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。     

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pcDNA3.0-23重组质粒免疫效果评价

目的 比较微小隐孢子虫表面抗原CP23真核表达载体pcDNA3.0-23经不同免疫途径产生的免疫效果。方法 提取微小隐孢子虫基因组DNA,PCR扩增CP23基因片段,克隆至真核表达载体pcDNA3.0,构建pcD-NA3.0-23重组质粒,分别通过肌肉注射和滴鼻(粘膜)免疫2种途径免疫BALB/c小鼠,免疫3次,2周后检测抗CP23特异性抗体IgG滴度、小鼠脾脏、血清中CD4⁺和CD8⁺T细胞、细胞因子γ干扰素(IFN-γ);用微小隐孢子虫攻击感染被免疫小鼠,收集小鼠粪便,计算小鼠排出的卵囊量。结果 肌注组与滴鼻组小鼠血清抗CP23特异性抗体IgG滴度随免疫次数增加均明显升高,高于对照组及空质粒组(P<0.05),肌注组高于滴鼻组(P<0.05);肌注组与滴鼻组小鼠的CD4⁺T细胞、CD4⁺/CD8⁺比值均高于磷酸缓冲液(PBS)对照组及pcDNA3.0空质粒组(P<0.05),但2种免疫途径的差异无统计学意义(P>0.05);肌注组和滴鼻组脾细胞培养上清中IFN-γ明显高于对照组及空质粒组(P<0.05);微小隐孢子虫攻击小鼠后,2种免疫途径的小鼠排出卵囊量明显少于对照组,且排出时间缩短,2种途径的差异无统计学意义。结论 pcDNA3.0-23重组质粒作为基因疫苗,可产生较好的细胞及体液免疫反应;不同的免疫途径可产生不同的免疫反应。     

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。     

两种机会性寄生原虫——微小隐孢子虫和蓝氏贾第鞭毛虫基因检测方法的建立

目的建立两种机会性寄生原虫——微小隐孢子虫和蓝氏贾第鞭毛虫（贾第虫）基因检测方法。方法从微小隐孢子虫和蓝氏贾第鞭毛虫感染者粪便标本内分离纯化卵囊和包囊，提取基因组DNA。根据微小隐孢子虫18S rRNA基因和贾第虫磷酸丙糖异构酶（tim）基因各设计或合成两对特异性引物，采用PCR技术分别对从卵囊和包囊提取的和6种对照DNA样本（日本血吸虫、刚地弓形虫、溶组织内阿米巴、旋毛虫和阴道毛滴虫以及人体血细胞DNA等），以及此二种虫相互间的DNA样本进行检测，以确定该法的特异性和敏感性。方法建立后，取艾滋病患者粪便标本对之进行检测。结果从微小隐孢子虫和贾第虫的DNA样本中分别扩增出各自相应基因的500bp和683bp长度的片段；最少可检测出20pg隐孢子虫和0．4pg贾第虫DNA；几种对照DNA样本均不发生交叉反应；受检的30例艾滋病患者粪便标本中，7例显示微小隐孢子虫阳性，未检出贾第虫。结论建立的基因检测方法，对微小隐孢子虫和贾第虫的检测具有高度的特异性和敏感性，可用于临床艾滋病合并感染的早期诊断和人群流行病学筛查。     

098 隐孢子虫病的病原、表现、诊断与治疗

隐孢子虫病是由一种卵囊为球形、直径4～6 μm、对加氯消毒有极强抵抗力的肠道寄生性原虫--微小隐孢子虫所致的、经粪-口途径传播、人畜共患急性传染病.在免疫功能正常的人中常表现为潜伏期2～28d,持续1～2周,以水样腹泻为主,伴腹痛、低热和恶心呕吐等症状的自限性疾病;而在艾滋病患者等免疫功能低下的人群中往往表现为严重而持久的、有时甚至致命的顽固性腹泻.目前尚无特效治疗.其确诊有赖于病原学检测,如直接涂片法、抗酸染色法、酶联免疫吸附法、免疫荧光法及PCR技术等.据研究,摄入10～100个微小隐孢子虫卵囊或呛一口受污染的水就可能感染发病,使50%健康志愿者感染的卵囊量(ID5o)约为132个.其感染率在艾滋病病毒阳性且伴腹泻者中约为13.8%～23.9%,在普通腹泻者中约为2.1%～6.1%.有关该病暴发流行的报道正呈渐增之势,其危害的广泛性和严重性,尤其是对供水安全造成的威胁越来越不容忽视.     

隐孢子虫和环孢菌与旅行者腹泻

腹泻是旅行中常见的医学问题。致病原以大肠杆菌为主,还有沙门氏杆菌、霍乱弧菌等。近年来发现环孢菌与隐孢子虫,也是一种引起腹泻的致病原。虽然它们不是旅行者腹泻的主要致病原,但是,它们引起腹泻的病程较长,尤其对有免疫缺陷的患者,可引起较严重的后果,故须对它们要有进一步的认识。     

隐孢子虫病诊断与治疗研究进展

隐孢子虫病是一种全球性的人兽共患寄生虫病,是免疫功能低下或免疫功能缺陷患者致死的主要原因之一.此文就隐孢子虫病的实验室诊断与治疗的研究进展作了综述.     

饮用水隐孢子虫污染与感染研究现状分析

隐孢子虫是可以感染人体和其他脊椎动物的肠道病原体,在水源性疾病流行中其占有重要的位置,是引起人类腹泻疾病常见因素之一。自上世纪80—90年代发达国家因饮用水隐孢子虫污染导致流行以来,对饮用水的隐孢子虫污染与人感染的研究不断深入,尽管多数感染者症状轻微或无症状,但由于对隐孢子虫病患者的治疗缺乏针对性的药物,因而更受关注,被世界卫生组织和美国疾控中心列为新发传染病。本文对人隐孢子虫病流行现状、饮用水隐孢子虫污染与感染风险和监测等进行系统回顾和深入分析,讨论相关制约因素,并提出了近期需要关注和亟待研究解决的主要问题。     

江苏省某地农村集中式供水隐孢子虫污染与儿童感染现状研究

目的了解江苏省某地农村集中式供水隐孢子虫污染与儿童感染现状,探讨可能的传播途径及影响因素。方法对调查地区集中式供水设施进行卫生学调查,按照《生活饮用水标准检验方法》（GB/T5750–2006）检测24份水源水和出厂水中隐孢子虫卵囊含量。采取整群抽样方法调查供水地区1 651名儿童,通过问卷调查了解儿童一般人口学资料、生活习惯和环境卫生情况等,采集儿童粪便,用金胺酚─改良抗酸染色法检查隐孢子虫卵囊。结果水源水及出厂水水样中均未检出隐孢子虫卵囊,儿童粪便中隐孢子虫卵囊阳性率为1.09%,感染率幼儿园儿童大于小学生（1~3年级）,儿童感染者多无自觉症状,未发现与感染有关的影响因素。结论该地农村儿童存在隐孢子虫感染,需要进一步对可能的感染途径和影响因素进行研究。     

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。     

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。     

Inactivation of Cryptosporidium parvum under chlorinated recreational water conditions

Cryptosporidium is a chlorine-resistant protozoan parasite and the etiological agent in many disinfected recreational water outbreaks. While previous studies have reported disinfection Ct values for Cryptosporidium parvum using sodium hypochlorite, these studies have employed conditions and procedures which are not ideal for establishing public health remediation recommendations for chlorinated recreational water venues. In the present study, free chlorine Ct values were measured at pH 7.5 using young oocysts (<1 month old) and tissue culture to determine oocyst viability. Two different oocyst isolates were used: one originating from Iowa and one from Maine (USA). This study determined that the Ct values for a 3-log reduction in oocyst viability were 10,400 (Iowa) and 15,300 (Maine) at pH 7.5. These Ct values are higher than the Centers for Disease Control and Prevention (USA) currently recommends (Ct=9,600) for achieving a 3.0-log inactivation of Cryptosporidium oocysts during remediation of recreational water venues following fecal diarrhea accidents.     

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.     

Time and Temperature Effects on the Viability and Infectivity of Cryptosporidium parvum Oocysts in Chlorinated Tap Water

The authors compared the viability and infectivity of Cryptosporidium parvum oocysts in chlorinated tap water at various storage durations (i.e., 2 wk, 4 wk, 6 wk, or 8 wk) and at 2 cool temperatures (i.e., 10[ddot]C and 4[ddot]C), using in vitro (excystation) and in vivo (suckling mouse) methods. After 8 wk, mean oocyst excystation decreased to 33.4% and 26.7% at 10[ddot]C and 4[ddot]C, respectively. Suckling mice infectivity was higher after storage at 10[ddot]C than after storage at 4[ddot]C. These data suggest that Cryptosporidium parvum oocysts can survive and remain infectious for 8 wk in cool chlorinated tap water.     

Prevalence of Cryptosporidium parvum in private drinking water cisterns in Bani-Kenanah district, northern Jordan

Due to water scarcity in Jordan, the water authority only pump the water once or twice a week to the population. Thus people in rural areas, including the Bani-Kenanah district, make the most of their water resources by storing rainwater in private reservoirs for use during periods of water shortage. These reservoirs include; underground cisterns and concrete or metal tanks. The water collected in these reservoirs is at risk of contamination. During the period March-July 2002, the three types of reservoirs from 368 households were surveyed for presence of Escherichia coli and Cryptosporidium parvum, indicators of contamination. The cistern was the most contaminated reservoir with 17% (95% CI: 13,22) for E. coli (significant, P<0.05), and 2% (95% CI: 1,4) for C. parvum. Only 1% (95% CI: 1,6) of the metal reservoirs had E. coli, while concrete reservoirs were free. No C. parvum oocysts were detected in either the concrete or metal reservoirs. Reservoirs opening at floor level and the bucket kept outside the reservoir were significant (P<0.05) enhancing risk factors for contamination with C. parvum.     

GIS-based analysis of the fate of waste-related pathogens Cryptosporidium parvum, Giardia lamblia and Escherichia coli in a tropical canal network

Urban canals play a major socio-economic role in many tropical countries and, particularly, Thailand. One of the overlooked functions that they perform is a significant attenuation of waste-related pathogens posing considerable health risk, as well as pollution attenuation in general. The study dealt with a comparison of three canals receiving: (i) municipal, (ii) mainly industrial and (iii) mainly agricultural wastewater, listed in order of progressively decreasing organic loading. The occurrence and fate of waterborne Cryptosporidium parvum, Giardia lamblia and Escherichia coli were monitored in the canals by both real-time PCR and conventionally for 12 months. The pathogens are etiological agents of an estimated 38% and 47% of diarrhea cases worldwide and in Thailand, respectively. The geographic information system (GIS) was used to evaluate and map point and, particularly, non-point pollution sources which allowed differentiating the canal sections in terms of predominant pathogen sources. The flowthrough canals, which can be viewed as waste stabilization ponds, were found to be efficiently removing the pathogens at the following generalized specific rates: 0.3 (C. parvum), 1.2 (G. lamblia), 1.8 (E. coli) log₁₀/km.d in the dry season. The rates decreased in the rainy season for E. coli and G. lamblia, but increased for C. parvum which indicated different removal mechanisms. Data suggest that E. coli and G. lamblia were mainly removed through sedimentation and sunlight (UV) irradiation, while the likely mechanism for C. parvum was predation. Overall, the specific pathogen removal rates positively correlated with the canal organic loading rates in the rainy season. As an important result, an estimate of the municipal pollution mitigation by over 2,280 km canals in the Greater Bangkok suggests that concomitant to the pathogens at least 36–95 tons of BOD₅ is being removed daily, thereby saving the receiving Chao Phraya River and Bight of Bangkok, by far exceeding current, from major eutrophication problems.     

5种分离纯化粪便中隐孢子虫卵囊方法的比较

[目的]比较5种分离纯化粪便中隐孢子虫卵囊的方法。[方法]分别用饱和氯化钠漂浮法、Sheatherg蔗糖漂浮法、甲醛一乙醚沉淀法、不连续蔗糖密度梯度离心法和Percoll密度梯度离心法对感染小鼠粪样中的隐孢子虫卵囊进行分离纯化,浓集回收的卵囊利用鼠抗隐孢子虫卵囊壁单克隆抗体间接免疫荧光法检测卵囊数,作定量比较,同时定性比较回收卵囊液的纯度。[结果]Percoll密度梯度回收的卵囊数量最多,而且杂质少;不连续蔗糖密度梯度离心法和Sheatherg蔗糖漂浮法次之,但前者回收的卵囊液中杂质较少;饱和氯化钠漂浮法获得的卵囊液中杂质也较少,但卵囊数次之;甲醛一乙醚沉淀法获得的卵囊液中检出的数量最少,且杂质最多。[结论]不连续蔗糖密度梯度离心法是一种经济、有效的卵囊分离纯化方法。