the detection and quantification of Giardia spp. and Cryptosporidium spp. in filter backwash water samples.     

深圳市地表水贾第鞭毛虫和隐孢子虫污染状况的调查

目的:了解深圳市地表水贾第鞭毛虫和隐孢子虫的污染状况,为饮用水水质卫生、安全供水提供科学依据。方法:采集以水库水为源水的8家村镇水厂水样及3家污水处理厂排出水,共21份样品,应用美国环保局(EPA)的标准检测方法,对水样作抽滤、淘洗、磁分离、染色鉴定的处理,检测贾第鞭毛虫和隐孢子虫卵囊含量。结果:深圳市8家村镇级水厂有6家源水检出贾第鞭毛虫包囊,1家水厂的源水检出隐孢子虫卵囊,3家污水处理厂中有2家污水处理后排出水检出贾第鞭毛虫和隐孢子虫卵囊。结论:深圳市地表水可检出致病性原虫,源水受到污染的可能性存在,要保证水质卫生,做到优质安全供水,必须加强卫生管理。     

微小隐孢子虫CRIP基因克隆与分析

目的 比对分析微小隐孢子虫南京(NJ)株亲环素-RNA相互作用蛋白(CRIP)与其他隐孢子虫株CRIP基因序列的差异。方法 根据GenBank微小隐孢子虫Iowa Ⅱ株CRIP基因序列设计并合成2对引物,应用巢式聚合酶链反应(PCR)技术从微小隐孢子虫NJ株基因组DNA中扩增CRIP基因,并将其克隆到pMD18-T载体上,将重组质粒pMD18-T-CpCRIP进行PCR和双酶切鉴定后测序,应用生物信息学方法分析微小隐孢子虫NJ株CRIP基因与其他种属的CRIP基因序列同源性。结果 巢式PCR扩增获得的特异性CRIP基因序列,经PCR及双酶切鉴定确为pMD18-T-CpCRIP重组质粒;测序结果显示,该序列有119 bp,微小隐孢子虫NJ株CRIP基因全长为909 bp,编码302个氨基酸;测序结果和同源性分析显示,中国微小隐孢子虫NJ株CRIP基因序列与国外的微小隐孢子虫Iowa II株同源性为98%;该隐孢子虫NJ株CRIP基因序列获GenBank登录号JQ396883。结论 微小隐孢子虫NJ株CRIP基因与其他微小隐孢子虫株存在基因变异。     

Comparison of current methods used to detect Cryptosporidium oocysts in stools

In this review all of the methods that are currently in use for the investigation of Cryptosporidium in stool material are highlighted and critically discussed. It appears that more qualifications and background knowledge in this field regarding the diagnosis of the Cryptosporidium parasite is required. Furthermore, there is no standardization for the protocols that are commonly used to either detect oocysts in faeces or to diagnose the Cryptosporidium infection. It is therefore necessary to initiate further education and research that will assist in improving the accuracy of the diagnosis of Cryptosporidium oocysts in the faecal micro-cosmos. Where ambient concentrations of oocysts are low in stool material, detection becomes a formidable task. Procedures for ring tests and the standardization of multi-laboratory testing are recommended. It is also necessary to enhance the routine surveillance capacity of cryptosporidiosis and to improve the safety against it, considering the fact that this disease is under diagnosed and under reported. This review is intended to stimulate research that could lead to future improvements and further developments in monitoring the diagnostic methodologies that will assist in harmonizing Cryptosporidium oocysts in stool diagnosis.     

2008年深圳市集中式供水中贾第鞭毛虫和隐孢子虫污染现状

目的了解深圳市集中式供水企业水处理工艺情况以及原水、出厂水中贾第鞭毛虫、隐孢子虫的污染现状,为防治原虫介水传染病提供科学依据。方法于2008年5—7月,对19户供水企业的水源、水处理工艺进行现场卫生学调查。采集每户供水企业原水、出厂水水样各1件,检测水样中贾第鞭毛虫孢囊和隐孢子虫卵囊。结果深圳市19户市、区级集中式供水企业的原水均采用混凝沉淀、石英砂过滤、加氯消毒的常规水处理工艺。2008年深圳市供水企业原水、出厂水中均未检出贾第鞭毛虫孢囊和隐孢子虫卵囊。结论深圳市集中式供水未受贾第鞭毛虫和隐孢子虫污染。     

微小隐孢子虫腺苷酸激酶基因克隆及分析

目的获得微小隐孢子虫(Cryptosporidium parvum,Cp)南京(NJ)株的腺苷酸激酶(adenylate kinase,AK)基因的核苷酸和氨基酸序列,比对分析与其他隐孢子虫株AK基因序列的差异。方法采用昆明种小鼠建立微小隐孢子虫NJ株感染模型,根据GenBank微小隐孢子虫Iowa II株AK基因已知序列设计合成2对引物,应用巢式PCR方法从微小隐孢子虫NJ株基因组DNA中扩增AK基因,并将其克隆入pMD18-T载体;对重组质粒pMD18-T-CpAK经PCR及酶切鉴定后测序,应用生物信息学方法分析微小隐孢子虫NJ株AK基因与其他虫株的核苷酸和氨基酸序列差异。结果巢式PCR扩增获得特异的AK基因序列,酶切及PCR鉴定为正确的pMD18-T-CpAK重组质粒,扩增序列为903bp,含隐孢子虫AK全长基因663 bp;核苷酸序列测定及同源性分析表明,微小隐孢子虫NJ株AK基因与人隐孢子虫TU502 type 2株AK的同源性为99%,与微小隐孢子虫Iowa II株AK序列同源性为98%;进化树分析表明,微小隐孢子虫NJ株的AK基因与人隐孢子虫TU502 type 2株AK基因亲缘关系最近。结论成...     

微小隐孢子虫20K亲环蛋白基因克隆及分析

目的克隆微小隐孢子虫(Cp)南京株(NJ)的20K 亲环蛋白(CyP)基因,并对其核苷酸和氨基酸序列进行分析。方法采用昆明种小鼠建立微小隐孢子虫NJ株感染模型,根据微小隐孢子虫Iowa II株20K CyP 基因序列设计合成2对引物,应用巢式PCR技术从微小隐孢子虫NJ株基因组DNA 中扩增20K CyP基因,并将其克隆到pMD18-T载体上,将阳性克隆重组质粒进行菌落PCR及双酶切鉴定;应用生物信息学方法分析微小隐孢子虫NJ株20K CyP基因与其他虫株核苷酸和氨基酸序列差异,并分析该基因蛋白结构域。结果巢式PCR 扩增得到特异的20K CyP基因,经PCR及双酶切鉴定获得了正确的pMD18-T-20K CyP重组质粒;测序结果显示,微小隐孢子虫NJ株20K CyP 基因全长519 bp,编码173个氨基酸,该基因已登录GenBank,登录号为JQ284431;氨基酸序列分析表明,微小隐孢子虫NJ株与Iowa II株20K CyP基因编码的氨基酸序列具有100%同源性;对20K Cyp基因进行蛋白结构域分析,显示该基因序列所编码的蛋白具有特异性类亲环蛋白A、B、H的肽脯氨酰顺反异构酶(PPIase)区域,该区域与人的亲环蛋白A、B、H具有相似性。结论成功克隆微小隐孢子虫NJ株20K CyP基因,该基因具有高度保守性。     

Relative importance of the various environmental sources of Cryptosporidium oocysts in three watersheds

This study was conducted to guide the prioritisation of efforts to manage Cryptosporidum contamination of drinking water supplies in Trinidad, W.I. The main objective was to investigate the relative importance of three main types of sources of Cryptosporidium oocysts: urban, agriculture and wildlife. Weekly surface water samples were collected from 19 sites distributed among three watersheds, and examined for the presence of oocysts. A stratified random sampling design was used with each watershed representing one of the three main sources of oocysts listed above. Results showed a significant association between watershed and the occurrence of positive samples (c²=16.523, d.f. =2, p = 0.000), indicating that land use influenced the presence of oocysts. Urban and forested lands were the two most important sources of oocysts. There was no apparent association between agriculture and the presence of oocysts, and there was no significant difference between the percentage of positive samples at sites below agricultural facilities and sites not associated with agriculture within a single watershed (c²=2.45, d.f. =1, p = 0.117). We conclude that urban and wildlife are the main types of sources of Cryptosporidium contamination of surface water, whereas the contribution of agriculture is minor.     

Contamination of water supplies with Cryptosporidium parvum and Giardia lamblia and diarrheal illness in selected Russian cities

Cryptosporidium parvum and Giardia lamblia are important agents of waterborne diarrheal illness worldwide. While giardiasis is routinely diagnosed in Russia with a chemical staining technique, data on the prevalence of cryptosporidiosis are scarce. Monitoring of the respective parasites in water supplies in Russia is very limited. A health survey conducted in the city of Cherepovets and three other cities in the European part of Russia using enzyme-linked immunosorbent assays (ELISA) demonstrated that 6.9% of diarrheal patients tested had C. parvum antigens in their fecal samples; 9.4% had G. lamblia antigens. A survey of occurrence of these parasites in water supplies in Cherepovets and seven other cities demonstrated that source and finished water samples from several of these cities were contaminated with either C. parvum oocysts or G. lamblia cysts. The surveys were not designed to assess associations between presence or concentrations of C. parvum and G. lamblia in water and related gastrointestinal diseases in exposed populations. Rather, the goals were to demonstrate the presence of disinfection-resistant protozoan parasites in untreated and treated waters, and the importance of these pathogens as causative agents of diarrheal illnesses in a number of Russian cities.     

Cryptosporidium hominis infection of the human respiratory tract

Cryptosporidium oocysts, observed in a natural sputum sample of a patient with HIV, were further studied by using DNA markers to determine the species of the parasite. C. hominis was identified as the species infecting the patient's respiratory tract, a finding that strengthens evidence regarding this pathogen's role in human disease.