Additional haplogroups of Toxoplasma gondii out of Africa: population structure and mouse-virulence of strains from Gabon

Abstract

Background: Toxoplasma gondii is found worldwide, but distribution of its genotypes as well as clinical expression of human toxoplasmosis varies across the continents. Several studies in Europe, North America and South America argued for a role of genotypes in the clinical expression of human toxoplasmosis. Genetic data concerning T. gondii isolates from Africa are scarce and not sufficient to investigate the population structure, a fundamental analysis for a better understanding of distribution, circulation, and transmission.

Methodology/principal findings: Seropositive animals originating from urban and rural areas in Gabon were analyzed for T. gondii isolation and genotyping. Sixty-eight isolates, including one mixed infection (69 strains), were obtained by bioassay in mice. Genotyping was performed using length polymorphism of 13 microsatellite markers located on 10 different chromosomes. Results were analyzed in terms of population structure by Bayesian statistical modeling, Neighbor-joining trees reconstruction based on genetic distances, F(ST) and linkage disequilibrium. A moderate genetic diversity was detected. Three haplogroups and one single genotype clustered 27 genotypes. The majority of strains belonged to one haplogroup corresponding to the worldwide Type III. The remaining strains were distributed into two haplogroups (Africa 1 and 3) and one single genotype. Mouse virulence at isolation was significantly different between haplogroups. Africa 1 haplogroup was the most virulent.

Conclusion: Africa 1 and 3 haplogroups were proposed as being new major haplogroups of T. gondii circulating in Africa. A possible link with strains circulating in South and Central America is discussed. Analysis of population structure demonstrated a local spread within a rural area and strain circulation between the main cities of the country. This circulation, favored by human activity could lead to genetic exchanges. For the first time, key epidemiological questions were addressed for the West African T. gondii population, using the high discriminatory power of microsatellite markers, thus creating a basis for further epidemiological and clinical investigations.

Figure 1. Determination of the optimal value of K (i.e. the optimal number of clusters in the dataset).

A. Mean (± SD) of Ln P(D) over 10 STRUCTURE runs for successive K values ranging from 1 to 10, B. Variations of ΔK as calculated by Evanno and colleagues and C. Variations of L(K+1)-L(K) as calculated by Garnier and colleagues for successive K values.

Figure 2. Neighbor-joining tree and clusters as inferred from Cavalli-Sforza distances and by STRUCTURE calculated for 13 microsatellite markers and on all 69 Gabonese T. gondii strains.

A: Neighbor-joining tree as inferred from Cavalli-Sforza distances on all 69 Gabonese T. gondii strains and seven Type reference strains. Midpoint rooting was applied for the Neighbor-joining tree; no outgroup was used. Geographical origin and genotype membership (# genotype number) for each strain are reported. Colors correspond to colors of populations found with STRUCTURE for K = 3 (blue, green and red for respectively cluster C3.1, C3.2 and C3.3 and black for the single isolate strain: GAB4-2007-GAL-DOM1 and for Type reference strains), except for Type III-like isolates which are in purple. B: Clusters as inferred by STRUCTURE on all 69 Gabonese T. gondii strains. K = 2, K = 3 and K = 5 are indicated by bars in different colors next to the tree. The partition (i.e. relative q_ik) of each of the 69 T. gondii strain genotypes within each cluster for the most likely number of inferred clusters K = 2, 3 and 5 is presented.

Figure 3. Neighbor-joining tree of genotypes inferred from Cavalli-Sforza distances calculated for the data of 13 microsatellite markers and all 69 Gabonese isolates, including also all the reference strains.

[Seven Type reference strains: Type I (GT1, ENT, and B1), Type II (Me49 and PRU), Type III (CTG, VEG and NED), Africa 2 (CCH002-2004-NIA) (Ajzenberg et al. 2009), and Africa 1 (DPHT) (Ajzenberg et al., 2009), and seven reference isolates originating from different countries: France (GPHT), Brazil (TgCkBr93, TgCkBr59, TgCkBr40), and Caribbean islands (ENVL-2002-MAC)]. Note: Midpoint rooting was applied for the Neighbor-joining tree; no outgroup was used. Colors correspond to colors of populations found with STRUCTURE for K = 3 (blue, green and red for respectively cluster C3.1, C3.2 and C3.3 and black for the single isolate strain: GAB4-2007-GAL-DOM1 and for Type reference strains), except for Type III-like isolates which are in purple.

Figure 4. Proportion of surviving mice infected with T. gondii haplogroups.

Figure 5. Proportion of membership of each geographic population in each of the 5 clusters (STRUCTURE, K = 5).

Note: Adapted from the Central Intelligence Agency Web site . Colors correspond to the populations in Figure 2.