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。     

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。     

隐孢子虫肠内外感染及组织病理变化

<正>隐孢子虫病是由隐孢子虫引起的一种以腹泻为特征的人畜共患原虫病。隐孢子虫具有广泛的宿主类型,可以寄生于哺乳类、鸟类、爬行类及两栖类等。1986年,世界卫生组织将本病列为人的艾滋病(AIDS)怀疑指标之一。近年来,随着免役缺陷性     

艾滋病慢性腹泻患者隐孢子虫及病原菌感染调查

目的 调查北京地区艾滋病相关性慢性腹泻患者中隐孢子虫及其他病原微生物感染状况.方法 收集67例北京地区艾滋病相关性慢性腹泻患者粪便,对患者粪便标本进行集卵,检测隐孢子虫卵囊,同时对留取的粪便检测隐孢子虫卵囊片段;用流式细胞仪检测患者血液中CD4⁺细胞计数;检测其是否存在引起腹泻的病原菌和艰难梭菌毒素.结果 4例粪便标本检测隐孢子虫卵囊阳性,且PCR检测均为阳性,2种方法阳性符合率100%,确定感染率为5.97%(4/67),感染者均为获得性免疫缺陷综合征中晚期患者,其血液中CD4⁺细胞计数<200、200～499、≥500 cells/μL的患者感染率分别为5.9%(2/34)、7.4%(2/27)、0;被感染者均系无业人员,生活来源无保障,生存环境较差;对67份标本同期进行细菌培养及涂片革兰染色,其中60份标本真菌培养阳性,经鉴定均为念珠菌,艰难梭菌毒素A/B检测有1例阳性.结论 北京地区艾滋病慢性腹泻患者中存在着隐孢子虫感染,其感染率与患者性别无关,与患者生存环境有一定关系;患者CD4⁺细胞计数水平降低及疾病发展到中晚期的患者易发生隐孢子虫感染;北京地区艾滋病慢性腹泻患者中普遍存在真菌肠道感染.     

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。     

家蝇热休克蛋白HSP20基因克隆、表达和序列分析

目的对家蝇热休克蛋白20(HSP20)基因进行生物信息学分析, 并进行克隆、表达研究。方法利用美国国家生物技术信息中心和瑞士生物信息学研究所的蛋白分析专家系统中有关基因和蛋白序列的分析工具, 结合其他生物信息学分析软件包, 从GenBank上家蝇基因组序列识别出编码HSP20的基因, 分析预测该蛋白质的结构功能;设计引物PCR扩增HSP20基因, 将其克隆到原核表达质粒PEASY-E1中, 重组质粒在大肠杆菌OrigmiB/DE3中经用异丙硫代-β-D半乳糖苷(IPTG)诱导表达, 表达产物用十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)鉴定。结果HSP20基因序列全长865bp, 最大开放阅读框(ORF)为567bp, 编码188个氨基酸, 理论分子量为21443.2 Da, 等电点为5.96;所构建的重组质粒经PCR、双酶切及测序鉴定成功;SDS-PAGE分析结果显示, 重组质粒在OrigmiB/DE3中表达并纯化, 得到的融合蛋白相对分子质量约为21443.2 Da, 诱导12 h蛋白表达量最高, SDS-PAGE法得到的蛋白条带与目的蛋白大小相符。结论成功构建PEASY-E1-HSP20重组原核表达质粒并表达出融合HSP20蛋白, 为进一步研究该蛋白的功能奠定基础。     

金坛市农村集中式供水隐孢子虫污染及儿童感染现状

目的了解金坛市农村集中式供水隐孢子虫污染及人群感染等情况。方法采集12家农村集中式供水单位出厂水,用Filta-Max方法检测隐孢子虫包囊和卵囊;采集农村儿童粪便,采用金胺-酚改良抗酸染色法检查隐孢子虫感染情况。采用问卷调查法了解农村集中式供水单位情况;了解儿童家庭情况、生活习惯、最近健康状况、饮食情况等资料。结果农村集中式供水单位的出厂水未检出隐孢子虫。儿童隐孢子虫感染率为1.09%,小学生和幼儿分别为0.51%和1.61%,差异有统计学意义(P0.05)。低年龄特征、近1年家人有寄生虫感染和最近3个月家人患腹泻或痢疾是隐孢子虫感染的危险因素(P0.05)。结论在预防隐孢子虫污染饮水的同时,应加强防治知识宣传,改善环境,养成良好的卫生习惯。     

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。     

The effect of cyanuric acid on the disinfection rate of Cryptosporidium parvum in 20-ppm free chlorine

Cyanuric acid is used to stabilize free chlorine to reduce photodegradation in outdoor swimming pools. While there have been numerous studies examining its effect on the disinfection rates of bacteria and viruses, it is not known whether cyanuric acid can significantly impact the effectiveness of hyperchlorination for inactivating Cryptosporidium oocysts present in fecally-contaminated swimming pools. This study examined the effect of cyanuric acid on the disinfection rate of Cryptosporidium parvum under swimming pool hyperchlorination conditions (20 mg/ml free chlorine). When 50 mg/L cyanuric acid was present there was a 0.70-log₁₀ reduction in oocyst viability after 10 hours as compared to a 3.7-log₁₀ reduction without cyanuric acid. Aids to remediation, such as decreasing the pH to enhance the germicidal efficiency of the free chlorine and doubling the amount of free chlorine residual, were still unable to achieve a 3-log₁₀ reduction. Current public health recommendations for hyperchlorination and pool remediation are insufficient for pools using cyanurate-stabilized chlorine to achieve a three log inactivation of the parasite.     

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.