Development of a Multilocus Sequence Tool for Typing Cryptosporidium muris and Cryptosporidium andersoni

State Key Laboratory of Bioreactor Engineering, School of Resource and Environmental Engineering, East China University of Science and Technology, Shanghai, China.
Journal of clinical microbiology (Impact Factor: 3.99). 10/2010; 49(1):34-41. DOI: 10.1128/JCM.01329-10
Source: PubMed


Although widely used for the characterization of the transmission of intestinal Cryptosporidium spp., genotyping tools are not available for C. muris and C. andersoni, two of the most common gastric Cryptosporidium spp. infecting mammals. In this study, we screened the C. muris whole-genome sequencing data for microsatellite and minisatellite sequences. Among the 13 potential loci (6 microsatellite
and 7 minisatellite loci) evaluated by PCR and DNA sequencing, 4 were eventually chosen. DNA sequence analyses of 27 C. muris and 17 C. andersoni DNA preparations showed the presence of 5 to 10 subtypes of C. muris and 1 to 4 subtypes of C. andersoni at each locus. Altogether, 11 C. muris and 7 C. andersoni multilocus sequence typing (MLST) subtypes were detected among the 16 C. muris and 12 C. andersoni specimens successfully sequenced at all four loci. In all analyses, the C. muris isolate (TS03) that originated from an East African mole rat differed significantly from other C. muris isolates, approaching the extent of genetic differences between C. muris and C. andersoni. Thus, an MLST technique was developed for the high-resolution typing of C. muris and C. andersoni. It should be useful for the characterization of the population genetics and transmission of gastric Cryptosporidium spp.

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    • "Oocysts of C. andersoni LI03 originally obtained from naturally infected cattle in South Bohemia, Czech Republic were used for this study. C. andersoni LI03 was previously characterized by Kváč et al. (2008) and Feng et al. (2011). Oocysts were purified from fecal samples using sucrose and cesium chloride gradient centrifugation (Arrowood and Sterling, 1987; Kilani and Sekla, 1987). "
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    ABSTRACT: We describe the course of infection of Cryptosporidium andersoni LI03, originally isolated from cattle, in outbred Gerbillus gerbillus (Lesser Egyptian Gerbil), Meriones unguiculatus (Mongolian gerbil), and Meriones tristrami (Tristram's jird). While both Meriones spp. partially cleared the infection and shed a low number of oocysts (less than 15,000 oocysts per gram (OPG)), chronic infection with a mean infection intensity reaching 200,000 OPG was observed in G. gerbillus. These data suggest that G. gerbillus can be used as a laboratory model for the long-term maintenance and study of C. andersoni without the need for host immunosuppression.
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    • "Cryptosporidium species and genotypes were determined by nested PCR amplification of the small subunit ribosomal RNA (SSU rRNA) gene [16] and by single PCR amplification of the Cryptosporidium oocyst wall protein (COWP) gene [30]. Subtyping was achieved by amplifying the minisatellite targets (four loci, namely CM-MS1 coding for hypothetical protein; CM-MS2 coding for 90 kDa heat shock protein; CM-MS3 coding for hypothetical protein; CM-MS16 coding for leucine rich repeat family protein) according to Feng et al [21] and Wang et al [22]. DNA of Cryptosporidium andersoni was used as the positive control for each target gene-based PCR analyses, and samples without DNA and host (cattle) DNA were included in each amplification run to exclude contamination. "
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    ABSTRACT: The present study examined the prevalence and genotypes of Cryptosporidium andersoni in cattle in Shaanxi province, China. A total of 2071 fecal samples (847 from Qinchuan cattle and 1224 from dairy cattle) were examined for the presence of Cryptosporidium oocysts, and 70 samples (3.4%) were C. andersoni-positive and those positive samples were identified by PCR amplification of the small subunit ribosomal RNA (SSU rRNA) and the Cryptosporidium oocyst wall protein (COWP) genes. C. andersoni was the only species found in the examined cattle in this province. Fifty-seven C. andersoni isolates were characterized into 5 MLST subtypes using multilocus sequence typing analysis, including a new subtype in the native beef breed Qinchuan cattle. All of these C. andersoni isolates presented a clonal genetic structure. These findings provide new insights into the genetic structure of C. andersoni isolates in Shaanxi province and basic data of Cryptosporidium prevalence status, which in turn have implications for controlling cryptosporidiosis in this province.
    Full-text · Article · Apr 2013 · PLoS ONE
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    • "In contrast, other eight MLST subtypes had 1–4 isolates (Table 1). Thus, a total of 14 C. muris and 17 C. andersoni MLST subtypes have been identified, including those reported previously [31] (Table S1). "
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    ABSTRACT: In this study, nine C. muris and 43 C. andersoni isolates from various animals in China were subtyped by a multilocus sequence typing (MLST) tool. DNA sequence analyses showed the presence of 1-2 subtypes of C. muris and 2-6 subtypes of C. andersoni at each of the four loci (MS1, MS2, MS3, and MS16), nine of which represented new subtypes. Altogether, two C. muris and 10 C. andersoni MLST subtypes were detected. Linkage disequilibrium analysis indicated although the overall population structure of the two parasites was clonal, the Chinese C. andersoni in cattle has an epidemic structure. Three and two clusters were produced in the C. muris and C. andersoni populations by Structure 2.3.3 analysis, with Chinese C. muris and C. andersoni substructures differing from other countries. Thus, this study suggested the prevalence of C. andersoni in China is not attributed to the introduction of dairy cattle. More studies involving more genetic loci and systematic sampling are needed to better elucidate the population genetic structure of C. muris and C. andersoni in the world and the genetic basis for the difference in host specificity among the two most common gastric parasites.
    Full-text · Article · Aug 2012 · PLoS ONE
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