Bats (Chiroptera) are the only mammals naturally able to fly. Due to this characteristic they play a relevant ecological role in the niches they inhabit. These mammals spread infectious diseases from enzootic to domestic foci. Rabbies, SARS, fungi, ebola and trypanosomes are the most common pathogens these animals may host. We conducted intensive sampling of bats from the phyllostomidae, vespertilionidae and emballonuridae families in six localities from Casanare department in eastern Colombia. Blood-EDTA samples were obtained and subsequently submitted to analyses of mitochondrial and nuclear genetic markers in order to conduct barcoding analyses to discriminate trypanosome species. The findings according to the congruence of the three molecular markers suggest the occurrence of Trypanosoma cruzi cruzi (51%), T. c. marinkellei (9%), T. dionisii (13%), T. rangeli (21%), T. evansi (4%) and T. theileri (2%) among 107 positive bat specimens. Regarding the T. cruzi DTUs, we observed the presence of TcI (60%), TcII (15%), TcIII (7%), TcIV (7%) and TcBAT (11%) being the first evidence to our concern of the foreseen genotype TcBAT in Colombia. These results allowed us to propose reliable hypotheses regarding the ecology and biology of the bats circulating in the area including the enigmatic question whether TcBAT should be considered a novel DTU. The epidemiological and evolutionary implications of these findings are herein discussed.

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BackgroundChagas disease is a systemic pathology caused by Trypanosoma cruzi. This parasite reveals remarkable genetic variability, evinced in six Discrete Typing Units (DTUs) named from T. cruzi I to T. cruzi VI (TcI to TcVI). Recently newly identified genotypes have emerged such as TcBat in Brazil, Colombia and Panama associated to anthropogenic bats. The genotype with the broadest geographical distribution is TcI, which has recently been associated to severe cardiomyopathies in Argentina and Colombia. Therefore, new studies unraveling the genetic structure and natural history of this DTU must be pursued.ResultsWe conducted a spatial and temporal analysis on 50 biological clones of T. cruzi I (TcI) isolated from humans with different clinical phenotypes, triatomine bugs and mammal reservoirs across three endemic regions for Chagas disease in Colombia. These clones were submitted to a nuclear Multilocus Sequence Typing (nMLST) analysis in order to elucidate its genetic diversity and clustering. After analyzing 13 nuclear housekeeping genes and obtaining a 5821 bp length alignment, we detected two robust genotypes within TcI henceforth named TcIDOM (associated to human infections) and a second cluster associated to peridomestic and sylvatic populations. Additionaly, we detected putative events of recombination and an intriguing lack of linkage disequilibrium.ConclusionsThese findings reinforce the emergence of an enigmatic domestic T. cruzi genotype (TcIDOM), and demonstrates the high frequency of recombination at nuclear level across natural populations of T. cruzi. Therefore, the need to pursue studies focused on the diferential virulence profiles of TcI strains. The biological and epidemiological implications of these findings are herein discussed.

Vaccine development is an expensive and time-consuming process that heavily relies on animal models. Yet, vaccine candidates that have previously succeeded in animal experiments often fail in clinical trials questioning the predictive value of animal models. Alternative assay systems that can add to the screening and evaluation of functional characteristics of vaccines in a human context before embarking on costly clinical trials are therefore urgently needed. In this study, we have established an in vitro system consisting of long-term cultures of unfractionated peripheral blood mononuclear cells (PBMCs) from healthy volunteers to assess (recall) T cell responses to vaccine candidates. We observed that different types of influenza vaccines (whole inactivated virus (WIV), split, and peptide vaccines) were all able to stimulate CD4 and CD8 T cell responses but to different extents in line with their reported in vivo properties. In-depth analyses of different T cell subsets revealed that the tested vaccines evoked mainly recall responses as indicated by the fact that the vast majority of the responding T cells had a memory phenotype. Furthermore, we observed vaccine-induced activation of T follicular helper cells, which are associated with the induction of humoral immune responses. Our results demonstrate the suitability of the established PBMC-based system for the in vitro evaluation of memory T cell responses to vaccines and the comparison of vaccine candidates in a human immune cell context. As such, it can help to bridge the gap between animal experiments and clinical trials and assist in the selection of promising vaccine candidates, at least for recall antigens.

Vaccine development relies on testing vaccine candidates in animal models. However, results from animals cannot always be translated to humans. Alternative ways to screen vaccine candidates before clinical trials are therefore desirable. Dendritic cells (DCs) are the main orchestrators of the immune system and the link between innate and adaptive responses. Their activation by vaccines is an essential step in vaccine-induced immune responses. We have systematically evaluated the suitability of two different human DC-based systems, namely the DC-cell line MUTZ-3 and primary monocyte-derived DCs (Mo-DCs) to screen immunopotentiating properties of vaccine candidates. Two different influenza vaccine formulations, whole inactivated virus (WIV) and subunit (SU), were used as model antigens as they represent a high immunogenic and low immunogenic vaccine, respectively. MUTZ-3 cells were restricted in their ability to respond to different stimuli. In contrast, Mo-DCs readily responded to WIV and SU in a vaccine-specific way. WIV stimulation elicited a more vigorous induction of activation markers, immune response-related genes and secretion of cytokines involved in antiviral responses than the SU vaccine. Furthermore, Mo-DCs differentiated from freshly isolated and freeze/thawed peripheral blood mononuclear cells (PBMCs) showed a similar capacity to respond to different vaccines. Taken together, we identified human PBMC-derived Mo-DCs as a suitable platform to evaluate vaccine-induced immune responses. Importantly, we show that fresh and frozen PBMCs can be used indistinctly, which strongly facilitates the routine use of this system. In vitro vaccine pre-screening using human Mo-DCs is thus a promising approach for evaluating the immunopotentiating capacities of new vaccine formulations that have not yet been tested in humans.