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Retrospective distribution of Trypanosoma cruzi I genotypes in Colombia
research-article
Author(s):
Cielo M León
1
,
2 ,
Carolina Hernández
1
,
2 ,
Marleny Montilla
3 ,
Juan David Ramírez
2
,
+
Publication date (Print):
May 2015
Journal:
Memórias do Instituto Oswaldo Cruz
Publisher:
Instituto Oswaldo Cruz, Ministério da Saúde
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Abstract
Trypanosoma cruzi is the aetiological agent of Chagas disease, which affects approximately eight million people in the Americas. This parasite exhibits genetic variability, with at least six discrete typing units broadly distributed in the American continent. T. cruzi I (TcI) shows remarkable genetic diversity; a genotype linked to human infections and a domestic cycle of transmission have recently been identified, hence, this strain was named TcIDom. The aim of this work was to describe the spatiotemporal distribution of TcI subpopulations across humans, insect vectors and mammalian reservoirs in Colombia by means of molecular typing targeting the spliced leader intergenic region of mini-exon gene. We analysed 101 TcI isolates and observed a distribution of sylvatic TcI in 70% and TcIDom in 30%. In humans, the ratio was sylvatic TcI in 60% and TcIDom in 40%. In mammal reservoirs, the distribution corresponded to sylvatic TcI in 96% and TcIDom in 4%. Among insect vectors, sylvatic TcI was observed in 48% and TcIDom in 52%. In conclusion, the circulation of TcIDom is emerging in Colombia and this genotype is still adapting to the domestic cycle of transmission. The epidemiological and clinical implications of these findings are discussed herein.
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Most cited references72
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The revised Trypanosoma cruzi subspecific nomenclature: rationale, epidemiological relevance and research applications.
Bianca Zingales, Michael Miles, David A. Campbell … (2012)
The protozoan Trypanosoma cruzi, its mammalian reservoirs, and vectors have existed in nature for millions of years. The human infection, named Chagas disease, is a major public health problem for Latin America. T. cruzi is genetically highly diverse and the understanding of the population structure of this parasite is critical because of the links to transmission cycles and disease. At present, T. cruzi is partitioned into six discrete typing units (DTUs), TcI-TcVI. Here we focus on the current status of taxonomy-related areas such as population structure, phylogeographical and eco-epidemiological features, and the correlation of DTU with natural and experimental infection. We also summarize methods for DTU genotyping, available for widespread use in endemic areas. For the immediate future multilocus sequence typing is likely to be the gold standard for population studies. We conclude that greater advances in our knowledge on pathogenic and epidemiological features of these parasites are expected in the coming decade through the comparative analysis of the genomes from isolates of various DTUs. Copyright © 2012 Elsevier B.V. All rights reserved.
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A new genotype of Trypanosoma cruzi associated with bats evidenced by phylogenetic analyses using SSU rDNA, cytochrome b and Histone H2B genes and genotyping based on ITS1 rDNA.
Késsia Paiva, Alexandre Teixeira, M Cavazzana … (2009)
We characterized 15 Trypanosoma cruzi isolates from bats captured in the Amazon, Central and Southeast Brazilian regions. Phylogenetic relationships among T. cruzi lineages using SSU rDNA, cytochrome b, and Histone H2B genes positioned all Amazonian isolates into T. cruzi I (TCI). However, bat isolates from the other regions, which had been genotyped as T. cruzi II (TC II) by the traditional genotyping method based on mini-exon gene employed in this study, were not nested within any of the previously defined TCII sublineages, constituting a new genotype designated as TCbat. Phylogenetic analyses demonstrated that TCbat indeed belongs to T. cruzi and not to other closely related bat trypanosomes of the subgenus Schizotrypanum, and that although separated by large genetic distances TCbat is closest to lineage TCI. A genotyping method targeting ITS1 rDNA distinguished TCbat from established T. cruzi lineages, and from other Schizotrypanum species. In experimentally infected mice, TCbat lacked virulence and yielded low parasitaemias. Isolates of TCbat presented distinctive morphological features and behaviour in triatomines. To date, TCbat genotype was found only in bats from anthropic environments of Central and Southeast Brazil. Our findings indicate that the complexity of T. cruzi is larger than currently known, and confirmed bats as important reservoirs and potential source of T. cruzi infections to humans.
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Genome-Scale Multilocus Microsatellite Typing of Trypanosoma cruzi Discrete Typing Unit I Reveals Phylogeographic Structure and Specific Genotypes Linked to Human Infection
Martin Llewellyn, Michael Miles, Hernán Carrasco … (2009)
Introduction T. cruzi, the etiological agent of Chagas disease, is a vector borne zoonosis and considered the most important parasitic infection in Latin America. In excess of 10 million people are thought to carry the parasite, with ten times that number at risk (http://www.who.int). Consistent with a long history on the continent [1], T. cruzi ecology in the silvatic environment is highly complex. Over 73 mammalian genera and just over half of 137 described species of haematophagous triatomine bug are involved with parasite carriage and transmission [2],[3]. T. cruzi has an endemic range that stretches from the Southern USA to Northern Argentina. Most human infection is found in Central and South America and occurs primarily through contact with the contaminated faeces of domiciliated triatomine vector species. Genotypic data support the existence of six stable discrete typing units (DTUs) in T. cruzi: TcI, TcIIa, TcIIb, TcIIc, TcIId, and TcIIe [4]. Greatest molecular divergence is observed between TcI and TcIIb [1],[4]. TcIIa and TcIIc have distinct genotypes but their affinities to other DTUs are inadequately understood [4],[5]. TcIId and TcIIe are hybrids, and have haplotypes shared across TcIIb and TcIIc [1],[6]. The ecological and epidemiological relevance of different T. cruzi DTUs have been the subject of considerable debate. Using a retrospective analysis of all available genotype records, we recently showed that diversification in the silvatic environment is likely to be driven by ecological niche as well as host species, with arboreal Didelphimorpha (opossums) the principal hosts of TcI, and terrestrial Cingulata (armadillos) the principal hosts of TcIIc [7]. TcI is a major agent for human disease north of the Amazon Basin [8],[9], but is also ubiquitous in silvatic transmission cycles throughout the Americas [10],[11]. In the Southern Cone region of South America, DTUs TcIIb, TcIId, and TcIIe cause most human infection [10]. With the exception of putative epizootic outbreaks [12], TcIIb, TcIId, and TcIIe are so far rare in the silvatic cycle [7]. The current six-genotype classification of T. cruzi is likely to provide a poor reflection of the total diversity present. Abundant evidence from nucleotide sequence [13],[14], microsatellite [5],[15], RAPD [16] and MLEE [11],[17] data exists to suggest that considerable genetic variation is hidden at the sub-DTU level. Combining an adequate sample size with a genetic marker of sufficient resolution to unravel fine-scale relationships, however, remains a significant challenge. Indeed few, if any, detailed studies exist to document the population genetic diversity of a mammalian protozoan parasite in its true silvatic cycle. For many zoonotic infections, e.g. Cryptosporidium spp, Trypanosoma brucei sspp, Leishmania spp, and Toxoplasma gondii, domestic mammals and (where applicable) associated vectors are the obvious target for population-level studies of parasite genetic variation since these are the most likely source of human outbreaks. For T. cruzi, this rationale must also extend to wild reservoir hosts. Many, especially opportunistic scavengers like D. marsupialis, also come into close contact with humans, either directly, or via infected silvatic vector species. In areas now free or without a history of vectorial domestic transmission, oral outbreaks are a growing concern [18]. High-resolution population genetic studies of other parasitic zoonoses have facilitated epidemiological tracking of human disease outbreaks, with obvious implications for the planning of effective disease control [19],[20]. Molecular methods transformed our early understanding of T. cruzi epidemiology, with the revelation that distinct transmission cycles (domestic/silvatic) could harbour different major lineages of parasite [21]. Predominantly clonal propagation observed in T. cruzi is in keeping with this result, where micro-endemic clones with characteristic host propensities, geographic distribution, medical significance and biological attributes should exist within the parasite population [22]. However, widespread multi-host T. cruzi lineages like TcI persist outside of this paradigm. With the advent of the T. cruzi genome [23], the stage is now set to re-examine the micro-epidemiology of human disease outbreaks in TcI in the context of ultra-high resolution genetic analysis and, crucially, silvatic parasite populations. In this study we have developed a multilocus microsatellite typing (MLMT) system for TcI and applied it to parasite isolates from throughout the Americas. While this is among the largest panel of isolates from a single DTU ever analysed, sample sizes are still restrictive. Similarly, widespread deviation from Mendelian sexuality in T. cruzi limits the inferences that can be made from standard population genetic analyses. To circumvent these issues, we largely avoided model-based population assignment protocols (e.g. Structure [24]). In spite of these limitations, we are able to identify key features of silvatic TcI populations and highlight population genetic processes that accompany a switch to the human host in two endemic areas. In doing so we show that the pattern of within-DTU parasite genetic diversity may contain vital epidemiological information in terms of control strategies, parasite pathogenesis and ultimately human disease. Results A final dataset comprising 12,329 alleles (excluding missing data) from 135 isolates was subjected to analysis. Most strains presented one or two alleles at each locus. Multiple (≥3) alleles were observed at a small proportion of loci (0.98%) and only among strains not biologically cloned. Multiclonality, rather than aneuploidy, was determined to be the major source of this phenomenon by reference to analysis of a subset of nine microsatellite loci across 211 clones taken from a subset of eight strains that demonstrated multiple alleles at individual loci in the uncloned state (data not shown). Samples were allocated to seven populations: North and Central American (AM North/Cen), Venezuelan silvatic (VEN silv), North Eastern Brazil (BRAZ North-East), Northern Bolivia (BOL North), Northern Argentina (ARG North), Bolivian and Chilean Andes (ANDES Bol/Chile) and Venezuelan domestic (VEN dom). A full list of sample allocations is included in Table S2 and the rationale for the assignment of individuals to populations is detailed in the Methods section. Genetic diversity and rare allele frequency distributions Greatest genetic diversity was observed in populations drawn from palm and lowland moist forest associated ecotopes in VEN silv, BRAZ North-East and BOL North (Allelic richness (Ar) = 2.229–2.344, Table 1). Small, genetic-drift prone populations lose rare alleles at a faster rate than they can be replenished by mutation. Poisson-distributed rare allele frequency plots for VEN silv, BRAZ North-East and BOL North are, instead, characteristic of populations with a large, stable N e at mutation-drift equilibrium (Figure S1) [25]. It is of note that patterns of both allelic richness and rare allele distribution are consistent across VEN silv (n = 37) BRAZ North-East (n = 39) and BOL North (n = 16), largely independent of sample size (Table 1, Figure S1). Additionally, the size of geographic focus had little relevance in determining the amount of diversity present in these populations. A marginal reduction in allelic richness, for example, was observed between BRAZ North-East and BOL North (Ar = 2.344–2.229, Table 1), despite a massive reduction in sampling area (∼4,500,000 km2–10 km2). 10.1371/journal.ppat.1000410.t001 Table 1 Population genetic parameters for seven TcI populations. Population N/G MNA Ar a HO b HE b % HDc %HEd IA e P-Valuef AM North/Cen 7/7 1.92 1.532 0.332 0.445 0.00 0.00 2.39 0.005 VEN silv 37/37 6.45 2.337 0.449 0.637 44.19 0.00 1.38 ANDES Bol+VEN dom) generated by random shuffling of alleles between groups, was negative (p = 0.0639), albeit marginally, but suggests that direct comparisons of overall heterozygosity levels between these population groups should be approached with caution. 10.1371/journal.ppat.1000410.g001 Figure 1 Unrooted neighbour-joining D AS tree showing TcI population structure across the Americas. Based on the multilocus microsatellite profiles of 135 TcI isolates. D AS values were calculated as the mean across 1,000 random diploid re-samplings of the dataset to accommodate multi-allelic loci. The presence of more than two alleles per locus did not disrupt the delineation of major clades (>90% majority consensus support). D AS-based bootstrap values were calculated over 10,000 trees from 100 re-sampled datasets, and those >75% are shown on major clades. Branch colour codes indicate strain origin. Black: Didelphis species; purple: non-Didelphis mammalian reservoir; green: silvatic triatomine; red: human; blue: domestic triatomine. Colored block arrows and circles indicate broad population types. Yellow: Venezuelan domestic and North/Central American groups; green: major silvatic populations; blue: South-Western clade. Black arrow indicates Colombian outlier assigned to Brazilian population. Human symbol indicates putative genetic association with domestic transmission. Closed red circle area is proportionate to sampling density. See text for details of population codes. F IS values were also analysed by syntenous sequence fragment (SSF) (as defined by the CL-Brener genome project; no chromosomal assembly is currently available), of which nine are represented in our panel with ≥2 microsatellite loci (Table S3, Figure 2). Calculations included both large and small (‘true’) population groupings for comparison. Mean F IS values per SSF were consistently positive across major silvatic populations BOL North, VEN silv & BRAZ North-East. This provides support for heterozygote deficiency at the population level, but also for a consistent level of heterozygosity between fragments. The same is broadly true for AM North/Cen, concomitant with an increase in error associated with both a reduction in genetic diversity and sample size. F IS values for sub-population groupings from BOL North (BOL North 1 & BOL North 2) and VEN silv (VEN silv 3) reflect those of their source populations. A marginal decrease in F IS across some SSFs could be attributed to a Wahlund effect, and not uniquely to error, but major inconsistencies were not observed. In contrast, high inter-SSF variance was observed in both ANDES Bol/Chil and VEN dom, and to a lesser extent ARG North, with some strongly negative values regardless of an increase in error about the mean. These data provide support for a distinction between these populations and those exclusively from the silvatic environment. At the sub-population level, the exclusion of Chilean isolates from ANDES Bol did not have a major impact on the derived values, although error in this case was extremely high. 10.1371/journal.ppat.1000410.g002 Figure 2 Mean F IS values across loci on nine syntenous sequence fragments (SSFs) examined in eleven populations. Values suggest that gene conversion is a genomically diffuse process in homozygous silvatic populations. Error bars represent +/−standard error about the mean. Values without error bars correspond to SSFs containing only a single variable locus. Missing values correspond to SSFs containing no variable loci. Populations with postfix 1,2,3,4 are subsamples of larger populations. Numbers in parentheses indicate population size (n). Pair-wise measure of genetic distance Figure 1 shows a Neighbor-joining tree based on pair-wise D AS measures between individual isolates. Good bootstrap support was found for the grouping of isolates from VEN dom and AM North/Cen (88.5%), for subdivision within Argentinean isolates (100%), for subdivision within BOL North (92.5%), as well as for the grouping of isolates obtained from the Bolivian and Chilean Andes. In the silvatic environment no clear diversification was observed by reservoir host, a phenomenon supported by a non-significant estimate of F ST between Didelphis sp. and non-Didelphis sp. reservoir hosts in BRAZ North-East (F ST = 0.006, p = 0.594). Sample size restricts similar comparisons in other silvatic populations. A portion of the pair-wise genetic diversification observed in the dataset could be attributed to isolation by distance (IBD). A Mantels test for matrix correspondence between pair-wise genetic (D AS) and geographic distance (km) revealed a highly significant positive correlation between these two measures (RXY = 0.394, p 5000 km apart. Linkage disequilibrium in TcI populations Accounting for known physical linkage and excluding loci of unknown linkage group, the level of multilocus linkage disequilibrium was assessed using the IA, and was found to be statistically greater than a null distribution generated from 1000 random permutations in all populations (Table 1). Thus, the current dataset is consistent with predominant clonality in this parasite. Discussion This study represents the most comprehensive attempt to document within-DTU diversity in T. cruzi to date. Nonetheless, some sample sizes remain limiting in population genetic terms, although efforts were made to correct for any confounding effects. Similarly, caution is required given the deviation of T. cruzi from the assumptions of most standard population genetic models due to clonality. Certainly, high levels of genetic diversity in the principal silvatic TcI populations examined in this study are consistent with the putative ancient (3–16 MYA) origin of this DTU [1]. Similarly, rare allele frequency plots are consistent with a large, stable Ne [25]. Furthermore, we have shown that similar diversity indices could be derived from a study area of 10 km2 (BOL North) as from one of 4,500,000 km2 (BRAZ North-East), which suggests that this study has barely scraped the surface of the total circulating diversity present. In the silvatic environment, no apparent component of this diversity is partitioned by host. Thus, a constrained, extant co-evolutionary relationship is not compatible with the current dataset; contrary to a recent study using mini-exon sequence data from a limited number of Didelphis TcI strains [13]. Previously, we have suggested the ecological niche, rather than reservoir host, plays the dominant role in driving T. cruzi diversification [7]. This reflects a current model for wider trypanosome evolution, where “ecological host-fitting” is thought to define parasite clades [27]. Low levels of subdivision (F ST) between three populations sharing a similar ecotope across Amazonia are consistent with this supposition. While we demonstrate that TcI is eclectic in terms of host in arboreal lowland silvatic cycles, significant documentary evidence exists to suggest that D. marsupialis is the major carrier throughout much of lowland tropical South and Central America [7]. The majority of isolates examined here originate from this host. Tolerance by this species of high circulating parasitemia [28], as well as a possible propensity for non-vectorial transmission via infected territorial anal scent gland secretions [29], may predispose D. marsupialis to particularly intense T. cruzi transmission. Nonetheless, numerous vectors and secondary hosts are also implicated in TcI transmission and carriage [7],[30], and parasite dispersal between geographic foci is unlikely to be linked to D. marsupialis alone. Continental scale spatial structure in silvatic TcI (Figure 3) fits with the general ecology of undisturbed wild transmission. Most triatomine vectors, for example, are ill-adapted to long-range flight, and are thus incapable of rapid parasite dispersal between distant foci, providing ample time for spatial differentiation to occur among parasite populations. Sample size corrected genetic diversity estimates suggest a considerable reduction in genetic differentiation in AM North/Cen with respect to core silvatic populations. Furthermore, IBD breaks down among these isolates and a loss of rare alleles in this population could be interpreted as evidence of a recent population bottleneck [25]. Until recently, genetic studies of TcI diversity have failed to detect the signature of a rapid biogeographic expansion of this DTU into the USA [31]. Our findings are bolstered by low genetic diversity identified among new mini-exon sequence data derived from North and Central American TcI isolates [13], but greater sampling from this region would confirm our observations. The expansion of TcI into North and Central America is likely to have occurred since the formation of the Isthmus of Panama 2–4 MYA, providing a useful phylogeographic calibration point for future studies, and may correspond to the northerly migration of didelphid marsupials [32]. In this study, TcI strains from infected humans were sampled widely in Venezuela (Table S2). Although their sample size is currently limited (n = 15 for the domestic clade – includes one vector isolate (Table S2)), their robust genetic clustering, by comparison to the extensively sampled and genetically diverse parasite population from the silvatic environment, serves to make them representative and important. There are suggestions that Chagas disease is locally resurgent [33], and genetic discontinuity between the domestic population and most silvatic isolates raises significant questions regarding human disease transmission. Molecular data from the low-lying west of the country demonstrates that most silvatic and domestic populations of the principal vector, Rhodnius prolixus, are indistinguishable [34] and it follows that the parasite should also be invasive. However, in our study, the predominant T. cruzi strains infecting humans in the same and nearby areas bear little resemblance to those in the silvatic environment. Intriguingly, however, silvatic TcI genotypes prevail among almost all adult intradomiciliary triatomines sampled. All three triatomine species, Triatoma maculata, Panstrongylus geniculatus, and R. prolixus are also described from the silvatic environment in Venezuela [3] and could, therefore, be invasive, and the parasite strains infecting them not of human origin. The occurrence of a domestic TcI clade in Venezuela, in spite of the presence of silvatic strains inside houses, presents an interesting problem. Among African trypanosomes (T. brucei sspp.), human infective forms display only a limited array of genotypes (T. b. rhodesiense & gambiense [20],[35]). Detailed studies of T. b. brucei population genetics in the silvatic environment are, however, lacking. Some evidence suggests that vectors and domestic mammalian reservoirs in T. b. brucei populations sympatric with human T. b. rhodesiense outbreaks support a greater diversity of strains [20]. However, no specific genes associated with human infectivity are known in T. cruzi, unlike in T. b. rhodesiense [36], that might drive the domestic expansion of an epidemic clone. Furthermore, silvatic-type TcI strains were capable of sustaining long-term, symptomatic infection in a subset of patients studied (Table S2). One possible confounder in our sampling, as in a recent population study of strains from West African T. b. gambiense symptomatic human infections [35], is a lack of samples from asymptomatic patients, which are required to refute an association between parasite genotype and virulence or pathogenicity. In the absence of a clear adaptive explanation for the lack of diversification among Venezuelan domestic isolates on the basis of current data, an ecological one may be more parsimonious. Low transmission of the parasite to the human host by invasive adult triatomines may reflect the inefficient stercorarian route by which T. cruzi is normally spread [2]. Instead, repeated blood meals taken by domestic triatomine colonies may be necessary to ensure infective contact with the human host. In this case, other humans or domestic reservoirs will be the primary sources of human infection, human and domestic vector migration the main driver of parasite dispersal, and a widespread, uniform domestic parasite genotype the result. This is an observation supported by a lack of IBD among domestic strains. The distribution of this genotype may be wider than described here, and there is now preliminary mini-exon sequence evidence that a domestic TcI genotype may also occur in Colombia [14]. The origin of the divergent Venezuelan human TcI population remains enigmatic. Isolates bear closest resemblance, by all measures employed in this study, to the North and Central American clade. In all likelihood, TcI populations migrated to the North prehistorically in conjunction with invasive mammalian reservoir hosts during the Great American Interchange [32]. Low genetic diversity is also identified in domestic R. prolixus populations from Central America [37], although presumably their northerly migration occurred many thousands of years later alongside human populations. It is highly improbable that domestic TcI strains carried northwards with R. prolixus subsequently dispersed so widely into the silvatic environment. The source of the domestic outbreak identified here probably remains sequestered among silvatic transmission cycles somewhere in the northerly distribution of TcI in South America. A greater sampling effort is required around Cochabamba (ANDES Bol) from both human and wild reservoirs before satisfactory conclusions can be drawn regarding local parasite transmission. Intriguingly, temporal heterogeneity seems to be negligible, and ∼20 years separate the isolation of human and rodent strains (Table S2). Epidemiologically, congruence between populations from these two hosts is not unexpected. Local domestic and silvatic T. infestans populations match genetically and morphologically [38], and rodent isolates were collected within two kilometres of a major suburb of Cochabamba, where active urban transmission still occurs [39]. It is not clear, however, whether the parasite is invasive to the domestic setting, or whether domestic strains have re-invaded the silvatic cycle. A major observation of this study, and in others examining genetic diversity in T. cruzi [1],[4],[15], is the deficiency of heterozygosity with respect to Hardy-Weinberg expectations observed in most populations. Similar observations are frequently made in the Leishmania spp. populations [40]–[42]. These levels of homozygosity are atypical with respect to other clonally reproducing diploids [35],[43],[44], where diversity is known to accumulate between alleles within the individual in the absence of recombination, leading to extreme levels of heterozygosity at homologous loci (the ‘Meselson effect’ [45]). Heterozygous deficiency in silvatic populations in our dataset cannot be uniquely attributed to hidden subdivision (Walhund effect). We still find positive F IS values in non-subdivided sub-samples of isolates within populations. Here, some increase in heterozygosity was observed (Figure 2), but not to the extent predicted by the Meselson effect. Multilocus linkage disequilibrium suggests that recombination is at most infrequent in the current dataset, although the Index of Association [46] is a relatively insensitive measure [44]. Thus, widespread loss of heterozygosity due to homologous recombination or gene conversion, not inbreeding, is the most likely genetic phenomenon that would result in the observed diversity in our data. Importantly, we can show that these events are apparently genomically diffuse, in silvatic populations at least. Most SSFs show similar levels of heterozygosity within populations, rather than some showing strong evidence of the Meselson effect (strongly negative F IS) and others showing complete homozygosity, as would be expected of larger scale effects like ploidy cycles [47] or those following genome fusion events in yeast [48]. Populations ANDES Bol/Chil and VEN Dom share many features in population genetic terms: reduced diversity; non-equilibrium rare allele frequencies; and high inter-SSF variance in F IS values where strongly negative values on some SSFs reflect marginally raised overall heterozygosity at the population level. It remains to be seen whether these are unique characteristics of human TcI clades, whether they reflect possible past recombination events or some form of balancing selection, and we could not attribute significance to a decrease in F IS from background levels. DTUs TcIId and TcIIe both show fixed heterozygosity at most loci because they are almost certainly hybrids [1],[5], not due to the Meselson effect, and far in excess of heterozygosity levels observed in our dataset. Confirmation of the characteristics we have observed will come with more intensive sampling from domestic foci in both regions, as well as others across South America. Our data now show, with increasing support from other studies [13],[14],[49],[50], that most T. cruzi lineages actually represent highly heterogeneous populations across their distribution, heterogeneity that may be highly informative in epidemiological terms. Control strategies would now greatly benefit from high density parasitological surveys in and around individual endemic disease foci, especially if a pathogenic human TcI genotype does exist, signalling a return in study design, if not methodology, to the early investigations of the 1970s [21]. Such studies should include parasite samples from silvatic mammals and vectors, as well as domestic sources, including both symptomatic and asymptomatic (or indeterminate) human cases. To this extent, using microsatellite markers developed here, T. cruzi population genetics can be observed at the finest scale and provide real insights into the true nature of Chagas disease transmission. Methods We assembled a panel of 135 T. cruzi samples belonging to TcI from throughout the silvatic distribution of this lineage (Table S2). DTU-level genotyping was achieved through analysis of the non-transcribed spacer region of the mini-exon gene, as described previously [51]. Microsatellite motifs were extracted from the draft sequence of the T. cruzi genome available at http://www.genedb.org. Four Mb of sequence, including at least 13 syntenous sequence fragments, were scanned for di- and tri-nucleotide repeats using a pattern matching script written in sed. An extension of the algorithm was included to extract the up and downstream flanking regions of the microsatellite sequence (∼200 bp). Primer design was achieved in PRIMER3 [52]. Among 200 microsatellite loci identified, 45 were polymorphic. A further three were included from two previous studies [6],[15]. Primers and binding sites are listed in Table S3. The following reaction cycle was implemented across all loci: a denaturation step of 4 minutes at 95°C, then 30 amplification cycles (95°C for 20 seconds, 57°C for 20 seconds, 72°C for 20 seconds) and a final 20 minute elongation step at 72°C. With a final volume of 10 ul, 1× ThermoPol Reaction Buffer (New England Biolabs (NEB), UK), 4 mM MgCl2, 34 uM dNTPs; 0.75 pmols of each primer, 1 unit of Taq polymerase (NEB, UK) and 1 ng of genomic DNA were added. Five fluorescent dyes were used to label forward primers – 6-FAM & TET (Proligo, Germany), NED, PET & VIC (Applied Biosystems, UK). Allele sizes were determined using an automated capillary sequencer (AB3730, Applied Biosystems, UK), manually checked for errors and typed “blind” to control for user bias. Microsatellite diversity analysis Allelic richness estimates were calculated in FSTAT 2.9.3.2 [53] and corrected for sample size using Hurlbert's rarefaction method [54] in MolKin v3.0 [55]. Pair-wise estimates of population subdivision (F ST, Table S1) and heterozygosity indices (Table 1) were estimated in ARLEQUIN 3.0 [56]. P-values for multiple tests were corrected using a sequential Bonferroni correction [57]. F IS provides an alternative measure of heterozygosity by assessing the level of identity of alleles within individuals compared to that between individuals where +1 represents all individuals homozygous for different alleles, and −1 all individuals heterozygous for the same alleles. Mean F IS estimates over loci in selected groups of sub-populations were calculated in FSTAT 2.9.3.2 using Weir and Cockerman's (1984) unbiased estimators [58]. Confidence intervals for F IS estimates were calculated by bootstrapping over loci and tests for significant differences between values also in FSTAT 2.9.3.2 using 10,000 random permutations. Mean F IS values per sequence fragment per population were calculated across standard (not Weir and Cockerman's) F IS values in FSTAT 2.9.3.2. To assess the level of multilocus linkage disequilibrium, the Index of Association (IA, multilocus) was calculated in MULTILOCUS 1.3b [46],[59] (Table 1). Genetic distances between isolates were evaluated in MICROSAT under an infinite alleles model of microsatellite evolution using D AS (1-proportion of shared alleles at all loci / n) [60] (Figure 1). To accommodate multi-allelic loci, a script was written in Microsoft Visual Basic to make multiple random diploid re-samplings of each multilocus profile (software available on request). Individual-level genetic distances were calculated as the mean across multiple re-sampled datasets. A single randomly sampled dataset was used for population-level analysis. A Mantel's test for matrix correspondence was executed in GENALEX 6 to compare pair-wise geographical (km) and genetic distance (D AS) [61] (Figure 3). Samples were assigned to populations on an a priori basis according to geography and transmission cycle. D AS - defined sample clustering was also used to inform population identity, and obvious outliers assigned to the correct genetic group (Figure 1). Rare allele frequency plots were calculated as in Luikart et al., 1998 [25], to detect perturbation following putative population events (e.g. population bottlenecks). Supporting Information Figure S1 Allele frequency classes among seven TcI populations. (6.79 MB TIF) Click here for additional data file. Table S1 FST estimates of interpopulation differentiation for seven TcI subpopulations based on microsatellite data. (0.04 MB DOC) Click here for additional data file. Table S2 Panel of T. cruzi TcI genotype isolates assembled for microsatellite analysis. (0.31 MB DOC) Click here for additional data file. Table S3 Microsatellite loci and primers employed in this study. (0.12 MB DOC) Click here for additional data file.
Author and article information
Journal
Journal ID (nlm-ta): Mem Inst Oswaldo Cruz
Journal ID (iso-abbrev): Mem. Inst. Oswaldo Cruz
Title:
Memórias do Instituto Oswaldo Cruz
Publisher:
Instituto Oswaldo Cruz, Ministério da Saúde
ISSN
(Print):
0074-0276
ISSN
(Electronic):
1678-8060
Publication date
(Print and electronic):
May
2015
Publication date
(Print):
May
2015
Volume: 110
Issue: 3
Pages: 387-393
Affiliations
Author notes
Article
DOI: 10.1590/0074-02760140402
PMC ID: 4489476
PubMed ID: 25946157
SO-VID: f37bae8e-62b3-45cd-a202-cd2adbde09cb
License:
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License, which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
History
Date
received
: 30
October
2014
Date
accepted
: 25
February
2015
Page count
Figures: 4,
Tables: 1,
Equations: 0,
References: 38,
Pages: 1
Funding
Funded by: COLCIENCIAS, Red Chagas
Award ID: 380-2011/5014-537-30398
Financial support: COLCIENCIAS, Red Chagas
(380-2011/5014-537-30398)
Categories
Subject:
Articles
Data availability: