Seroprevalence of Toxoplasma gondii and direct genotyping using minisequencing in free-range pigs in Burkina Faso.

BACKGROUND: Swine are a major source of meat for humans. As such, they can play an important role in the epidemiology of human toxoplasmosis. Therefore, we performed an epidemiological study to determine the prevalence and genotypes of Toxoplasma gondii in Burkina Fasan swine. METHODS: The prevalence of T. gondii infection was evaluated in a 3-month prospective study at the slaughterhouse of Bobo-Dioulasso, Burkina Faso. Anti-Toxoplasma IgG titers were determined on meat juices from pig diaphragms using a commercially available ELISA assay. The DNA was extracted from 25mg of heart biopsies of seropositive animals (IgG ≥50% of the control) and the presence of T. gondii DNA was detected using a quantitative PCR assay. Genotyping was performed directly on DNA from PCR-positive biopsies using high-resolution melting and minisequencing analyses of the repeated B1 gene. RESULTS: The prevalence of carcasses positive for anti-Toxoplasma IgG was 29% (87/300) with no difference according to sex and age in contrast to the village of origin (p=0.018). Of the 87 seropositive animals, two were PCR positive (parasitic load at 64 and 128 parasites/mg of heart biopsy). Two new genotypes belonging to Type II and Type III and different from the genotypes previously described using minisequencing were identified. CONCLUSION: Our study provides the first T. gondii seroprevalence data in Burkina Fasan swine. In addition, this direct typing method suggests diversity of the T. gondii genotypes circulating in domestic animals in Burkina Faso. This needs to be confirmed on a wider sampling of subjects.

Direct genotyping of Toxoplasma gondii from amniotic fluids based on B1 gene polymorphism using minisequencing analysis.

BACKGROUND: Because some Toxoplasma gondii genotypes may be more virulent in pregnant women, discriminating between them appears valuable. Currently, the main genotyping method is based on single copy microsatellite markers, which limit direct genotyping from amniotic fluids (AFs) to samples with a high parasitic load. We investigated whether the multicopy gene B1 could type the parasite with a higher sensitivity. To estimate the amplifiable DNA present in AFs, we first compared three different PCR assays used for Toxoplasma infection diagnosis: the P30-PCR, targeting the single copy gene P30; the B1-PCR, targeting the repeated B1 gene; and RE-PCR, targeting the repeated element. RESULTS: Of the 1792 AFs analyzed between 2008 and 2011, 73 were RE-PCR positive. Of those, 49 (67.1%) were P30-PCR and B1-PCR positive, and 14 (19.2%) additional AFs were B1-PCR positive only.All 63 BI-positive AFs (France n = 49; overseas n = 14) could be genotyped based on an analysis of eight nucleotide polymorphisms (SNPs) located within the B1 gene. Following high-resolution melting (HRM) analysis, minisequencing was carried out for each of the eight SNPs. DNA from six reference strains was included in the study, and AFs were assigned to one of the three major lineages (Types I, II, and III). In total, 26 genotypes were observed, and the hierarchical clustering distinguished two clades in lineages II (IIa, n = 30 and IIb, n = 4) and III (IIIa n = 23 and IIIb n = 6). There was an overrepresentation of overseas isolates in Clade IIb (4/4, 100%) and Clade IIIa (8/22; 36.4%) (p <0.0001), whereas medical interruption and fetal death were overrepresented in Clade IIb (2/4, 50%) and Clade IIIa (4/23, 17.4%) (p = 0.049). CONCLUSIONS: Although the current genotyping system cannot pretend to replace multilocus typing, we clearly show that targeting the multicopy B1 gene yields a genotyping capacity of AFs around 20% better than when single copy targets are used. The present genotyping method also allows clear identification of genotypes of potential higher virulence.

Genotyping of the protozoan pathogen Toxoplasma gondii using high-resolution melting analysis of the repeated B1 gene.

Genetic studies of the protozoan parasite Toxoplasma gondii have identified three main distinct types according to virulence in some hosts. Several methods have been developed to differentiate genotypes currently dominated by microsatellite markers targeting single-copy loci. We analyzed the possibility of using the 35-fold repetitive B1 gene via high-resolution melting (HRM) curve analysis. Sequencing of the B1 gene of 14 reference strains (four Type I, six Type II, and four Type III strains) identified 18 single nucleotide polymorphisms (SNP). Primers were designed to amplify eight of them for HRM analysis and for relative quantification of each nucleotide variation using SNaPshot mini-sequencing. Genotyping with five microsatellite markers was performed for comparison. Two to four HRM profiles were obtained depending on the SNP tested. The differences observed relied on the different ratios of nucleotides at the SNP locus as evidenced via SNaPshot mini-sequencing. The three main lineages could be distinguished by using several HRM profiles. Some HRM profiles proved more informative than the analysis based on five microsatellite markers, showing additional differences in Type I and Type II strains. Using HRM analysis, we obtained at least an equally good discrimination of the main lineages than that based on five microsatellite markers.