Molecular Identification of Toxoplasma gondii and Other Coccidia in Fecal Samples from Shelter Cats

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Abstract
The objective was to detect T. gondii and various protozoan oocysts in the feces of sheltered cats in Thailand using two molecular approaches, Sanger sequencing and un-targeted Next-generation sequencing (NGS). A total of 166 fecal samples from shelter cats, in 26 samples, Toxoplasma gondii oocyst–like structures were detected. The harvested oocysts were grouped into nine pooled samples. DNA was extracted from all pooled samples and tested using quantitative PCR (qPCR), employing coc1 and coc2 primers, which are commonly used to amplify Apicomplexan DNA. The sequenced of the qPCR products were analyzed by Sanger sequencing. Sequence from all pooled samples had similarity to Cystoisospora spp. To further characterize the oocyst species, NGS was performed. Bioinformatic analysis was conducted using a de novo assembler to generate scaffolds, which were then aligned against a custom database of coccidian whole-genome references. This analysis revealed the presence of T. gondii DNA in three pooled samples. In addition, DNA from other protozoan parasites—including Eimeria spp., Cystoisospora spp., Besnoitia besnoiti, Hammondia hammondi, and Cryptosporidium parvum—was also detected. These findings indicate that T. gondii is circulating among shelter cats, many of which were formerly stray. Moreover, NGS sequencing provided more comprehensive information on the diversity of coccidian species in cat feces compared with Sanger sequencing of PCR-amplified targets.

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