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.

Seroprevalence and isolation of Toxoplasma gondii from free-range chickens from Espírito Santo state, southeastern Brazil.

Prevalence of Toxoplasma gondii infection in 510 free-range (FR) chickens (380 from 33 small farms, and 130 from a slaughter house for FR chickens) from Espírito Santo state, southeastern Brazil, was investigated. Antibodies to T. gondii were sought using commercial indirect haemagglutination (IHAT, Imuno-HAI Toxo(®), Wama Diagnóstica, São Paulo, Brazil, cut-off 1:16) and the modified agglutination test (MAT, cut-off 1:25) tests. Attempts were made to isolate viable T. gondii from seropositive chickens by bioassay in mice. Pooled samples of brain, heart and quadriceps muscle of one thigh (total 40 g) from 64 chickens with IHAT titers of ≥ 1:16 were minced, digested in pepsin and bioassayed in mice. Antibodies to T. gondii were found in 40.4% (206/510) FR chickens by IHAT (titer ≥ 1:16) and 38.8% (198/510) by MAT (titer ≥ 1:25); concordance between IHAT and MAT was 81.6% (kappa index=0.614). Viable T. gondii was isolated (designated TgCkBr234-281) from 48 of 64 (75%) seropositive (IHAT titers ≥ 1:32) FR chickens. Most isolates of T. gondii were virulent for mice; 100% of mice inoculated with 44 of 48 isolates died of toxoplasmosis within 30 days post inoculation (p.i). An epidemiological investigation revealed that people living in rural areas have little knowledge about the parasite and about the risk of acquiring it from raw meat. Results indicated that the locally available IHAT was useful for screening of chicken sera for T. gondii antibodies.