Presence of Cryptosporidium parvum in pre-washed vegetables from different supermarkets in South East England: A pilot study.

Cryptosporidium is an important water-borne and food-borne parasite with a high burden of disease. This organism has been shown to contaminate various leafy vegetables; however, studies assessing the presence of Cryptosporidium spp in pre-washed and ready-to-eat vegetables are limited. Routine surveillance in the UK revealed a nationwide exceedance of human cases of Cryptosporidium. Therefore, this study aims to assess the presence of this parasite in pre-washed vegetables from supermarkets in the UK. A total of 36 samples were purchased from four different supermarkets. A nested PCR targeting the SSU rRNA was carried out on 24 samples, 58% were PCR-positive for Cryptosporidium. Sanger sequencing confirmed that, of these sequences, 4/24 (17%) produced significant similarities to Cryptosporidium parvum. This study provides evidence for the presence of C. parvum in pre-washed and ready-to-eat vegetables. Future work to identify the point of contamination is required.

Increased diversity and novel subtypes among clinical Cryptosporidium parvum and Cryptosporidium hominis isolates in Southern Ireland.

Reported incidence rates of cryptosporidiosis in Ireland are consistently among the highest in Europe. Despite the national prevalence of this enteric parasite and the compulsory nature of incidence surveillance and reporting, in-depth analyses seeking to genotype clinical isolates of Cryptosporidium on an intra-species level are rarely undertaken in Ireland. This molecular epidemiology study of 163 clinical Cryptosporidium isolates was conducted in Southern Ireland, from 2015 to 2018, in order to ascertain population subtype heterogeneity. Analysis was conducted via real-time PCR amplification and gp60 gene sequencing, which successfully determined the subtype designation of 149 of the 163 (91.4%) tested isolates. Overall, 12 C. parvum and five C. hominis subtypes were identified, with the incidence of the regionally predominant C. parvum species found to primarily occur during springtime months, while C. hominis incidence was largely confined to late summer and autumnal months. Additionally, one C. parvum and four C. hominis subtypes were newly reported by this study, having not been previously identified in clinical or livestock infection in Ireland. Overall, these data give insight into the diversification of the Cryptosporidium population and emergent subtypes, while also allowing comparisons to be made with clinical epidemiological profiles reported previously in Ireland and elsewhere.

First report of zoonotic Cryptosporidium parvum GP60 subtypes IIaA15G2R1 and IIaA16G3R1 in wild ponies from the northern Iberian Peninsula.

Studies on the prevalence and molecular characterization of Cryptosporidium spp. affecting feral horses are scarce. The highland areas of the northern Iberian Peninsula are home to a large population of wild ponies which generally roam free in the ancient natural range and are subjected to a traditional exploitation regime. In the present study, a total of 79 non-diarrhoeal faecal samples from the wild ponies were collected from the ground immediately after defecation. Cryptosporidium was detected in 10 of the samples (12.6%) by a direct immunofluorescence antibody test and DNA amplification and sequencing. Analysis of partial sequences of the small subunit ribosomal RNA (SSU-rRNA) and heat shock protein (hsp70) loci revealed the presence of Cryptosporidium parvum. In addition, amplification and sequencing of a fragment of the 60-kDa glycoprotein (GP60) locus identified C. parvum subtypes IIaA15G2R1 and IIaA16G3R1. This study reports, for the first time, the occurrence of C. parvum in wild ponies in Europe, specifically in the northern Iberian Peninsula. Identification of the common subtype IIaA15G2R1 and also subtype IIaA16G3R1 (first description) indicates that these hosts may play a role in the sylvatic transmission of C. parvum and that they may act as a reservoir of zoonotic cryptosporidiosis.

Cryptosporidium parvum as a risk factor of diarrhea occurrence in neonatal alpacas in Peru.

Cryptosporidiosis has been reported as an important cause of neonatal diarrhea and mortality in cattle, sheep, and other ruminants, but its impact on alpaca health has not been studied thoroughly. In this study, we have determined the prevalence and evaluated the role of cryptosporidiosis as a risk factor for diarrhea occurrence in newborn alpacas. During the calving season (January-March) of 2006, stool specimens (N = 1312) were collected from 24 herds of newborn alpacas in Puno and Cuzco, departments that account for the largest populations of alpacas in Peru. All the specimens were microscopically screened for Cryptosporidium spp. using the acid-fast technique. The association between Cryptosporidium detection and diarrhea was analyzed using χ2 test and generalized lineal model. Cryptosporidium species were determined by PCR-RFLP analysis of the small subunit rRNA gene. Cryptosporidium oocysts were detected in 159 of 1312 (12.4%) newborn alpacas. Results of the analyses demonstrated that crypstosporidiosis was significantly associated with diarrhea (PR = 3.84; CI95% 2.54-5.81; p < 0.0001). Only Cryptosporidium parvum was detected in the 153 Cryptosporidium-infected animals. Thus, there is an association of C. parvum infection with diarrhea in neonatal alpacas.

An apple a day: an outbreak of cryptosporidiosis in Norway associated with self-pressed apple juice.

In the autumn of 2018, an outbreak of cryptosporidiosis affected adult employees from the same company in Western Norway. The organism was Cryptosporidium parvum, GP60 subtype IIaA14G1R1. All those infected had drunk from the same container of self-pressed apple juice. Incubation period (1 week) and clinical signs were similar among those infected, although some experienced a more prolonged duration of symptoms (up to 2-3 weeks) than others. The infections resulted after consumption from only one of 40 containers of juice and not from any of the other containers. It seems that although Cryptosporidium oocysts were detected in a sample from another container, the contamination did not affect the whole batch. This is perhaps indicative of a restricted contamination event, either from contaminated ground in the orchard, or during collection of the fruit, or during processing. Although outbreaks of food-borne cryptosporidiosis have previously been associated with consumption of contaminated apple juice, most of the more recent outbreaks of food-borne cryptosporidiosis have been associated with salad vegetables or herbs. This outbreak, the first outside USA reported to be associated with apple juice, is a timely reminder that such juice is a suitable transmission vehicle for Cryptosporidium oocysts, and that appropriate hygienic measures are essential in the production of such juice, including artisanal (non-commercial) production.

Molecular epidemiology of Cryptosporidium spp. in calves in Estonia: high prevalence of Cryptosporidium parvum shedding and 10 subtypes identified.

We investigated the molecular epidemiology of Cryptosporidium spp. in Estonia by testing fecal samples from 486 calves aged <2 months, raised on 53 cattle farms, for the presence of Cryptosporidium DNA. The parasites were identified and characterized by sequencing of the 18S rRNA gene and of the 60 kDa glycoprotein (gp60) gene. Moreover, using a questionnaire, we surveyed factors that could be relevant for animal-to-human and human-to-animal transmission of Cryptosporidium spp. on the farms. Cryptosporidium spp. were shed by 23% of the investigated calves and at least one shedding calf was found on 66% of the farms. Cryptosporidium parvum was the most common species shed, while C. bovis and C. ryanae were also detected. More than half of the calves aged 8-14 days shed C. parvum. Nine previously described C. parvum subtypes (IIaA14G1R1, IIaA16G1R1, IIaA17G1R1, IIaA18G1R1, IIaA19G1R1, IIaA20G1R1, IIaA21G1R1, IIaA22G1R1 and IIaA16G2R1) and an apparently novel subtype IIlA21R2 were found. Calves from farms that reported spreading manure on fields during spring had 10 times higher odds to shed Cryptosporidium spp. in their feces than calves from farms that did not. Calves aged 8-14 days had higher odds to shed IIa18G1R1 as well as IIaA16G1R1 than younger calves.

Development and application of DNA-aptamer-coupled magnetic beads and aptasensors for the detection of Cryptosporidium parvum oocysts in drinking and recreational water resources.

Environmentally stable and disinfectant-resistant oocysts of Cryptosporidium spp. shed in the feces of infected humans and animals frequently contaminate water resources and are subsequently spread via potable and recreational waters. The current monoclonal-antibody-based methods for detecting them in water are slow, labor-intensive, and demand skills to interpret the results. We have developed DNA-aptamer-based aptasensors, coupled with magnetic beads, to detect and identify the oocysts of C. parvum for monitoring recreational and drinking water sources. A sensitive and specific electrochemical aptasensor (3'-biotinylated R4-6 aptamer) was used as a secondary ligand to bind the streptavidin-coated magnetic beads. This was incorporated into a probe using gold nanoparticle modified screen-printed carbon electrodes. Square wave voltammetry allowed for specific recognition of C. parvum oocysts. The aptamer-coated probes had an oocyst detection limit of 50. It did not bind to the cysts of Giardia duodenalis, another common waterborne pathogen, thus indicating its high specificity for the target pathogen. The system could successfully detect C. parvum oocysts in spiked samples of the raw lake and river waters. Therefore, the combined use of the aptasensor and magnetic beads has the potential to monitor water quality for C. parvum oocysts in field samples without relying on monoclonal antibodies and skill-demanding microscopy.

Distribution of Cryptosporidium parvum gp60 subtypes in calf herds of Saxony, Germany.

Cryptosporidiosis is a common protozoan parasitic infection that causes diarrhoea in neonatal calves. The high shedding of environmentally resistant oocysts facilitates outbreaks of cryptosporidiosis in humans. In total, 58 farms (512 calves) in Germany (Saxony and Brandenburg) were visited three times each. Faecal samples of pre-weaned calves were microscopically examined for oocysts of Cryptosporidium spp. using Heine staining and were scored with regard to their consistency. Overall, 88.9% of calves tested microscopically positive for Cryptosporidium spp. in at least one sample, and the excretion of oocysts was significantly (P < 0.01) associated with a higher faecal score (diarrhoea). After DNA extraction from pooled farm isolates, 47 samples were successfully subtyped by sequence analysis of the 60 kDa glycoprotein gene (gp60). All isolates belonged to subtype family IIa. IIaA15G2R1 was the most common subtype (present on 66% of the farms), followed by IIaA16G3R1 (13%). Subtypes IIaA14G1R1, IIaA14G2R1, IIaA1612R1, IIaA16G2R1, IIaA17G1R1, IIaA17G2R1, IIaA17G4R1 and IIaA19G2R1 were found sporadically. This is the first description of gp60 subtype IIaA17G4R1 in cattle in Germany.

Prevalence of Cryptosporidium parvum in dairy calves and GP60 subtyping of diarrheic calves in central Argentina.

Cryptosporidiosis of calves is caused by the enteroprotozoan Cryptosporidium spp. The disease results in intense diarrhea of calves associated with substantial economic losses in dairy farming worldwide. The aim of this study was to determine calf, herd, and within-herd Cryptosporidium prevalence and identify Cryptosporidium species and subtypes in calves with diarrhea in intensive dairy herds in central Argentina. A total of 1073 fecal samples were collected from 54 randomly selected dairy herds. Cryptosporidium-oocysts were isolated and concentrated from fecal samples using formol-ether and detected by light microscopy with the modified Ziehl-Neelsen technique. Overall prevalence of oocyst-excreting calves was found to be 25.5% (274/1073) (95% C.I. 22.9; 28.1%). Of the herds studied, 89% (48/54) included at least one infected calf, whereas within-herd prevalence ranged from the absence of infection to 57% (20/35). A highly significant association was found between the presence of diarrhea and C. parvum infection (χ2 = 55.89, p < 0.001). For species determination, genomic DNA isolated from oocyst-positive fecal samples was subjected to PCR-RFLP of the 18S rRNA gene resulting exclusively in Cryptosporidium parvum identification. C. parvum isolates of calves displaying diarrhea and high rate of excretion of oocysts were subtyped by PCR amplification and direct sequencing of the 60 kDa glycoprotein (GP60) gene. Altogether five GP60 subtypes, designated IIaA18G1R1, IIaA20G1R1, IIaA21G1R1, IIaA22G1R1, and IIaA24G1R1 were identified. Interestingly, IIaA18G1R1 and IIaA20G1R1 were predominant in calves with diarrhea and high infection intensity. Notably, IIaA24G1R1 represents a novel, previously unrecognized C. parvum subtype. The subtype IIaA18G1R1, frequently found in this study, is strongly implicated in zoonotic transmission. These results suggest that calves might be an important source for human cryptosporidiosis in Argentina.

Molecular Prevalence and Genotypes of Cryptosporidium parvum and Giardia duodenalis in Patients with Acute Diarrhea in Korea, 2013-2016.

Cryptosporidium parvum and Giardia duodenalis are the main diarrhea-causing parasitic pathogens; however, their prevalence in Korea is unknown. Here, we conducted a survey to determine the prevalence and genotype distribution of these 2 pathogens causing acute diarrhea in 8,571 patients hospitalized in 17 Regional Institute of Health Environment sites in Korea, during 2013-2016. C. parvum and G. duodenalis were detected and genotyped by nested PCR, and the isolate were molecularly characterized by sequencing the glycoprotein 60 (Gp60) and ß-giardin genes, respectively. The overall prevalence of C. parvum and G. duodenalis was 0.37% (n=32) and 0.55% (n=47), respectively, and both pathogens were more prevalent in children under 9 years old. Molecular epidemiological analysis showed that the C. parvum isolates belonged to the IIa family and were subtyped as IIaA13G2R1, IIaA14G2R1, IIaA15G2R1, and IIaA18G3R1. Analysis of the ß-giardin gene fragment from G. duodenalis showed that all positive strains belong to assemblage A. This is the first report on the molecular epidemiology and subtyping of C. parvum and G. duodenalis in such a large number of diarrheal patients in Korea. These results highlight the need for continuous monitoring of these zoonotic pathogens and provide a basis for implementing control and prevention strategies. Further, the results might be useful for epidemiological investigation of the source of outbreak.

Identification of a novel piscine Cryptosporidium genotype and Cryptosporidium parvum in cultured rainbow trout (Oncorhynchus mykiss).

This study reports for the first time the presence and molecular characterization of Cryptosporidium in farmed rainbow trout (Oncorhynchus mykiss Walbaum, 1792). A total of 360 fish, with no apparent clinical signs of disease, were collected and classified into groups according to their size. Cryptosporidium oocysts were detected by immunofluorescence microscopy in 33 specimens (9.2%), which were located in pyloric caeca samples (42.4%), intestinal scrapings (39.4%), or at both locations (18.2%). In the smallest (youngest) fish group, a higher percentage of positive samples were detected in the pyloric caeca relative to the intestinal location (58.8 vs. 17.6%; P = 0.01), including a cluster with more than 10 oocysts observed in the pyloric caeca of one specimen. PCR amplification and sequencing of fragments of SSU-rDNA and hsp70 genes identified a novel Cryptosporidium piscine genotype (genotype 9) in two specimens and Cryptosporidium parvum in seven fish, including the specimen in which the oocyst cluster was observed. Moreover, Cryptosporidium oocysts were detected in farm water samples (41.7 and 16.7% from influent and effluent, respectively). Although Giardia was not found in gastrointestinal samples, Giardia cysts were observed in 50.0 and 33.3% of the influent and effluent water samples, respectively. The results support the existence of natural infections by C. parvum in freshwater cultured fish, suggesting that the rainbow trout could shed infectious oocysts in aquatic environments and it may be a potential source of human infection when this edible fish is handled.

Genetic uniqueness of Cryptosporidium parvum from dairy calves in Colombia.

Fecal specimens from 432 pre-weaned calves younger than 35 days were collected over a 2-year period (2010-2012) from 74 dairy cattle farms in the central area of Colombia. These samples were microscopically examined for the presence of Cryptosporidium oocysts, and positive specimens were selected for molecular examination. Microscopy revealed that 115 calves (26.6%) from 44 farms (59.5%) tested positive. Oocyst shedding was recorded in calves aged 3-day-old onwards, although the infection rate peaked at 8-14 days (40.7%). Infection rates were higher in diarrheic (52.2%) than in non-diarrheic calves (19.9%) (p < 0.0001, χ2), and infected calves had up to seven times more probability of having diarrhea than non-infected calves. Cryptosporidium species and subtypes were successfully identified in 73 samples from 32 farms. Restriction and sequence analyses of the SSU rRNA gene revealed C. parvum in all but two isolates identified as Cryptosporidium bovis. Sequence analyses of the 60-KDa glycoprotein (gp60) gene revealed eight subtypes within the IIa family. An unusual subtype (IIaA18G5R1) was the most prevalent and widely distributed (more than 66% specimens and 68% farms) while the subtype most frequently reported in cattle worldwide (IIaA15G2R1) was found in less than 13% of specimens and 16% farms. The remaining subtypes (IIaA16G2R1, IIaA17G4R1, IIaA20G5R1, IIaA19G6R1, IIaA20G6R1, and IIaA20G7R1) were restricted to 1-3 farms. This is the first large-sample size study of Cryptosporidium species and subtypes in Colombia and demonstrates the genetic uniqueness of this protozoan in cattle farms in this geographical area.

Epidemiological observations on cryptosporidiosis and molecular characterization of Cryptosporidium spp. in sheep and goats in Kuwait.

Molecular epidemiological analysis of cryptosporidiosis in Middle Eastern countries suggests that small ruminants could play a major role in the transmission of Cryptosporidium spp. to humans, with a dominance of Cryptosporidium parvum, especially its IId subtypes. However, little information is available on the epidemiology and risk factors of cryptosporidiosis as well the distribution of Cryptosporidium species/genotypes and subtypes in small ruminants in this area, including Kuwait. In the present study, 47 farms from several areas in Kuwait were visited once during October 2014 to September 2015 to collect data on risk factors associated with Cryptosporidium infection. Fecal samples from 334 sheep and 222 goats were examined for Cryptosporidium oocysts by Ziehl-Neelsen staining (ZN) and antigens by enzymatic immunoassay (EIA). The Cryptosporidium prevalence was higher when samples were examined by EIA than ZN (11.4 and 7.2% in sheep and goats by EIA, compared with 4.2 and 3.6% by ZN, respectively). Young age (less than 3 months) and closed housing system are risk factors of Cryptosporidium infection. A correlation between fecal consistency and the occurrence of Cryptosporidium spp. was observed; non-formed fecal samples were often found positive. Molecular characterization of 30 ovine and caprine samples using PCR-RFLP analysis of the small subunit rRNA gene revealed the presence of C. parvum in 23 samples, Cryptosporidium ubiquitum in five samples, and Cryptosporidium xiaoi in two samples. Sequence analysis of C. parvum at the 60 KDa glycoprotein gene locus identified two subtypes, IIaA15G2R1 and IIdA20G1, with the latter being more common (in 2 and 20 successfully subtyped samples, respectively). Only one subtype of C. ubiquitum (XIIa) was recorded. Cryptosporidiosis in small ruminants apparently poses public health problem in Kuwait.

The Ultra-Structural Similarities between Cryptosporidium parvum and the Gregarines.

Using a transmission electron microscopy-based approach, this study details the striking similarities between Cryptosporidium parvum and the gregarines during in vitro axenic development at high ultra-structural resolution. C. parvum zoites displayed three unusual regions within uninucleated parasites: epimerite-like, protomerite-like, and the cell body; these regions exhibited a high degree of morphological similarity to gregarine-like trophozoites. The presence of a mucron-like bulging structure at the side of the free ovoid gregarine-like zoites was observed after 2 h of cultivation. An irregular pattern of epicytic-like folds were found to cover the surface of the parasites 24 h postcultivation. Some extracellular stages were paired in laterocaudal or side-side syzygy, with the presence of a fusion zone between some of these zoites. The present findings are in agreement with phylogenetic studies that have proposed a sister relationship with gregarines. Cryptosporidium appears to exhibit tremendous variety in cell structure depending on the surrounding environment, thereby mimicking the "primitive" gregarines in terms of the co-evolution strategy between the parasites and their environments. Given this degree of similarity, different aspects of the evolutionary biology of Cryptosporidium need to be examined, considering the knowledge gained from the study of gregarines.

Cryptosporidium parvum and Enterocytozoon bieneusi in American Mustangs and Chincoteague ponies.

The prevalence of Cryptosporidium and microsporidia in feral horses, which have minimal contact with livestock and humans, is not currently known. We report the findings of a study on Cryptosporidium and microsporidia in 34 Mustangs and 50 Chincoteague ponies in the USA. Fecal samples were screened for presence of Cryptosporidium spp. by analysis of the small-subunit rRNA (SSU) and 60-kDa glycoprotein (gp60) genes, and Enterocytozoon bieneusi and Encephalitozoon spp. by analysis of the ribosomal internal transcribed spacer region (ITS). Cryptosporidium spp. and E. bieneusi were detected in 28/84 (33.3%) and 7/84 (8.3%) samples, respectively. Sequence analysis of SSU and ITS revealed the presence of Cryptosporidium parvum (n = 20) and E. bieneusi genotype horse 1 (n = 7), respectively. Subtyping of C. parvum isolates at the gp60 locus showed the presence of subtype IIaA17G2R1 in Mustangs and subtypes IIaA13G2R1 and IIaA15G2R1 in Chincoteague ponies. Enterocytozoon bieneusi genotype horse 1 was detected in Mustangs (n = 2) and Chincoteague ponies (n = 5). No Cryptosporidium or E. bieneusi positive animals had diarrhea. The finding that Mustangs and Chincoteague ponies are host to the zoonotic pathogen C. parvum suggests that their infrequent contact with humans and livestock is sufficient to maintain transmission; however, we should also consider the possibility that C. parvum is an established parasite of Mustangs and Chincoteague ponies that persists in these animals independently of contact with humans or livestock.

Cryptosporidium species and Cryptosporidium parvum subtypes in dairy calves and goat kids reared under traditional farming systems in Turkey.

Molecular characterizations of Cryptosporidium spp. in ruminants reared under traditional animal management systems are scarce and studies conducted thus far have revealed largely an absence of the pathogenic and zoonotic species Cryptosporidium parvum in pre-weaned animals. In this study, we examined Cryptosporidium species and subtype distribution in free-range pre-weaned dairy calves and goat kids with diarrhea. Cryptosporidium-positive specimens from pre-weaned calves on 10 farms and goat kids on 4 farms in Ankara, Balikesir, Corum, Kirikkale, and Kirsehir Provinces, Turkey were genotyped by PCR-restriction length polymorphism analysis of the small subunit rRNA gene, which identified C. parvum in 27 calves and 9 goat kids and Cryptosporidium ryanae in 1 calf. Among the C. parvum isolates successfully subtyped by DNA sequence analysis of the 60 kDa glycoprotein gene, three subtypes were detected in calves, including IIaA13G2R1 (20/23), IIdA18G1 (2/23), and IIdA20G1b (1/23), and four subtypes were detected in goat kids, including IIaA13G2R1 (3/8), IIaA15G1R1 (2/8), IIdA22G1 (2/8), and IIdA18G1 (1/8). Data of the study suggest that dairy calves reared in a traditional cow-calf system in Turkey are mainly infected with a C. parvum subtype rarely seen elsewhere, whereas goat kids are infected with diverse subtypes. As all five C. parvum subtypes found in this study are known human pathogens, pre-weaned farm animals could play a potential role in the transmission of human cryptosporidiosis.

Detection by PCR of pathogenic protozoa in raw and drinkable water samples in Colombia.

We evaluated the presence of DNA of Giardia, Toxoplasma, and Cryptosporidium by PCR, and of Giardia and Cryptosporidium genera by immunofluorescence antibody test (IFAT), in water samples, before, during, and after plant treatment for drinkable water. We applied this method in 38 samples of 10 l of water taken from each of the water treatment steps and in 8 samples taken at home (only for Toxoplasma PCR) in Quindio region in Colombia. There were 8 positive samples for Cryptosporidium parvum (21 %), 4 for Cryptosporidium hominis (10.5 %), 27 for Toxoplasma gondii (58.6 %), 2 for Giardia duodenalis assemblage A (5.2 %), and 5 for G. duodenalis assemblage B (13.1 %). By IFAT, 23 % were positive for Giardia and 21 % for Cryptosporidium. An almost perfect agreement was found between IFAT and combined results of PCR, by Kappa composite proportion analysis. PCR positive samples were significantly more frequent in untreated raw water for C. parvum (p = 0.02). High mean of fecal coliforms, high pH values, and low mean of chlorine residuals were strongly correlated with PCR positivity for G. duodenalis assemblage B. High pH value was correlated with PCR positivity for C. parvum. Phylogenetic analysis of DNA sequences was possible, showing water and human clinical sequences for Toxoplasma within the same phylogenetic group for B1 repeated sequence. PCR assay is complementary to IFAT assay for monitoring of protozoa in raw and drinkable water, enabling species identification and to look for phylogenetic analysis in protozoa from human and environmental sources.

Population structure of natural and propagated isolates of Cryptosporidium parvum, C. hominis and C. meleagridis.

The three protozoan species Cryptosporidium parvum, C. meleagridis and C. hominis (phylum Apicomplexa) are enteric pathogens of humans. The former two species are zoonotic and the latter is thought to infect only humans. To better characterize the structure and transmission of natural and laboratory-propagated isolates, we analyzed a collection of archived human and animal isolates of these three species by deep-sequencing polymerase chain reaction products amplified from a polymorphic sequence on chromosome 1. Thousands of screened 200-nucleotide sequences were analyzed to compare the diversity among samples, to assess the impact of laboratory propagation on population complexity and to identify taxonomically mixed isolates. Contrary to our expectation, repeated propagation in animals did not reduce intra-isolate diversity nor was diversity associated with host species. Significantly, in most samples, sequences characteristic of a different species were identified. The presence of C. hominis alleles in C. parvum and C. meleagridis isolates confirms earlier reports of mixed isolates and raises the possibility that the host range of C. hominis is broader than typically assumed. In a genetically divergent isolate of C. parvum, a majority of sequences was found to be recombinant, suggesting that this genotype originated from a C. parvum × C. hominis recombination event.

Multiple Cryptosporidium parvum subtypes detected in a unique isolate of a Chilean neonatal calf with diarrhea.

To further understand the composition of population of parasite in a single host, we analyzed the GP60 gene of Cryptosporidium parvum amplified from DNA of a randomly selected isolate found in the feces of a diarrheic calf from a dairy farm in Central Chile. Direct sequencing of the amplicon yield the IIaA17G4R1 C. parvum subtype. The same amplicon was cloned in Escherichia coli (22 clones) and sequenced, yielding three different GP60 subtypes, IIaA17G4R1 (16/22), IIaA16G4R1 (1/22), and IIaA15G4R1 (1/22), and four sequences with nucleotide substitutions in the serine repeats, which subtype would be otherwise IIaA17G4R1. It is thus possible to determine allelic polymorphism using Sanger sequencing with an additional step of bacterial cloning. The results also indicate the necessity to further characterize parasite populations in a single host to better understand the dynamics of Cryptosporidium epidemiology.

Emergence of novel subtypes of Cryptosporidium parvum in calves in Poland.

The aim of the study was to identify the Cryptosporidium parvum subtypes circulating in Polish cattle and their distribution in relation to the age and health status of tested animals. In total, 779 fecal samples were obtained from young cattle originating from 237 farms. C. parvum strains were identified at the 18 small-subunit ribosomal RNA (SSU rRNA), COWP, and LIB13 loci and were subsequently analyzed by sequencing at the 60-kDa glycoprotein (GP60) locus for subtype determination. The presence of 71 C. parvum strains belonging to IIa, IId, or IIl subtype families was shown. The strains from the IIa allele family prevailed with IIaA17G1R1, IIaA17G2R1, and IIaA15G2R1 subtypes occurring frequently. Two novel subtypes IIaA10G1R1 and IIlA19R3 were detected for the first time in a bovine host. The highest C. parvum prevalence (22.5 %, confidence interval (CI) = 2.5 %) was observed among the youngest animals up to 2 weeks of age, followed by the prevalence among those aged 2 to 4 weeks (6.6 %, CI = 2.6 %) and then among older cattle (4.9 %, CI = 2.1). The occurrence of diarrhea in animals was associated with the presence of the IIaA16G1R1b subtype, while infections caused by IIaA15G2R1 strains were more likely to be asymptomatic. The geographical distribution of subtypes revealed that strains from the IIa subtype family were detected all over the country frequently compared to the IId and IIl subtypes, the sporadic appearances of which confirmed their endemic occurrence. Subtype analysis revealed the presence of zoonotic strains indicating cattle as a reservoir for human cryptosporidiosis.