Isolation and Genotyping of Toxoplasma gondii in Brazilian Dogs

Abstract

Strains of Toxoplasma gondii in Brazil are highly genetically diverse compared to strains from North America and Europe. Dogs are epidemiologically important because they act as sentinels for T. gondii infections in humans and are good indicators of environmental contamination. The aim of this study was to isolate and genetically characterize T. gondii strains from tissues of naturally infected Brazilian dogs. For this study, 21 blood samples were collected from dogs at the Zoonosis Control Centers of Ilhéus and Itabuna cities, Bahia, Brazil. The sera were examined for T. gondii antibodies using the indirect hemagglutination test. Brains and hearts of seropositive dogs were bioassayed in mice to isolate and characterize T. gondii parasites by PCR-RFLP using 10 genetic markers (SAG1, newSAG2, SAG3, BTUB, c22-8, c29-2, GRA6, PK1, APICO, and L358). However, T. gondii was isolated from only 4 (57.1%) dogs, designated TgDgBr6, 13, 17, and 21. All strains were virulent, causing clinical changes (rough hair coat, lethargy, and abdominal distention) and the death of all mice within 8-20 days after inoculation. Genetic analysis of these 4 T. gondii isolates revealed 4 distinct genotypes with different clonal lineage combinations (types I, II, and III) and 2 atypical alleles. Using PCR-RFLP with several markers, this study contributes to evaluations of the genetic diversity of strains circulating in Brazil.

Keywords: Brazil; Toxoplasma gondii; dog; genotype; toxoplasmosis.

Fig. 1

Resolution on agarose gel of the products from PCR with specific primers for Toxoplasma gondii. Lane 1, molecular weight, 100-bp DNA ladder; lane 2, positive control; lanes 3, negative control; lane 4–7, positive animals.

Fig. 2

Resolution on agarose gel of the products from nested-PCR with genomic genetic markers SAG1 (A), SAG3 (B), and L358 (C) for Toxoplasma gondii. PM, molecular weight, 100-bp DNA ladder; 1–4, positive samples; CN, negative control; I, positive control type I (RH strain); II, positive control type II (PTG strain); III, positive control type III (CTG strain).

Fig. 3

Phylogram of Toxoplasma gondii strains as determined by multilocus sequence analysis using 10 genomic genetic markers (SAG1, SAG2 novo, SAG3, BTUB, c22-8, c29-2, GRA6, L358, PK1, and APICO). The tree was constructed using the neighbor-joining method after bootstrapping with 1,000 repetitions. The distances were computed using the Tajima-Nei method. Strains from dogs (of this study), sheep and pigs from Bahia State of Brazil, and the Brazilian reference strains were highlighted. RH strain was used as positive control (PC) of the sequencing reaction.