Toxoplasma gondii, the causal agent of toxoplasmosis, is an important water and food borne protozoan parasite belonging to the phylum Apicomplexa. T. gondii research is an important area of study because of its ability to infect a wide range of hosts and more specifically, humans. Toxoplasma gondii is a highly prevalent obligate intracellular parasite that has no host specificity and infects all warm-blooded vertebrates including mammals and birds. It is the only known species in the genus Toxoplasma and is considered one of the most successful eukaryotic pathogen in the world in terms of the number of host species and percentage of animals infected worldwide (54, 20, 71). Up to one-third of the human population in the world is chronically infected and more than 60 million people in United States itself are believed to be infected (18, 73, Toxoplasmosis: Fact Sheet - CDC DPD).
Transmission: Human infections are primarily caused by ingesting uncooked meat containing viable tissue cysts or by ingesting food or water contaminated with oocysts shed in the feces of infected cats. Symptoms: Primary infections in normal healthy adults are mostly asymptomatic but in some patients lymphadenopathy or ocular toxoplasmosis can occur (18, 10, 76). In immunocompromised individuals, T. gondii can cause life-threatening encephalitis and in pregnant women symptoms range from congenital blindness, mental retardation to even death of the fetus (18, 44). Life-cycle: T. gondii has a complex life cycle that includes sexual and asexual replication (43, 19, 73). Sexual replication of parasite occurs in felids which are the definitive hosts and all warm-blooded vertebrates can serve as intermediate hosts in which asexual replication takes place. Oocysts are shed by cats into the environment and they find their way to the intermediate hosts via contaminated food and water. After several days of replication as rapidly growing highly infective tachyzoites during acute infection, the parasites switch to slowly growing bradyzoites, which are encysted to form tissue cysts and reside in host cells for the life of the host. These bradyzoites may reactivate and convert to tachyzoites if host immune response is compromised, such as in AIDS patients (20, 73). T. gondii is the only known apicomplexan that can transmit directly among intermediate hosts and cause infection without cycling through its definitive feline host which empowers it to parasitize and infect virtually any bird or mammal (8).
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Population Structure: T. gondii was previously believed to have a distinctly clonal structure composed of Types I, II and III lineages (14, 40, 2).While clonality is the predominant pattern in North America and Europe, recombinant strains representing mixtures of the three clonal types were also occasionally found. Moreover, a small number of isolates were highly divergent from the three major lineages, and these were referred to as 'exotic or atypical genotypes' (40). Recent population genetic studies of Toxoplasma gondii from animals and humans world-wide have revealed high frequency of non type I, II and III genotypes (4, 52). Moreover, recent studies found that isolates of T.gondii from Brazil and parts of S. America are biologically and genetically different from those in North America and Europe thereby challenging the present clonal structure and generating the need for a detailed study to redefine the existing population structure (52, 53, 32, 49, 70, 24).
Biological Significance: The correlation of parasite genotypes with human toxoplasmosis has important implications for development of better diagnostic protocols, vaccines and drugs for more effective prevention, treatment and control strategies. In mice, the various strains of the parasite differ enormously in their virulence and disease presentation with type I being acutely virulent (9). In humans too, disease manifestations are highly variable, ranging from asymptomatic to severe, especially in cases of brain and eye infection (9).Cases of acute disseminated toxoplasmosis caused by atypical T. gondii strain have been reported even in immunocompetent individuals from S. America (60). It is of great interest to study the biological differences among different genotypes of T. gondii and to investigate whether the genotypes are related to disease manifestations in human toxoplasmosis. This requires better ways to accurately determine the genotype of an infecting strain in order to test the relationship between the type of strain and its disease causing potential in different geographical regions and in various disease scenarios. Clear correlations will then substantially improve the management of human disease, matching an aggressive infection with an equally aggressive treatment.
Methods for genotyping
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A variety of different methods exist to genotype T. gondii isolates, and they have distinct advantages and disadvantages. Early studies of strain typing were based on multilocus enzyme electrophoresis (MLEE). Several polymorphic enzymes were used to characterize T. gondii isolates largely collected from France, grouping them into three major zymodemes Z1, Z2 and Z3 (14). While MLEE is quite specific, it requires a large number of purified parasites to perform.
Later typing methods focused on microsatellite (MS) markers (2, 7), which are short repeated segments of DNA that tend to occur in non-coding DNA. MS markers are sensitive, reliable and amenable to high-throughput analyses. But though MSs are highly polymorphic, they can be prone to homoplasy as the number of repeats can expand and contract during replication.
Randomly amplified polymorphic DNA (RAPD) was also used to characterize strains of T. gondii (38, 31); however, this technique is highly influenced by contaminating host DNA, which can be a significant source of variability.
Of late, restriction fragment length polymorphism (RFLP) analysis of specific genetic loci has been widely used to characterize T. gondii isolates (66, 40, 42). RFLP markers are amenable to high-throughput analysis using PCR amplification, followed by restriction digestion and gel electrophoresis, are easy to use and have a high resolution in identifying T. gondii isolates (70). Genotyping T. gondii by the multilocus PCR-RFLP markers has generated invaluable information in revealing the parasite's diversity. But the existing multilocus PCR-RFLP markers were originally developed based on DNA sequence polymorphisms in the clonal Type I, II and III lineages and they primarily distinguish the three clonal lineages. So they might, to some extent under-estimate the true diversity of the parasite population on a global scale which is elucidated by the fact that non-clonal alleles (denoted u-1, u-2) have been revealed for half of these markers, including SAG1, SAG2 (new), c22-8, c29-2 and PK1. This suggests that many T. gondii isolates are highly diverged from the clonal Type I, II and III lineages at the DNA sequence level. Moreover, it requires a significant investment of time to develop and optimize each marker and RFLP markers are limited to capturing changes that alter restriction enzyme sites, and many sequence polymorphisms are missed by this analysis.
All of the above-mentioned methods underestimate the true rate of polymorphism and hence may misclassify variants owing to homoplasy or insufficient resolving power. In contrast, direct sequencing of genomic regions reveals the complete genetic diversity including single nucleotide polymorphisms (SNPs) and small insertions and deletions (e.g. indels). Direct sequencing generally detects much greater genetic diversity than other methods. For example, a high degree of polymorphism was observed at the GRA6 locus by sequencing (nine allelic sequences from 30 strains), whereas the PCR-RFLP analysis only detected three groups (30). Thus, sequence-based methods provide the best approach for detecting polymorphisms in new isolates or from previously unsampled populations. A variety of different loci have been used for sequence-based analysis, including both coding regions for housekeeping genes, antigens and selectively neutral introns (50). Highly polymorphic antigens provide maximum resolution for detecting recent divergence within populations. In contrast, selectively neural regions provide the best source of data to calculate the age of common ancestry between different lineages and to predict common ancestry (50). The obvious disadvantage of sequence-based typing is its increased cost and need for access to sophisticated technology.
However, once the population structure is known for a given region, more cost-effective typing methods can be developed to detect the major alleles (i.e. MS or RFLP typing). For example- the sequence information generated will be useful to select additional restriction endonucleases that recognise non-clonal Type I, II and III alleles, therefore eliminating the bias of these genetic markers.
Population structure in North America and Europe
Toxoplasma gondii possesses a well-characterized sexual cycle, yet the population structure of T. gondii in most regions reflects a high frequency of asexual replication. A large number of toxoplasma gondii isolates collected from human disease cases and chronic animal infections from Europe and North America have shown remarkably little diversity and have been classified into one of three clonal genetic lineages (Types I, II, III) based on multilocus enzymes electrophoresis, microsatellite typing and PCR-RFLP with acutely virulent strains comprising a single clonal type (14, 40, 66, 2). Identical multilocus genotypes, a high degree of linkage disequilibrium between markers and a relative absence of recombinants exhibited by the majority of isolates were strong indicators of clonality. The predominant clonal lineages differ by only 1-2% at the nucleotide level (36).
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Another striking feature of North American and European clonal lineages of T. gondii is the presence of biallelic polymorphisms at each locus, extending throughout the genome. Such biallelism was evident from early RFLP studies and was further validated by sequencing genes from a variety of isolates (36). Biallelism was thought to have surfaced by genetic drift in the ancestral population, followed by a severe genetic bottleneck that resulted in the emergence of three predominant lineages. (1, 55, 11).
While clonality is the predominant pattern in North America and Europe, recombinant strains representing mixtures of the three clonal types were also occasionally found. Moreover, a small number of isolates were highly divergent from the three major lineages, and these were referred to as 'exotic or atypical genotypes' (40).
The population structure of the clonal lineages of T. gondii can be best explained by a genetic bottleneck followed by a rapid expansion (67). This pattern might reflect an unusual combination of genes, shared by the three lineages, that is responsible for their successful expansion. Several adaptive traits, including evolution of oral transmission between intermediate hosts (69), enhanced transmission by domestic cats or adaptations to domestic rodents (53) might explain this expansion. Alternatively, the pattern of clonality may simply reflect an unusual demographic process that dramatically restricted the gene pool. Testing these models will require a better understanding of population structure and modes of transmission in the wild.
Population structure in South America and other geographical regions
While clonality clearly predominates in much of North America and Europe, additional sampling of animals or humans from a wider range of locations indicates that strains from other geographical regions have more genetic variability than previously reported (4). For example, highly unusual genotypes were detected in individuals who contracted toxoplasmosis while in the jungles of French Guyana (15, 12). Studies from Brazil based on RFLP markers, and similar studies using MS markers, showed that animal isolates of T. gondii are genetically different from those in North America and Europe (52, 53, 32, 49, 70, 24, 63).
Also, sequencing studies comparing intron sequences from a collection of T. gondii isolates from Brazil and French Guyana to previously characterized reference strains from North America revealed that in Brazil, T. gondii lineages were highly diverged from the clonal Type I, II and III lineages that predominate in North America and Europe (50).This conclusion has subsequently been echoed by numerous other studies of isolates from various animal hosts in Central and South America. Similar diverse genotypes were also observed in human samples in Brazil (49, 33), suggesting the divergence of isolates in South America is not due to differences in host range but rather due to geographical differences. Interestingly, both clonal Type I and III lineages were found in Brazil, however not a single Type II isolate was identified. The prevalence of genetically more diverse strains in South America raises the question of whether they are distinct lineages or merely recombinants of the genotypes that are prevalent in the North. More recent sampling in parts of North America also suggests that isolates from wild animals have more diverse genotypes, as tested by multilocus RFLP analysis (28). Limited studies from Asia suggest predominance of genetically mixed strains (68, 24).
Major differences in population structure between North and South America
Comparative intron sequencing studies between the north and south American strains showed separate sets of mutually exclusive biallelic polymorphisms which indicated that North and South American populations were reproductively isolated and had evolved in the absence of genetic exchange over a long period of time (48, 50).
Phylogenetic network reconstruction based on intron sequences clustered all T. gondii strains into 11 distinct haplogroups representing the major lineages (50, Fig 1). These 11 haplogroups were also supported by the Bayesian model for predicting population STRUCTURE 29). 3 of these groups correspond to the previously recognized clonal lineages in North America and Europe (i.e. I, II and III or 1, 2 and 3). Of the new groups, 4 are almost exclusively found in South America (i.e. 4, 5, 8 and 9). These groups generally show more deeply branching phylogenies and low levels of linkage disequilibrium, consistent with greater extent of sexual recombination (i.e. group 4), but there are also pockets of clonality (i.e. groups 8 and 9).
Fig1: T.gondii strains show marked geographic separation and clonality. (A) Phylogenetic analysis of T. gondii strains based on intron sequences identified11 separate haplogroups (numbered in boxes), with striking geographic separation between NA and E (blue lettering) and SA (red lettering). Unrooted phylogram generated by using neighbor-joining analysis; bootstrap values are given by the percent at each node
This suggests an epidemic population structure of the parasite in Brazil, in which frequent genetic exchange has generated a variety of recombinants and a few successful clonal lineages have expanded into wider geographical areas. This is in sharp contrast to the clonal population structure in North America and Europe, where only three clonal lineages predominate and genetic exchanges among these lineages are rare (14, 40, 2).
Seropositive rates of T. gondii infection in humans and animals are also much higher in Brazil. About 50-80% of adult humans had antibodies to T. gondii, about 35% of 237 cats from São Paulo state and 84% of 58 cats from Paraná were seropositive to T. gondii (5, 62, 23). The high seropositive rate in cats could result in heavy contamination of the environment by T. gondii oocysts, which will lead to contamination of food and water supplies for humans (5, 16). The highly contaminated environment will inevitably lead to a high rate of T. gondii infection in intermediate hosts and the consequence is the increased opportunity of genetic recombination in cats. This unique epidemiology of T. gondii transmission might be responsible for creating and maintaining the predicted epidemic population structure of the parasite in Brazil.
Origin and Evolution
Despite the presence of mutually exclusive biallelic polymorphisms, T. gondii strains from North and South America have been believed to share a common ancestry. However, they were separated from each other over a long period of time during which they accumulated characteristic SNPs by random mutation and drift. Coalescence analysis supports a model whereby the three predominant lineages evolved recently from a common ancestor within the last 10,000 years, expanding very rapidly to populate a variety of hosts (69). This is around the same time as the domestication of agricultural animals, as well as the adoption of companion animals such as domestic cats (17). A number of sexually differentiated parasites of agricultural animals have been found to exhibit similar low genetic diversity, reflecting genetic bottlenecks and anthropogenic expansions in their recent ancestry (64). By calculating the extent of geographical allelic diversity, it was estimated that this split occurred approximately 106 years ago. One possible explanation for this split is that T. gondii may have migrated into South America with cats when the Panamanian land bridge was established (45, 50) Reconnection of this land bridge approximately 1-3 Myr ago has been suggested to explain the diversification of plants and animals once introduced into the South (57).
The genome of T. gondii bears another signature feature related to origin and ancestry: existence of a single monomorphic version of chromosome 1a (Chr1a*), as revealed by comparative genomic sequencing (48). Based on the rate of somatic mutations between isolates within the lineages, it was estimated that Chr1a* arose approximately 104 years ago, probably coincident with the origin of the lineages (48). The odds of all three lineages acquiring this same exact Chr1a* by chance have been estimated to be at least 1:1000 (48), suggesting this pattern arose owing to a selective advantage. Further sampling will be necessary to test whether Chr1a is driving this expansion owing to a selective advantage or whether it is due to some other underlying demographic factor.
Association of genotypes with biological hosts and phenotypes
In North America and Europe, type II strains are most commonly associated with human toxoplasmosis, both in congenital infections and in patients with AIDS (40, 42, 39, 2, 3 ). Several studies have indicated that the majority of isolates from agricultural animals are also type II, including pigs in the USA (58, 27, 25) and sheep from Britain (61). But chickens in North America show a higher prevalence of type III strains than type II (21, 22), consistent with an early survey that indicated both type II and type III strains are common in animals (40). The reasons for the apparent differences between animal (types II and III) and human (largely type II) infections are unclear but might be due to differences in susceptibility or differences in disease-causing potential because human isolates were largely collected from disease cases, while animal infections were largely subclinical. More recent surveys, using an expanded set of RFLP markers, have indicated that while the majority of isolates from sheep in North America were type II, a number of distinct new genotypes were found (27). Such new variants might arise by somatic mutations, or result from sexual recombination between the major lineages, or they might represent entirely new genotypes. Deciphering these different models would require further sequence-based analysis of haplotypes.
The T. gondii clonal lineages differ in a number of phenotypes such as growth, migration and transmigration and importantly, virulence in laboratory mice (6, 41, 59). Type I strains cause lethal infection in all strains of laboratory mice even at low inocula (lethal dose (LD100) approx. 1), whereas types II and III strains are much less virulent (median lethal dose (LD50) ≥105). Using forward genetic mapping studies to identify genes that determine natural differences in the virulence of T. gondii in the mouse model, secretory protein kinases discharged from apical organelles, called rhoptries (ROPs) have been implicated as the key determinant of acute virulence by independent screening experiments (65, 72). Many South American strains are also virulent in murine models (50) and similar approaches have demonstrated that ROPs also contribute to acute virulence in these lineages (51).
Predicting the population structure of T. gondii in other parts of the world is uncertain and difficult given the markedly different population structure of T. gondii in North and South America. Limited sampling based largely on RFLP markers suggests that African populations are similar to those in Europe and North America (56, 75). These surveys focused primarily on domestic animals and in particular chickens, so they might be influenced by introduction of parasite strains from Europe along with importation of domestic animals. Further testing of feral animals will be necessary to properly define the population structure in these regions. In contrast, studies from Asia suggest genetically mixed strains predominant (68, 24), yet the extent of genetic diversity in these regions has not been properly estimated. Sequencing of introns and other loci from isolates in new regions is needed to capture the full genetic diversity and hence accurately estimate local population structure.
The correlation, if any, between the parasite genotypes and the disease manifestation in human toxoplasmosis is another area of considerable interest and needs future investigation. Previous studies reported that certain strains were more frequently associated with a particular type of toxoplasmosis in human patients (34, 37, 46, 49 and 74). However, as there is no large scale epidemiological study to reveal the diversity of T. gondii genotypes both in human and animal populations, it is not clear if the bias of disease manifestations is due to the background genotypes in the environment where these patients reside, or the consequence of different biological traits that make certain genotypes more virulent in causing a particular type of human disease. Therefore, it is necessary to have a thorough epidemiological study to reveal T. gondii population diversity in the environment.
The life cycle of T. gondii included modes for sexual and asexual transmission, hence the population structure may vary dramatically in different localities. By understanding these patterns, we can predict the risk of spread through the food chain and the potential for zoonotic infection. Defining the contribution of population structure to the spread of traits like immunogenicity and pathogenesis is highly significant to human health. Modelling such relationships in a model parasite like T. gondii may also provide insight into other parasites of animals that pose risk to humans.