Cryptosporidiosis: a brief literature review and update regarding Cryptosporidium in feces of Canada geese (Branta canadensis)

Canada geese are increasingly abundant in Ohio, with large nesting populations throughout the state, and goose feces contaminate grassy areas and pavements in many public, commercial, and residential sites. In 1999 the authors found a high prevalence of Giardia, Campylobacter, and especially Cryptosporidium in collected feces of Canada geese. The purpose of this follow-up study was to survey known Canada geese sites in three counties in Ohio (Lucas, Ottawa, and Wood) and to determine the prevalence of sites testing positive for Cryptosporidium. The sites included golf courses, cemeteries, public parks, and health care and teaching facilities. At each of 11 sites, 12 goose feces of wet and loose appearance were collected and manually compressed into one composite sample representing that site. The samples were tested for Cryptosporidium with a sensitive monoclonal enzyme immunoassay (EIA) method. In 2000 and 2001, nine of 11 sites (81.8 percent) and nine of 10 sites (90 percent), respectively, were positive for Cryptosporidium. The species or genotypes of Cryptosporidium found in the geese feces and their potential to infect humans is unknown. A survey of the literature indicates, however, that while C. parvum (human genotype) is the main cause of cryptosporidiosis in humans, C. parvum (zoonotic genotypes), C. meleagridis (bird genotype), and C. felis (cat genotype) have occasionally been isolated from infected people. Further research is required to define the public health importance of Cryptosporidium in feces of Canada geese and other bird species.     

Identifying host sources, human health risk and indicators of Cryptosporidium and Giardia in a Canadian watershed influenced by urban and rural activities

Cryptosporidium and Giardia were characterized in a watershed in southern Ontario, Canada, over a 2? year period. River samples were collected every two weeks, primarily near a municipal drinking water treatment plant intake. Cryptosporidium and Giardia were frequently detected with an overall occurrence rate of 88 and 97%, respectively. Giardia concentrations were higher than Cryptosporidium, with median values of 80 cysts 100 L(-1) and 12 oocysts 100 L(-1), respectively. Although pathogens rarely show a significant relationship with fecal or water quality indicators, this study determined that Cryptosporidium, but not Giardia, was significantly correlated with Escherichia coli, turbidity and river flow. There was no correlation between the two types of protozoa, and only Giardia showed a seasonal trend with higher concentrations at cold water temperatures. Cryptosporidium genotyping of all samples found that farm animals and wildlife were an important contributor of oocysts in the watershed, and that Cryptosporidium strains/genotypes of medium to high risk for human infection (C. hominis, C. parvum and C. ubiquitum) were detected in 16% of samples. This study was able to identify Cryptosporidium host sources and human health risk, and to identify differences between Cryptosporidium and Giardia occurrence in the watershed.     

New cryptosporidium genotypes in HIV-infected persons.

Using DNA sequencing and phylogenetic analysis, we identified four distinct Cryptosporidium genotypes in HIV-infected patients: genotype 1 (human), genotype 2 (bovine) Cryptosporidium parvum, a genotype identical to C. felis, and one identical to a Cryptosporidium sp. isolate from a dog. This is the first identification of human infection with the latter two genotypes.     

Monitoring of Cryptosporidium and Giardia in the Vantaa river basin, southern Finland

We compared two sampling methods to assess the contamination of the Vantaa river basin by Giardia cysts and Cryptosporidium oocysts: 10-1 grab samples, the common river mussel Anadonta piscinalis, were analysed for concentration of (oo)cysts from river water. The samples were collected 2-5 times in autumn 2001 from four wastewater treatment plants and four river water sites located downstream of the plants, and six times from raw water of a drinking water plant using the river as water source. The presence of Giardia and Cryptosporidium was analysed by IF microscopy and PCR. Both cysts and oocysts were detected at all sampling sites, but oocysts were more common than cysts in river water samples. In contrast, cysts were more common in A. piscinalis. Most Cryptosporidium-positive samples were of genotype 2 and Giardia were assemblage B. In river water, MPN of Escherichia coli did not correlate to the presence of (oo)cysts. In conclusion, low (oo)cyst counts were regularly identified in the Vantaa river basin which is contaminated by discharges of treated wastewater of human origin. In general, both methods to appropriate to detect (oo)cysts, but grab samples yielded more positive results. Grab sampling is also more practical and less expensive than analysis of A. piscinalis.     

Multilocus genotyping of Cryptosporidium parvum Type 2: population genetics and sub-structuring.

Cryptosporidium parvum is an intracellular protozoan parasite that infects the gastrointestinal tract of humans and other mammals. It has significant economic importance as a pathogen of livestock and, as there is no effective treatment or vaccine available, understanding transmission routes and identifying sources of infection is key to preventing future outbreaks and controlling this disease. In this study we have determined the multilocus genotype (MLG) of 240 C. parvum Type 2 (bovine) isolates using a combination of seven micro- and minisatellite markers. These isolates were collected over a period of 19 months and are from three different geographical locations within Scotland and three different host species. The results of this study have enabled us to address questions concerning C. parvum population genetics in relation to host, temporal and geographical sub-structuring. We identified 48 multilocus genotypes within the Type 2 C. parvum isolates and found no evidence to support geographic or temporal sub-structuring of the populations. However host sub-structuring was identified within the human Type 2 population highlighting the potential use of such a typing system in understanding the epidemiology of this parasite in addition to raising interesting questions with regard to its population genetic structure. We also isolated two C. parvum 'monkey type' isolates from two separate human cases indicating that this genotype is not restricted to monkey hosts with the multilocus genotypes of these isolates distinguishing them from all other isolates.     

Host-shaped segregation of the Cryptosporidium parvum multilocus genotype repertoire

Cattle are among the major reservoirs of Cryptosporidium parvum in nature. However, the relative contribution of C. parvum oocysts originating from cattle to human disease burden is difficult to assess, as various transmission pathways -- including the human to human route -- can co-occur. In this study, multilocus genotype richness of representative samples of human and bovine C. parvum are compared statistically using analytical rarefaction and non-parametric taxonomic richness estimators. Results suggest that in the time and space frames underlying the analysed data, humans were infected with significantly wider spectra of C. parvum genotypes than cattle, and consequently, a significant fraction of human infections may not have originated from the regional bovine reservoirs. This study provides statistical support to the emerging idea of the existence of distinct anthroponotic C. parvum cycles that do not involve cattle.     

The epidemiology of sporadic human infections with unusual cryptosporidia detected during routine typing in England and Wales, 2000-2008

Routine typing of 14 469 isolates from human cryptosporidiosis cases between 2000 and 2008 revealed that 7439 (51·4%) were Cryptosporidium (C.) hominis, 6372 (44·0%) C. parvum, 51 (0·4%) both C. hominis and C. parvum, 443 (3·1%) were not typable and 164 (1·1%) were other Cryptosporidium species or genotypes. Of the latter, 109 were C. meleagridis, 38 C. felis, 11 C. ubiquitum, one C. canis, two horse, two novel and one skunk genotype. C. hominis monkey genotype and C. cuniculus were identified in a separate study. Patients with unusual infections were older than those with C. hominis (P<0·01) or C. parvum (P<0·01) and were more likely to be immunocompromised (Fisher's exact P<0·01). Forty-one percent of unusual cases had travelled abroad, mainly to the Indian subcontinent. Significant risk factors in those with unusual species were travel abroad (C. meleagridis, P<0·01), being immunocompromised (C. felis, Fisher's exact P=0·02), and contact with cats (C. felis, Fisher's exact P=0·02).     

Occurrence of Giardia and Cryptosporidium in water supplies of Russia and Bulgaria

The aim of the present study was to investigate water supplies in southern Russia and Bulgaria, in order to estimate the occurrence of Giardia and Cryptosporidium in drinking water resources from these countries. A total of 166 water samples of different origin (surface, tap, bottled, well, spring and waste water) were collected from Rostov (southern Russia), Sofia and Varna (Bulgaria) Greater Areas and screened for the detection of Giardia cysts and Cryptosporidium oocysts. The method incorporated concentration of water samples by filtration and flocculation, sucrose purification, (oo)cyst detection/identification by immunofluorescence test and differential interference contrast. Sixteen out of 166 samples (9.6%) were positive for Giardia and 30 (18.1%) positive for Cryptosporidium. Both Giardia and Cryptosporidium were detected in tap, river, well and waste waters. Giardia cysts were additionally detected in bottled water. Particularly some river, waste and well water samples were highly contaminated with (oo)cysts. This study has shown that drinking water supplies in Russia and Bulgaria are subject to contamination with Giardia and Cryptosporidium, with potential hazards for the public health.     

Cryptosporidium parvum in oysters from commercial harvesting sites in the Chesapeake Bay.

Oocysts of Cryptosporidium parvum, a zoonotic waterborne pathogen, can be removed by bivalve molluscs from contaminated water and retained on gills and in hemolymph. We identified oocysts of C. parvum in oysters from seven sites in the Chesapeake Bay area. These findings document the presence of C. parvum infectious for humans in oysters intended for human consumption.     

Detection and characterisation of Giardia and Cryptosporidium in Hungarian raw, surface and sewage water samples by IFT, PCR and sequence analysis of the SSUrRNA and GDH genes

We investigated the prevalence of Giardia and Cryptosporidium species and analysed the genotypes in 36 samples collected from different water sources and various geographic areas in Hungary. Samples were collected from drinking water and sewage treatment plants and from the recreation area of Lake Balaton. The (oo)cysts were purified according to the US EPA 1623 method and they were detected by immunofluorescence test (IFT). Genomic DNA was extracted from all samples and then the GDH target gene for Giardia and the SSUrDNA for both Giardia and for Cryptosporidium species were amplified by PCR. 24 out of 36 samples (67%) were Giardia positive and 15 (42%) were Cryptosporidium positive by IFT. PCR confirmed that 13 out of 36 samples (36%) were Giardia positive and 10 (28%) contained Cryptosporidium. Twelve Giardia and two Cryptosporidium PCR products were successfully sequenced. In seven samples G. lamblia Assemblage A and in one sample Assemblage B and in four cases Assemblages A and B have been found. In one sample C. parvum and in the other separate sample C. meleagridis were detected. Sequence analysis revealed a new subtype of G. duodenalis complex, clustered close to the Assemblage A group. This study provides the first report on simultaneous detection and genotyping of G. duodenalis and Cryptosporidium species from water supplies in Hungary.     

Genetic polymorphism among Cryptosporidium parvum isolates: evidence of two distinct human transmission cycles.

We report the results of molecular analysis of 39 isolates of Cryptosporidium parvum from human and bovine sources in nine human outbreaks and from bovine sources from a wide geographic distribution. All 39 isolates could be divided into either of two genotypes, on the basis of genetic polymorphism observed at the thrombospondin-related adhesion protein (TRAP-C2) locus. Genotype 1 was observed only in isolates from humans. Genotype 2, however, was seen in calf isolates and in isolates from a subset of human patients who reported direct exposure to infected cattle or consumed items thought to be contaminated with cattle faces. Furthermore, experimental infection studies showed that genotype 2 isolates were infective to mice or calves under routine laboratory conditions, whereas genotype 1 isolates were not. These results support the occurrence of two distinct transmission cycles of C. parvum in humans.     

Hyperimmune bovine colostrum specific for recombinant Cryptosporidium parvum antigen confers partial protection against cryptosporidiosis in immunosuppressed adult mice.

Preparturient cows were immunized three times over a six-week period with recombinant plasmid DNA encoding the Cryptosporidium parvum CP15/60 antigen by injecting the DNA in the mammary gland. Serum was collected at each immunization and first colostrum was collected after parturition; all were assayed for Cryptosporidium-specific antibodies (Ab). A serological response to C. parvum sporozoite and oocyst antigen was detected in cows immunized with pCP15/60 plasmid DNA. Colostrum from these cows, unlike colostrum from normal controls, contained Ab specific for C. parvum sporozoites and oocysts as indicated by immunofluorescence Ab (IFA) staining. Colostrum was also tested for conferring passive immunity against C. parvum infection by oral administration to immunosuppressed adult inbred mice. Immune colostrum and control colostrum were administered to separate groups of dexamethasone (DEX)-treated adult C57BL/6NCr mice beginning 12 h before and at 12 h intervals for 3 days after oral C. parvum oocyst infection. Cryptosporidium development was assayed in ilea of immune- and control-colostrum-treated mice 96 h postinfection by semiquantitative PCR. Mice receiving immune colostrum showed partial protection (about 50% reduction) against intestinal C. parvum development compared to mice receiving control colostrum. This protection was evident at a challenge dose of 10(3) C. parvum oocysts per mouse; no differences were noted in parasite development between groups receiving immune or control colostrum and infected with 10(4) oocysts. This study showed that serum and colostrum Ab response to C. parvum can be elicited in preparturient cows by direct injection of recombinant pCP15/60 plasmid DNA and that passive protection against cryptosporidiosis can be obtained by treating immunosuppressed mice with immune colostrum before and after C. parvum infection.     

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。     

Comparative genome analysis of two Cryptosporidium parvum isolates with different host range

Parasites of the genus Cryptosporidium infect the intestinal and gastric epithelium of different vertebrate species. Some of the many Cryptosporidium species described to date differ with respect to host range; whereas some species' host range appears to be narrow, others have been isolated from taxonomically unrelated vertebrates. To begin to investigate the genetic basis of Cryptosporidium host specificity, the genome of a Cryptosporidium parvum isolate belonging to a sub-specific group found exclusively in humans was sequenced and compared to the reference C. parvum genome representative of the zoonotic group. Over 12,000 single-nucleotide polymorphisms (SNPs), or 1.4 SNP per kilobase, were identified. The genome distribution of SNPs was highly heterogeneous, but non-synonymous and silent SNPs were similarly distributed. On many chromosomes, the most highly divergent regions were located near the ends. Genes in the most diverged regions were almost twice as large as the genome-wide average. Transporters, and ABC transporters in particular, were over-represented among these genes, as were proteins with predicted signal peptide. Possibly reflecting the presence of regulatory sequences, the distribution of intergenic SNPs differed according to the function of the downstream open reading frame. A 3-way comparison of the newly sequenced anthroponotic C. parvum, the reference zoonotic C. parvum and the human parasite Cryptosporidium hominis identified genetic loci where the anthroponotic C. parvum sequence is more similar to C. hominis than to the zoonotic C. parvum reference. Because C. hominis and anthroponotic C. parvum share a similar host range, this unexpected observation suggests that proteins encoded by these genes may influence the host range.     

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.     

Genotyping of Cryptosporidium spp. isolated from human stool samples in Switzerland

In a study to estimate the frequency of Cryptosporidium infections in Switzerland, stool samples from patients found to be positive for Cryptosporidium spp. by modified Ziehl-Neelson staining and fluorescence microscopy were used for genotyping experiments. With 9 of 12 samples, DNA extraction and subsequent genotyping was successful. All Cryptosporidium-isolates belonged to the bovine genotype. In one stool sample, two strains of Cryptosporidium were demonstrated, suggesting a mixed infection. In comparison with reference strains from calves, one of the isolates showed a full sequence identity and the other a similarity of 97.5%. The fact that only bovine genotypes were detected suggests, that cryptosporidiosis must primarily be considered as a zoonotic disease in Switzerland. This is in contrast to other countries, where the human genotype of C. parvum was shown to dominate the epidemiological situation. The results of our study are supported by the previous finding, that two of the analysed strains originated from patients who used to consume raw milk or raw cream, a known risk factor for cryptosporidiosis.     

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。     

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 degrees C and 4 degrees 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 degrees C and 4 degrees C, respectively. Suckling mice infectivity was higher after storage at 10 degrees C than after storage at 4 degrees C. These data suggest that Cryptosporidium parvum oocysts can survive and remain infectious for 8 wk in cool chlorinated tap water.     

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。     

Incidence of cryptosporidiosis species in paediatric patients in Malawi

We determined the incidence of cryptosporidiosis in children aged <5 years presenting with diarrhoea in an urban and rural hospital-based setting in Malawi. Stools were collected over a 22-month period during both rainy and dry seasons. A range of microscopic methods were used to determine the presence of Cryptosporidium spp. oocysts. Species determination was by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of oocyst-extracted DNA using 18S rRNA and COWP gene loci. Cryptosporidium spp. oocysts were seen in 5.9% (50/848) of samples, of which 43 amplified by PCR-RFLP indicated the following species: C. hominis, C. parvum, C. hominis/C. parvum, C. meleagridis and C. andersoni. Seven samples could not be amplified by PCR. Wider species diversity was found in the rural setting, and may be a result of increased malnutrition and zoonotic exposure in this area. Improvements in water, sanitation, household hygiene and animal control are required to reduce the incidence of infection in this population.