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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:BackgroundThelaziosis is a neglected vector-borne disease caused by parasitic nematode worms of the genus Thelazia which affects various hosts. Limited attention has been given to ungulate-associated Thelazia species. Current diagnosis of thelaziosis and the identification/differentiation of species heavily relies on morphological features. Therefore, we conducted an epidemiological study in Romanian cattle, with the aim to obtain morphological and molecular data that can be used for species identification.MethodsThe eyes of 705 slaughtered cattle were sampled and subjected to morphological identification, morphometric analysis, and molecular characterization. PCR amplification and sequence analysis were performed based on the cytochromec oxidase subunit 1 (COI) gene. Statistical tests assessed the correlations between infection parameters and ecological or biogeographical factors. A novel PCR method was developed based on the consensus sequence from each species. Specific forward primers were designed for each of the three species, and a reverse primer (COIintR) was used for all reactions. A consensus thermal profile was established by gradient PCR amplification of each species separately.ResultsOf the sampled cattle, 19.3% were infected with Thelazia spp. Prevalence varied significantly with ecogeographical factors. A total of 585 Thelazia nematodes were recovered, with T. rhodesi being the most abundant, followed by T. skrjabini and T. gulosa. Morphometric and molecular analyses supported the morphological identification, yielding unique sequences for each species. From the 59 T. rhodesi specimens sequenced, 29 unique sequences were obtained, with a 99.1-99.85% nucleotide identity to the only other COI sequence present in GenBank®. All nine T. gulosa isolates were unique (99.37-100% nucleotide identity to other sequences), while T. skrjabini specimens displayed 98.47-100% nucleotide identity to the sole available sequence.ConclusionsBovine thelaziosis is prevalent in Romania, raising concerns for animal welfare and potential economic impacts. Infected cattle grazing alongside vulnerable wild ruminants, such as the European bison, may affect conservation efforts. Our newly developed multiplex PCR shows promise as a valuable surveillance tool, enabling the detection of occult infections in apparently healthy animals through lachrymal secretion testing.

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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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