Toxoplasma gondii, the causal agent of toxoplasmosis, is an of import H2O and nutrient borne protozoon parasite belonging to the phylum Apicomplexa. T. gondii research is an of import country of survey because of its ability to infect a broad scope of hosts and more specifically, worlds. Toxoplasma gondii is a extremely prevailing obligate intracellular parasite that has no host specificity and infects all warm-blooded craniates including mammals and birds. It is the lone known species in the genus Toxoplasma and is considered one of the most successful eucaryotic pathogen in the universe in footings of the figure of host species and per centum of animate beings infected worldwide ( 54, 20, 71 ) . Up to tierce of the human population in the universe is inveterate septic 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 chiefly caused by consuming uncooked meat incorporating feasible tissue cysts or by consuming nutrient or H2O contaminated with oocysts shed in the fecal matters of septic cats. Symptoms: Primary infections in normal healthy grownups are largely symptomless but in some patients lymphadenopathy or optic toxoplasmosis can happen ( 18, 10, 76 ) . In immunocompromised persons, T. gondii can do dangerous phrenitis and in pregnant adult females symptoms range from inborn sightlessness, mental deceleration to even decease of the foetus ( 18, 44 ) . Life-cycle: T. gondii has a complex life rhythm that includes sexual and nonsexual reproduction ( 43, 19, 73 ) . Sexual reproduction of parasite occurs in felines which are the unequivocal hosts and all warm-blooded craniates can function as intermediate hosts in which nonsexual reproduction takes topographic point. Oocysts are shed by cats into the environment and they find their manner to the intermediate hosts via contaminated nutrient and H2O. After several yearss of reproduction as quickly turning extremely morbific tachyzoites during acute infection, the parasites switch to slowly turning bradyzoites, which are encysted to organize tissue cysts and reside in host cells for the life of the host. These bradyzoites may reactivate and change over to tachyzoites if host immune response is compromised, such as in AIDS patients ( 20, 73 ) . T. gondii is the lone known apicomplexan that can convey straight among intermediate hosts and do infection without cycling through its unequivocal felid host which empowers it to parasitize and infect virtually any bird or mammal ( 8 ) .
Population Structure: T. gondii was antecedently believed to hold a distinctly clonal construction composed of Types I, II and III line of descents ( 14, 40, 2 ) .While clonality is the prevailing form in North America and Europe, recombinant strains stand foring mixtures of the three clonal types were besides on occasion found. Furthermore, a little figure of isolates were extremely divergent from the three major line of descents, and these were referred to as ‘exotic or untypical genotypes ‘ ( 40 ) . Recent population familial surveies of Toxoplasma gondii from animate beings and worlds global have revealed high frequence of non type I, II and III genotypes ( 4, 52 ) . Furthermore, recent surveies 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 disputing the present clonal construction and bring forthing the demand for a elaborate survey to redefine the bing population construction ( 52, 53, 32, 49, 70, 24 ) .
Biological Significance: The correlativity of parasite genotypes with human toxoplasmosis has of import deductions for development of better diagnostic protocols, vaccinums and drugs for more effectual bar, intervention and control schemes. In mice, the assorted strains of the parasite differ tremendously in their virulency and disease presentation with type I being acutely virulent ( 9 ) . In worlds excessively, disease manifestations are extremely variable, runing from symptomless to severe, particularly in instances of encephalon and oculus infection ( 9 ) .Cases of ague disseminated toxoplasmosis caused by untypical T. gondii strain have been reported even in immunocompetent persons from S. America ( 60 ) . It is of great involvement to analyze the biological differences among different genotypes of T. gondii and to look into whether the genotypes are related to disease manifestations in human toxoplasmosis. This requires better ways to accurately find the genotype of an infecting strain in order to prove the relationship between the type of strain and its disease doing possible in different geographical parts and in assorted disease scenarios. Clear correlativities will so well better the direction of human disease, fiting an aggressive infection with an every bit aggressive intervention.
Methods for genotyping
A assortment of different methods exist to genotype T. gondii isolates, and they have distinguishable advantages and disadvantages. Early surveies of strain typing were based on multilocus enzyme cataphoresis ( MLEE ) . Several polymorphous enzymes were used to qualify T. gondii isolates mostly collected from France, grouping them into three major zymodemes Z1, Z2 and Z3 ( 14 ) . While MLEE is rather specific, it requires a big figure of purified parasites to execute.
Subsequently typing methods focused on microsatellite ( MS ) markers ( 2, 7 ) , which are short perennial sections of Deoxyribonucleic acid that tend to happen in non-coding DNA. MS markers are sensitive, dependable and conformable to high-throughput analyses. But though MSs are extremely polymorphous, they can be prone to homoplasy as the figure of repetitions can spread out and contract during reproduction.
Randomly amplified polymorphous DNA ( RAPD ) was besides used to qualify strains of T. gondii ( 38, 31 ) ; nevertheless, this technique is extremely influenced by polluting host DNA, which can be a important beginning of variableness.
Of late, limitation fragment length polymorphism ( RFLP ) analysis of specific familial venue has been widely used to qualify T. gondii isolates ( 66, 40, 42 ) . RFLP markers are conformable to high-throughput analysis utilizing PCR elaboration, followed by limitation digestion and gel cataphoresis, are easy to utilize and hold a high declaration in placing T. gondii isolates ( 70 ) . Genotyping T. gondii by the multilocus PCR-RFLP markers has generated priceless information in uncovering the parasite ‘s diverseness. But the bing multilocus PCR-RFLP markers were originally developed based on DNA sequence polymorphisms in the clonal Type I, II and III line of descents and they chiefly separate the three clonal line of descents. So they might, to some extent under-estimate the true diverseness of the parasite population on a planetary graduated table which is elucidated by the fact that non-clonal allelomorphs ( 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 extremely diverged from the clonal Type I, II and III line of descents at the DNA sequence degree. Furthermore, it requires a important investing of clip to develop and optimise each marker and RFLP markers are limited to capturing alterations that alter limitation enzyme sites, and many sequence polymorphisms are missed by this analysis.
All of the above-named methods underestimate the true rate of polymorphism and hence may misclassify discrepancies owing to homoplasy or insufficient deciding power. In contrast, direct sequencing of genomic parts reveals the complete familial diverseness including individual nucleotide polymorphisms ( SNPs ) and little interpolations and omissions ( e.g. indels ) . Direct sequencing by and large detects much greater familial diverseness than other methods. For illustration, a high grade of polymorphism was observed at the GRA6 venue by sequencing ( nine allelomorphic sequences from 30 strains ) , whereas the PCR-RFLP analysis merely detected three groups ( 30 ) . Therefore, sequence-based methods provide the best attack for observing polymorphisms in new isolates or from antecedently unsampled populations. A assortment of different venues have been used for sequence-based analysis, including both coding parts for housekeeping cistrons, antigens and selectively impersonal noncoding DNAs ( 50 ) . Highly polymorphous antigens provide maximal declaration for observing recent divergency within populations. In contrast, selectively nervous parts provide the best beginning of informations to cipher the age of common lineage between different line of descents and to foretell common lineage ( 50 ) . The obvious disadvantage of sequence-based typewriting is its increased cost and demand for entree to sophisticated engineering.
However, one time the population construction is known for a given part, more cost-efficient typewriting methods can be developed to observe the major allelomorphs ( i.e. MS or RFLP typing ) . For example- the sequence information generated will be utile to choose extra limitation endonucleases that recognize non-clonal Type I, II and III allelomorphs, hence extinguishing the prejudice of these familial markers.
Toxoplasma gondii possesses a well-characterized sexual rhythm, yet the population construction of T. gondii in most parts reflects a high frequence of nonsexual reproduction. A big figure of toxoplasma gondii isolates collected from human disease instances and chronic animate being infections from Europe and North America have shown unusually small diverseness and have been classified into one of three clonal familial line of descents ( Types I, II, III ) based on multilocus enzymes cataphoresis, microsatellite typewriting and PCR-RFLP with acutely deadly strains consisting a individual clonal type ( 14, 40, 66, 2 ) . Identical multilocus genotypes, a high grade of linkage disequilibrium between markers and a comparative absence of recombinants exhibited by the bulk of isolates were strong indexs of clonality. The prevailing clonal line of descents differ by merely 1-2 % at the nucleotide degree ( 36 ) .
Another dramatic characteristic of North American and European clonal line of descents of T. gondii is the presence of biallelic polymorphisms at each venue, widening throughout the genome. Such biallelism was apparent from early RFLP surveies and was farther validated by sequencing cistrons from a assortment of isolates ( 36 ) . Biallelism was thought to hold surfaced by familial impetus in the hereditary population, followed by a terrible familial constriction that resulted in the outgrowth of three prevailing line of descents. ( 1, 55, 11 ) .
While clonality is the prevailing form in North America and Europe, recombinant strains stand foring mixtures of the three clonal types were besides on occasion found. Furthermore, a little figure of isolates were extremely divergent from the three major line of descents, and these were referred to as ‘exotic or untypical genotypes ‘ ( 40 ) .
The population construction of the clonal line of descents of T. gondii can be best explained by a familial constriction followed by a rapid enlargement ( 67 ) . This form might reflect an unusual combination of cistrons, shared by the three line of descents, that is responsible for their successful enlargement. Several adaptative traits, including development of unwritten transmittal between intermediate hosts ( 69 ) , enhanced transmittal by domestic cats or versions to domestic gnawers ( 53 ) might explicate this enlargement. Alternatively, the form of clonality may merely reflect an unusual demographic procedure that dramatically restricted the cistron pool. Testing these theoretical accounts will necessitate a better apprehension of population construction and manners of transmittal in the natural state.
While clonality clearly predominates in much of North America and Europe, extra sampling of animate beings or worlds from a wider scope of locations indicates that strains from other geographical parts have more familial variableness than antecedently reported ( 4 ) . For illustration, extremely unusual genotypes were detected in persons who contracted toxoplasmosis while in the jungles of Gallic Guyana ( 15, 12 ) . Surveies from Brazil based on RFLP markers, and similar surveies utilizing MS markers, showed that carnal isolates of T. gondii are genetically different from those in North America and Europe ( 52, 53, 32, 49, 70, 24, 63 ) .
Besides, sequencing surveies comparing noncoding DNA sequences from a aggregation of T. gondii isolates from Brazil and Gallic Guyana to antecedently characterized mention strains from North America revealed that in Brazil, T. gondii line of descents were extremely diverged from the clonal Type I, II and III lineages that predominate in North America and Europe ( 50 ) .This decision has later been echoed by legion other surveies of isolates from assorted carnal hosts in Central and South America. Similar diverse genotypes were besides observed in human samples in Brazil ( 49, 33 ) , proposing the divergency of isolates in South America is non due to differences in host scope but instead due to geographical differences. Interestingly, both clonal Type I and III line of descents were found in Brazil, nevertheless non a individual Type II isolate was identified. The prevalence of genetically more diverse strains in South America raises the inquiry of whether they are distinguishable line of descents or simply recombinants of the genotypes that are prevailing in the North. More recent sampling in parts of North America besides suggests that isolates from wild animate beings have more diverse genotypes, as tested by multilocus RFLP analysis ( 28 ) . Limited surveies from Asia suggest predomination of genetically assorted strains ( 68, 24 ) .
Comparative noncoding DNA sequencing surveies between the North and south American strains showed separate sets of reciprocally sole biallelic polymorphisms which indicated that North and South American populations were reproductively stray and had evolved in the absence of familial exchange over a long period of clip ( 48, 50 ) .
Phylogenetic web Reconstruction based on noncoding DNA sequences clustered all T. gondii strains into 11 distinguishable haplogroups stand foring the major line of descents ( 50, Fig 1 ) . These 11 haplogroups were besides supported by the Bayesian theoretical account for foretelling population STRUCTURE 29 ) . 3 of these groups correspond to the antecedently recognized clonal line of descents in North America and Europe ( i.e. I, II and III or 1, 2 and 3 ) . Of the new groups, 4 are about entirely found in South America ( i.e. 4, 5, 8 and 9 ) . These groups by and large show more profoundly ramifying evolutions and low degrees of linkage disequilibrium, consistent with greater extent of sexual recombination ( i.e. group 4 ) , but there are besides pockets of clonality ( i.e. groups 8 and 9 ) .
Fig1: T.gondii strains show pronounced geographic separation and clonality. ( A ) Phylogenetic analysis of T. gondii strains based on noncoding DNA sequences identified11 separate haplogroups ( numbered in boxes ) , with dramatic geographic separation between NA and E ( bluish inscription ) and SA ( ruddy inscription ) . Unrooted phylogram generated by utilizing neighbor-joining analysis ; bootstrap values are given by the per centum at each node
This suggests an epidemic population construction of the parasite in Brazil, in which frequent familial exchange has generated a assortment of recombinants and a few successful clonal line of descents have expanded into broad geographical countries. This is in crisp contrast to the clonal population construction in North America and Europe, where merely three clonal line of descents predominate and familial exchanges among these line of descents are rare ( 14, 40, 2 ) .
Seropositive rates of T. gondii infection in worlds and animate beings are besides much higher in Brazil. About 50-80 % of grownup worlds had antibodies to T. gondii, approximately 35 % of 237 cats from S & A ; atilde ; o Paulo province and 84 % of 58 cats from Paran & A ; aacute ; were seropositive to T. gondii ( 5, 62, 23 ) . The high seropositive rate in cats could ensue in heavy taint of the environment by T. gondii oocysts, which will take to taint of nutrient and H2O supplies for worlds ( 5, 16 ) . The extremely contaminated environment will necessarily take to a high rate of T. gondii infection in intermediate hosts and the effect is the increased chance of familial recombination in cats. This alone epidemiology of T. gondii transmittal might be responsible for making and keeping the predicted epidemic population construction of the parasite in Brazil.
Despite the presence of reciprocally sole biallelic polymorphisms, T. gondii strains from North and South America have been believed to portion a common lineage. However, they were separated from each other over a long period of clip during which they accumulated characteristic SNPs by random mutant and impetus. Coalescence analysis supports a theoretical account whereby the three predominant lineages evolved late from a common ascendant within the last 10,000 old ages, spread outing really quickly to dwell a assortment of hosts ( 69 ) . This is around the same clip as the domestication of agricultural animate beings, every bit good as the acceptance of comrade animate beings such as domestic cats ( 17 ) . A figure of sexually differentiated parasites of agricultural animate beings have been found to exhibit similar low familial diverseness, reflecting familial constrictions and anthropogenetic enlargements in their recent lineage ( 64 ) . By ciphering the extent of geographical allelomorphic diverseness, it was estimated that this split occurred about 106 old ages ago. One possible account for this split is that T. gondii may hold migrated into South America with cats when the Panamanian land span was established ( 45, 50 ) Reconnection of this land span about 1-3 Myr ago has been suggested to explicate the variegation of workss and animate beings one time introduced into the South ( 57 ) .
The genome of T. gondii bears another signature characteristic related to origin and ancestry: being of a individual monomorphic version of chromosome 1a ( Chr1a* ) , as revealed by comparative genomic sequencing ( 48 ) . Based on the rate of bodily mutants between isolates within the line of descents, it was estimated that Chr1a* arose about 104 old ages ago, likely coincident with the beginning of the line of descents ( 48 ) . The odds of all three line of descents geting this same exact Chr1a* by opportunity have been estimated to be at least 1:1000 ( 48 ) , proposing this form arose owing to a selective advantage. Further sampling will be necessary to prove whether Chr1a is driving this enlargement owing to a selective advantage or whether it is due to some other implicit in demographic factor.
In North America and Europe, type II strains are most normally associated with human toxoplasmosis, both in inborn infections and in patients with AIDS ( 40, 42, 39, 2, 3 ) . Several surveies have indicated that the bulk of isolates from agricultural animate beings are besides type II, including hogs 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 study that indicated both type II and type III strains are common in animate beings ( 40 ) . The grounds for the evident differences between animate being ( types II and III ) and human ( mostly type II ) infections are ill-defined but might be due to differences in susceptibleness or differences in disease-causing potency because human isolates were mostly collected from disease instances, while carnal infections were mostly subclinical. More recent studies, utilizing an expanded set of RFLP markers, have indicated that while the bulk of isolates from sheep in North America were type II, a figure of distinguishable new genotypes were found ( 27 ) . Such new discrepancies might originate by bodily mutants, or consequence from sexual recombination between the major line of descents, or they might stand for wholly new genotypes. Decoding these different theoretical accounts would necessitate farther sequence-based analysis of haplotypes.
The T. gondii clonal line of descents differ in a figure of phenotypes such as growing, migration and transmigration and significantly, virulency in research lab mice ( 6, 41, 59 ) . Type I strains do deadly infection in all strains of research lab mice even at low inoculant ( deadly dosage ( LD100 ) approx. 1 ) , whereas types II and III strains are much less deadly ( average lethal dosage ( LD50 ) ?105 ) . Using frontward familial function surveies to place cistrons that determine natural differences in the virulency of T. gondii in the mouse theoretical account, secretory protein kinases discharged from apical cell organs, called rhoptries ( ROPs ) have been implicated as the cardinal determiner of acute virulency by independent testing experiments ( 65, 72 ) . Many South American strains are besides deadly in murine theoretical accounts ( 50 ) and similar attacks have demonstrated that ROPs besides contribute to acute virulency in these line of descents ( 51 ) .
Predicting the population construction of T. gondii in other parts of the universe is unsure and hard given the markedly different population construction of T. gondii in North and South America. Limited trying based mostly on RFLP markers suggests that African populations are similar to those in Europe and North America ( 56, 75 ) . These studies focused chiefly on domestic animate beings and in peculiar poulets, so they might be influenced by debut of parasite strains from Europe along with importing of domestic animate beings. Further testing of ferine animate beings will be necessary to decently specify the population construction in these parts. In contrast, surveies from Asia suggest genetically assorted strains predominant ( 68, 24 ) , yet the extent of familial diverseness in these parts has non been decently estimated. Sequencing of noncoding DNAs and other venue from isolates in new parts is needed to capture the full familial diverseness and hence accurately estimate local population construction.
The correlativity, if any, between the parasite genotypes and the disease manifestation in human toxoplasmosis is another country of considerable involvement and needs future probe. Previous surveies reported that certain strains were more often associated with a peculiar type of toxoplasmosis in human patients ( 34, 37, 46, 49 and 74 ) . However, as there is no big graduated table epidemiological survey to uncover the diverseness of T. gondii genotypes both in homo and animate being populations, it is non clear if the prejudice of disease manifestations is due to the background genotypes in the environment where these patients reside, or the effect of different biological traits that make certain genotypes more deadly in doing a peculiar type of human disease. Therefore, it is necessary to hold a thorough epidemiological survey to uncover T. gondii population diverseness in the environment.
The life rhythm of T. gondii included manners for sexual and nonsexual transmittal, therefore the population construction may change dramatically in different vicinities. By understanding these forms, we can foretell the hazard of spread through the nutrient concatenation and the potency for zoonotic infection. Specifying the part of population construction to the spread of traits like immunogenicity and pathogenesis is extremely important to human wellness. Modeling such relationships in a theoretical account parasite like T. gondii may besides supply insight into other parasites of animate beings that pose hazard to worlds.