Molecular epidemiology of cryptosporidiosis: An update

Molecular tools have been developed to detect and differentiate Cryptosporidium at the species/genotype and subtype levels. These tools have been increasingly used in characterizing the transmission of Cryptosporidium spp. in humans and animals. Results of these molecular epidemiologic studies have led to better appreciation of the public health importance of Cryptosporidium species/genotypes in various animals and improved understanding of infection sources in humans. Geographic, seasonal and socioeconomic differences in the distribution of Cryptosporidium spp. in humans have been identified, and have been attributed to differences in infection sources and transmission routes. The transmission of C. parvum in humans is mostly anthroponotic in developing countries, with zoonotic infections play an important role in developed countries. Species of Cryptosporidium and subtype families of C. hominis have been shown to induce different clinical manifestations and have different potential to cause outbreaks. The wide use of a new generation of genotyping and subtyping tools in well designed epidemiologic studies should lead to a more in-depth understanding of the epidemiology of cryptosporidiosis in humans and animals.     

Development of fluorescent in situ hybridisation for Cryptosporidium detection reveals zoonotic and anthroponotic transmission of sporadic cryptosporidiosis in Sydney

Cryptosporidium, is the most common non-viral cause of diarrhea worldwide. Of the 5 described species that contribute to the majority of human infections, C. parvum is of major interest due to its zoonotic potential. A species-specific fluorescence in situ hybridisation probe was designed to the variable region in the small subunit of the 18S rRNA of C. parvum and labeled with Cy3. Probe specificity was validated against a panel of 7 other Cryptosporidium spp. before it was applied to 33 human faecal samples positive for cryptosporidiosis which were obtained during the period from 2006–2007. Results were compared to PCR-RFLP targeting the 18S rDNA. FISH results revealed that 19 of the 33 isolates analysed were identified as C. parvum. Correlation of PCR-RFLP and FISH was statistically significant (P < 0.05), resulting in a calculated correlation coefficient of 0.994. In this study, species identification by FISH and PCR-RFLP provided preliminary evidence to support both anthroponotic and zoonotic transmission of sporadic cases of cryptosporidiosis in the Sydney basin. In conclusion, FISH using a C. parvum-specific probe provided an alternative tool for accurate identification of zoonotic Cryptosporidium which will be applied in the future to both epidemiological and outbreak investigations.     

Prevalence of Cryptosporidium genotypes in pre and post-weaned pigs in Australia

A total of 289 pig faecal samples were collected from pre-weaned and post-weaned piglets and sows from 1 indoor and 3 outdoor piggeries located in the south-west region of Western Australia and screened at the 18S rRNA locus for the presence of Cryptosporidium. An overall prevalence of 22.1% (64/289) was identified. Cryptosporidium was more prevalent in post-weaned animals (p < 0.05); 32.7% (51/156) versus 10.6% (13/123) for pre-weaned animals. Sequence analysis identified Cryptosporidium suis in all pre-weaned isolates genotyped (7/13). In post-weaned pigs that were genotyped (n = 38), the non-zoonotic Cryptosporidium species, pig genotype II was identified in 32 samples and C. suis in 6 samples. The zoonotic species Cryptosporidium parvum was not detected, suggesting that domestic pigs do not pose a significant public health risk. Pig genotype II was significantly (p < 0.05) associated with ‘normal’ stools, indicating an asymptomatic nature in the porcine host.     

Genetic Diversity within Cryptosporidium parvum and Related Cryptosporidium Species

To assess the genetic diversity in Cryptosporidium parvum, we have sequenced the small subunit (SSU) rRNA gene of seven Cryptosporidium spp., various isolates of C. parvum from eight hosts, and a Cryptosporidium isolate from a desert monitor. Phylogenetic analysis of the SSU rRNA sequences confirmed the multispecies nature of the genus Cryptosporidium, with at least four distinct species (C. parvum, C. baileyi, C. muris, and C. serpentis). Other species previously defined by biologic characteristics, including C. wrairi, C. meleagridis, and C. felis, and the desert monitor isolate, clustered together or within C. parvum. Extensive genetic diversities were present among C. parvum isolates from humans, calves, pigs, dogs, mice, ferrets, marsupials, and a monkey. In general, specific genotypes were associated with specific host species. A PCR-restriction fragment length polymorphism technique previously developed by us could differentiate most Cryptosporidium spp. and C. parvum genotypes, but sequence analysis of the PCR product was needed to differentiate C. wrairi and C. meleagridis from some of the C. parvum genotypes. These results indicate a need for revision in the taxonomy and assessment of the zoonotic potential of some animal C. parvum isolates.     

Cryptosporidium spp., Giardia intestinalis,and Enterocytozoon bieneusi in Captive Non-Human Primates in Qinling Mountains

Non-human primates (NHPs) are confirmed as reservoirs of Cryptosporidium spp., Giardia intestinalis, and Enterocytozoon bieneusi. In this study, 197 fresh fecal samples from 8 NHP species in Qinling Mountains, northwestern China, were collected and examined using multilocus sequence typing (MLST) method. The results showed that 35 (17.8%) samples were positive for tested parasites, including Cryptosporidium spp. (3.0%), G. intestinalis (2.0%), and E. bieneusi (12.7%). Cryptosporidium spp. were detected in 6 fecal samples of Macaca mulatta, and were identified as C. parvum (n=1) and C. andersoni (n=5). Subtyping analysis showed Cryptosporidium spp. belonged to the C. andersoni MLST subtype (A4, A4, A4, and A1) and C. parvum 60 kDa glycoprotein (gp60) subtype IId A15G2R1. G. intestinalis assemblage E was detected in 3 M. mulatta and 1 Saimiri sciureus. Intra-variations were observed at the triose phosphate isomerase (tpi), beta giardin (bg), and glutamate dehydrogenase (gdh) loci, with 3, 1, and 2 new subtypes found in respective locus. E. bieneusi was found in Cercopithecus neglectus (25.0%), Papio hamadrayas (16.7%), M. mulatta (16.3%), S. sciureus (10%), and Rhinopithecus roxellana (9.5%), with 5 ribosomal internal transcribed spacer (ITS) genotypes: 2 known genotypes (D and BEB6) and 3 novel genotypes (MH, XH, and BSH). These findings indicated the presence of zoonotic potential of Cryptosporidium spp. and E. bieneusi in NHPs in Qinling Mountains. This is the first report of C. andersoni in NHPs. The present study provided basic information for control of cryptosporidiosis, giardiasis, and microsporidiosis in human and animals in this area.     

Distribution and Clinical Manifestations of Cryptosporidium Species and Subtypes in HIV/AIDS Patients in Ethiopia

Background

Cryptosporidiosis is an important cause for chronic diarrhea and death in HIV/AIDS patients. Among common Cryptosporidium species in humans, C. parvum is responsible for most zoonotic infections in industrialized nations. Nevertheless, the clinical significance of C. parvum and role of zoonotic transmission in cryptosporidiosis epidemiology in developing countries remain unclear.

Methodology/Principal Findings

In this cross-sectional study, 520 HIV/AIDS patients were examined for Cryptosporidium presence in stool samples using genotyping and subtyping techniques. Altogether, 140 (26.9%) patients were positive for Cryptosporidium spp. by PCR-RFLP analysis of the small subunit rRNA gene, belonging to C. parvum (92 patients), C. hominis (25 patients), C. viatorum (10 patients), C. felis (5 patients), C. meleagridis (3 patients), C. canis (2 patients), C. xiaoi (2 patients), and mixture of C. parvum and C. hominis (1 patient). Sequence analyses of the 60 kDa glycoprotein gene revealed a high genetic diversity within the 82 C. parvum and 19 C. hominis specimens subtyped, including C. parvum zoonotic subtype families IIa (71) and IId (5) and anthroponotic subtype families IIc (2), IIb (1), IIe (1) and If-like (2), and C. hominis subtype families Id (13), Ie (5), and Ib (1). Overall, Cryptosporidium infection was associated with the occurrence of diarrhea and vomiting. Diarrhea was attributable mostly to C. parvum subtype family IIa and C. hominis, whereas vomiting was largely attributable to C. hominis and rare Cryptosporidium species. Calf contact was identified as a significant risk factor for infection with Cryptosporidium spp., especially C. parvum subtype family IIa.

Conclusions/Significance

Results of the study indicate that C. parvum is a major cause of cryptosporidiosis in HIV-positive patients and zoonotic transmission is important in cryptosporidiosis epidemiology in Ethiopia. In addition, they confirm that different Cryptosporidium species and subtypes are linked to different clinical manifestations.     

Advances in molecular epidemiology of cryptosporidiosis in dogs and cats

The use of molecular tools has led to the identification of several zoonotic Cryptosporidium spp. in dogs and cats. Among them, Cryptosporidium canis and Cryptosporidium felis are dominant species causing canine and feline cryptosporidiosis, respectively. Some Cryptosporidium parvum infections have also been identified in both groups of animals. The identification of C. canis, C. felis and C. parvum in both pets and owners suggests the possible occurrence of zoonotic transmission of Cryptosporidium spp. between humans and pets. However, few cases of such concurrent infections have been reported. Thus, the cross-species transmission of Cryptosporidium spp. between dogs or cats and humans has long been a controversial issue. Recently developed subtyping tools for C. canis and C. felis should be very useful in identification of zoonotic transmission of both Cryptosporidium spp. Data generated using these tools have confirmed the occurrence of zoonotic transmission of these two Cryptosporidium spp. between owners and their pets, but have also shown the potential presence of host-adapted subtypes. Extensive usage of these subtyping tools in epidemiological studies of human cryptosporidiosis is needed for improved understanding of the importance of zoonotic transmission of Cryptosporidium spp. from pets.     

Tracking Host Sources of Cryptosporidium spp. in Raw Water for Improved Health Risk Assessment

Recent molecular evidence suggests that different species and/or genotypes of Cryptosporidium display strong host specificity, altering our perceptions regarding the zoonotic potential of this parasite. Molecular forensic profiling of the small-subunit rRNA gene from oocysts enumerated on microscope slides by U.S. Environmental Protection Agency method 1623 was used to identify the range and prevalence of Cryptosporidium species and genotypes in the South Nation watershed in Ontario, Canada. Fourteen sites within the watershed were monitored weekly for 10 weeks to assess the occurrence, molecular composition, and host sources of Cryptosporidium parasites impacting water within the region. Cryptosporidium andersoni, Cryptosporidium muskrat genotype II, Cryptosporidium cervine genotype, C. baileyi, C. parvum, Cryptosporidium muskrat genotype I, the Cryptosporidium fox genotype, genotype W1, and genotype W12 were detected in the watershed. The molecular composition of the Cryptosporidium parasites, supported by general land use analysis, indicated that mature cattle were likely the main source of contamination of the watershed. Deer, muskrats, voles, birds, and other wildlife species, in addition to sewage (human or agricultural) may also potentially impact water quality within the study area. Source water protection studies that use land use analysis with molecular genotyping of Cryptosporidium parasites may provide a more robust source-tracking tool to characterize fecal impacts in a watershed. Moreover, the information is vital for assessing environmental and human health risks posed by water contaminated with zoonotic and/or anthroponotic forms of Cryptosporidium.     

Biological and Genetic Characterization of Cryptosporidium spp. and Giardia duodenalis Isolates from Five Hydrographical Basins in Northern Portugal

To understand the situation of water contamination with Cryptosporidium spp. and Giardia spp. in the northern region of Portugal, we have established a long-term program aimed at pinpointing the sources of surface water and environmental contamination, working with the water-supply industry. Here, we describe the results obtained with raw water samples collected in rivers of the 5 hydrographical basins. A total of 283 samples were analyzed using the Method 1623 EPA, USA. Genetic characterization was performed by PCR and sequencing of genes 18S rRNA of Cryptosporidium spp. and β-giardin of Giardia spp. Infectious stages of the protozoa were detected in 72.8% (206 of 283) of the water samples, with 15.2% (43 of 283) positive for Giardia duodenalis cysts, 9.5% (27 of 283) positive for Cryptosporidium spp. oocysts, and 48.1% (136 of 283) samples positive for both parasites. The most common zoonotic species found were G. duodenalis assemblages A-I, A-II, B, and E genotypes, and Cryptosporidium parvum, Cryptosporidium andersoni, Cryptosporidium hominis, and Cryptosporidium muris. These results suggest that cryptosporidiosis and giardiasis are important public health issues in northern Portugal. To the authors'' knowledge, this is the first report evaluating the concentration of environmental stages of Cryptosporidium and Giardia in raw water samples in the northern region of Portugal.     

High diversity of Cryptosporidium subgenotypes identified in Malaysian HIV/AIDS individuals targeting gp60 gene

Background

Currently, there is a lack of vital information in the genetic makeup of Cryptosporidium especially in developing countries. The present study aimed at determining the genotypes and subgenotypes of Cryptosporidium in hospitalized Malaysian human immunodeficiency virus (HIV) positive patients.

Methodology/Principal Findings

In this study, 346 faecal samples collected from Malaysian HIV positive patients were genetically analysed via PCR targeting the 60 kDa glycoprotein (gp60) gene. Eighteen (5.2% of 346) isolates were determined as Cryptosporidium positive with 72.2% (of 18) identified as Cryptosporidium parvum whilst 27.7% as Cryptosporidium hominis. Further gp60 analysis revealed C. parvum belonging to subgenotypes IIaA13G1R1 (2 isolates), IIaA13G2R1 (2 isolates), IIaA14G2R1 (3 isolates), IIaA15G2R1 (5 isolates) and IIdA15G1R1 (1 isolate). C. hominis was represented by subgenotypes IaA14R1 (2 isolates), IaA18R1 (1 isolate) and IbA10G2R2 (2 isolates).

Conclusions/Significance

These findings highlighted the presence of high diversity of Cryptosporidium subgenotypes among Malaysian HIV infected individuals. The predominance of the C. parvum subgenotypes signified the possibility of zoonotic as well as anthroponotic transmissions of cryptosporidiosis in HIV infected individuals.     

Genotypes and subtypes of Cryptosporidium spp. in diarrheic lambs and goat kids in northern Greece

Inconsistent data exist on the distribution of zoonotic Cryptosporidium species and subtypes in sheep and goats in European countries, and few such data are available from Greece. In this study, 280 fecal specimens were collected from 132 diarrheic lambs and 148 diarrheic goat kids aged 4 to 15?days on 15 farms in northern Greece, and examined for Cryptosporidium spp. using microscopy of Ziehl-Neelsen-stained fecal smears. Cryptosporidium spp. in 80 microscopy-positive fecal specimens (39 from lambs and 41 from goat kids) were genotyped by PCR-RFLP analysis of the small subunit rRNA gene and subtyped by sequence analysis the 60?kDa glycoprotein gene. Among the 33 specimens successfully genotyped, C. parvum was found in 32 and C. xiaoi in one. Seven subtypes belonging to two subtype families (IIa and IId) were identified among the 29 C. parvum specimens successfully subtyped, including IIaA14G2R1 (1/29), IIaA15G2R1 (6/29), IIaA20G1R1 (7/29), IIdA14G2 (1/29), IIdA15G1 (9/29), IIdA16G1 (3/29), and IIdA23G1 (2/29). Lambs were more commonly infected with C. parvum IIa subtypes, whereas goat kids were more with IId subtypes. The results illustrate that C. parvum is prevalent in diarrheic lambs and goat kids in northern Greece and these animals could potentially play a role in epidemiology of human cryptosporidiosis.     

Novel Cryptosporidium Genotype in Wild Australian Mice (Mus domesticus)

A total of 250 mouse fecal specimens collected from crop farms in Queensland, Australia, were screened for the presence of Cryptosporidium spp. using PCR. Of these, 19 positives were detected and characterized at a number of loci, including the 18S rRNA gene, the acetyl coenzyme A gene, and the actin gene. Sequence and phylogenetic analyses identified two genotypes: mouse genotype I and a novel genotype (mouse genotype II), which is likely to be a valid species. Cryptosporidium parvum, which is zoonotic, was not detected. The results of the study indicate that wild Australian mice that are not in close contact with livestock are probably not an important reservoir of Cryptosporidium infection for humans and other animals.     

Molecular detection of genotypes and subtypes of Cryptosporidium infection in diarrheic calves,lambs, and goat kids from Turkey

The studies on Cryptosporidium infections of animals in Turkey mostly rely on microscopic observation. Few data are available regarding the prevalence of Cryptosporidium genotypes and subtypes infection. The aim of this study is to analyse the detection of Cryptosporidium genotypes and subtypes from young ruminants. A total of 415 diarrheic fecal specimens from young ruminants were examined for the Cryptosporidium detection by use of nested PCR of the small subunit ribosomal RNA (SSU rRNA) gene and the highly polymorphic 60 kDa glycoprotein (gp60) gene followed by sequence analyses. The results of this study revealed that 25.6% (106 of 415) of the specimens were positive for Cryptosporidium spp. infection. We identified 27.4% (91/333), 19.4% (13/67), and 13.4% (2/15) of positivity in calves, lambs and goat kids, respectively. Genotyping of the SSU rRNA indicated that almost all positive specimens were of C. parvum, except for one calf which was of C. bovis. Sequence analysis of the gp60 gene revealed the most common zoonotic subtypes (IIa and IId) of C. parvum. We detected 11 subtypes (IIaA11G2R1, IIaA11G3R1, IIaA12G3R1, IIaA13G2R1, IIaA13G4R1, IIaA14G1R1, IIaA14G3R1, IIaA15G2R1, IIdA16G1, IIdA18G1, IIdA22G1); three of them (IIaA12G3R1, IIaA11G3R1 and IIaA13G4R1) was novel subtypes found in calves and lambs. Additionally, three subtypes (IIaA11G2R1, IIaA14G3R1, and IIdA16G1) were detected in young ruminants for the first time in Turkey. These results indicate the high infection of Cryptosporidium in Turkey and propose that young ruminants are likely a major reservoir of C. parvum and a potential source of zoonotic transmission.     

Prevalence and Molecular Characterization of Cryptosporidium in Goats across Four Provincial Level Areas in China

This study assessed the prevalence, species and subtypes of Cryptosporidium in goats from Guangdong Province, Hubei Province, Shandong Province, and Shanghai City of China. Six hundred and four fecal samples were collected from twelve goat farms, and the overall infection rate was 11.4% (69/604). Goats infected with Cryptosporidium were found in eleven farms across four provincial areas, and the infection rate ranged from 2.9% (1/35) to 25.0% (9/36). Three Cryptosporidium species were identified. Cryptosporidium xiaoi (45/69, 65.2%) was the dominant species, followed by C. parvum (14/69, 20.3%) and C. ubiquitum (10/69, 14.5%). The infection rate of Cryptosporidium spp. was varied with host age and goat kids were more susceptible to be infected than adult goats. Subtyping C. parvum and C. ubiquitum positive samples revealed C. parvum subtype IIdA19G1 and C. ubiquitum subtype XIIa were the most common subtypes. Other C. parvum subtypes were detected as well, such as IIaA14G2R1, IIaA15G1R1, IIaA15G2R1 and IIaA17G2R1. All of these subtypes have also been detected in humans, suggesting goats may be a potential source of zoonotic cryptosporidiosis. This was the first report of C. parvum subtypes IIaA14G2R1, IIaA15G1R1 and IIaA17G2R1 infecting in goats and the first molecular identification of C. parvum and its subtypes in Chinese goats.     

Assessment of Zoonotic Transmission of Giardia and Cryptosporidium between Cattle and Humans in Rural Villages in Bangladesh

Giardia and Cryptosporidium are important causes of diarrhoea in Bangladesh. The high prevalence of both parasites in humans and cattle in rural Bangladesh and the common use of water ponds by village inhabitants and their animals suggest a potential for zoonotic transmission. Direct transmission of Giardia and Cryptosporidium between cattle and their handlers and indirect transmission through water ponds was investigated. Faecal/stool samples were collected from 623 calves and 125 calf handlers in a cross-sectional survey. In two villages, water samples were collected monthly from water ponds and faecal/stool samples were collected monthly from inhabitants and their cattle. Giardia cysts and Cryptosporidium oocysts were detected in water samples and in faecal/stool samples and positive samples were genotyped, to determine their human or animal origin. The prevalence of Giardia and Cryptosporidium in calves was 22% and 5% respectively. In calf handlers, the prevalence of Giardia and Cryptosporidium was 11.2% and 3.2% respectively. Both in the cross-sectional survey and in the longitudinal study in the villages, G. duodenalis assemblage E was most prevalent in calves, while in humans assemblage AII, BIII and BIV were found. In cattle, Cryptosporidium parvum, C. bovis and C. andersoni were identified, but no Cryptosporidium sequences were obtained from humans. Giardia and Cryptosporidium were detected in 14/24 and 12/24 water samples respectively. G. duodenalis assemblage E and BIV (-like), as well as C. andersoni and C. hominis were identified. Although the presence of Giardia and Cryptosporidium in both water ponds suggests that water-borne transmission of Giardia and Cryptosporidium is possible, the genotyping results indicate that there is no significant direct or indirect (water-borne) transmission of Giardia between cattle and people in this area of rural Bangladesh. No conclusions could be drawn for Cryptosporidium, because of the low number of sequences that were obtained from human and water samples.     

Detection and Molecular Characterization of Cryptosporidium spp. from Wild Rodents and Insectivores in South Korea

In order to examine the prevalence of Cryptosporidium infection in wild rodents and insectivores of South Korea and to assess their potential role as a source of human cryptosporidiosis, a total of 199 wild rodents and insectivore specimens were collected from 10 regions of South Korea and screened for Cryptosporidium infection over a period of 2 years (2012-2013). A nested-PCR amplification of Cryptosporidium oocyst wall protein (COWP) gene fragment revealed an overall prevalence of 34.2% (68/199). The sequence analysis of 18S rRNA gene locus of Cryptosporidium was performed from the fecal and cecum samples that tested positive by COWP amplification PCR. As a result, we identified 4 species/genotypes; chipmunk genotype I, cervine genotype I, C. muris, and a new genotype which is closely related to the bear genotype. The new genotype isolated from 12 Apodemus agrarius and 2 Apodemus chejuensis was not previously identified as known species or genotype, and therefore, it is supposed to be a novel genotype. In addition, the host spectrum of Cryptosporidium was extended to A. agrarius and Crosidura lasiura, which had not been reported before. In this study, we found that the Korean wild rodents and insectivores were infected with various Cryptosporidium spp. with large intra-genotypic variationa, indicating that they may function as potential reservoirs transmitting zoonotic Cryptosporidium to livestock and humans.     

Prevalence and Genetic Characterization of Cryptosporidium Isolates from Common Brushtail Possums (Trichosurus vulpecula) Adapted to Urban Settings

The common brushtail possum (Trichosurus vulpecula) is one of the most abundant native marsupials in urban Australia, having successfully adapted to utilize anthropogenic resources. The habituation of possums to food and shelter available in human settlements has facilitated interaction with people, pets, and zoo animals, increasing the potential for transmission of zoonotic Cryptosporidium pathogens. This study sought to examine the identity and prevalence of Cryptosporidium species occurring in possums adapted to urban settings compared to possums inhabiting remote woodlands far from urban areas and to characterize the health of the host in response to oocyst shedding. Findings indicated that both populations were shedding oocysts of the same genotype (brushtail possum 1 [BTP1]) that were genetically and morphologically distinct from zoonotic species and genotypes and most closely related to Cryptosporidium species from marsupials. The urban population was shedding an additional five Cryptosporidium isolates that were genetically distinct from BTP1 and formed a sister clade with Cryptosporidium parvum and Cryptosporidium hominis. Possums that were shedding oocysts showed no evidence of pathogenic changes, including elevated levels of white blood cells, diminished body condition (body mass divided by skeletal body length), or reduced nutritional state, suggesting a stable host-parasite relationship typical of Cryptosporidium species that are adapted to the host. Overall, Cryptosporidium occurred with a higher prevalence in possums from urban habitat (11.3%) than in possums from woodland habitat (5.6%); however, the host-specific nature of the genotypes may limit spillover infection in the urban setting. This study determined that the coexistence of possums with sympatric populations of humans, pets, and zoo animals in the urban Australian environment is unlikely to present a threat to public health safety.     

Genotyping of Cryptosporidium Isolates from Chamelea gallina Clams in Italy

Chamelea gallina clams collected from the mouths of rivers along the Adriatic Sea (central Italy) were found to harbor Cryptosporidium parvum (genotype 2), which is the lineage involved in zoonotic transmission. The clams were collected from the mouths of rivers near whose banks ruminants are brought to graze. This paper reports the environmental spread of C. parvum in Italy and highlights the fact that genotyping of seaborne Cryptosporidium isolates is a powerful tool with which to investigate the transmission patterns and epidemiology of this microorganism.     

Molecular Characterization of Cryptosporidium Oocysts in Samples of Raw Surface Water and Wastewater

Recent molecular characterizations of Cryptosporidium parasites make it possible to differentiate the human-pathogenic Cryptosporidium parasites from those that do not infect humans and to track the source of Cryptosporidium oocyst contamination in the environment. In this study, we used a small-subunit rRNA-based PCR-restriction fragment length polymorphism (RFLP) technique to detect and characterize Cryptosporidium oocysts in 55 samples of raw surface water collected from several areas in the United States and 49 samples of raw wastewater collected from Milwaukee, Wis. Cryptosporidium parasites were detected in 25 surface water samples and 12 raw wastewater samples. C. parvum human and bovine genotypes were the dominant Cryptosporidium parasites in the surface water samples from sites where there was potential contamination by humans and cattle, whereas C. andersoni was the most common parasite in wastewater. There may be geographic differences in the distribution of Cryptosporidium genotypes in surface water. The PCR-RFLP technique can be a useful alternative method for detection and differentiation of Cryptosporidium parasites in water.     

Spatiotemporal Analysis of Cryptosporidium Species/Genotypes and Relationships with Other Zoonotic Pathogens in Surface Water from Mixed-Use Watersheds

Nearly 690 raw surface water samples were collected during a 6-year period from multiple watersheds in the South Nation River basin, Ontario, Canada. Cryptosporidium oocysts in water samples were enumerated, sequenced, and genotyped by detailed phylogenetic analysis. The resulting species and genotypes were assigned to broad, known host and human infection risk classes. Wildlife/unknown, livestock, avian, and human host classes occurred in 21, 13, 3, and <1% of sampled surface waters, respectively. Cryptosporidium andersoni was the most commonly detected livestock species, while muskrat I and II genotypes were the most dominant wildlife genotypes. The presence of Giardia spp., Salmonella spp., Campylobacter spp., and Escherichia coli O157:H7 was evaluated in all water samples. The greatest significant odds ratios (odds of pathogen presence when host class is present/odds of pathogen presence when host class is absent) for Giardia spp., Campylobacter spp., and Salmonella spp. in water were associated, respectively, with livestock (odds ratio of 3.1), avian (4.3), and livestock (9.3) host classes. Classification and regression tree analyses (CART) were used to group generalized host and human infection risk classes on the basis of a broad range of environmental and land use variables while tracking cooccurrence of zoonotic pathogens in these groupings. The occurrence of livestock-associated Cryptosporidium was most strongly related to agricultural water pollution in the fall (conditions also associated with elevated odds ratios of other zoonotic pathogens occurring in water in relation to all sampling conditions), whereas wildlife/unknown sources of Cryptosporidium were geospatially associated with smaller watercourses where urban/rural development was relatively lower. Conditions that support wildlife may not necessarily increase overall human infection risks associated with Cryptosporidium since most Cryptosporidium genotypes classed as wildlife in this study (e.g., muskrat I and II genotype) do not pose significant infection risks to humans. Consequently, from a human health perspective, land use practices in agricultural watersheds that create opportunities for wildlife to flourish should not be rejected solely on the basis of their potential to increase relative proportions of wildlife fecal contamination in surface water. The present study suggests that mitigating livestock fecal pollution in surface water in this region would likely reduce human infection risks associated with Cryptosporidium and other zoonotic pathogens.