The majority of known Toxoplasma gondii isolates from Europe and North America belong to three clonal lines that differ dramatically in their virulence, depending on the host. To identify the responsible genes, we mapped virulence in F1 progeny derived from crosses between type II and type III strains, which we introduced into mice. Five virulence (VIR) loci were thus identified, and for two of these, genetic complementation showed that a predicted protein kinase (ROP18 and ROP16, respectively) is the key molecule. Both are hypervariable rhoptry proteins that are secreted into the host cell upon invasion. These results suggest that secreted kinases unique to the Apicomplexa are crucial in the host-pathogen interaction.Toxoplasma gondii is an obligate intracellular parasite capable of infecting a wide variety of warm-blooded animals. Infections are widespread in humans and can lead to severe disease in utero or in individuals with a suppressed immune system. The majority of European and North American isolates belong to three distinct clonal lines, referred to as types I, II, and III (1,2). Types I and III appear to be the result of just one or two matings between an ancestral type II strain and, respectively, one or other of a pair of closely related strains that are distinct from type II (3-6). The three major Toxoplasma lines differ in a number of phenotypes (7), the best described of which is virulence in mice: type I strains are the most virulent with a lethal dose (LD 100 ) of one parasite (8,9), whereas types II and III have values for median lethal dose (LD 50 ) that range from 10 2 to 10 5 . There may also be differences in the virulence of the three strains in humans (10-12).Previously (3), we demonstrated that a cross between a type II and a type III strain produced F 1 progeny (S23 and CL11) that were more virulent (up to 3 logs) than 14 of their siblings (3). Because only two of the 16 progeny showed this difference, it was likely that multiple loci controlled virulence in these strains, and to identify these loci, we phenotyped 23 additional recombinant F 1 progeny from II × III crosses (13,14). Progeny with high virulence were identified by infecting mice with 100 tachyzoites; progeny with very low virulence were identified by infecting mice with 100,000 parasites. †To whom correspondence should be addressed.

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Marked phenotypic variation characterizes isolates of Toxoplasma gondii, a ubiquitous zoonotic parasite that serves as an important experimental model for studying apicomplexan parasites. Progress in identifying the heritable basis for clinically and epidemiologically significant differences requires a robust system for describing and interpreting evolutionary subdivisions in this prevalent pathogen. To develop such a system, we have examined more than 950 isolates collected from around the world and genotyped them using three independent sets of polymorphic DNA markers, sampling 30 loci distributed across all nuclear chromosomes as well as the plastid genome. Our studies reveal a biphasic pattern consisting of regions in the Northern Hemisphere where a few, highly clonal and abundant lineages predominate; elsewhere, and especially in portions of South America are characterized by a diverse assemblage of less common genotypes that show greater evidence of recombination. Clustering methods were used to organize the marked genetic diversity of 138 unique genotypes into 15 haplogroups that collectively define six major clades. Analysis of gene flow indicates that a small number of ancestral lineages gave rise to the existing diversity through a process of limited admixture. Identification of reference strains for these major groups should facilitate future studies on comparative genomics and identification of genes that control important biological phenotypes including pathogenesis and transmission.

The global predominance of three clonal Toxoplasma gondii lineages suggests that they are endowed with an exceptional trait responsible for their current parasitism of nearly all warm-blooded vertebrates. Genetic polymorphism analyses indicate that these clonal lineages emerged within the last 10,000 years after a single genetic cross. Comparison with ancient strains (approximately 1 million years) suggests that the success of the clonal lineages resulted from the concurrent acquisition of direct oral infectivity. This key adaptation circumvented sexual recombination, simultaneously promoting transmission through successive hosts, hence leading to clonal expansion. Thus, changes in complex life cycles can occur rapidly and can profoundly influence pathogenicity.

Toxoplasma gondii is among the most prevalent parasites worldwide, infecting many wild and domestic animals and causing zoonotic infections in humans. T. gondii differs substantially in its broad distribution from closely related parasites that typically have narrow, specialized host ranges. To elucidate the genetic basis for these differences, we compared the genomes of 62 globally distributed T. gondii isolates to several closely related coccidian parasites. Our findings reveal that tandem amplification and diversification of secretory pathogenesis determinants is the primary feature that distinguishes the closely related genomes of these biologically diverse parasites. We further show that the unusual population structure of T. gondii is characterized by clade-specific inheritance of large conserved haploblocks that are significantly enriched in tandemly clustered secretory pathogenesis determinants. The shared inheritance of these conserved haploblocks, which show a different ancestry than the genome as a whole, may thus influence transmission, host range and pathogenicity.

Toxoplasma gondii is a highly prevalent protozoan parasite that infects a wide range of animals and threatens human health by contaminating food and water. A markedly limited number of clonal parasite lineages have been recognized as predominating in North American and European populations, whereas strains from South America are comparatively diverse. Here, we show that strains from North America and Europe share distinct genetic polymorphisms that are mutually exclusive from polymorphisms in strains from the south. A striking exception to this geographic segregation is a monomorphic version of one chromosome (Chr1a) that characterizes virtually all northern and many southern isolates. Using a combination of molecular phylogenetic and phenotypic analyses, we conclude that northern and southern parasite populations diverged from a common ancestor in isolation over a period of Ϸ10 6 yr, and that the monomorphic Chr1a has swept each population within the past 10,000 years. Like its definitive feline hosts, T. gondii may have entered South America and diversified there after reestablishment of the Panamanian land bridge. Since then, recombination has been an infrequent but important force in generating new T. gondii genotypes. Genes unique to a monomorphic version of a single parasite chromosome may have facilitated a recent population sweep of a limited number of highly successful T. gondii lineages.biogeography ͉ evolution ͉ pathogen ͉ transmission ͉ virulence

Toxoplasma gondii is a widespread parasite of animals that causes zoonotic infections in humans. Previous studies have revealed a strongly clonal population structure in North America and Europe, while strains from South America are genetically separate and more diverse. However, the composition within North America has been questioned by recent descriptions of genetically more variable strains from this region. Here, we examined an expanded set of isolates using sequencedbased phylogenetic and population analyses to re-evaluate the population structure of T. gondii in North America. Our findings reveal that isolates previously defined by atypical restriction fragment length polymorphism patterns fall into two discrete groups. In one case, these new isolates represent variants of an existing lineage, from which they differ only by minor mutational drift. However, in the second case, it is evident that these isolates define a completely new lineage that is common in North America. Support for this new lineage was based on phylogeny, principle components analysis, STRUCTURE analyses, and statistical analysis of gene flow between groups. This new group, referred to as haplogroup 12, contains divergent genotypes previously referred to as A and X, isolated from sea otters. Consistent with this, group 12 was found primarily in wild animals, as well as occasionally in humans. This new lineage also has a highly clonal population structure. Analysis of the inheritance of multilocus genotypes revealed that different strains within group 12 are the products of a single recombination event between type 2 and a unique parental lineage. Collectively, the archetypal type 2 has been associated with clonal expansion of a small number of lineages in the North, as a consequence of separate but infrequent genetic crosses with several different parental lines.

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