Use of VNTRS within coding sequences to genotype Toxoplasma gondii

MORETTA ROSALIA; RUYBAL PAULA; MARTIN VALENTINA

CABA

Otro; Reunion Conjunta de Sociedades de Biociencias; 2017

Toxoplasma gondii is an intracellular protozoan with a worldwide prevalence in human and animal populations. Infection occurs as a result of ingestion of resistant forms present in meat products and exposure to cat faeces. In immunocompetent individuals is generally asymptomatic. Severe disease may occur in immunocompromised subjects and in congenital toxoplasmosis, which is caused by transplacental acquisition of Toxoplasma gondii. Genetic diversity of T. gondii has been studied using a PCR-RFLP scheme based on nine molecular markers. These studies led to the description of a clonal population structure with three main lineages, designated as type I, II and III. The aim of this study was to develop molecular markers that allowed the discrimination of genetic variants within each clonal linage and therefore describe T. gondii population variability closer to strain level. We analyzed the genome of Toxoplasma gondii to identify genes containing variable number tandem repeats (VNTRs). The coding sequences of T. gondii ME49 genome (www.toxodb.org) were processed with Tandem Repeat Finder software. A panel of candidate markers was selected based on the following parameters: the repeat period (<9), the number of repeats copies (>20), the repeat module composition (to avoid single and dinucleotide runs) and the absence of introns within the repeat region. The selected panel of eight molecular markers was analyzed in PRU (type II) and RH (type I) strains. As a first step, the variability of the PCR product size allowed us to differentiate PRU from ME49 (both type II strains) and RH from GT1 (both type I strains). Additionally, amplification products from PRU and RH strains were sequenced to study intra-lineage variability. Polymorphic markers between type I and type II strains presented specific arranges of the VNTR pattern. Nonetheless, those markers that didn´t present size polymorphisms were also conserved at the sequence level.