A Novel Genotype and First Record of Trypanosoma lainsoni in Argentina.

Trypanosomes are a group of parasitic flagellates with medical and veterinary importance. Despite many species having been described in this genus, little is known about many of them. Here, we report a genetic and morphological characterization of trypanosomatids isolated from wild mammals from the Argentine Chaco region. Parasites were morphologically and ultrastructurally characterized by light microscopy and transmission electron microscopy. Additionally, 18s rRNA and gGAPDH genes were sequenced and analyzed using maximum likelihood and Bayesian inference. Morphological characterization showed clear characteristics associated with the Trypanosoma genus. The genetic characterization demonstrates that the studied isolates have identical sequences and a pairwise identity of 99% with Trypanosoma lainsoni, which belongs to the clade of lizards and snakes/rodents and marsupials. To date, this species had only been found in the Amazon region. Our finding represents the second report of T. lainsoni and the first record for the Chaco region. Furthermore, we ultrastructurally described for the first time the species. Finally, the host range of T. lainsoni was expanded (Leopardus geoffroyi, Carenivora, Felidae; and Calomys sp., Rodentia, Cricetidae), showing a wide host range for this species.

Evidence of hybridization, mitochondrial introgression and biparental inheritance of the kDNA minicircles in Trypanosoma cruzi I.

BACKGROUND: Genetic exchange in Trypanosoma cruzi is controversial not only in relation to its frequency, but also to its mechanism. Parasexual genetic exchange has been proposed based on laboratory hybrids, but population genomics strongly suggests meiosis in T. cruzi. In addition, mitochondrial introgression has been reported several times in natural isolates although its mechanism is not fully understood yet. Moreover, hybrid T. cruzi DTUs (TcV and TcVI) have inherited at least part of the kinetoplastic DNA (kDNA = mitochondrial DNA) from both parents. METHODOLOGY/PRINCIPAL FINDINGS: In order to address such topics, we sequenced and analyzed fourteen nuclear DNA fragments and three kDNA maxicircle genes in three TcI stocks which are natural clones potentially involved in events of genetic exchange. We also deep-sequenced (a total of 6,146,686 paired-end reads) the minicircle hypervariable region (mHVR) of the kDNA in such three strains. In addition, we analyzed the DNA content by flow cytometry to address cell ploidy. We observed that most polymorphic sites in nuclear loci showed a hybrid pattern in one cloned strain and the other two cloned strains were compatible as parental strains (or nearly related to the true parents). The three clones had almost the same ploidy and the DNA content was similar to the reference strain Sylvio (a nearly diploid strain). Despite maxicircle genes evolve faster than nuclear housekeeping ones, we detected no polymorphisms in the sequence of three maxicircle genes showing mito-nuclear discordance. Lastly, the hybrid stock shared 66% of its mHVR clusters with one putative parent and 47% with the other one; in contrast, the putative parental stocks shared less than 30% of the mHVR clusters between them. CONCLUSIONS/SIGNIFICANCE: The results suggest a reductive division, a natural hybridization, biparental inheritance of the minicircles in the hybrid and maxicircle introgression. The models including such phenomena and explaining the relationships between these three clones are discussed.

Elucidating diversity in the class composition of the minicircle hypervariable region of Trypanosoma cruzi: New perspectives on typing and kDNA inheritance.

BACKGROUND: Trypanosoma cruzi, the protozoan causative of Chagas disease, is classified into six main Discrete Typing Units (DTUs): TcI-TcVI. This parasite has around 105 copies of the minicircle hypervariable region (mHVR) in their kinetoplastic DNA (kDNA). The genetic diversity of the mHVR is virtually unknown. However, cross-hybridization assays using mHVRs showed hybridization only between isolates belonging to the same genetic group. Nowadays there is no methodologic approach with a good sensibility, specificity and reproducibility for direct typing on biological samples. Due to its high copy number and apparently high diversity, mHVR becomes a good target for typing. METHODOLOGY/PRINCIPAL FINDINGS: Around 22 million reads, obtained by amplicon sequencing of the mHVR, were analyzed for nine strains belonging to six T. cruzi DTUs. The number and diversity of mHVR clusters was variable among DTUs and even within a DTU. However, strains of the same DTU shared more mHVR clusters than strains of different DTUs and clustered together. In addition, hybrid DTUs (TcV and TcVI) shared similar percentages (1.9-3.4%) of mHVR clusters with their parentals (TcII and TcIII). Conversely, just 0.2% of clusters were shared between TcII and TcIII suggesting biparental inheritance of the kDNA in hybrids. Sequencing at low depth (20,000-40,000 reads) also revealed 95% of the mHVR clusters for each of the analyzed strains. Finally, the method revealed good correlation in cluster identity and abundance between different replications of the experiment (r = 0.999). CONCLUSIONS/SIGNIFICANCE: Our work sheds light on the sequence diversity of mHVRs at intra and inter-DTU level. The mHVR amplicon sequencing workflow described here is a reproducible technique, that allows multiplexed analysis of hundreds of strains and results promissory for direct typing on biological samples in a future. In addition, such approach may help to gain knowledge on the mechanisms of the minicircle evolution and phylogenetic relationships among strains.

Experimental evidence of biological interactions among different isolates of Trypanosoma cruzi from the Chaco Region.

Many infectious diseases arise from co-infections or re-infections with more than one genotype of the same pathogen. These mixed infections could alter host fitness, the severity of symptoms, success in pathogen transmission and the epidemiology of the disease. Trypanosoma cruzi, the etiological agent of Chagas disease, exhibits a high biological variability often correlated with its genetic diversity. Here, we developed an experimental approach in order to evaluate biological interaction between three T. cruzi isolates belonging to different Discrete Typing Units (DTUs TcIII, TcV and TcVI). These isolates were obtained from a restricted geographical area in the Chaco Region. Different mixed infections involving combinations of two isolates (TcIII + TcV, TcIII + TcVI and TcV + TcVI) were studied in a mouse model. The parameters evaluated were number of parasites circulating in peripheral blood, histopathology and genetic characterization of each DTU in different tissues by DNA hybridization probes. We found a predominance of TcVI isolate in blood and tissues respect to TcIII and TcV; and a decrease of the inflammatory response in heart when the damage of mice infected with TcVI and TcIII + TcVI mixture were compared. In addition, simultaneous presence of two isolates in the same tissue was not detected. Our results show that biological interactions between isolates with different biological behaviors lead to changes in their biological properties. The occurrence of interactions among different genotypes of T. cruzi observed in our mouse model suggests that these phenomena could also occur in natural cycles in the Chaco Region.

Trypanosoma cruzi diversity in the Gran Chaco: mixed infections and differential host distribution of TcV and TcVI.

The transmission cycles of Trypanosoma cruzi in the Gran Chaco are complex networks involving domestic and wild components, whose interrelationships are not well understood. Knowing the circuit of transmission of the different Discrete Typing Units (DTUs) of T. cruzi in the complex environment of the Chaco region is relevant to understanding how the different components (reservoirs, vectors, ecotopes) interact. In the present study we identified the DTUs infecting humans and dogs in two rural areas of the Gran Chaco in Argentina, using molecular methods which avoid parasite culture. Blood samples of humans and dogs were typified by PCR-DNA blotting and hybridization assays with five specific DNA probes (TcI, TcII, TcIII, TcV and TcVI). PCR analyses were performed on seropositive human and dog samples and showed the presence of T. cruzi DNA in 41.7% (98/235) and 53% (35/66) samples, respectively. The identification of infective DTUs was determined in 83.6% (82/98) and 91.4% (32/35) in human and dog samples, respectively. Single infections (36.7% - 36/98) and a previously not detected high proportion of mixed infections (47.9% - 47/98) were found. In a 15.3% (15/98) of samples the infecting DTU was not identified. Among the single infections TcV was the most prevalent DTU (30.6% - 30/98) in human samples; while TcVI (42.8% - 15/35) showed the highest prevalence in dog samples. TcV/TcVI was the most prevalent mixed infection in humans (32.6% - 32/98); and TcI/TcVI (14.3% - 5/35) in dogs. Significant associations between TcV with humans and TcVI with dogs were detected. For the first time, the presence of TcIII was detected in humans from this region. The occurrence of one human infected whit TcIII (a principally wild DTU) could be suggested the emergence of this, in domestic cycles in the Gran Chaco.

Preponderant clonal evolution of Trypanosoma cruzi I from Argentinean Chaco revealed by Multilocus Sequence Typing (MLST).

Trypanosoma cruzi has been historically classified as a species with preponderant clonal evolution (PCE). However, with the advent of highly polymorphic markers and studies at geographically reduced scales, the PCE in T. cruzi was challenged. In fact, some studies have suggested that recombination in T. cruzi lineage I (TcI) is much more frequent than previously believed. Further analyses of TcI populations from different geographical regions of Latin America are needed to examine this hypothesis. In the present study, we contribute to this topic by analyzing the population structure of TcI from a restricted geographical area in the Chaco region, Argentina. We analyzed TcI isolates from different hosts and vectors using a Multilocus Sequence Typing (MLST) approach. These isolates were previously characterized by sequencing the spliced leader intergenic region (SL-IR). Low levels of incongruence and well-supported clusters for MLST dataset were obtained from the analyses. Moreover, high linkage disequilibrium was found and five repeated and overrepresented genotypes were detected. In addition, a good correspondence between SL-IR and MLST was observed which is expected under PCE. However, recombination is not ruled out because five out of 28 pairs of loci were incompatible with strict clonality and one possible genetic exchange event was detected. Overall, our results represent evidence of PCE in TcI from the study area. Finally, considering our findings we discuss the scenario for the genetic structure of TcI.

Biological behavior of different Trypanosoma cruzi isolates circulating in an endemic area for Chagas disease in the Gran Chaco region of Argentina.

The biological behavior of the different Trypanosoma cruzi strains is still unclear and the importance of exploring the relevance of these differences in natural isolates is of great significance. Herein we describe the biological behavior of four T. cruzi isolates circulating sympatrically in a restricted geographic area in Argentina endemic for Chagas Disease. These isolates were characterized as belonging to the Discrete Typing Units (DTUs) TcI, TcIII, TcV and TcVI as shown by Multilocus Enzyme Electrophoresis and Multilocus Sequence Typing. In order to study the natural behavior of the different isolates and to preserve their natural properties, we developed a vector transmission model that allows their maintenance in the laboratory. The model consisted of serial passages of these parasites between insect vectors and mice. Vector-derived parasite forms were then inoculated in C57BL/6J mice and number of parasite in peripheral blood, serological response and histological damage in acute and chronic phases of the infection were measured. Parasites from DTUs TcI, TcIII and TcVI were detected by direct fresh blood examination, while TcV parasites could only be detected by Polimerase Chain Reaction. No significant difference in the anti-T. cruzi antibody response was found during the chronic phase of infection, except for mice infected with TcV parasites where no antibodies could be detected. Histological sections showed that TcI isolate produced more damage in skeletal muscle while TcVI induced more inflammation in the heart. This work shows differential biological behavior among different parasite isolates obtained from the same cycle of transmission, permitting the opportunity to formulate future hypotheses of clinical and epidemiological importance.

Candidate targets for Multilocus Sequence Typing of Trypanosoma cruzi: validation using parasite stocks from the Chaco Region and a set of reference strains.

A Multilocus Sequence Typing (MLST) scheme was designed and applied to a set of 20 Trypanosoma cruzi stocks belonging to three main discrete typing units (T. cruzi I, V and VI) from a geographically restricted Chagas disease endemic area in Argentina, 12 reference strains comprising two from each of the six main discrete typing units of the parasite (T. cruzi I-VI), and one T. cruzi marinkellei strain. DNA fragments (≅400-bp) from 10 housekeeping genes were sequenced. A total of 4178 bp were analyzed for each stock. In all, 154 polymorphic sites were identified. Ninety-five sites were heterozygous in at least one analyzed stock. Seventeen diploid sequence types were identified from 32 studied T. cruzi stocks (including the reference strains). All stocks were correctly assigned to their corresponding discrete typing units. We propose this MLST scheme as provisional, with scope for improvement by studying new gene targets on a more diverse sample of stocks, in order to define an optimized MLST scheme for T. cruzi. This approach is an excellent candidate to become the gold standard for T. cruzi genetic typing. We suggest that MLST will have a strong impact on molecular epidemiological studies of Chagas disease and the phylogenetics of its causative agent.

Interest and limitations of Spliced Leader Intergenic Region sequences for analyzing Trypanosoma cruzi I phylogenetic diversity in the Argentinean Chaco.

Internal and geographical clustering within Trypanosoma cruzi I (TcI) has been recently revealed by using Multilocus Microsatellite Typing and sequencing of the Spliced-Leader Intergenic Region (SL-IR). In the present work, 14 isolates and 11 laboratory-cloned stocks obtained from a geographically restricted area in Chaco Province, Argentina, were analyzed by PCR and sequencing of SL-IR. We were able to differentiate 8 different genotypes that clustered into 4 groups. One of these groups was classified within the formerly described haplotype A and another one within the recently described SL-IR group E. Both were phylogenetically well-supported. In contrast, none of the stocks from the Chaco province were grouped within the cluster previously named haplotype D despite the fact that they shared a similar microsatellite motif in the SL-IR. No evidence of recombination or gene conversion within these stocks was found. On the other hand, multiple ambiguous alignments in the microsatellite region of SL-IR, affecting the tree topology and relationships among groups were detected. Finally, since there are multiple copies of the SL-IR, and they are arranged in tandem, we discuss how molecular processes affecting this kind of sequences could mislead phylogenetic inference.