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。     

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。     

AIDS合并隐孢子虫感染性腹泻的病原学检查

[目的]探讨获得性免疫缺陷综合症(AIDS)合并隐孢子虫感染性腹泻的病原学检查方法. [方法]对5例AIDS合并隐孢子虫感染性腹泻的住院患者,应用新鲜粪便标本进行不同染色方法的对比检查. [结果]经碘液染色法卵囊的结构呈圆形或卵圆形折光颗粒;金胺-酚染色后的卵囊在荧光镜高倍镜下观察,卵囊呈乳白色或略带绿色的荧光;改良抗酸染色后,背景为蓝绿色,卵囊内隐约可见4个玫瑰红色子孢子;经金胺-酚-改良抗酸复染法染色后,非特异性颗粒染成蓝黑色,卵囊结构同改良抗酸染色法.[结论]临床上可以先采用粪便直接涂片碘液染色观察,如发现可疑卵囊标本,再涂片作金胺-酚改良抗酸复染法,即可获得快速、准确的诊断.     

上海某污水厂出水及其排放水体中隐孢子虫和贾第鞭毛虫的分析检测

目的：检测上海某污水厂处理出水及其排放水体黄浦汀上游中隐孢子虫和贾第鞭毛虫存在水平。方法：微孔滤膜过滤、乙酸乙酯福尔马林分离、Giemsa染色、油镜及相差显微镜观察。结果：对污水厂所取八次水样中，贾第氏虫孢囊检出6次，阳性率为75％，检出贾第氏虫孢囊最高含量为56个／10L；隐孢子虫卵囊共检出4次，阳性率为50％，检出隐孢子虫卵囊的最高含量为63个／10L；贾第氏虫孢囊与隐孢子虫卵囊的共同阳性率为12．5％。从其排放水体黄浦江上游所取的8份水样中，贾第氏虫孢囊共检出2次，阳性率为25％，最高含量为45个／10L；隐孢子虫卵囊只检出过1次，阳性率为12．5％，卵囊含量为30个／10L；同时检测出贾第氏虫孢囊和隐孢子虫卵囊的共同阳性率为0％。结论：上海城市污水处理厂应加强对两虫的去除工艺改进，减少上海城市供水生物安全风险。     

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.     

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。     

Percoll密度梯度离心法在血液组分分离及元素测定中的应用

目的探讨元素在大鼠血液经Percoll密度梯度离心法分离后的不同组分(全血、血浆、红细胞、外周血单个核细胞)中的分布特点。方法将SD大鼠随机分成对照组和不同剂量受试组,共9组。采用4种元素(Hg、I、Mn、Sr)的混合溶液气管滴注染毒,连续染毒10 d后股动脉取血。用Percoll液分离血液,分别收集血浆、红细胞、外周血单个核细胞(PBMC),并用电感耦合等离子体质谱仪(ICP-MS)测定全血及上述3种血液成分中4种染毒元素的含量。结果随染毒剂量增加,全血和血浆组分中的元素呈直线回归关系,红细胞与白细胞中部分元素含量与染毒剂量无线性回归关系。结论元素在血液的不同组分中的含量受呼吸道染毒的影响各不相同,利用Percoll密度梯度离心法选取合适的组分可以实现实验目的。     

生活饮用水中贾第鞭毛虫和隐孢子虫定量检测的质量控制

[目的]对定量检测生活饮用水中贾第鞭毛虫和隐孢子虫的方法进行质量控制,分析其关键控制点,评估方法的可用性.[方法]测定"滤囊过滤→免疫磁分离→荧光染色法"实验各阶段的加标回收率和不同类型水样的加标总回收率,应用危害因素分析及关键控制点HACCP分析实验过程中影响定量检测的关键因素.[结果]实验各阶段"富集浓缩→免疫磁分离→染色计数"的平均加标回收率为:贾第鞭毛虫50%→80%→97%:隐孢子虫52%→88%→95%.不同类型水样平均加标回收率为:蒸馏水加标贾第鞭毛虫39%,隐孢子虫43%;水源水加标贾第鞭毛虫38%,隐孢子虫42%;管网水加标贾第鞭毛虫50%,隐孢子虫50%.方法总计贾第鞭毛虫加标回收率为42%,隐孢子虫加标回收率为45%.[结论]滤囊过滤/免疫磁分离/荧光染色法定量检测水中贾第鞭毛虫和隐孢子虫,在采取质量控制措施以避免实验各环节的危险源后,其回收率满足美国EPA1623和我国GB/T5750方法的要求,可用于各类水样的检测.     

福州地区腹泻病人与肠道原虫感染的关系

[目的]了解福州地区腹泻病人与肠道原染的关系。[方法]通过生理盐水直接涂片法、卢戈氏碘液染色涂片法或改良抗酸染色法对3116名腹泻病人的新鲜粪便进行镜检。[结果]在3116名腹泻病人中，肠道原虫感染率为21.66%；弃除63例结肠内阿米巴致病性未获证实外，肠道致病原虫的感染率为19.64%。其中以溶组织内阿米巴感染率17.65%为最高，其次隐孢子虫为0.64%，人毛滴虫0.61%，人芽囊原虫0.42%，蓝贾第鞭毛虫0.32%。[结论]福州地区腹泻病人有19.64%是由肠道致病原虫所引起，其中主要是溶组织内阿米巴，在儿童中主要是由隐孢子虫、人毛滴虫、人芽囊原虫和蓝贾第鞭毛虫。     

濮阳市鼠类沙门氏菌感染状况研究

目的：研究我市鼠类沙门氏菌感染状况及特征，为制订防治措施提供科学依据。方法：取不同种类鼠粪１０ 克按《卫生防疫检验》微生物学部分及《寄生虫检验》进行沙门氏菌培养分离及鉴定，同时采用饱和生理盐水漂浮法及淘洗沉淀法涂片镜检寄生虫卵及幼虫。结果：沙门氏菌检出率为３５ ．５ ％ ，全部为鼠伤寒沙门氏菌，噬菌体分型均为Ⅰ型。虫卵及幼虫总检出率为６９ ．８ ％ ，其中线虫阳性率３１ ．７５ ％ ，蛔虫卵阳性率１０ ．１ ％ ，结肠内阿米巴包囊阳性率９ ．５ ％ ，绦虫卵阳性率２ ．６ ％ ，两种虫卵混合感染者阳性率为１２ ．２ ％ 。ＨＢ ｓＡｇ 监测结果全部阴性。     

Preparation of Human Spermatozoa for In Vitro Fertilization by Isopycnic Centrifugation on Self-Generating Density Gradients

A simple and rapid method is described for the retrieval of highly motile, morphologically normal spermatozoa from human semen. This method is a modification of a technique described previously [3] and employs the process of isopycnic centrifugation of semen on self-generating Percoll density gradients. The procedure is carried out under sterile conditions and has no detectable deleterious effects on the fertilizing capacity of spermatozoa. Spermatozoa may be recovered from semen samples of widely differing quality and can be used successfully for in vitro fertilization (IVF). This technique may be useful not only for the preparation of spermatozoa for IVF and possibly for artificial insemination by husband (AIH) but also for the investigation and analysis of the causes of infertility associated with oligozoospermia.     

Detection of Cryptosporidium parvum oocysts in experimentally contaminated lettuce using filtration, immunomagnetic separation, light microscopy, and PCR

Recovery of Cryptosporidium parvum oocysts in a fecal suspension that experimentally contaminated onto lettuce leaves was investigated. Material recovered from the lettuce samples by washing in detergent solutions were concentrated by filtration using the Envirochek Sampling Capsule. Oocysts were concentrated by immunomagnetic separation (IMS) and detected by microscopy following modified Ziehl-Neelsen (MZN) staining. Cryptosporidal DNA was detected using a nested-PCR assay for amplification of a fragment of the Cryptosporidium oocyst wall protein (COWP) gene, which was applied to DNA extracted from both filtrates, and material recovered from MZN stained smears on glass slides after microscopy. No Cryptosporidium were detected by microscopy or by PCR of un-inoculated lettuce leaves. After IMS, means of 0-6.5% of the total numbers of oocysts inoculated were recovered and detected by microscopy. Detection by PCR was less sensitive than microscopy. There was a strong association between successful PCR amplification, the numbers of oocysts detected by microscopy and the numbers of oocysts in the inoculum. This study confirms that C. parvum oocysts can be recovered from contaminated lettuce using filtration and IMS, and detected by microscopy and PCR. However, further developments are required to improve recovery of this parasite.     

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.     

Comparison of two methodologies for detection of Giardia spp. cysts and Cryptosporidium spp. oocysts in activated sludge samples from a sewage treatment plant in the city of Campinas, São Paulo State, Brazil

Giardia spp. and Cryptosporidium spp. are recognized worldwide as highly infectious protozoan parasites that can cause severe gastrointestinal disease in humans and animals. The detection of these pathogens in activated sludge samples becomes interesting since there is an increasing trend for the use of sewage sludge (biosolids) in agriculture. A total of 22 samples were collected and evaluated by means of Centrifugal - Concentration, followed or not followed by a purification process (ether clarification and sucrose flotation). Student t tests for comparison of the two procedures indicated a higher recovery rate of Giardia cysts with Centrifugal - Concentration; with regard to Cryptosporidium oocysts, no significant differences were found between the two methods, as only two samples were positive. The Centrifugal - Concentration procedure was shown to be the simplest and cheapest to perform, as emphasized by the efficiency recovery results.     

Comparison of two methodologies for detection of Giardia spp. cysts and Cryptosporidium spp. oocysts in activated sludge samples from a sewage treatment plant in the city of Campinas, Sao Paulo State, Brazil

Giardia spp. and Cryptosporidium spp. are recognized worldwide as highly infectious protozoan parasites that can cause severe gastrointestinal disease in humans and animals. The detection of these pathogens in activated sludge samples becomes interesting since there is an increasing trend for the use of sewage sludge (biosolids) in agriculture. A total of 22 samples were collected and evaluated by means of Centrifugal – Concentration, followed or not followed by a purification process (ether clarification and sucrose flotation). Student t tests for comparison of the two procedures indicated a higher recovery rate of Giardia cysts with Centrifugal – Concentration; with regard to Cryptosporidium oocysts, no significant differences were found between the two methods, as only two samples were positive. The Centrifugal – Concentration procedure was shown to be the simplest and cheapest to perform, as emphasized by the efficiency recovery results.     

Occurrence of Cryptosporidium oocysts and Giardia cysts in the Nakdong River and their removal during water treatment

This study was conducted in preparation of a pending Cryptosporidium regulation in Korea. The study had two main objectives: 1) to examine the occurrence of Cryptosporidium oocysts in the Nakdong River; and 2) to evaluate their removal during water treatment. Occurrence of Giardia cysts was also examined. Average (arithmetic mean) numbers of Cryptosporidium oocysts and Giardia cysts at the treatment intake site were 2.6 l^-1 and 4.8 l^-1, respectively. Generally, the number of Giardia cysts was higher than that of Cryptosporidium oocysts at more sites, but the difference was minimal. Comparison of tributaries indicated that livestock wastes were more serious pollutants than sewage in terms of protozoa contamination. In general, fewer oocysts and cysts were detected during winter. No correlation was found for such water quality parameters as T-N, T-P, TOC, DO, pH and temperature with the numbers of oocysts and cysts except for suspended solids, which showed the highest correlation (R²=0.55). Removal of Cryptosporidium oocysts was evaluated using a Cryptosporidium tracer, which has similar characteristics to Cryptosporidium oocysts. The tracer removal depended on turbidity removal. Coagulation followed by sedimentation resulted in 1.2–1.5 log removal of the tracer under optimal conditions. Filtration resulted in 1.3–1.5 log removal of the tracer. These treatability experiments showed that traditional water treatment processes could achieve 2.5–3.0 log removal of the oocysts.     

Meeting report: Application of genotyping methods to assess risks from cryptosporidium in watersheds

A workshop titled "Application of Genotyping Methods to Assess Pathogen Risks from Cryptosporidium in Drinking Water Catchments" was held at the International Water Association biennial conference, Marrakech, Morocco, 23 September 2004. The workshop presented and discussed the findings of an interlaboratory trial that compared methods for genotyping Cryptosporidium oocysts isolated from feces. The primary goal of the trial and workshop was to assess the utility of current Cryptosporidium genotyping methods for determining the public health significance of oocysts isolated from feces in potable-water-supply watersheds. An expert panel of 16 watershed managers, public health practitioners, and molecular parasitologists was assembled for the workshop. A subordinate goal of the workshop was to educate watershed management and public health practitioners. An open invitation was extended to all conference delegates to attend the workshop, which drew approximately 50 interested delegates. In this report we summarize the peer consensus emerging from the workshop. Recommendations on the use of current methods by watershed managers and public health practitioners were proposed. Importantly, all the methods that were reported in the trial were mutually supporting and found to be valuable and worthy of further utility and development. Where there were choices as to which method to apply, the small-subunit ribosomal RNA gene was considered to be the optimum genetic locus to target. The single-strand conformational polymorphism method was considered potentially the most valuable for discriminating to the subtype level and where a large number of samples were to be analyzed. A research agenda for protozoan geneticists was proposed to improve the utility of methods into the future. Standardization of methods and nomenclature was promoted.     

Detection System of Cryptosporidium parvum Oocysts by Brackish Water Benthic Shellfish (Corbicula japonica) as a Biological Indicator in River Water

The brackish water benthic shellfish, Corbicula japonica, was experimentally exposed to Cryptosporidium parvum oocysts at 1.51 × 10⁴ oocysts/clam/day for 7 or 14 days. Oocysts were predominantly eliminated through the feces of Corbicula japonica in both cases by microscopic and PCR methods. The fecal excretion rates of oocysts within 4 days after the last exposure to Corbicula japonica were 87.6% for the 7-day exposure group and 86.0% for the 14-day exposure group. The tissue residue level of oocysts in the gastrointestinal tract 3 days after the last exposure was 2.7% of total exposed oocysts and that of 7 days was 1.1% for the 7-day exposure case and 1.6 and 0.5% for the 14-day exposure case, respectively, maintaining infectivity to cultured cells (HCT-8) in vitro. At the same time, field tests of Corbicula japonica for collecting oocysts showed that this clam could certainly collect Cryptosporidium parvum oocysts in the natural river and, furthermore, the gene type of C. parvum could be also identified proving its effectiveness as a biological indicator. The present study showed that the brackish water benthic shellfish Corbicula japonica may be capable of gathering and preserving Cryptosporidium parvum oocysts to a considerable extent under the natural ecological conditions, and further suggests the effectiveness of Corbicula japonica as a practical and general bioindicator for estimates of river water contamination by oocysts of Cryptosporidium parvum.