Molecular identification of Cryptosporidium species in Canadian post-weaned calves and adult dairy cattle.

Cryptosporidium is a zoonotic protozoan parasite that is distributed globally and impacts both human and animal health. There are over 40 species of Cryptosporidium described to date, of which four (C. parvum, C. bovis, C. ryanae and C. andersoni) are routinely reported in cattle. The goal of this study was to identify the Cryptosporidium species infecting dairy cattle from across Canada using cow fecal samples and post-weaned calf rectal swabs obtained through the Canadian National Dairy Study. A total of 353 cattle samples (117 pooled rectal fecal swabs from post-weaned calves and 236 cow fecal samples) from 175 herds across the 10 Canadian provinces were analysed by targeting Cryptosporidium's small subunit ribosomal RNA (SSU rRNA or 18S) gene. Herd prevalence of Cryptosporidium was 27.4% nationally, ranging from 0% in Saskatchewan (SK) to 62% in Prince Edward Island (PE). The national prevalence of Cryptosporidium cattle infections was 15.4% in pooled rectal fecal swab samples from post-weaned calves and 16.1% in adult cows. Sanger sequence analysis of the SSU rRNA gene target revealed that C. bovis, C. andersoni and C. ryanae occurred in both adults and post-weaned calves, with C. bovis as the predominant species detected in pooled fecal swab samples of post-weaned calves (9/18, 50%) and C. andersoni as the predominant species in cows (25/38, 66%). Cryptosporidium parvum was not observed in any of the pooled rectal swab samples from post-weaned calves but was observed in one mixed infection of C. bovis/C. parvum in an adult cow. The fifth species identified in this study was C. muris and was present in two adult cows. Low concentrations of oocyst equivalents, as measured by quantitative real-time PCR (qPCR) of the SSU rRNA gene copy number, were observed in a subset of cattle samples. Cryptosporidium andersoni concentrations varied from province to province, with the widest range and highest counts in cows from PE. In conclusion, oocysts from Cryptosporidium species observed in this study are shed into the environment, contributing to the environmental load. However, the Cryptosporidium species in the post-weaned calves and cows found in this study pose a lower risk to the dairy cattle than if they were infected with C. parvum. Similarly, these Cryptosporidium species pose only a small risk to public health as the three species are infrequently reported in humans globally and have not been reported in Canadians to date.

Draft Hybrid Genome Assembly of a Canadian Cyclospora cayetanensis Isolate.

The apicomplexan parasite Cyclospora cayetanensis causes foodborne gastrointestinal disease in humans. Here, we report the first hybrid assembly for C. cayetanensis, which uses both Illumina MiSeq and Oxford Nanopore Technologies MinION platforms to generate genomic sequence data. The final genome assembly consists of 44,586,677 bases represented in 313 contigs.

Genotyping Canadian Cyclospora cayetanensis Isolates to Supplement Cyclosporiasis Outbreak Investigations.

Cyclospora cayetanensis is an emerging foodborne parasite that causes cyclosporiasis, an enteric disease of humans. Domestically acquired outbreaks have been reported in Canada every spring or summer since 2013. To date, investigations into the potential sources of infection have relied solely on epidemiological data. To supplement the epidemiological data with genetic information, we genotyped 169 Canadian cyclosporiasis cases from stool specimens collected from 2010 to 2021 using an existing eight-marker targeted amplicon deep (TADS) scheme specific to C. cayetanensis as previously described by the US Centers for Disease Control and Prevention (CDC). This is the first study to genotype Canadian Cyclospora cayetanensis isolates, and it focuses on evaluating the genotyping performance and genetic clustering. Genotyping information was successfully collected with at least part of one of the markers in the TADS assay for 97.9% of specimens, and 81.1% of cyclosporiasis cases met the minimum requirements to genetically cluster into 20 groups. The performance of the scheme suggests that examining cyclosporiasis cases genetically will be a valuable tool for supplementing epidemiological outbreak investigations and to minimize further infections. Further research is required to expand the number of discriminatory markers to improve genetic clustering.

CryptoGenotyper: A new bioinformatics tool for rapid Cryptosporidium identification.

Cryptosporidium is a protozoan parasite that is transmitted to both humans and animals through zoonotic or anthroponotic means. When a host is infected with this parasite, it causes a gastrointestinal disease known as cryptosporidiosis. To understand the transmission dynamics of Cryptosporidium, the small subunit (SSU or 18S) rRNA and gp60 genes are commonly studied through PCR analysis and conventional Sanger sequencing. However, analyzing sequence chromatograms manually is both time consuming and prone to human error, especially in the presence of poorly resolved, heterozygous peaks and the absence of a validated database. For this study, we developed a Cryptosporidium genotyping tool, called CryptoGenotyper, which has the capability to read raw Sanger sequencing data for the two common Cryptosporidium gene targets (SSU rRNA and gp60) and classify the sequence data into standard nomenclature. The CryptoGenotyper has the capacity to perform quality control and properly classify sequences using a high quality, manually curated reference database, saving users' time and removing bias during data analysis. The incorporated heterozygous base calling algorithms for the SSU rRNA gene target resolves double peaks, therefore recovering data previously classified as inconclusive. The CryptoGenotyper successfully genotyped 99.3% (428/431) and 95.1% (154/162) of SSU rRNA chromatograms containing single and mixed sequences, respectively, and correctly subtyped 95.6% (947/991) of gp60 chromatograms without manual intervention. This new, user-friendly tool can provide both fast and reproducible analyses of Sanger sequencing data for the two most common Cryptosporidium gene targets.

Molecular characterization of Cryptosporidium isolates from humans in Ontario, Canada.

BACKGROUND: Cryptosporidiosis is a gastrointestinal disease with global distribution. It has been a reportable disease in Canada since 2000; however, routine molecular surveillance is not conducted. Therefore, sources of contamination are unknown. The aim of this project was to identify species and subtypes of Cryptosporidium in clinical cases from Ontario, the largest province in Canada, representing one third of the Canadian population, in order to understand transmission patterns. METHODS: A total of 169 frozen, banked, unpreserved stool specimens that were microscopy positive for Cryptosporidium over the period 2008-2017 were characterized using molecular tools. A subset of the 169 specimens were replicate samples from individual cases. DNA was extracted directly from the stool and nested PCR followed by Sanger sequencing was conducted targeting the small subunit ribosomal RNA (SSU) and glycoprotein 60 (gp60) genes. RESULTS: Molecular typing data and limited demographic data were obtained for 129 cases of cryptosporidiosis. Of these cases, 91 (70.5 %) were due to Cryptosporidium parvum and 24 (18.6%) were due to Cryptosporidium hominis. Mixed infections of C. parvum and C. hominis occurred in four (3.1%) cases. Five other species observed were Cryptosporidium ubiquitum (n = 5), Cryptosporidium felis (n = 2), Cryptosporidium meleagridis (n = 1), Cryptosporidium cuniculus (n = 1) and Cryptosporidium muris (n = 1). Subtyping the gp60 gene revealed 5 allelic families and 17 subtypes of C. hominis and 3 allelic families and 17 subtypes of C. parvum. The most frequent subtype of C. hominis was IbA10G2 (22.3%) and of C. parvum was IIaA15G2R1 (62.4%). CONCLUSIONS: The majority of isolates in this study were C. parvum, supporting the notion that zoonotic transmission is the main route of cryptosporidiosis transmission in Ontario. Nonetheless, the observation of C. hominis in about a quarter of cases suggests that anthroponotic transmission is also an important contributor to cryptosporidiosis pathogenesis in Ontario.