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The model of predominant clonal evolution (PCE) proposed for micropathogens does not state that genetic exchange is totally absent, but rather, that it is too rare to break the prevalent PCE pattern. However, the actual impact of this "residual" genetic exchange should be evaluated. Multilocus Sequence Typing (MLST) is an excellent tool to explore the problem. Here, we compared online available MLST datasets for seven eukaryotic microbial pathogens: Trypanosoma cruzi, the Fusarium solani complex, Aspergillus fumigatus, Blastocystis subtype 3, the Leishmania donovani complex, Candida albicans and Candida glabrata. We first analyzed phylogenetic relationships among genotypes within each dataset. Then, we examined different measures of branch support and incongruence among loci as signs of genetic structure and levels of past recombination. The analyses allow us to identify three types of genetic structure. The first was characterized by trees with well-supported branches and low levels of incongruence suggesting well-structured populations and PCE. This was the case for the T. cruzi and F. solani datasets. The second genetic structure, represented by Blastocystis spp., A. fumigatus and the L. donovani complex datasets, showed trees with weakly-supported branches but low levels of incongruence among loci, whereby genetic structuration was not clearly defined by MLST. Finally, trees showing weakly-supported branches and high levels of incongruence among loci were observed for Candida species, suggesting that genetic exchange has a higher evolutionary impact in these mainly clonal yeast species. Furthermore, simulations showed that MLST may fail to show right clustering in population datasets even in the absence of genetic exchange. In conclusion, these results make it possible to infer variable impacts of genetic exchange in populations of predominantly clonal micro-pathogens. Moreover, our results reveal different problems of MLST to determine the genetic structure in these organisms that should be considered.

Background: Trypanosoma cruzi, the etiological agent of Chagas Disease, exhibits remarkable genetic diversity and is classified into different Discrete Typing Units (DTUs). Strain typing techniques are crucial for studying T. cruzi, because their DTUs have significant biological differences from one another. However, there is currently no methodological strategy for the direct typing of biological materials that has sufficient sensitivity, specificity, and reproducibility. The high diversity and copy number of the minicircle hypervariable regions (mHVRs) makes it a viable target for typing. Methodology/Principal findings: Approximately 24 million reads obtained by amplicon sequencing of the mHVR were analyzed for 62 strains belonging to the six main T. cruzi DTUs. To build reference databases of mHVR diversity for each DTU and to evaluate this target as a typing tool. Strains of the same DTU shared more mHVR clusters than strains of different DTUs, and clustered together. Different identity thresholds were used to build the reference sets of the mHVR sequences (85% and 95%, respectively). The 95% set had a higher specificity and was more suited for detecting co-infections, whereas the 85% set was excellent for identifying the primary DTU of a sample. The workflow's capacity for typing samples obtained from cultures, a set of whole-genome data, under various simulated PCR settings, in the presence of co-infecting lineages and for blood samples was also assessed. Conclusions/Significance: We present reference databases of mHVR sequences and an optimized typing workflow for T. cruzi including a simple online tool for deep amplicon sequencing analysis (https://ntomasini.github.io/cruzityping/). The results show that the workflow displays an equivalent resolution to that of the other typing methods. Owing to its specificity, sensitivity, relatively low cost, and simplicity, the proposed workflow could be an alternative for screening different types of samples. Author summary: Chagas disease, caused by the parasite Trypanosoma cruzi, is a significant public health concern in Latin America. This parasite is genetically diverse and classified into different lineages. Proper strain typing techniques are necessary to study T. cruzi, because their lineages have significant biological differences. Several typing methods have been proposed, each of which has its own strengths and limitations. However, most of these methods lack sensitivity or fail for discriminating some lineages. Genetic markers with high copy numbers are required to gain sensitivity. Here, we deep sequenced DNA regions present in the large mitochondrion of the parasite (mHVRs) from strains belonging to the six main lineages to obtain reference mHVR sequences and develop a typing workflow. Amplicon sequencing of mHVR was conducted on 62 T. cruzi strains. Despite high sequence diversity, strains of the same lineage shared more sequences than strains of different lineages. Two reference sets of mHVR sequences were generated and evaluated for their ability to typify distinct types of T. cruzi samples. The workflow presented in this study could serve as a valuable resource for T. cruzi typing in future studies. [ABSTRACT FROM AUTHOR]

Kinetoplastids are a diverse group of flagellates which exhibit editing by insertion/deletion of Us in the mitochondrial mRNAs. Some mRNAs require editing to build most of their coding sequences, a process known as pan-editing. Evidence suggests that pan-editing is an ancestral feature in kinetoplastids. Here, we investigate how the transition from nonedited to pan-edited states occurred. The mitochondrial mRNAs and protein sequences from nine kinetoplastids and related groups (diplonemids, euglenids, and jakobids) were analyzed. RNA editing increased protein hydrophobicity to extreme values by introducing Us in the second codon position, despite the absence of editing preferences related to codon position. In addition, hydrophobicity was maintained by purifying selection in species that lost editing by retroposition of the fully edited mRNA. Only a few hydrophobic to hydrophilic amino acid changes were inferred for such species. In the protein secondary structure, these changes occurred spatially close to other hydrophilic residues. The analysis of coevolving sites showed that multiple changes are required together for hydrophobicity to be lost, which suggest the proteins are locked into extended hydrophobicity. Finally, an analysis of the NAD7 protein–protein interactions showed they can also influence hydrophobicity increase in the protein and where editing can occur in the mRNA. In conclusion, our results suggest that protein hydrophobicity has influenced editing site selection and how editing expanded in mRNAs. In effect, the hydrophobicity increase was entrenched by a neutral ratchet moved by a mutational pressure to introduce Us, thus helping to explain both RNA editing increase and, possibly, persistence. [ABSTRACT FROM AUTHOR]

Trypanosoma cruzi , as other kinetoplastids, has a complex mechanism of editing of mitochondrial mRNAs that requires guide RNAs (gRNAs) coded in DNA minicircles in the kinetoplast. There are many variations on this mechanism among species. mRNA editing and gRNA repertoires are almost unknown in T. cruzi. Here, gRNAs were inferred based on deep-sequenced minicircle hypervariable regions (mHVRs) and editing cascades were rebuilt in strains belonging to the six main T. cruzi lineages. Inferred gRNAs were clustered according to their sequence similarity to constitute gRNA classes. Extreme diversity of gRNA classes was observed, which implied highly divergent gRNA repertoires among different lineages, even within some lineages. In addition, a variable gRNA class redundancy (i.e., different gRNA classes editing the same mRNA region) was detected among strains. Some strains had upon four times more gRNA classes than others. Such variations in redundancy affected gRNA classes of all mRNAs in a concerted way, i.e., there are correlated variations in the number of gRNAs classes editing each mRNA. Interestingly, cascades were incomplete for components of the respiratory complex I in several strains. Finally, gRNA classes of different strains may potentially edit mitochondrial mRNAs from other lineages in the same way as they edit their own mitochondrial mRNAs, which is a prerequisite for biparental inheritance of minicircle in hybrids. We propose that genetic exchange and biparental inheritance of minicircles combined with minicircle drift due to (partial) random segregation of minicircles during kDNA replication is a suitable hypothesis to explain the divergences among strains and the high levels of gRNA redundancy in some strains. In addition, our results support that the complex I may not be required in some stages in the life cycle as previously shown and that linkage (in the same minicircle) of gRNAs that edit different mRNAs may prevent gRNA class lost in such stage. [ABSTRACT FROM AUTHOR]

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. Author summary: Chagas disease, an important public health problem in Latin America, is caused by the parasite Trypanosoma cruzi. Despite being a widely studied parasite, several questions on the biology of genetic exchange remain unanswered. Population genomic studies have inferred meiosis in T. cruzi, but this cellular division mechanism has not been observed in laboratory yet. In addition, previous results suggest that mitochondrial DNA (called kDNA) may be inherited from both parents in hybrids. Here, we analyzed a hybrid strain and its potential parents to address the mechanisms of genetic exchange at nuclear and mitochondrial levels. We observed that the hybrid strain had heterozygous patterns and DNA content compatible with a meiosis event. Also, we observed that the evolutionary histories of nuclear DNA and kDNA maxicircles were discordant and that the three strains shared identical DNA sequences. Mitochondrial introgression of maxicircle DNA from one genotype to another may explain this observation. In addition, we demonstrated that the hybrid strain shared kDNA minicircles with both parental strains. Our results suggest that hybridization implied meiosis and biparental inheritance of the kDNA. Further research is required to address such phenomena in detail. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

People living in areas with active vector-borne transmission of Chagas disease have multiple contacts with its causative agent, Trypanosoma cruzi. Reinfections by T. cruzi are possible at least in animal models leading to lower or even hardly detectable parasitaemia. In humans, although reinfections are thought to have major public health implications by increasing the risk of chronic manifestations of the disease, there is little quantitative knowledge about their frequency and the timing of parasite re-inoculation in the course of the disease. Here, we implemented stochastic agent-based models i) to estimate the rate of re-inoculation in humans and ii) to assess how frequent are reinfections during the acute and chronic stages of the disease according to alternative hypotheses on the adaptive immune response following a primary infection. By using a hybrid genetic algorithm, the models were fitted to epidemiological data of Argentinean rural villages where mixed infections by different genotypes of T. cruzi reach 56% in humans. To explain this percentage, the best model predicted 0.032 (0.008–0.042) annual reinfections per individual with 98.4% of them occurring in the chronic phase. In addition, the parasite escapes to the adaptive immune response mounted after the primary infection in at least 20% of the events of re-inoculation. With these low annual rates, the risks of reinfection during the typically long chronic stage of the disease stand around 14% (4%-18%) and 60% (21%-70%) after 5 and 30 years, with most individuals being re-infected 1–3 times overall. These low rates are better explained by the weak efficiency of the stercorarian mode of transmission than a highly efficient adaptive immune response. Those estimates are of particular interest for vaccine development and for our understanding of the higher risk of chronic disease manifestations suffered by infected people living in endemic areas. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Phylogenetic relationships among major lineages of Trypanosoma cruzi are still debatable. Particularly, it is controversial the origin of two main lineages: TcIII and TcIV. Some authors proposed that these lineages have been the result of an ancient hybridization between TcI and TcII, and this was one of the most accepted evolutionary models in the scientific community for several years. In the present paper we analyse several genomes of T . cruzi in order to examine if there is evidence supporting that TcIII is an ancient TcI/TcII hybrid. Our results show that TcIII is mainly related to TcI and not to TcII and there is few evidence of mosaicism for TcIII. Our results challenge the hypothesis of the ancient TcI/TcII hybridization. [ABSTRACT FROM AUTHOR]

Trypanosoma cruzi, the aetiological agent of Chagas disease possess extensive genetic diversity. This has led to the development of a plethora of molecular typing methods for the identification of both the known major genetic lineages and for more fine scale characterization of different multilocus genotypes within these major lineages. Whole genome sequencing applied to large sample sizes is not currently viable and multilocus enzyme electrophoresis, the previous gold standard for T. cruzi typing, is laborious and time consuming. In the present work, we present an optimized Multilocus Sequence Typing (MLST) scheme, based on the combined analysis of two recently proposed MLST approaches. Here, thirteen concatenated gene fragments were applied to a panel of T. cruzi reference strains encompassing all known genetic lineages. Concatenation of 13 fragments allowed assignment of all strains to the predicted Discrete Typing Units (DTUs), or near-clades, with the exception of one strain that was an outlier for TcV, due to apparent loss of heterozygosity in one fragment. Monophyly for all DTUs, along with robust bootstrap support, was restored when this fragment was subsequently excluded from the analysis. All possible combinations of loci were assessed against predefined criteria with the objective of selecting the most appropriate combination of between two and twelve fragments, for an optimized MLST scheme. The optimum combination consisted of 7 loci and discriminated between all reference strains in the panel, with the majority supported by robust bootstrap values. Additionally, a reduced panel of just 4 gene fragments displayed high bootstrap values for DTU assignment and discriminated 21 out of 25 genotypes. We propose that the seven-fragment MLST scheme could be used as a gold standard for T. cruzi typing, against which other typing approaches, particularly single locus approaches or systematic PCR assays based on amplicon size, could be compared. Author Summary: The single-celled parasite Trypanosoma cruzi occurs in mammals and insect vectors in the Americas. When transmitted to humans it causes Chagas disease (American trypanosomiasis) a major public health problem. T. cruzi is genetically diverse and currently split into six groups, known as TcI to TcVI. Multilocus sequence typing (MLST) is a method used for studying the population structure and diversity of pathogens and involves sequencing DNA of several different genes and comparing the sequences between isolates. Here, we assess 13 T. cruzi genes and select the best combination for diversity studies. Outputs reveal that a combination of 7 genes can be used for both lineage assignment and high resolution studies of genetic diversity, and a reduced combination of four loci for lineage assignment. Application of MLST for assigning field isolates of T. cruzi to genetic groups and for detailed investigation of diversity provides a valuable approach to understanding the taxonomy, population structure, genetics, ecology and epidemiology of this important human pathogen. [ABSTRACT FROM AUTHOR]

Highlights: [•] We developed a software for multi-locus sequence data analysis (MLSTest). [•] It was designed for typing and clustering in Eukaryotes (particularly diploids). [•] It includes tools to analyze population genetic structure. [•] It includes tools to develop and to optimize typing schemes. [ABSTRACT FROM AUTHOR]

Abstract: 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 4178bp 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. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

Apoptosis is necessary for the regulation of growth during development, but the precise details of this regulation have not been completely characterized. In this study, we used the Drosophila genital disc as a model to analyze the contribution of apoptosis to growth regulation, We studied the expression or activity of several elements of the apoptotic death pathway such as Drosophila inhibitor of apoptosis, caspases, the apoptotic genes reaper (rpr) and head involution defective, as well as elements of the Jun-NH2-terminal kinase (JNK) pathway. We found that the JNK pathway is active in a dynamic, asymmetric and genitalia-specific manner. Apoptosis, as measured in terms of the expression of a variety of apoptotic molecules, occurs in a JNK-dependent and -independent manner and was detected among engrailed (en) expressing cells. JNK regulation of apoptotic genes is necessary to control growth in both sexes and rotation in males; this regulatory role is necessary to execute en+ cell death and to activate expression of rpr in these ceils, rpr is up-regulated at antero-posterior borders, and this expression appears to be of particular importance in the control of growth, since the balance between cell proliferation and death in those regions appears to depend on the equilibrium between pro- and anti-apoptotic factors at s cellular level. [ABSTRACT FROM AUTHOR]

Background: We have previously shown that the transcription factor c-Fos is also capable of associating to endoplasmic reticulum membranes (ER) and activating phospholipid synthesis. Herein we examined phospholipid synthesis status in brain tumors from human patients and from NPcis mice, an animal model of the human disease Neurofibromatosis Type 1 (NF1). Principal Findings: In human samples, c-Fos expression was at the limit of detection in non-pathological specimens, but was abundantly expressed associated to ER membranes in tumor cells. This was also observed in CNS of adult tumor-bearing NPcis mice but not in NPcis fos(-/-) KO mice. A glioblastoma multiforme and a malignant PNS tumor from a NF1 patient (MPNST) showed a 2- and 4- fold c-Fos-dependent phospholipid synthesis activation, respectively. MPNST samples also showed increased cell proliferation rates and abundant c-Fos expression. Conclusions: Results highlight a role of cytoplasmic c-Fos as an activator of phospholipid synthesis in events demanding high rates of membrane biogenesis as occurs for the exacerbated growth of tumors cells. They also disclose this protein as a potential target for controlling tumor growth in the nervous system. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Leishmaniasis is a vector-borne protozoan infection affecting over 350 million people around the world. In Argentina cutaneous leishmaniasis is endemic in nine provinces and visceral leishmaniasis is spreading from autochthonous transmission foci in seven provinces. However, there is limited information about the diversity of the parasite in this country. Implementation of molecular strategies for parasite typing, particularly multilocus sequence typing (MLST), represents an improved approach for genetic variability and population dynamics analyses. We selected six loci as candidates implemented in reference strains and Argentinean isolates. Phylogenetic analysis showed high correlation with taxonomic classification of the parasite. Autochthonous Leishmania ( Viannia ) braziliensis showed higher genetic diversity than L . ( Leishmania ) infantum but low support was obtained for intra- L . braziliensis complex variants suggesting the need of new loci that contribute to phylogenetic resolution for an improved MLST or nested-MLST scheme. This study represents the first characterization of genetic variability of Leishmania spp. in Argentina. [ABSTRACT FROM AUTHOR]

Highlights: [•] A reassessment of Leptospira spp. MLST schemes was performed. [•] The reassessed scheme includes 7 of 13 loci of the most common used MLST schemes. [•] The new scheme strongly support Leptospira species phylogenetic relations. [•] The Argentinian isolates exhibited congruence between allelic profile and serogroup. [•] This 7 loci scheme provide an alternative to typing by serological methods. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

Genome-based phylogeny has been proposed to be more accurate than phylogeny based in a few genes as MLST-based phylogeny. However, much is not always better. Here we analyzed 368 complete genomes corresponding to 9 bacterial species in order to address intraspecific phylogeny. The studied species were: Burkholderia pseudomallei, Campylobacter jejuni, Chlamydia trachomatis, Helicobacter pylori, Klebsiella pneumoniae, Listeria monocytogenes, Salmonella enterica, Staphylococcus aureus and Streptococcus pyogenes. The intra-specific phylogenies were inferred using the complete genome sequences of different strains of these species and their MLST schemes. A supermatrix approach was used to infer maximum likelihood phylogenies in both cases. The phylogenetic incongruence between the supermatrix-based genome or MLST tree and individual trees (constructed from genome fragments or MLST genes, respectively) was analyzed. In supermatrix-based trees for genomes, most branches showed a high branch support; however, a high number of branches also showed high percentage of topologically incongruent individual trees. Interestingly, genome and MLST trees showed similar levels of incongruence in the phylogeny for each bacteria specie. Both genome and MLST approaches showed that C. trachomatis and S. aureus have a tree-like evolutionary history (low levels of internal incongruence). Instead, B. pseudomallei and S. pyogenes show high levels of incongruence (network-like evolutionary story) probably caused by HGT (horizontal gene transfer). Concluding, our analysis showed that: high branch supports obtained in genome phylogenies could be an artifact probably caused by data size; MLST is valid to address intraspecific phylogenetic structure; and, each species has its own evolutionary history, which could be affected by HGT to different extents. • Genome trees hide extreme incongruence in intraspecific phylogeny in bacteria • Genomic data can cause artifactual statistical support for branches • MLST unveils the same intraspecific genetic structure patterns than genomes [ABSTRACT FROM AUTHOR]

Acidovorax spp. cause a wide range of economically important diseases in monocotyledonous and dicotyledonous plants, including sugarcane, corn, rice, oat, millet, foxtail watermelon, and orchid. In Argentina, the red stripe disease of sugarcane caused by Acidovorax avenae affects 30% of the milling stems with important economic losses. To explore the genetic diversity of this bacterium associated with red stripe in Argentina, multilocus sequence typing (MLST) was applied. This study included 15 local strains isolated from four different sugarcane planting regions and selected after random amplified polymorphic DNA analysis and reference strains of A. citrulli, A. avenae, and A. oryzae to investigate their phylogenetic relationships. MLST analysis resulted in five sequence types among the sugarcane A. avenae strains which constitute a clonal complex, meaning a common and close origin. Sugarcane strains were related to A. avenae from other hosts and distant to A. citrulli. Signals of frequent recombination in several lineages of A. avenae was detected and we observed that A. oryzae is closely related to A. avenae strains. This study provides valuable data in the field of epidemiological and evolutionary investigations of novel clone of A. avenae strains causing sugarcane red stripe. The knowledge of the genetic diversity and strain-host specificity are important to select the genotypes with the best response to the red stripe disease.

Introduction: Phylogenetic relationships between different lineages of Trypanosoma cruzi, the agent of Chagas disease, have been controversial for several years. However, recent phylogenetic and phylogenomic analyses clarified the nuclear relationships among such lineages. However, incongruence between nuclear and kinetoplast DNA phylogenies has emerged as a new challenge. This incongruence implies several events of mitochondrial introgression at evolutionary level. However, the mechanism that gave origin to introgressed lineages is unknown. Here, I will review and discuss how maxicircles of the kinetoplast were horizontally and vertically transferred between different lineages of T. cruzi., Conclusion: Finally, I will discuss what we know - and what we don't - about the kDNA transference and inheritance in the context of sexual reproduction in this parasite.

Several different models of Trypanosoma cruzi evolution have been proposed. These models suggest that scarce events of genetic exchange occurred during the evolutionary history of this parasite. In addition, the debate has focused on the existence of one or two hybridisation events during the evolution of T. cruzi lineages. Here, we reviewed the literature and analysed available sequence data to clarify the phylogenetic relationships among these different lineages. We observed that TcI, TcIII and TcIV form a monophyletic group and that TcIII and TcIV are not, as previously suggested, TcI-TcII hybrids. Particularly, TcI and TcIII are sister groups that diverged around the same time that a widely distributed TcIV split into two clades (TcIVS and TcIVN). In addition, we collected evidence that TcIII received TcIVS kDNA by introgression on several occasions. Different demographic hypotheses (surfing and asymmetrical introgression) may explain the origin and expansion of the TcIII group. Considering these hypotheses, genetic exchange should have been relatively frequent between TcIII and TcIVS in the geographic area in which their distributions overlapped. In addition, our results support the hypothesis that two independent hybridisation events gave rise to TcV and TcVI. Consequently, TcIVS kDNA was first transferred to TcIII and later to TcV and TcVI in TcII/TcIII hybridisation events.

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., (Copyright © 2014 Elsevier B.V. All rights reserved.)