水源性原虫病流行病学与分子检测研究进展

水源性原虫病是由经水传播的原虫所引起的一类寄生虫病。近年来,由水污染原虫造成大量疾病的爆发引起了对水源性原虫病的高度重视。本文就近年来国内外对隐孢子虫、贾第虫和环孢子虫等水源性原虫病流行病学与分子检测研究进展作 一概述。     

河南地区鸭隐孢子虫病流行病学调查

隐孢子虫病是重要的人兽共患原虫病,广泛分布于世界各地.隐孢子虫(Crptosporidium)宿主种类广泛,可以寄生于240多种动物,隐孢子虫有效种目前已达到16个,另有40多个基因型.其中已确定的禽类有效种有3个,即贝氏隐孢子虫(C.baileyi)、火鸡隐孢子虫(C.meleagridis)和鸡隐孢子虫(C.galli),还可能存在新的有效种和基因型,至少存在有2个未命名种(分别寄生于鹌鹑和鸵鸟[1])和3个新基因型(从黑鸭分离到的鸭基因型和加拿大鹅分离到的两个鹅基因型)[2].……     

山东部分地区猪源隐孢子虫卵囊的分离与基因型鉴定

应用抗酸染色技术对山东地区部分猪场隐孢子虫感染情况进行检测,通过PCR扩增部分18s r RNA序列,分析同源性,并绘制基因进化树,鉴定其基因型。结果表明,648份猪粪样品的感染率为12.04%,隐孢子虫卵囊有两种形态,18s r RNA序列分析发现与C.parvum"mouse"型和C.muris有100%和99.8%的同源性,并分别处于同一分支。说明山东地区猪隐孢子虫感染率较高,感染的隐孢子虫基因型是C.parvum"mouse"型和C.muris,提示猪与鼠之间存在交叉传播的可能。     

隐孢子虫不同基因型P23基因的克隆及序列比较

为克隆隐孢子虫不同基因型子孢子表面抗原P23基因,比较其序列差异,提取上海地区分离的隐孢子虫鼠基因型(Cryptosporidiummouse genotype)、隐孢子虫兔基因型(Cryptosporidiumrabbit geno-type)、隐孢子虫猪基因型Ⅱ(Cryptosporidiumpig genotypeⅡ)总RNA,经RT-PCR扩增P23基因,克隆到pMD18-T载体中,进行序列测定,并与GenBank上下载的微小隐孢子虫(Cryptosporidium parvum)序列进行同源性比对。结果显示,从隐孢子虫3个基因型中均扩增出了P23基因。与微小隐孢子虫P23基因核苷酸序列比较,隐孢子虫鼠基因型、兔基因型、猪基因型ⅡP23基因同源性分别为97.6%、97.3%、97.3%,氨基酸序列同源性分别为97.3%、97.3%和96.4%。获得了隐孢子虫鼠基因型、兔基因型、猪基因型Ⅱ子孢子表面抗原P23基因。     

基于18S rRNA和HSP70基因序列的隐孢子虫种系发育分析

为阐明河南区域隐孢子虫分子流行病学特点,用PCR技术扩增分离虫株的18S rRNA基因全序列和HSP70基因序列,并对扩增片段进行测序。用PAUP 4.0和TREEPUZZLE 4.1构建进化树,试图从分子水平证明河南省不同地区不同宿主来源隐孢子虫的遗传特征,以阐明隐孢子虫病的分子流行病学特点。通过18S rRNA基因全序列和HSP70基因序列分析,其结果：河南人源隐孢子虫分离株为Cryptosporidium parvum鼠基因型;河南鹿源隐孢子虫分离株为C. parvum鹿基因型;河南猪源隐孢子虫的2个分离株均为C. parvum猪基因I型,即C. suis;河南鹌鹑源的隐孢子虫2个分离株分别为C. baileyi和C. meleagridis;河南乌鸡源隐孢子虫和鸵鸟源隐孢子虫分离株均为C. baileyi;河南牛源隐孢子虫分离株为C.andersoni。     

河南省规模化鸡场隐孢子虫感染情况调查

隐孢子虫（Cryptosporidium）是一种引起人兽共患病的机会性原虫,能感染包括人在内的240多种动物,引起动物机体不同程度的消化道和呼吸道症状。目前已鉴定出16个有效种,40多个基因型。感染禽类的隐孢子虫主要有3个种：贝氏隐孢子虫（C.baileyi）、火鸡隐孢子虫（C．meleagridis）和鸡隐孢子虫（C．galli）。其中C．baileyi为感染鸡的优势虫种,主要寄生在鸡的喉头、气管、法氏囊、泄殖腔等部位;C．meleagridis主要寄生于小肠,属于人兽共患病原;     

新疆某医院粪便样品中隐孢子虫种类的鉴定

为了解新疆某医院粪便样本中人源性隐孢子虫种系基因型,将收集来的腹泻病人粪便样本采用乙酸乙酯-改良抗酸染色法进行卵囊鉴定,同时提取隐孢子虫感染阳性样本的核酸,设计特异性引物扩增隐孢子虫的18 S rRNA基因和HSP70基因,依据所获得的目的基因序列构建系统发生分析。结果表明,新疆地区人粪便中隐孢子虫的阳性率为16.5%,高于全国平均水平。系统进化分析显示,其分子生物学特征主要为微小隐孢子虫(C.parvum)和人隐孢子虫(C.hominis)。说明新疆地区人源性隐孢子虫种系主要为C.parvum及C.hominis,具备人兽共患传播的可能性。     

隐孢子虫P23基因的原核表达及间接ELISA检测方法的建立

将隐孢子虫鼠基因型（Cryptosporidium mouse genotype）P23基因亚克隆到pGEX-4T-1载体中,并用大肠杆菌BL21（DE3）为宿主菌诱导表达。以纯化的rP23为诊断抗原,建立隐孢子虫间接ELISA检测方法,观察其敏感性、特异性和重复性,并对临床血清样品进行检测。结果隐孢子虫鼠基因型P23基因在大肠杆菌中获得了高效表达,Western blot检测显示rP23能被隐孢子虫感染兔血清识别。以纯化rP23为诊断抗原,成功地建立了检测兔隐孢子虫病的间接ELISA技术。对23份临床血清检测结果显示,该方法检出率高于Sheather＇s蔗糖漂浮法、套式PCR法。本研究结果为研制兔隐孢子虫病诊断试剂盒打下了基础。     

隐孢子虫卵囊形态学观察及动物交叉感染试验

作者观察了从湖南６种动物分离出的隐孢子虫卵囊的光镜下形态，并以鸡源、牛源和猪源隐孢子虫卵囊人工感染实验动物作交叉感染性研究。结果鉴定出隐孢子虫３个种，即贝氏隐孢子虫（Ｃ．ｂａｉｌｅｙｉ），寄生于鸡、鸭；微小隐孢子虫（Ｃ．Ｐａｒｖｕｍ），寄生于牛、山羊、猪、家兔和小鼠；鼠隐孢子虫（Ｃ．ｍｕｒｉｓ），寄生于牛。交叉感染结果表明，来自鸡的隐孢子虫可以感染雏鸡和雏鸭，而不能感染小鼠和家兔；来自牛的隐孢子虫可以感染小鼠和家兔而不能感染雏鸡，来自猪的隐孢子虫可以感染小鼠而对雏鸡无感染性。作者认为，哺乳类和鸟类的隐孢子虫可能在宿主纲的水平上具有宿主持异性。     

青海地区绵羊羔小隐孢子虫病的血清学调查

寄生于羊的隐孢子虫主要对羔羊致病,轻者消瘦、阻碍生长,重者引发腹泻,甚至死亡,造成严重的经济损失.寄生于绵羊和山羊的隐孢子虫中,微小隐孢子虫(Cryptosporidium parvum)、人隐孢子虫(C. hominis)、猪隐孢子虫(C. suis)和隐孢子虫鹿基因型(Cervinegenotype)为人兽共患虫种和基因型[1].     

Cryptosporidium and Giardia as foodborne zoonoses

Cryptosporidium and Giardia are major causes of diarrhoeal disease in humans, worldwide and are major causes of protozoan waterborne diseases. Both Cryptosporidium and Giardia have life cycles which are suited to waterborne and foodborne transmission. There are 16 'valid'Cryptosporidium species and a further 33+ genotypes described. Parasites which infect humans belong to the Giardia duodenalis "type", and at least seven G. duodenalis assemblages are recognised. Cryptosporidium parvum is the major zoonotic Cryptosporidium species, while G. duodenalis assemblages A and B have been found in humans and most mammalian orders. In depth studies to determine the role of non-human hosts in the transmission of Cryptosporidium and Giardia to humans are required. The use of harmonised methodology and standardised and validated molecular markers, together with sampling strategies that provide sufficient information about all contributors to the environmental (oo)cyst pool that cause contamination of food and water, are recommended. Standardised methods for detecting (oo)cysts in water are available, as are optimised, validated methods for detecting Cryptosporidium in soft fruit and salad vegetables. These provide valuable data on (oo)cyst occurrence, and can be used for species and subspecies typing using appropriate molecular tools. Given the zoonotic potential of these organisms, epidemiological, source and disease tracking investigations involve multidisciplinary teams. Here, the role of the veterinarian is paramount, particularly in understanding the requirement for adopting comprehensive sampling strategies for analysing both sporadic and outbreak samples from all potential non-human contributors. Comprehensive sampling strategies increase our understanding of parasite population biology and structure and this knowledge can be used to determine what level of discrimination is required between isolates. Genetic exchange is frequent in C. parvum populations, leading to recombination between alleles at different loci, the generation of a very large number of different genotypes and a high level of resolution between isolates. In contrast, genetic exchange appears rare in Cryptosporidium hominis and populations are essentially clonal with far fewer combinations of alleles at different loci, resulting in a much lower resolution between isolates with many being of the same genotype. Clearly, more markers provide more resolution and high throughput sequencing of a variety of genes, as in multilocus sequence typing, is a way forward. Sub-genotyping tools offer increased discrimination, specificity and sensitivity, which can be exploited for investigating the epidemiology of disease, the role of asymptomatic carriers and contaminated fomites and for source and disease tracking for food and water contaminated with small numbers of (oo)cysts.     

The first report on Cryptosporidium suis and Cryptosporidium pig genotype II in Eurasian wild boars (Sus scrofa) (Czech Republic)

A total of 193 faecal samples of adult Eurasian wild boars were collected at 12 enclosures across the Czech Republic and examined for Cryptosporidium infection using both microscopic and molecular tools. Cryptosporidium oocysts were not detected in any of the 193 faecal samples examined using the aniline-carbol-methyl violet staining method. Thirty-two positive cases of Cryptosporidium infection were detected using either genus- or species-specific nested PCR. Mono-infection with Cryptosporidium suis and Cryptosporidium pig genotype II were found in 13 and 7 cases, respectively. Five mixed infections of C. suis and Cryptosporidium pig genotype II were detected using PCR/RFLP with genus specific primers. The number of detected mixed infections increased 2.4 fold when a species-specific PCR was employed. No other Cryptosporidium spp. was detected. Unlike cryptosporidiosis of domestic pigs, C. suis was detected as a dominant species infecting adult Eurasian wild boars. There was no association between diarrhoea and the presence of Cryptosporidium infection in the Eurasian wild boars studied. This is the first report on the Cryptosporidium infection caused by C. suis and Cryptosporidium pig genotype II in Eurasian wild boars (Sus scrofa).     

Cryptosporidium infection in dogs in Osaka,Japan

Cryptosporidium parvum is a zoonotic pathogen composed of genetically distinct but morphologically identical genotypes. Recent molecular study indicates that dogs may transmit the cattle genotype, which is known to be pathogenic to humans. Although large-scale studies of Cryptosporidium infection in dogs have been performed in several countries, the isolates were not accurately identified because of the lack of a method for molecular analysis. It is important to identify the isolates harbored in dogs, which come in close contact with humans, in order to control human cryptosporidiosis. The aim of the present study was to calculate the prevalence of Cryptosporidium infection in dogs in Osaka city, Japan, and to characterize the isolates molecularly. The prevalence was determined to be 9.3% (13/140) by PCR. All isolates were found to be Cryptosporidium canis (previously known as the dog genotype), which is thought to be non-pathogenic in humans, based on the sequencing of diagnostic fragments. These results indicate that PCR-based diagnostic methods are a useful tool for the diagnosis and molecular epidemiology of Cryptosporidium infection in dogs, and that dogs living in Osaka are not a significant reservoir for human cryptosporidiosis. It is unclear why C. canis is dominant in dogs. Further study is required to understand this partial parasitism.     

Prevalence and molecular characterization of Cryptosporidium and Giardia species and genotypes in sheep in Maryland

In the United Kingdom and Australia sheep have been implicated as sources of Cryptosporidium and Giardia that infect humans, but no such studies have been conducted in North America. Therefore, a study was undertaken to investigate the prevalence of these parasites in sheep on a farm in Maryland. Feces were collected from 32 pregnant ewes 1, 2, and 3 days after parturition and from each of their lambs 7, 14, and 21 days after birth. The presence of Cryptosporidium oocysts and Giardia cysts was determined by both immunofluorescence microscopy and PCR/gene sequence analysis. PCR was consistently more sensitive than microscopy. The prevalence, by PCR, of Cryptosporidium in ewes and lambs was 25 and 77.4%, respectively. Three species/genotypes of Cryptosporidium were identified: C. parvum, a novel C. bovis-like genotype, and Cryptosporidium cervine genotype. Cryptosporidium parvum and the cervine genotype have been reported worldwide in human infections. The novel C. bovis-like genotype is reported here for the first time. The prevalence of Giardia in ewes and lambs was 12 and 4%, respectively. Most infections were Assemblage E which is not zoonotic; however, one ewe was infected with zoonotic Assemblage A. The identification of only two lambs infected with C. parvum and one ewe infected with G. duodenalis Assemblage A suggests a low prevalence of these zoonoses. However, the high prevalence of the zoonotic cervine genotype indicates that sheep should be considered a potential environmental source of this human pathogen.     

Molecular identification of Cryptosporidium spp. in animal and human hosts from the Czech Republic

To study the diversity of Cryptosporidium spp. in various hosts, we used the variability of the small-subunit rRNA gene and the Cryptosporidium oocyst wall protein genes. Oocysts from humans, cattle, horses, dogs, field mice, chickens, reptiles, deer, goat, cat, antelope and from a sample of water reservoir were assayed. The zoonotic C. parvum bovine genotype sequence was found to be present in the most of isolates. This study shows a complex epidemiology pattern for C. parvum bovine genotype infections. The identification of cattle, horse, and deer isolates emphasizes a transmission route for C. parvum via these hosts, and identifies a potential source for human infection in the Czech Republic. Furthermore, C. andersoni from a cow, C. baileyi from a chicken, C. felis from a cat, C. meleagridis from a dog, and C. saurophilum and C. serpentis from reptiles were also identified in the isolates from the Czech Republic.     

Risk of infection with Cryptosporidium parvum and Cryptosporidium hominis in dairy cattle in the New York City watershed

OBJECTIVE: To determine the risk posed by Cryptosporidium parvum and Cryptosporidium hominis from dairy cattle in the New York City watershed (NYCW). SAMPLE POPULATION: Samples from cattle at risk for shedding Cryptosporidium organisms on randomly selected dairy farms in the NYCW. PROCEDURE: Feces were collected for 4 years from calves at risk for infection on 37 dairies. Oocysts were detected by use of centrifugation concentration-flotation microscopy. The DNA was directly isolated from fecal samples and used to amplify fragments of the small subunit ribosomal RNA and thrombospondin-related adhesion protein C-2 genes by use of nested polymerase chain reaction assays. Small subunit ribosomal RNA fragments were restriction digested by the enzyme Vspl and thrombospondin-related adhesion protein C-2 fragments were digested by Eco91l to distinguish between C hominis (formerly known as genotype 1) and C parvum (formerly known as genotype 2). RESULTS: Of 437 fecal samples examined, 214 contained oocysts. Amplicons were generated for 200 samples. We can be certain, with 95% confidence, that cattle in the NYCW did not harbor C hominis. CONCLUSIONS AND CLINICAL RELEVANCE: Cryptosporidium infections in cattle are under examination because of the potential contamination of public waters by manure. Although cattle may be the source of zoonotic infection via C parvum, they pose little risk for C hominis (the strain commonly isolated from humans in waterborne outbreaks of disease). Other sources of oocysts should be considered when investigating outbreaks attributable to contaminated urban drinking water because cattle pose only a small risk via shedding of C hominis.     

Cryptosporidium ryanae n. sp. (Apicomplexa: Cryptosporidiidae) in cattle (Bos taurus)

A new species, Cryptosporidium ryanae, is described from cattle. Oocysts of C. ryanae, previously identified as the Cryptosporidium deer-like genotype and recorded as such in GenBank (AY587166, EU203216, DQ182597, AY741309, and DQ871345), are similar to those of Cryptosporidium parvum and Cryptosporidium bovis but smaller. This genotype has been reported to be prevalent in cattle worldwide. Oocysts obtained from a calf for the present study are the smallest Cryptosporidium oocysts reported in mammals, measuring 2.94-4.41micromx2.94-3.68microm (mean=3.16micromx3.73microm) with a length/width shape index of 1.18 (n=40). The pre-patent period for two Cryptosporidium-na?ve calves fed C. ryanae oocysts was 11 days and the patent period was 15-17 days. Oocysts were not infectious for BALB/c mice or lambs. Fragments of the SSU-rDNA, HSP-70, and actin genes amplified by PCR were purified and PCR products were sequenced. Multi-locus analysis of the three unlinked loci demonstrated the new species to be distinct from all other species and also demonstrated a lack of recombination, providing further evidence of species status. Based on morphological, molecular and biological data, this geographically widespread parasite found only in Bos taurus calves is recognized as a new species and is named C. ryanae.     

First record of Cryptosporidium infection in a raccoon dog (Nyctereutes procyonoides viverrinus)

Cryptosporidium species have been found in more than 150 species of mammals, but there has been no report in raccoon dogs. Here we found the Cryptosporidium organism in a raccoon dog, Nyctereutes procyonoides viverrinus, and identified this isolate using PCR-based diagnostic methods. Cryptosporidium diagnostic fragments of the 18S ribosomal RNA, Cryptosporidium oocyst wall protein and 70-kDa heat shock protein genes were amplified from the isolate and sequenced to reveal the phylogenetic relationships between it and other Cryptosporidium species or genotypes reported previously. The results showed that the raccoon dog isolate represented the C. parvum cattle genotype which could be a causative agent in human cryptosporidiosis.     

奶牛隐孢子虫Nested PCR—RFLP方法的建立及初步应用

为快速检测并准确鉴别奶牛隐孢子虫种,以隐孢子虫18S rRNA基因的特殊区域为基础,设计内、外引物,并根据软件分析确定相应的内切酶EcoT141,采用Nested PCR-RFLP方法进行虫种种型鉴别分析。在Nested PCR两次PCR反应中,以微小隐孢子虫（Cryptos poridium parvum,C．p）和安氏隐孢子虫（Cryptosporidium andersoni,C．an）卵囊提取的DNA为模板,均能扩增出长约800bp和500bp的明亮条带,且特异性强,其他虫种不能扩增出条带,该方法最低可检测到5个卵囊／g粪便;对于由内引物扩增出的500bp的条带,C.an的PCR产物能被内切酶EcoT141酶切,酶切后的片段分别为416bp和92bp,C.p的PCR产物不能被此酶酶切。用所建立的Nested PCR-RFLP法对上海奶牛389头和进口奶牛200头的共计589份粪样进行检测,Nested PCR的结果表明上海奶牛和进口奶牛的隐孢子虫阳性率分别为19．02％和3．5％,RFLP的结果表明上海奶牛感染的主要是Cp和C．an,进口奶牛感染的主要是C．p。研究结果表明,本研究建立的检测奶牛粪便中的隐孢子虫的NestedPCR-RFLP法,可用于奶牛隐孢子虫流行病学调查并有效鉴别奶牛隐孢子虫种。