Major Strain Haplotypes

Although Howe and Sibley (1995) suggested the presence of three main lineages, type I, II and III, of T. gondii, phylogenetic studies conducted at that time revealed that Toxoplasma has only two major clonal groups (Ajzenberg et al., 2002a; Lehmann et al., 2000). In fact, group 2 is genetically heterogeneous and type III is considered as a sub-group of group 2 (Ajzenberg et al., 2002a). The observation by Grigg et al. (2001a) that many loci of T. gondii

isolates display only 2 alleles (biallelic), suggested that the T. gondii population had only two different ancestries and that the three predominant lineages only resulted from successful recombination between them.

In newly-explored regions, analysis of the population structure may be difficult for various reasons. Firstly, it is predicted that there will be failure of markers to detect genetic diversity in a new area where those markers have been used to identify polymorphisms in existing populations. Secondly, the problematic requirement for intensive sampling strategies to detect allelic frequencies. Thirdly, the absence of exact definitions of the extent to which lineages should be divergent in order to be considered as unique. Although individual isolates within a clonal lineage reveal high levels of linkage disequilibrium and limited recombination, it is not suggested that they are identical (Tibayrenc et al., 1991; Tibayrenc et al., 1993). This is in agreement with minor differences that occur in common alleles due to occasional mutations. Therefore, considering new isolates as unique, solely because they differ in a certain trait of SNPs, is not sufficient to classify them according to genetic diversity. If all isolates genomes were sequenced, they would all be genetically unique. Thus, suitable criteria for classification of strains are required to permit meaningful comparison.

Methods which are used for allocating the genotypes of new isolates into suitable groups include STRUCTURE analysis (Falush et al., 2003), based on multilocus genotype data, other phylogenetic methods, and network analysis (Templeton et al., 1992). For such analysis, the choice of loci involves both regions that are selectively neutral, for common

ancestry estimation, and regions that are under strong selection (antigens) for estimation of diversity. These comparative analyses have the capability to differentiate between minor variants that result from mutation and to identify new lineages which are unique to new regions, or recombinant genotypes that developed from sexual reproduction. Analysis of these local variations in genetic population structure is highly related to phenotypes like pathogenesis and transmission.

Khan et al. (2007) analyzed T. gondii population structure based on phylogenetic reconstruction of 46 strains from Europe, North and South America based on 8 intron sequences of 5 unlinked loci and apicoplast genomes. He found 11 haplogroups representing the major lineages which reflected the geographical distribution of T. gondii. Most of the strains from North America and Europe were grouped in 1, 2 and 3 haplogroups, which corresponded to the main three clonal lineages, which were recognized in North America and Europe; however, South American strains were distributed between most of the other haplogroups, reflecting the high level of diversity of these strains.

It was suggested that entire of 11 haplogroups might be reconstructed from only four ancestral lineages through very few genetic crosses (Khan et al., 2007). This is similar to the previous prediction that the main three North American lineages arose from only a few genetic crosses (Boyle et al., 2006). Therefore, the population structure of T. gondii had been shaped through infrequent random recombination over a long period of time.

The population structure of T. gondii was re-evaluated through using sequence-based phylogenetic and population analysis (Khan et al., 2011). This study was based on 5 introns within 5 unlinked loci in addition to 3 antigen encoding genes (SAG1, GRA6 and 7) on expanded sets of isolates of 66 strains from Europe, North and South America. It revealed clustering of 12 haplogroups within T. gondii strains. Additionally, a fourth clonal lineage was reported in North America, referred to as haplogroup 12. This study confirmed the previous findings, showing a significant geographical separation between European and North American strains compared with the higher diversity South American strains.

A comprehensive study by Su et al. (2012) set out to genotype 138 isolates of T. gondii

collected from around the world by application of three different sets of polymorphic DNA markers, including RFLP, microsatellites, and sequencing of introns from housekeeping

genome. These markers included 11 RFLP genetic markers distributed across 8 of 14 chromosomes in addition to one marker for the apicoplast, 15 microsatellite markers allocated among 10 of 14 chromosomes and 4 introns of 3 different genes. The marked genetic diversity of 138 unique genotypes was arranged, by using clustering methods, into 15 different haplogroups that joined together to form six major clades (Figure 2.1). This study confirmed the predominance of clonal lineages in Northern Hemisphere regions, while in regions of South America it showed higher genetic diversity.

It is obvious that, based on the above studies, we have a good understanding of global diversity; however, it is still difficult to apply this understanding to a more local or regional context. There is evidence that isolates from one geographical area, which might be predicted to be very similar, actually have very different phenotype. In a study by Bontell et al. (2009), 8 T. gondii strains that had been collected from Ugandan chickens were genotyped as type II strains (TgCkUg1, 3, 5, 7, 8 and 9), type III (TgCkUg6) and recombinant strains (TgCkUg2) based on five PCR-RFLP markers (SAG1, SAG2, SAG3, GRA6 and BTUB). Additionally, in vitro and in vivo growth criteria were assessed at the time of isolation. These Ugandan strains were avirulent in mice but while none of the infected mice died or manifested disease symptoms from infection, the parasite burden in the brain, heart, lung and muscle of mice showed significant variations among isolates. The parasite density in the brain of each infected mouse was 15 times the density in quadriceps muscle, 100 times the quantity in heart muscle and 1000 times the amount in lung tissues. In addition, there was shown to be an association between the parasite genotype and tissue burden in mice, where the type III (TgCkUg6) strain produced greater parasite densities compared with the type II strains (TgCkUg1, 3, 5, 7, 8 and 9), while the recombinant strain (TgCkUg2) had an intermediate phenotype. However, this relationship between parasite genotype and densities in mice tissues did not correlate with the growth rate in tissue culture. In this assay, 3 of type II (TgCkUg1, 3 and 8) strains had higher growth rates compared with the type III (TgCkUg6) and recombinant (TgCkUg2) strain with intermediate rates, while the remaining type II strains (TgCkUg5, 7 and 9) had low growth rates.

Figure 2.1: Population genetic structure of Toxoplasma gondii (Su et al., 2012)