We have compiled some recently reported data to help illustrate factors that likely will need to be accounted for when addressing this complex but important question. First, it is evident that genotype prevalence can vary both between host species and over geographic distributions. It has been noted previously that parasite genotype distributions differ between humans and domestic animals (Howe and Sibley, 1995), which is an important factor in determining which host species should be used to represent the domestic cycle. Additional evidence for this emerges when we re-analyze recently reported RFLP data for 182 isolates from pigs in the US (Velmurugan et al., 2009) using the SplitsTree4 software as described above. Comparing the network generated for the pig isolates (Figure 1D) to that for the data reported in the Howe and Sibley 1995 study (Figure 1B), in which the domestic cycle was largely represented by human isolates, reveals some important differences. “Type II’ and ‘Type III’ are also dominant clones in the pig population, but, in contrast to the human population, ‘Type I’ strains are absent, confirming what was reported originally (Howe and Sibley, 1995). It is also notable that other clones absent from the human sample set, including RFLP genotypes 3, 4 and 8, are quite prevalent in these pigs, but this difference may be attributable to the higher resolution typing applied to the pig sample set. Overall, the distribution of dominant clones in the pig data is more similar to that in the wildlife sample set (Figure 1C), but differences exist here as well, including the greater number of unique genotypes and the increased prevalence of ‘Type X’ strains (largely due to sea otter samples) in the wildlife data.

Geographic effects can also play a role in shaping our perception of which parasite genotypes are associated with which host species. This is certainly the case on a global level (e.g. North vs. South America), but has been noted on more local levels as well. For example, in Gabon, Mercier et al. (2010) provided evidence for distinct T. gondii populations between chickens acquired in different towns, which ranged from 105 to 510 kilometers distant. As mentioned above, Sundar et al. (2008) also found distinct strain compositions between the sea otters of Monterey Bay and Morro Bay, California, which are separated by approximately 200 kilometers. Similarly, when we re-examined the pig isolate data reported by Velmurugan et al. (2009) with pig genotypes classified by state of origin, different proportions of genotypes were found in different geographic sub-groupings (Figure 3).

In contrast to these data, however, Lehmann et al. (2004) found no difference measured by the F_st statistic between chicken isolates from two locations over 600 kilometers apart in Brazil, perhaps indicating more expansive transmission in this region. Brazil is also one of the few locations where recent sampling efforts have identified a significant number of wildlife and domestic animal isolates with a comparable typing scheme in a localized area. In the state of Sao Paulo, a standardized RFLP typing scheme has been applied to isolates from 36 capybaras (Yai et al., 2009), 19 dogs (Dubey et al., 2007a), 44 cats (Pena et al., 2008), 16 sheep and 10 goats (Ragozo et al., 2010). Using the capybara samples as representative of wildlife, we compared the proportions of shared genotypes between wildlife and domestic carnivores (cats and dogs) (Figure 4A) and between wildlife and domestic herbivores (sheep and goats) (Figure 4B). In both cases there were several genotypes that were unique at the host species level, but comparing capybaras to domestic carnivores revealed that the genotypes for 22 out of the 33 capybara samples (mixed infections excluded) were also found in the domestic animal samples in contrast to only five isolates sharing genotypes with domestic herbivore samples (Figure 4A and B). Though it is unclear whether these results could be attributed to geographic differences within the state of Sao Paulo, they do give weight to the notion that defining the T. gondii population circulating in domestic animals could be highly dependent upon which host species are chosen to represent the domestic cycle.

It will also be important to gather empirical and experimental evidence for the inferences drawn from these new genetic studies of T. gondii. This is especially true for the debates over the extent to which clonal expansion and sexual recombination occur in nature and over the mechanistic basis for how certain clones come to dominate in the population structure. As discussed above, there are many potential caveats in extrapolating from a clonal sample set to conclude that a population is clonal in nature (Feil and Spratt, 2001; Maynard Smith et al., 1993). Likewise, the extent of recombination can be over-estimated depending upon the sampling techniques used (Awadalla, 2003; Prugnolle and De Meeus, 2010). Given the vast and widespread population of definitive felid hosts (approx. 90 million in the US alone (Dabritz and Conrad, 2010)), it is very likely that a frequent and productive sexual cycle exists for this parasite, despite a predominance of certain clones. Support for or against this possibility could be gained by attempting to determine the extent of co-infections in future sampling efforts, especially those which represent prey species of felids, because a simultaneous infection of two or more T. gondii strains is necessary for a productive cross to occur. In this regard, it is worth noting that the number of studies that have reported at least some mixed strain infections in a wide variety of hosts, including cats, has increased greatly since more sensitive genotyping techniques have been applied (Al-Kappany et al., 2010; Aspinall et al., 2003; Boughattas et al., 2010; Dubey et al., 2007b; Dubey et al., 2009b; Dubey et al., 2006a; Dubey et al., 2006b; Dubey et al., 2006c; Dubey et al., 2005; Elbez-Rubinstein et al., 2009; Lindstrom et al., 2008; Mercier et al., 2010; Parameswaran et al., 2010; Ragozo et al., 2010; Sundar et al., 2008; Yai et al., 2009). This is in spite of the fact that the majority of studies still obtain isolates through bioassay, a technique that has been shown to preferentially isolate certain strains over others (Lindstrom et al., 2008), and only test tissues from a single organ, even though evidence exists that different strains localize to different organs (Dubey, 1997; Saeij et al., 2005). Techniques now exist that provide the sensitivity needed to allow extensive parasite genotyping directly from multiple host tissues to adequately address this question (Opsteegh et al., 2010).

Another possibility would be to directly test oocyst samples for multiple genotypes using these more sensitive typing techniques. It would also be of great benefit to experimentally verify whether a mixed strain infection in a single intermediate host is necessary for outcrossing in the felid host, or if consumption of multiple, singly-infected prey over the course of the one to three week patent period of oocyst shedding would suffice. For studies of T. gondii infections in wildlife, it is also highly relevant to determine sources of infection and examine whether differences exist between domestic and wild felid species in their fecundity and ability to promote sexual outcrossing and/or self-mating for T. gondii. Clearly there is still much information to be gathered that could shape our perception of the likelihood and frequency of productive sexual crosses in nature.

Regardless of the ambiguities surrounding the extent of sexual recombination in the T. gondii population, it is undeniable that certain clones have expanded and persist in both domestic and wildlife populations over time. How and why this has occurred has been the focus of several studies over the past decade due to concerns that the mechanistic basis of clonal expansion may represent a threat for the emergence of virulent genotypes. Of the many mechanisms that exist in the T. gondii lifecycle for clonal propagation, it was originally proposed that the three archetypal strains, Types I, II, and III (Howe and Sibley, 1995), were unique among T. gondii strains in that they had recently acquired the ability for enhanced oral transmission of tissue cysts among intermediate hosts, thus allowing for clonal transmission that bypassed the sexual stage in the definitive host (Su et al., 2003). This hypothesis was based on limited laboratory studies and showed that tissue cysts from a small sample (3 isolates) of non-archetypal strains had greatly reduced oral transmission among laboratory mice compared to Types I, II, and III (Su et al., 2003). However, with additional sampling, it has since been shown that virtually all isolates of T. gondii, even rare genotypes, are capable of oral transmission among intermediate hosts (Khan et al., 2007), suggesting this trait may not be solely responsible for the global dominance of certain genotypes.

A case has also been made suggesting that vertical transmission, either transplacentally or through ingestion of milk, may play an important role in maintaining clonal dominance of certain strains in nature (Johnson, 1997). This hypothesis was advanced based upon knowledge of the presumably important role transplacental transmission plays for infection with the related parasite, Neospora caninum, in domestic cattle and dogs (Johnson, 1997). Numerous studies have demonstrated that T. gondii is capable of vertical transmission in a variety of hosts, including humans, but few have examined the possibility that strict vertical transmission is maintained over several generations in natural host populations. High prevalence of Toxoplasma in certain hosts that are geographically isolated from definitive felid hosts, such as the arctic fox populations mentioned above (Prestrud et al., 2008), are intriguing scenarios to speculate whether vertical transmission is important, but evidence has yet to be gathered to support this hypothesis. Much remains to be done to delineate the relative roles of vertical versus oral, carnivorous transmission in maintaining clonality in nature.

Given the much greater infective potential inherent to the definitive host stage (a single infected cat can shed hundreds of millions of oocysts into the environment (Dubey, 2001)), we recently tested whether self-mating during this stage was a viable mechanism for the expansion of a single genotype in nature. We extensively genotyped oocyst samples recovered from a reservoir linked to a waterborne T. gondii outbreak in humans in Brazil and demonstrated that they were an identical genetic clone (Wendte et al., 2010a). Combining these results with serologic typing evidence from infected people (Vaudaux et al., 2010) confirmed that the outbreak, which was attributed to ingestion of oocyst contaminated water, was indeed clonal and apparently the result of a selfing event in a felid definitive host (Wendte et al., 2010a). Since well over a hundred people were affected by this outbreak, this result demonstrated the major role selfing in the definitive host can play as a potential mechanism for clonal expansion of a disease-producing genotype in nature as compared to vertical transmission or oral transmission via carnivory (Wendte et al., 2010a). Importantly, self-mating was also the cause of a devastating clonal outbreak of the related parasite Sarcocystis neurona that caused a point-source mass mortality event in a threatened Southern sea otters due to oocyst/sporocyst contamination of waterways; a result that expands the explanatory scope of selfing as a mechanism of clonality to other tissue cyst coccidia (Wendte et al., 2010a).

The genetic basis for the expansion of certain clones has also been proposed in population genetic studies. The archetypal clones have been associated with certain alleles for the rhoptry kinase protein, ROP18, associated with virulence in the mouse model (Khan et al., 2009) and a monomorphic chromosome Ia (Khan et al., 2007), leading to the conclusion that these loci may contribute to the success of these clonotypes in nature. Fortunately, the identification of a genetically distinct, dominant clone circulating in wildlife, ‘Type X’ (see Figure 1C), should allow future comparative genomic analyses to further refine potential candidate genes accounting for the success of certain genotypes to carry forward in experimental studies.

Notably, while this manuscript was under review, a study was published further characterizing many ‘Type X’ and apparently ‘Type X-like’ strains at five intron and 3 antigen loci with a variety of phylogenetic and population analyses techniques (Khan et al., 2011). This study grouped ‘Type X’ and many closely related strains into a new ‘haplogroup 12’ according to the previously published methodology of Khan et al. (2007). Similar to our discussion above of the 2007 study, close examination of the data reveals that consistent delineations between certain haplogroups break down to varying degrees depending on the analysis technique used (Khan et al., 2011). In fact, inconsistent delineations between ‘haplogroup 12’ (‘Type X’) and ‘haplogroup 2’ (‘Type II’) due to a bi-allelic inheritance pattern at many of the loci analyzed led to the conclusion that ‘haplogroup 12’ (‘Type X’) strains are the result of a cross between ‘Type II’ and a distinct ancestral type (Khan et al., 2011). This intriguing conclusion again speaks to the importance of the sexual cycle in the T. gondii population structure as a mechanism for the emergence of new strains that can go on to dominate clonally. It is also telling that what were once considered minor (and sometimes insignificant) genetic differences by RFLP and other typing schemes among ‘Type X’ genotypes compared against ‘Type II’ genotypes were indicative of major genomic level diversity. As the field moves forward with much more extensive, whole-genome level analyses, it is likely that several strains once thought to be identical clones will be found to be comprised of multiple, diverse genetic backgrounds.