Cryptosporidium Oocyst Wall Protein Gene Research Articles

Evaluation of a vaccine candidate isolated from Cryptosporidium parvum oocyst in mice.

Cryptosporidiosis is a leading cause of diarrheal disease worldwide and is an animal and public health burden. This study aimed to evaluate the protective potential of affinity-purified Cryptosporidium parvum oocyst antigen as a vaccine candidate according to fecal oocyst shedding, humoral and cellular immune responses, histopathological changes, and the number of parasite developmental stages in ileal and hepatic tissues. We isolated oocysts from naturally infected buffalo calves and identified them molecularly as C. parvum isolates (GenBank: ON730707 and ON730708) by targeting the Cryptosporidium oocyst wall protein gene. We propagated the C. parvum oocysts in mice. In addition, we prepared crude antigen from the isolated oocysts by purification using cyanogen bromide-activated Sepharose-4B affinity chromatography coupled with rabbit hyperimmune serum. Then, we divided 81 parasite-free mice into three groups: (1) non-vaccinated non-infected mice, (2) mice orally infected with 1 × 105 C. parvum oocysts on week 4 of the experiment, and (3) mice immunized twice with 40 μg/kg of the purified fraction at 2-week intervals. Then, we challenged the vaccinated group with C. parvum oocysts after 2 weeks, and the positive control group was infected at the same time. We observed a prolonged prepatent period and decreased oocyst shedding in the vaccinated infected mice compared with the non-vaccinated infected mice (t < 0.001). The vaccinated mice had significantly higher immunoglobulin G levels than those in the other two groups at all examined weeks. In addition, the production of cytokines interferon-gamma, interleukin (IL)-10, IL-12, and IL-15 was activated post-vaccination. After the challenge, all tested cytokines were significantly increased (p < 0.001) in the two infected groups compared with the non-vaccinated non-infected group, with the highest levels in the vaccinated infected group. Vaccinated infected mice exhibited significantly fewer pathological lesions in the ileum and liver than non-vaccinated infected mice, which showed prominent histopathological lesions. Endogenous developmental stages of C. parvum indicated that the ileum was more parasitized than the liver and that vaccination resulted in a lower number of oocysts in ileal and hepatic tissues (p < 0.05). Our prepared affinity-purified vaccine candidate could be promising in protecting against cryptosporidiosis.

Development of duplex real-time PCR for quick detection of cryptosporidiosis in goats.

Cryptosporidium spp. is the most important foodborne and waterborne pathogens and a leading cause of mortality from foodborne and waterborne gastrointestinal diseases. In neonates of domestic animals, it is associated with consistent diarrhea and dehydration. Cryptosporidium infection begins with the ingestion of sporulated oocytes disseminated by carrier animals that consistently contaminate the environment. Many diagnostic tests are available including microscopy and antigen trap-ELISA, but none of the diagnostic tests available currently cannot differentiate between active and passive infection in the host. In the current study, to address this challenge an mRNA-basedduplex TaqMan® probe PCR was developed to target the Cryptosporidium oocyst wall protein gene and 18SSU rRNA gene in a single tube that can detect metabolically active cryptosporidial oocysts. The mRNA transcripts are the direct indicator of any actively replicating cell and they will help decipher the active stages of its lifecycle in a host. This diagnostic assay was standardized by computing transcript copy number-based limit of detection (LOD). For COWP and 18SSU rRNA genes, the LOD was 7.08 × 1004 and 5.95 × 1005 , respectively. During active infections, the oocyst wall protein will be active and so its COWP gene transcripts will act as a marker for active infection. While transcripts for 18SSU rRNA are constitutively expressed in cryptosporidial life cycle. This current diagnostic assay will be a quantitative marker that will help assess the active stages of Cryptosporidium infection in neonates. The disease dynamics will help better understand to formulate the control strategies and contain infection among healthy animals.

Genotyping of Cryptosporidium species in children suffering from diarrhea in Sharkyia Governorate, Egypt.

The protozoan parasite Cryptosporidium is one of the principal reasons for childhood diarrhea around the world. This work aimed to differentiate Cryptosporidium species among children suffering from diarrhea in Sharkyia Governorate, Egypt. A total of 97 fecal specimens were taken from children suffering from diarrhea, attending Pediatric Clinics of Zagazig University and Al-Ahrar Hospitals. Full history was taken. Stool samples were examined microscopically using modified Ziehl-Neelsen stain for detection of Cryptosporidium oocysts. To identify Cryptosporidium genotypes, positive samples were then subjected to nested Polymerase chain reaction-restriction fragment length polymorphism targeting Cryptosporidium oocyst wall protein gene. The overall detection rate was 27.8% (27/97) using modified Ziehl-Neelsen stain staining method. Using nested polymerase chain reaction, the gene was amplified in 85.2% (23/27). Restriction fragment length polymorphism analysis revealed that 65.2% (15/23) were Cryptosporidium hominis, 30.4% (7/23) were Cryptosporidium parvum, and one sample was not typed (4.4%). The significant risk factors associated with Cryptosporidium infection in children were animal contact and residence in rural areas. Cryptosporidium is a common enteric parasite affecting children in Sharkyia Governorate, Egypt, with the predominance of C. hominis genotype in children.

Revisiting the infectivity and pathogenicity of Cryptosporidium avium provides new information on parasitic sites within the host

BackgroundCryptosporidium spp. are protozoans that cause diarrheal illness in humans and animals, including birds, worldwide. The present study was aimed to revisit the infectivity and pathogenicity of C. avium, recently considered to be a valid avian-infecting species of Cryptosporidium, and foster further understanding of its biological characteristics.ResultsResults showed that no Cryptosporidium oocysts were detected in the feces of experimentally inoculated BALB/c mice, Mongolian gerbils, quail or budgerigars within 30 days post-infection (dpi). Oocysts were first detected in feces of 3-day-old and 40-day-old hens at 8 and 9 dpi, respectively. In ducks infected with C. avium, oocysts were first detected at 9 dpi. Oocysts of infected animals were studied using a nested-polymerase chain reaction (PCR) technique for the SSU rRNA gene, actin gene, HSP70 gene and Cryptosporidium oocyst wall protein gene (COWP) detection. Restriction fragment length polymorphism (RFLP), using SspI and VspI restriction enzymes, was carried out to genotype the species and obtained amplification products were sequenced. Cryptosporidium developmental stages were found in the longitudinal plica of the bursa fabricii (BF) of hens, with high levels observed in histological sections and scanning electron microscopy. No pathological changes were observed.ConclusionsThese findings indicate that the bursa fabricii may be the primary site of C. avium infection. More biological data are needed to support the establishment of new species and contribute to the taxonomy of Cryptosporidium.

Intestinal Cryptosporidiosis in Renal Transplant Recipients: Prevalence, Species Detection and Comparative Evaluation of SSU rRNA and Cryptosporidium Oocyst Wall Protein Genes

Context: Cryptosporidiosis is intestinal opportunistic infection commonly occurring in immunocompromised patients including renal transplant (RT) recipients receiving continuous immunosuppressive therapy. Knowledge about species of Cryptosporidium-infecting RT recipients is necessary to know about mode of its transmission (anthroponotic or zoonotic). Various genes such as small subunit rRNA (SSU rRNA) and Cryptosporidium oocyst wall protein (COWP) genes may help in species identification though their sensitivity and specificity are highly variable. Subjects and Methods: A total of 993 and 575 stool samples were examined for Cryptosporidium by microscopy from 358 RT recipients and 200 healthy controls, respectively. Stool samples of RT recipients and healthy controls were subjected to polymerase chain reaction (PCR) followed by restriction fragment length polymorphism (RFLP) for species identification. Results:Cryptosporidium was more commonly detected amongst RT recipients than healthy controls (30/358, 8.4% vs. 0/200, respectively; P < 0.001). The infection was more common amongst patients with diarrhoea than those without (26/162, 16.1% vs. 4/145, 2.8%; P < 0.001). Cryptosporidium parvum was identified in 10/30 (33.3%) and Cryptosporidium hominis in 20/30 (66.7%) samples. SSU gene PCR-RFLP proved to be more sensitive (100%) than COWP (90%); however, specificity of both was same (100%). Conclusions: Cryptosporidiosis is common amongst RT recipients, particularly those with diarrhoea. C. hominis is the most common species in the studied population. SSU rRNA PCR was more sensitive molecular method for the differentiation of Cryptosporidium species.

Cryptosporidium testudinis sp. n., Cryptosporidium ducismarci Traversa, 2010 and Cryptosporidium tortoise genotype III (Apicomplexa: Cryptosporidiidae) in tortoises.

Understanding of the diversity of species of Cryptosporidium Tyzzer, 1910 in tortoises remains incomplete due to the limited number of studies on these hosts. The aim of the present study was to characterise the genetic diversity and biology of cryptosporidia in tortoises of the family Testudinidae Batsch. Faecal samples were individually collected immediately after defecation and were screened for presence of cryptosporidia by microscopy using aniline-carbol-methyl violet staining, and by PCR amplification and sequence analysis targeting the small subunit rRNA (SSU), Cryptosporidium oocyst wall protein (COWP) and actin genes. Out of 387 faecal samples from 16 tortoise species belonging to 11 genera, 10 and 46 were positive for cryptosporidia by microscopy and PCR, respectively. All samples positive by microscopy were also PCR positive. Sequence analysis of amplified genes revealed the presence of the Cryptosporidium tortoise genotype I (n = 22), C. ducismarci Traversa, 2010 (n = 23) and tortoise genotype III (n = 1). Phylogenetic analyses of SSU, COWP and actin gene sequences revealed that Cryptosporidium tortoise genotype I and C. ducismarci are genetically distinct from previously described species of Cryptosporidium. Oocysts of Cryptosporidium tortoise genotype I, measuring 5.8-6.9 µm × 5.3-6.5 µm, are morphologically distinguishable from C. ducismarci, measuring 4.4-5.4 µm × 4.3-5.3 µm. Oocysts of Cryptosporidium tortoise genotype I and C. ducismarci obtained from naturally infected Russian tortoises (Testudo horsfieldii Gray) were infectious for the same tortoise but not for Reeve's turtles (Mauremys reevesii [Gray]), common garter snake (Thamnophis sirtalis [Linnaeus]), zebra finches (Taeniopygia guttata [Vieillot]) and SCID mice (Mus musculus Linnaeus). The prepatent period was 11 and 6 days post infection (DPI) for Cryptosporidium tortoise genotype I and C. ducismarci, respectively; the patent period was longer than 200 days for both cryptosporidia. Naturally or experimentally infected tortoises showed no clinical signs of disease. Our morphological, genetic, and biological data support the establishment of Cryptosporidium tortoise genotype I as a new species, Cryptosporidium testudinis sp. n., and confirm the validity of C. ducismarci as a separate species of the genus Cryptosporidium.

Molecular characterization of bovine Cryptosporidium using Cryptosporidium oocyst wall protein (COWP) gene

Cryptosporidium is a globally distributed protozoan parasite and one of the most common causes of infection and diarrhea in humans and cattle. The aim of the present study was to determine the species of Cryptosporidium among cattle with diarrhea by a nested PCR-RFLP technique at Cryptosporidium oocyst wall protein ( COWP ). Fecal samples from 158 calves aged 1-20 weeks were collected from 10 dairy farms in Qazvin province, Iran. Initial identification of Cryptosporidium was carried out by Zeihl-Neelsen acid-fast staining method of stool samples. DNA was extracted from 26 (16.45 %) positive microscopically samples and Cryptosporidium genotypes were determined. Cryptosporidium parvum were identified in 80.8% of the positive samples and, Cryptosporidium andersoni in 19.2%. In conclusion the use of COWP primers could be sensitive enough to conduct a routine detection study. The nested PCR method using the COWP gene sequence can be an alternative diagnostic method to identify infected with Cryptosporidium and its genetic diversity.

A new genotype of Cryptosporidium from giant panda (Ailuropoda melanoleuca) in China

Fifty-seven fecal samples were collected from giant pandas (Ailuropoda melanoleuca) in the China Conservation and Research Centre for the Giant Panda (CCRCGP) in Sichuan and examined for Cryptosporidium oocysts by Sheather's sugar flotation technique. An 18-year-old male giant panda was Cryptosporidium positive, with oocysts of an average size of 4.60×3.99μm (n=50). The isolate was genetically analyzed using the partial 18S rRNA, 70kDa heat shock protein (HSP70), Cryptosporidium oocyst wall protein (COWP) and actin genes. Multi-locus genetic characterization indicated that the present isolate was different from known Cryptosporidium species and genotypes. The closest relative was the Cryptosporidium bear genotype, with 11, 10, and 6 nucleotide differences in the 18S rRNA, HSP70, and actin genes, respectively. Significant differences were also observed in the COWP gene compared to Cryptosporidium mongoose genotype. The homology to the bear genotype at the 18S rRNA locus was 98.6%, which is comparable to that between Cryptosporidium parvum and Cryptosporidium hominis (99.2%), or between Cryptosporidium muris and Cryptosporidium andersoni (99.4%). Therefore, the Cryptosporidium in giant pandas in this study is considered as a new genotype: the Cryptosporidium giant panda genotype.

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 FindingsDiagnostic 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.

Immunomagnetic Separation Significantly Improves the Sensitivity of Polymerase Chain Reaction in Detecting Giavdia Duodenalis and Cryptosporidium spp. in Dairy Cattle

The effectiveness of molecular methods for the detection of species of Giardia and Cryptosporidium in fecal samples is often reduced by low or intermittent cyst and oocyst shedding, and/or the presence of polymerase chain reaction (PCR) inhibitors. The present study investigates the use of immunomagnetic separation (IMS) as an additional concentration step before PCR in the detection of these common protozoan parasites in dairy cattle. The IMS-PCR assays were optimized for amplifying fragments of the 16S ribosomal RNA (rRNA), β-giardin, and glutamate dehydrogenase (GDH) genes of Giardia duodenalis, as well as fragments of the 18S rRNA, heat shock protein (HSP)-70, and Cryptosporidium oocyst wall protein (COWP) genes of Cryptosporidium spp. In all cases, IMS-PCR was more sensitive than PCR alone. A significantly greater number of Giardia-positive samples were identified using IMS-PCR of the 16S rRNA gene (P < 0.01) and of the GDH gene (P < 0.01), as compared with PCR without any additional concentration step. In the case of Cryptosporidium, IMS-PCR of the COWP gene (P = 0.02) resulted in a significantly greater number of positives than did PCR without the IMS concentration step. The greatest number of positives, however, was obtained using IMS-PCR to amplify a portion of the 16S rRNA gene of Giardia and a portion of the HSP-70 gene of Cryptosporidium. A further comparison of the optimized IMS-PCR assays to immunofluorescence microscopy suggested that the IMS-PCR assays were considerably more sensitive than microscopy was in the detection of Giardia cysts and Cryptosporidium oocysts in fecal samples.

Detection and differentiation of Cryptosporidium spp. in human clinical samples by use of real-time PCR.

Real-time PCR has the potential to streamline detection and identification of Cryptosporidium spp. in human clinical samples. In the present article, we report the first such assay to allow not only detection and differentiation of the most common human pathogens, Cryptosporidium hominis and Cryptosporidium parvum, but also simultaneous amplification of a region of the small subunit (SSU) rRNA gene, permitting direct sequence analysis to identify any Cryptosporidium species. An internal control is incorporated to identify the presence of PCR inhibitors. Analytical sensitivity was determined to be as low as 200 oocysts per gram of feces processed, equivalent to 2 oocysts per PCR. The C. hominis and C. parvum PCRs specifically detected only species/genotypes in their respective target clades. Diagnostic sensitivity and specificity, evaluated against a widely used conventional nested SSU rRNA gene PCR as a nominated gold standard using a panel of 258 (151 positive and 107 negative) samples, were 100% and 99.1%, respectively. The assay agreed with PCR-restriction fragment length polymorphism analysis of the Cryptosporidium oocyst wall protein gene for 134 of 136 (98.5%) samples tested prospectively and typed two additional isolates. The real-time PCR assay was sensitive, specific, and reproducible and significantly improved laboratory work flow and turnaround times.

Re-description of Cryptosporidiumcuniculus (Apicomplexa: Cryptosporidiidae): Morphology, biology and phylogeny

To provide re-description of Cryptosporidium cuniculus Inman and Takeuchi, 1979 (synonymous with rabbit genotype), a species closely related to Cryptosporidium hominis, the morphology, natural and experimental host specificity, and genetic characterisation were investigated. The morphology and diagnostic characteristics are typical of other intestinal species of Cryptosporidium, albeit with slightly larger oocysts (5.55–6.40 × 5.02–5.92 μm; mean 5.98 × 5.38 μm; length:width = 1.1; n = 50). Natural hosts appear to be European rabbits ( Oryctolagus cuniculus) and humans ( Homo sapiens). Experimental infections have been established in weanling rabbits ( O. cuniculus), immunosuppressed Mongolian gerbils ( Meriones unguiculatus) and immunosuppressed adult Porton strain mice ( Mus musculus), but not in neonatal mice. Patterns of infection measured by oocyst shedding are significantly different compared with C. hominis, particularly in rabbits. Histological examination reveals endogenous stages in the brush border of the epithelium of the small intestinal villi, but clinical signs are absent. Inoculation of human HCT-8 cells results in discrete clusters of endogenous stages. A close relationship with C. hominis is inferred from molecular analyses at the ssrRNA, 70 kDa heat shock protein (HSP70), actin, Cryptosporidium oocyst wall protein (COWP), 60 kDa glycoprotein (GP60) genes and a region encoding a product of unknown function (LIB13). Sequences contained limited, consistent polymorphisms at the ssrRNA, HSP70 and actin genes, were identical at the COWP and LIB13 genes and demonstrated two unique families at the GP60 gene. Although genetically closely related, there are significant biological differences between C. cuniculus and C. hominis that support these protozoa being separate species. This is based on the current understanding of these organisms and relies on the assumption that mating between these species would not normally occur. If this is subsequently demonstrated their categorisation may need to be re-addressed.

Cryptosporidium genotypes and subtypes in dairy calves in Egypt

Neonatal calves are prone to Cryptosporidium infection resulting in economic loss as well as a significant source for zoonotic infection. This study was devoted to ascertain the prevalence and molecular analysis of Cryptosporidium in dairy calves at Kafr El Sheikh Province, Egypt. Twenty-nine out of 96 faecal specimens collected from calves, less than 6 weeks of ages, microscopically showed cryptosporidia oocysts (prevalence 30.2%). Among 29 positives, 26 samples were clearly sequenced for the SSU rDNA gene and the Cryptosporidium oocyst wall protein gene (COWP). Homology search revealed that 2 samples were C. andersoni and 24 isolates were C. parvum genotype II. By sequence analysis of the high polymorphic 60-kDa glycoprotein gene, 23 samples of C. parvum belonged to the allele IId (subtypes IIdA20G1), and one sample belonged to the allele IIa (subtype IIaA15G2R1). Prevailing of allele family IId is a unique observation, contrasting the conception that IIa has been the most prevailing allele in calves and cattle generally in other countries such as in Europe and the USA. To our knowledge, this is the first molecular report about genotyping and subtyping of Cryptosporidium in Egypt.

Comparative Sensitivity of PCR Primer Sets for Detection of Cryptosporidium parvum

Improved methods for detection of Cryptosporidium oocysts in environmental and clinical samples are urgently needed to improve detection of cryptosporidiosis. We compared the sensitivity of 7 PCR primer sets for detection of Cryptosporidium parvum. Each target gene was amplified by PCR or nested PCR with serially diluted DNA extracted from purified C. parvum oocysts. The target genes included Cryptosporidium oocyst wall protein (COWP), small subunit ribosomal RNA (SSU rRNA), and random amplified polymorphic DNA. The detection limit of the PCR method ranged from 10(3) to 10(4) oocysts, and the nested PCR method was able to detect 10(0) to 10(2) oocysts. A second-round amplification of target genes showed that the nested primer set specific for the COWP gene proved to be the most sensitive one compared to the other primer sets tested in this study and would therefore be useful for the detection of C. parvum.

An outbreak of cryptosporidiosis in a collection of Stone curlews (Burhinus oedicnemus) in Dubai

We describe an outbreak of cryptosporidiosis in Stone curlews kept in a mixed-species rearing unit in Dubai. Cryptosporidium was the predominant intestinal pathogen detected, although microbiological investigations revealed a concurrent Salmonella infantis infection in two of the 29 Stone curlew chicks that died. Nineteen of 29 birds had catarrhal enteritis associated with histopathological findings of numerous Cryptosporidium developmental stages at the mucosal surface. Catarrhal enteritis was present without associated Cryptosporidium oocysts in five cases. Histology of the intestine, faecal examination by direct microscopy and antigenic detection by immunochromatography revealed the presence of Cryptosporidium spp. associated with catarrhal enteritis in intestinal sections and faeces. Clinical and histopathological outcomes of infection were severe, including disruption of intestinal epithelial integrity, the presence of numerous endogenous Cryptosporidium stages in intestinal epithelia and the excretion of large numbers of sporulated oocysts. The application of polymerase chain reaction and restriction fragment length polymorphism techniques at two 18S rRNA and one Cryptosporidium oocyst wall protein gene locus confirmed the presence of Cryptosporidium parvum DNA in faecal samples.

Molecular characterization of a new genotype of Cryptosporidium from American minks ( Mustela vison) in China

A total of 469 fecal samples were collected from American minks ( Mustela vison) on a farm in Hebei Province in China and examined for Cryptosporidium by Sheather's sugar flotation technique and 8 Cryptosporidim isolates were obtained. The partial 18S rRNA, 70 kDa heat shock protein (HSP70), Cryptosporidium oocyst wall protein (COWP) and actin genes of six isolates were sequenced. Sequence data were analyzed together with known Cryptosporidium spp. and genotypes. Results of this multi-locus genetic characterization indicated that the six Cryptosporidium isolates in this study shared the same sequences of the genes studied and were different from known Cryptosporidium species and genotypes. The closest relative was Cryptosporidium ferret genotype with 7, 22, 2 and 2 nucleotide differences in the 18S rRNA, HSP70, COWP and actin genes, respectively. The homology to ferret genotype at the 18S rRNA locus was 99.1%, which is comparable to that between C. parvum and C. hominis (99.2%), or between C. muris and C. andersoni (99.4%). Therefore, the Cryptosporidium in minks in this study is considered a new genotype, the Cryptosporidium mink genotype.

Detection of a mixed infection of a novel Cryptosporidium andersoni and its subgenotype in Japanese cattle

Cryptosporidium oocysts were detected in the feces of cattle in Saga, Japan. Isolates were morphologically large. We attempted to identify the species or genotypes of the isolates by analyzing the partial sequences of the 18S rRNA and Cryptosporidium oocyst wall protein (COWP) genes, and measuring the infectivity in mice. The isolates showed 100% homology with Cryptosporidium andersoni in the COWP gene sequence and it could be transmitted to mice, but in the 18S rRNA gene, there was an additional signal in the ABI sequence chromatogram. To examine the additional signal, we analyzed both the 18S rRNA and the COWP gene sequences of a single oocyst passaged from mice using a modified multiplex PCR that was able to amplify both genes. As a result, it was revealed that two distinct genotypes (Types A and B) of a novel C. andersoni type existed in the 18S rRNA gene, whereas the COWP gene sequences of both oocysts were identical to C. andersoni. Although the sequence of the 18S rRNA gene of Type A was identical to that of C. andersoni, that of Type B had a thymine insertion and was not identical to any sequence registered with GenBank. Here we report that this is a new type of C. andersoni.

Multilocus genetic analysis of Cryptosporidium in naturally contaminated bivalve molluscs

To evaluate the application of discriminatory multilocus PCR procedures for the characterization of Cryptosporidium in samples of naturally contaminated bivalve molluscan shellfish. Nucleic acid was extracted from 22 shellfish previously identified as contaminated with Cryptosporidium spp. and subjected to PCR-based analysis for two independent fragments of the Cryptosporidium oocyst wall protein (COWP) gene, three microsatellite markers (ML 1, GP 15 and MS 5) and an extra-chromosomal small double-stranded RNA (dsRNA). Overall, at least one COWP gene fragment was amplified from all 22 samples, 21 amplified the dsRNA and 14 at least one of the three microsatellite loci. More than one dsRNA or microsatellite allele was detected in 50% of samples. The majority of samples were contaminated with Cryptosporidium parvum types circulating in both humans and livestock. A novel dsRNA element was identified in one sample, which did not amplify any of the three microsatellite loci investigated. Multilocus analysis of Cryptosporidium can be applied to DNA extracted from naturally contaminated shellfish. This multilocus genetic analysis highlights that filter feeder molluscs are a potential source of cryptosporidial oocysts, which may be infectious to humans.

An outbreak of disease associated with cryptosporidia on a red-legged partridge (Alectoris rufa) game farm

An outbreak of disease associated with cryptosporidia on a red-legged partridge (Alectoris rufa) game farm is described. Morbidity (diarrhoea and cough) was between 60% and 70% during the first weeks of life (4 to 25 days) and mortality was higher than 50%. The results of bacteriological and virological analyses were negative. Histological examination and antigenic diagnosis by enzyme-linked immunosorbent analysis revealed the presence of Cryptosporidium spp. in respiratory and intestinal tracts. The application of polymerase chain reaction–restriction fragment length polymorphism techniques and sequencing of a Cryptosporidium oocyst wall protein gene fragment confirmed the existence of Cryptosporidium meleagridis in faecal samples. The results obtained suggest that avian cryptosporidiosis should be included among respiratory and enteric diseases routinely tested for in farmed birds.

Evidence supporting zoonotic transmission of Cryptosporidium spp. in Wisconsin.

Cryptosporidium hominis and Cryptosporidium parvum are the primary species of Cryptosporidium that infect humans. C. hominis has an anthroponotic transmission cycle, while C. parvum is zoonotic, infecting cattle and other ruminants, in addition to humans. Most cryptosporidiosis outbreaks in the United States have been caused by C. hominis, and this species is often reported as the primary cause of cryptosporidiosis in this country. However, outbreaks account for only 10% of the overall cryptosporidiosis cases, and there are few data on the species that cause sporadic cases. The present study identified the species/genotypes and subgenotypes of Cryptosporidium in 49 cases of sporadic cryptosporidiosis in Wisconsin during the period from 2003 to 2005. The species/genotype of isolates was determined by PCR restriction fragment length polymorphism analysis of the 18S rRNA and Cryptosporidium oocyst wall protein genes. The C. parvum and C. hominis isolates were subgenotyped by sequence analysis of the GP60 gene. Forty-four of 49 isolates were identified as C. parvum, and 1 was identified as C. hominis. Of the remaining isolates, one was identified as being of the cervine genotype, one was identified as being a cervine genotype variant, and two were identified as being of a novel human genotype, previously reported as W17. Nine different subgenotypes were identified within the C. parvum species, and two of these were responsible for 60% of the cases. In this study we found that most sporadic cases of cryptosporidiosis in Wisconsin are caused by zoonotic Cryptosporidium species, indicating that zoonotic transmission could be more frequently associated with sporadic cases in the United States.