Submitted to: International Journal for Parasitology
Publication Type: Peer reviewed journal
Publication Acceptance Date: 5/20/2005
Publication Date: 7/1/2005
Citation: Sreekumar, C., Hill, D.E., Miska, K.B., Vianna, M.B., Yan, L., Myers, R.L., Dubey, J.P. 2005. Genotyping and detection of multiple infections of Toxoplasma gondii using pyrosequencing. International Journal for Parasitology. 35:991-995. Interpretive Summary: Toxoplasma gondii is a single-celled parasite of all warm-blooded hosts worldwide. It causes mental retardation and loss of vision in children, and abortion in livestock. Cats are the main reservoir of T. gondii because they are the only hosts that can excrete the resistant stage (oocyst) of the parasite in the feces. Humans become infected by eating undercooked meat from infected animals and food and water contaminated with oocysts.Scientists at the Beltsville Agricultural Research Center describe a new method to distinguish isolates of T. gondii.The results will be of interest to biologists, parasitologists, and veterinarians.
Technical Abstract: A pyrosequencing assay, based on SAG2 gene polymorphisms, was designed for genotyping and detection of multiple infections of Toxoplasma gondii. The assay was tested on samples spiked with DNA from single and multiple genotypes of T. gondii and also on a DNA sample from the brain of a rat with multiple infections. To evaluate the comparative efficacy of the assay, identical samples were also analysed by PCR-restriction fragment length polymorphism (RFLP) and dideoxy sequencing. The pyrosequencing assay was found to be superior to the two conventional techniques. Genotyping and detection of multiple alleles were possible after a single PCR assay in duplex format, from both the spiked and direct samples. The simplex PCR assay enabled accurate quantification of the different alleles in the mix. In comparison, PCR-RFLP and dideoxy sequencing were neither able to unequivocally detect multiple genotype infections, nor quantify the relative concentrations of the alleles. We conclude that pyrosequencing offers a simple, rapid and efficient means for diagnosis and genotyping of T. gondii, as well as detection and quantification of multiple genotype infections of T. gondii.