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Molecular differentiation of cattle Sarcocystis spp. by multiplex PCR targeting 18S and COI genes following identification of Sarcocystis hominis in human stool samples.

ABSTRACT: Sarcocystis spp. are protozoan parasites which can infect a wide range of vertebrates, including humans; the latter can act as definitive hosts for two cattle Sarcocystis spp.: Sarcocystis hominis and Sarcocystis heydorni. Reports of intestinal sarcocystosis are well documented in the literature, but PCR-based methods have been scarcely used to identify Sarcocystis species in human stools, and have been limited to the molecular analysis of 18S ribosomal RNA (18S rRNA) gene sequences. Since the mitochondrial cytochrome c oxidase subunit I (COI) gene is one of the most promising tools for distinguishing between closely related Sarcocystis spp., and taking into account the lack of publicly available S. hominis COI sequences, in the present study we obtained the first partial COI sequence of S. hominis from human stool samples of patient with gastrointestinal symptoms. We designed specific COI primers to develop a multiplex PCR method for the identification of Sarcocystis spp. in cattle. The submission of the COI sequence described herein and the unambiguous identification of S. hominis through the application of the new multiplex PCR is important for determining the prevalence of this zoonotic Sarcocystis spp. in meat and the risk for consumers.

SUBMITTER: Rubiola S

PROVIDER: S-EPMC7058708 | biostudies-literature |

REPOSITORIES: biostudies-literature

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

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Molecular differentiation of cattle Sarcocystis spp. by multiplex PCR targeting 18S and COI genes following identification of Sarcocystis hominis in human stool samples.

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