PCR-RFLP genotyping of Toxoplasma gondii from chickens from Espírito Santo state, Southeast region, Brazil: New genotypes and a new SAG3 marker allele

Brazil is one of the regions with the highest prevalences of Toxoplasma gondii in humans and animals. Because free-range chickens become infected by feeding from ground contaminated with oocysts, the prevalence of T. gondii in this host has been widely used as an indicator of the strains prevalent in the environment. The genetic variability among T. gondii isolates from different healthy and sick hosts all over the world has been recently studied. Three clonal genetic lineages (Types I, II and III) were initially recognised as predominant in Western Europe and the United States. T. gondii strains are genetically diverse in South America. In Brazil, recombination plays an important role in strain diversification. The objective of this study was to genetically characterise T. gondii isolates from free-range chickens from Espírito Santo state, Southeast region, Brazil, using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). A total of 44 isolates among 47 previously described isolates (TgCkBr234-281) from free-range chickens were included in this study. Strain typing was performed using 12 PCR-RFLP markers: SAG1, SAG2, alt. SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, Apico and CS3. Eleven genotypes were identified. Ten isolates (23%) were grouped into four novel genotypes. Four isolates, distributed in four counties, corresponded to the Type BrI lineage, the genotype found most frequently in Brazil. No clonal Types I, II or III lineages were found. Two novel genotypes were represented by single isolates. Unique alleles were identified for the markers SAG1, c22-8 and CS3, and for the first time, a unique allele was found for the marker SAG3. Although a large number of T. gondii genotypes have already been identified from a variety of animal hosts in Brazil, new genotypes are continuously identified from different animal species. This study confirmed the diversity of T. gondii in Brazil and demonstrates clonal Type I, II and III lineages are rare in this country.