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.     

Genetic classification of Cryptosporidium isolates from humans and calves in Slovenia

To assess the importance of cattle as a source of human cryptosporidial infections in Slovenia, Cryptosporidium isolates from calves and humans with cryptosporidiosis were characterized genetically by direct DNA sequencing, targeting a variable region of the 60 subtypes', were identified, of which 7 were novel. In humans, C. hominis Ia (subtype IaA17R3) and Ib (IbA10G2) and Cryptosporidium parvum IIa (IIaA9G1R1, IIaA11G2R1, IIaA13R1, IIaA14G1R1, IIaA15G1R1, IIaA15G2R1, IIaA16G1R1, IIaA17G1R1 and IIaA19G1R1), IIc (IIcA5G3), and IIl (IIlA16R2) were recorded; this is the first record of the latter subtype in humans. In cattle, C. parvum IIa (IIaA13R1, IIaA15G2R1, IIaA16R1 and IIaA16G1R1) and IIl (IIlA16R2 and IIlA18R2) were recorded. Of the 15 subtypes identified, subtypes of C. parvum IIa were the most frequently encountered (>90%) in both humans and calves. The present findings suggest that zoonotic transmission plays an important role in sporadic human cryptosporidiosis in Slovenia.     

Molecular characterization of Cryptosporidium isolates from humans and animals in Iran

Isolates of Cryptosporidium spp. from human and animal hosts in Iran were characterized on the basis of both the 18S rRNA gene and the Laxer locus. Three Cryptosporidium species, C. hominis, C. parvum, and C. meleagridis, were recognized, and zoonotically transmitted C. parvum was the predominant species found in humans.     

牛源隐孢子虫分离株的分子鉴定

目的对安徽4个牛源隐孢子虫分离株进行鉴定,旨在阐明感染牛隐孢子虫主要虫种。方法采用PCR和Nested-PCR方法分别对4个分离株18S rRNA、HSP70基因进行扩增和测序,所测定的序列经生物信息学分析它们同源性和构建种系发育进化树,以确定各分离株与其他隐孢子虫虫种之间的亲缘关系。结果 （1）它们的18S rRNA基因和HSP70基因与安氏隐孢子虫（C.andersoni）AY954887相似性可达88.3%和98.3%;（2）在遗传进化树方面,它们与AY954892（河南株）亲缘关系接近。结论此次分离的4个牛源隐孢子虫分离株均为安氏隐孢子虫（Cryptosporidium andersoni）。     

Subtype analysis of Blastocystis sp. isolates from human and canine hosts in an urban community in the Philippines

Blastocystis sp. is a common gut-dwelling protist of both humans and animals. A cross-sectional survey among humans and their dogs was conducted to determine the prevalence of Blastocystis infection and to characterize the subtype (ST) distribution in an urban community in the Philippines. Fecal specimens from 1,271 humans and 145 dogs were collected and inoculated in diphasic culture medium. Prevalence of Blastocystis by culture was 13.0% (95% CI = 11.2–15.0) and 14.5% (95% CI = 9.6–21.2) for humans and dogs, respectively. A total of 168 culture isolates were genotyped using polymerase chain reaction (PCR) with seven pairs of ST-specific sequence-tagged-site (STS) primers. In humans, the ST present in this study were ST1 with 22.6% (95% CI = 17.2–29.0), ST2 with 3.1% (95% CI = 1.3–6.7), ST3 with 41.4% (95% CI = 34.9–48.6), ST4 with 14.8% (95% CI = 10.5–20.6), ST5 with 4.1% (95% CI = 2.0–8.0), and unknown ST with 13.9% (95% CI = 9.6–19.4). In dogs, the ST present in this study were ST1 with 4.3% (95% CI = 0.0–29.0), ST2 with 8.7% (95% CI = 1.3–28.0), ST3 with 17.4% (95% CI = 6.4–37.7), ST4 with 13.0% (95% CI = 3.7–33.0), ST5 with 13.0% (95% CI = 3.7–33.0), and unknown ST with 47.8% (95% CI = 29.2–67.0). This is the first study that reported Blastocystis ST4 in human and canine hosts in the Philippines.     

Generation of whole genome sequences of new Cryptosporidium hominis and Cryptosporidium parvum isolates directly from stool samples

Background

Whole genome sequencing (WGS) of Cryptosporidium spp. has previously relied on propagation of the parasite in animals to generate enough oocysts from which to extract DNA of sufficient quantity and purity for analysis. We have developed and validated a method for preparation of genomic Cryptosporidium DNA suitable for WGS directly from human stool samples and used it to generate 10 high-quality whole Cryptosporidium genome assemblies. Our method uses a combination of salt flotation, immunomagnetic separation (IMS), and surface sterilisation of oocysts prior to DNA extraction, with subsequent use of the transposome-based Nextera XT kit to generate libraries for sequencing on Illumina platforms. IMS was found to be superior to caesium chloride density centrifugation for purification of oocysts from small volume stool samples and for reducing levels of contaminant DNA.

Results

The IMS-based method was used initially to sequence whole genomes of Cryptosporidium hominis gp60 subtype IbA10G2 and Cryptosporidium parvum gp60 subtype IIaA19G1R2 from small amounts of stool left over from diagnostic testing of clinical cases of cryptosporidiosis. The C. parvum isolate was sequenced to a mean depth of 51.8X with reads covering 100 % of the bases of the C. parvum Iowa II reference genome (Bioproject PRJNA 15586), while the C. hominis isolate was sequenced to a mean depth of 34.7X with reads covering 98 % of the bases of the C. hominis TU502 v1 reference genome (Bioproject PRJNA 15585).The method was then applied to a further 17 stools, successfully generating another eight new whole genome sequences, of which two were C. hominis (gp60 subtypes IbA10G2 and IaA14R3) and six C. parvum (gp60 subtypes IIaA15G2R1 from three samples, and one each of IIaA17G1R1, IIaA18G2R1, and IIdA22G1), demonstrating the utility of this method to sequence Cryptosporidium genomes directly from clinical samples. This development is especially important as it reduces the requirement to propagate Cryptosporidium oocysts in animal models prior to genome sequencing.

Conclusion

This represents the first report of high-quality whole genome sequencing of Cryptosporidium isolates prepared directly from human stool samples.     

Preliminary molecular characterization of Cryptosporidium parvum isolates of wildlife rodents from Poland

Isolates of Cryptosporidium were collected from 3 species of woodland and field rodents (Clethrionomys glareolus, Microtus arvalis, and Apodemus flavicollis) and were characterized by polymerase chain reaction amplification and sequencing of fragments of the oocyst wall protein (COWP) gene and of the 18S ribosomal RNA gene. Sequence analysis of these markers revealed that the animals were infected with C. parvum, and that the genotype involved was almost identical to the mouse genotype previously described from Mus musculus. Thus, small rodents should be considered as an important reservoir of C. parvum genotypes closely related to the zoonotic genotype 2 and potentially hazardous to humans.     

Molecular Identification of a Rare Subtype of Cryptosporidium hominis in Infants in China

Two Cryptosporidium isolates from separate infants suffering from diarrhea were obtained from a hospital in Zhengzhou, China and were genotyped by PCR amplification and sequence analysis of the small-subunit ribosomal RNA (rRNA) (SSU rRNA), 70-kDa heat shock protein (HSP70), and actin genes. Further subtyping was performed by PCR amplification and sequence analysis of the 60-kDa glycoprotein (gp60) gene. Both the isolates were identified as Cryptosporidium hominis subtype IdA21, a rare subtype previously found only in a human immunodeficiency virus-infected child in South Africa and another child in Jordan.     

Prevalence and molecular identification of Cryptosporidium isolates from pet lizards and snakes in Italy

In order to acquire prevalence and genetic data on Cryptosporidium infections in captive lizards and snakes kept as pets, a survey was conducted on 150 individual reptiles from southern Italy. Fecal samples were preserved in 5% formalin and analyzed using a commercial immunofluorescence assay (IFA) for the detection of Cryptosporidium oocysts and Giardia cysts. IFA revealed the presence of Cryptosporidium oocysts in nine of the 150 samples examined (6.0%), precisely in 6/125 snakes (4.8%) and in 3/25 lizards (12.0%); all fecal samples tested negative for the presence of Giardia cysts. Molecular characterization based on nested PCR amplification and sequencing of the SSU-rRNA gene, revealed the presence of Cryptosporidium serpentis in three samples from snakes (Boa constrictor constrictor, Elapheguttata guttata guttata and Python molurus).     

Phylogenetic analysis of Cryptosporidium isolates from captive reptiles using 18S rDNA sequence data and random amplified polymorphic DNA analysis.

Sequence alignment of a polymerase chain reaction-amplified 713-base pair region of the Cryptosporidium 18S rDNA gene was carried out on 15 captive reptile isolates from different geographic locations and compared to both Cryptosporidium parvum and Cryptosporidium muris isolates. Random amplified polymorphic DNA (RAPD) analysis was also performed on a smaller number of these samples. The data generated by both techniques were significantly correlated (P < 0.002), providing additional evidence to support the clonal population structure hypothesis for Cryptosporidium. Phylogenetic analysis of both 18S sequence information and RAPD analysis grouped the majority of reptile isolates together into 1 main group attributed to Cryptosporidium serpentis, which was genetically distinct but closely related to C. muris. A second genotype exhibited by 1 reptile isolate (S6) appeared to be intermediate between C. serpentis and C. muris but grouped most closely with C. muris, as it exhibited 99.15% similarity with C. muris and only 97.13% similarity with C. serpentis. The third genotype identified in 2 reptile isolates was a previously characterized 'mouse' genotype that grouped closely with bovine and human C. parvum isolates.     

A technique for typing Cryptosporidium isolates.

Antigens extracted from Cryptosporidium oocysts, which had been purified from faeces or chick egg culture, were electrophoresed in sodium dodecyl sulfate-polyacrylamide gels, and blotted onto nitrocellulose membranes. A Cryptosporidium genus-specific monoclonal antibody MAb-C1 bound to multiple bands using several detection techniques, and these corresponded to bands detected using immune rabbit antisera. Using a detection system with fluorescein isothiocyanate (FITC)-labelled MAb-C1 and alkaline phosphatase-labelled anti-FITC, bands were detected between 50 and 300 kDa. Blots were examined directly and by using a laser scanner. The system was shown to be specific for Cryptosporidium spp., giving no staining with a variety of other pathogens, and with negative samples. The oocyst antigen which bound MAb-C1 was stable, and banding patterns were not significantly affected by pretreatment of oocysts with proteinase K, trypsin, formalin, or sodium hypochlorite, methods commonly used during preparation and storage of C. parvum oocysts. However, banding was reduced with potassium dichromate. Of 76 samples containing Cryptosporidium oocysts, 53 showed one or more MAb-C1 staining bands. Cryptosporidium baileyi and C. parvum could be clearly differentiated by their banding patterns, indicating that the system will distinguish between species. Some isolates, including a single isolate of C. muris, produced weak bands which made interpretation difficult. The technique showed differences between isolates of C. parvum, with two different banding types found in human isolates, and other banding types seen in calf and lamb isolates. This method provides a useful way of characterising isolates which may be new species.     

Restriction fragment length polymorphism analysis of Cryptosporidium parvum isolates of bovine and human origin.

Cryptosporidium parvum oocysts isolated from different hosts and geographical areas were compared by restriction endonuclease analysis of repetitive DNA: Iowa (bovine), Florida (bovine), New York (bovine), Peru (human), Brazil (human), and Mexico (human). Southern blot hybridization analysis was performed using the restriction endonuclease enzyme Eco RI and the DNA probe pV47-2. The probe hybridized with 18 bands present in all the isolates. The Brazilian, Mexican, and Peruvian human isolates had an additional common band of 4.3 kbp that was absent in the bovine isolates. Two extra bands of 14 and 12 kbp were present in the Brazilian isolate whereas the Mexican isolate had an extra band of 14 kbp. When the Iowa and Peru C. parvum isolates were passed twice through calves, oocysts recovered from both passages showed identical banding patterns, suggesting that recombination of the repetitive sequences was not altered during sexual reproduction. The DNA digested with other restriction endonucleases were tested confirming differences between isolates. A genomic DNA library is currently being produced to better define isolate variation in C. parvum.     

Epidemiology and molecular relationships of Cryptosporidium spp. in people, primates, and livestock from Western Uganda

Background

Cryptosporidium is one of the most common parasitic diarrheal agents in the world and is a known zoonosis. We studied Cryptosporidium in people, livestock, and non-human primates in the region of Kibale National Park, Uganda. Land use change near the park has resulted in fragmented forest patches containing small, remnant populations of wild primates that interact intensively with local people and livestock. Our goal was to investigate risk factors for Cryptosporidium infection and to assess cross-species transmission using molecular methods.

Methodology/Principal Findings

Diagnostic PCR revealed a prevalence of Cryptosporidium of 32.4% in humans, 11.1% in non-human primates, and 2.2% in livestock. In the case of humans, residence in one particular community was associated with increased risk of infection, as was fetching water from an open water source. Although 48.5% of infected people reported gastrointestinal symptoms, this frequency was not significantly different in people who tested negative (44.7%) for Cryptosporidium, nor was co-infection with Giardia duodenalis associated with increased reporting of gastrointestinal symptoms. Fecal consistency was no different in infected versus uninfected people or animals. DNA sequences of the Cryptosporidium oocyst wall protein gene placed all infections within a well-supported C. parvum/C. hominis clade. However, the only two sequences recovered from primates in the core of the park''s protected area fell into a divergent sub-clade and were identical to published sequences from C. parvum, C. hominis, and C. cuniculus, suggesting the possibility of a separate sylvatic transmission cycle.

Conclusions/Significance

Cryptosporidium may be transmitted frequently among species in western Uganda where people, livestock, and wildlife interact intensively as a result of anthropogenic changes to forests, but the parasite may undergo more host-specific transmission where such interactions do not occur. The parasite does not appear to have strong effects on human or animal health, perhaps because of persistent low-level shedding and immunity.     

Phenotypic and genotypic characterization of Cryptosporidium species and isolates

Recent outbreaks of cryptosporidiosis from contaminated water supplies have led to a need for the detection of Cryptosporidium oocysts from various hosts and contaminating sources. The presence of nonpathogenic species or strains of Cryptosporidium is important for diagnostic purposes as there is a potential for false-positive detection of pathogenic parasites. The present review focuses on phenotypic differences and recent advances in genotypic analyses of the genus Cryptosporidium with an emphasis on detecting various isolates and identifying differences in Cryptosporidium parvum and other species in this genus. The information currently available demonstrates important patterns in DNA sequences of Cryptosporidium, and our understanding of macro- and microevolutionary patterns has increased in recent years. However, current knowledge of Cryptosporidium genetic diversity is far from complete, and the large amount of both phenotypic and genotypic data has led to problems in our understanding of the systematics of this genus. Journal of Industrial Microbiology & Biotechnology (2001) 26, 95–106. Received 18 March 2000/ Accepted in revised form 13 August 2000     

Subtype distribution of Blastocystis isolates from synanthropic and zoo animals and identification of a new subtype

Blastocystis isolates from 56 Danish synanthropic and zoo animals, 62 primates primarily from United Kingdom (UK) collections and 16 UK primate handlers were subtyped by PCR, sequencing and phylogenetic analysis. A new subtype (ST) from primates and artiodactyls was identified and designated as Blastocystis sp. ST10. STs isolated from non-human primates (n = 70) included ST3 (33%), ST8 (21%), ST2 (16%), ST5 (13%), ST1 (10%), ST4 (4%) and ST10 (3%). A high prevalence of ST8 was seen among primate handlers (25%). This ST is normally very rare in humans, suggesting that acquisition of Blastocystis ST8 infections from primates by their handlers had occurred in these cases. Data from published studies of non-human primates, other mammals and birds were collected and interpreted to generate a comprehensive overview on the ST distribution in such animals. On the basis of information on 438 samples, it was found that Blastocystis from primates belong mainly to ST1, ST2, ST3, ST5 and ST8, ungulates and dogs mainly ST1, ST2, ST3, ST5 and ST10, rodents ST4 and birds mainly ST6 and ST7. The data indicate moderate host specificity, most clearly exemplified by the fact that STs isolated from avian and non-avian hosts rarely overlap.     

Cryptosporidium species and subtype analysis from dairy calves in Spain

Faecal specimens from 287 diarrhoeic calves younger than 21 days, collected over a 2-year period (2006-2007) from 82 dairy cattle farms in 14 provinces across the north of Spain, were examined for the presence of Cryptosporidium oocysts. Overall, 63 farms (76.8%) and 166 calves (57.8%) tested positive by microscopy. In order to elucidate the genetic diversity, selected positive specimens from 149 calves originating from 61 farms in the 14 provinces were examined by genotyping and subtyping techniques. Cryptosporidium parvum was the only species identified by PCR-RFLP of SSU rDNA from all 149 isolates and sequencing of a subset of 50 isolates, except for 2 specimens that were identified as C. bovis. Sequence analyses of the glycoprotein (GP60) gene revealed that most C. parvum isolates (98%) belonged to the subtype family IIa and 2 isolates were identified as the novel subtype IIdA23G1. Subtype IIaA15G2R1 was the most common and widely distributed (80.3% of the 61 farms), followed by subtype IIaA16G3R1 (14.7%), whereas the remaining IIa subtypes (IIaA16G2R1, IIaA17G2R1, IIaA18G3R1, IIaA19G3R1) were restricted to 1-3 farms. All these C. parvum IIa subtypes have previously been described in human patients, indicating that most isolates from diarrhoeic calves in northern Spain have zoonotic potential.     

Genetic characterization of Cryptosporidium isolates from ringed seals (Phoca hispida) in Northern Quebec, Canada

This study reports the molecular characterization of Cryptosporidium spp. isolates identified from intestinal contents of ringed seals (Phoca hispida) from Nunavik (Quebec, Canada). Cryptosporidium spp. fragments of 18S rRNA, HSP-70, and actin loci were amplified by PCR from seal intestinal contents. PCR-positive specimens were sequenced and compared with other Cryptosporidium species and genotypes reported previously. Sequence analysis showed the presence of C. muris and 2 novel genotypes in ringed seals.     

Evaluation of five membrane filtration methods for recovery of Cryptosporidium and Giardia isolates from water samples

We evaluated the efficiency of five membrane filters for recovery of Cryptosporidium parvum oocysts and Giardia lamblia cysts. These filters included the Pall Life Sciences Envirochek (EC) standard filtration and Envirochek high-volume (EC-HV) membrane filters, the Millipore flatbed membrane filter, the Sartorius flatbed membrane filter (SMF), and the Filta-Max (FM) depth filter. Distilled and surface water samples were spiked with 10 oocysts and 10 cysts/liter. We also evaluated the recovery efficiency of the EC and EC-HV filters after a 5-s backwash postfiltration. The backwashing was not applied to the other filtration methods because of the design of the filters. Oocysts and cysts were visualized by using a fluorescent monoclonal antibody staining technique. For distilled water, the highest percent recovery for both the oocysts and cysts was obtained with the FM depth filter. However, when a 5-s backwash was applied, the EC-HV membrane filter (EC-HV-R) was superior to other filters for recovery of both oocysts (n = 53 +/- 15.4 per 10 liters) and cysts (n = 59 +/- 11.5 per 10 liters). This was followed by results of the FM depth filter (oocysts, 28.2 +/- 8, P = 0.015; cysts, 49.8 +/- 12.2, P = 0.4260), and SMF (oocysts, 16.2 +/- 2.8, P = 0.0079; cysts, 35.2 +/- 3, P = 0.0079). Similar results were obtained with surface water samples. Giardia cysts were recovered at higher rates than were Cryptosporidium oocysts with all five filters, regardless of backwashing. Although the time differences for completion of filtration process were not significantly different among the procedures, the EC-HV filtration with 5-s backwash was less labor demanding.