Distribution of Trypanosoma cruzi DTUs in nature.
Ana Maria Jansen, Cristiane Varella Lisboa, Maria Augusta Dario and Samanta Cristina das Chagas Xavier.
Laboratory of Biology of Trypanosomatids, Instituto Oswaldo Cruz/Fiocruz
Email:firstname.lastname@example.org/ email@example.com/ firstname.lastname@example.org e email@example.com
The order Trypanosomatida is composed of trypanosomatids of 19 genera and which have different types of hosts and evolutionary cycles; are divided into two groups, according to their development in the insect vector and their form of transmission being anterior (Salivaria) or posterior (Stercoraria), which includes parasites that develop in the hindgut of their invertebrate host, in which the infective forms, metacyclic trypomastigotes, are eliminated in feces during blood feeding in vertebrates. Within this section is the medically important species Trypanosoma cruzi, the etiologic agent of Chagas’ disease (CD), a heteroxenic parasite capable of infecting all vertebrate species and mostly transmitted by different species of invertebrate vectors.
T. cruzi, the etiological agent of CD, is a multi-host parasite, capable of infecting hundreds of species of mammalian hosts, of eight different orders, transmitted by several species of hematophagous triatomines (family Reduviidae, subfamily Triatominae). Its occurrence is described throughout the American continent, from the southern United States to Argentina and Chile. The life cycle of T. cruzi is complex, since the parasite has four stages of development in its hosts: blood amastigote and trypomastigote in mammalian hosts; and epimastigote and metacyclic trypomastigote in the insect vector. The amastigote and trypomastigote forms (metacyclic and blood) are responsible for causing infection in their hosts.
T. cruzi is part of a clade called “T. cruzi clade” – composed of representatives of the subgenus Schizotrypanum and other mammalian trypanosomatid species. The T. cruzi clade (Figure 1) is heterogeneous, comprising a diversity of Trypanosomaspecies, with a wide diversity of mammalian hosts and worldwide distribution.
Figure 1: Representative phylogenetic tree of the T. cruzi clade. The five groups are represented using the following colors: wine – subgenus Schizotrypanum; purple – T. rangeli/T. conorhini; blue – Neotropical bats; orange – Australian marsupials; green – African bats. Source: Dario MA, 2017.
Since the International Symposium on the Advances of Chagas’ disease (1999) held in Rio de Janeiro, knowledge about the great genetic variability and phylogenetic relationships between the different lineages of the parasite has increased exponentially. This is due to the development of molecular tools used to identify different genetic subtypes of the parasite as well as to propose genetic relationships between different T. cruzi isolates. Currently, these subtypes are called Discrete Typing Units (DTU) – which has as its concept the set of populations that are genetically similar and can be identified by genetic, immunological and molecular markers in common.DTUs were named TcI (TcI), TcII (TcIIb), TcIII (IIa), TcIV (IIc), TcV (IId) and TcVI (IIe) and in the same year (2012) of the new naming consensus of genotypes of T. cruzi, a new genotype, named TcBat, was described. The DTU TcI and TcIIs represent the oldest T. cruzi genotypes and also known as parental genotypes. It is not known for sure when these DTUs separated, some studies point that this separation took place between 3-10 million years.
Table 1. Classification of Trypanosoma cruzi genotypes, according to a consensus held in 2009, changed the nomenclature of T. cruzi genotypes into six Discrete Typing Units (DTUs).
|T. cruzi I
|T. cruzi II
|T. cruzi III
|T. cruzi IV
|T. cruzi V
|T. cruzi VI
Adapted from Zingales et al., 2009.
More than 100 years after being recognized by Carlos Chagas himself that Chagas’ disease is primarily a sylvatic enzootic, many aspects of the enzootic cycle are still poorly understood. Among these, the variables that modulate the transmission of the different DTUs, which, as is known, occur in complex cycles, which may or may not overlap even in a single forest stratum; whose distribution patterns in nature and ecological and epidemiological importance still present numerous gaps that need to be known.
In Brazil, the seven DTUs (TcI to TcVI, TcBat) have already been described, but their distribution in nature, hosts and reservoirs and, mainly, their potential risk for human disease is still poorly understood. These gaps are probably due to the very difficulty of the collection work in the field, which results in subsampling and, therefore, in an incomplete view of the dispersion pattern of these genotypes.
It is known that TcI DTU is widely dispersed and frequent in nature with a wide spectrum of mammalian hosts and vectors, and can be found from the United States to Chile. Although it has been proposed that this TcI DTU is related to the cycle of sylvatic arboreal transmission and to marsupials of the genus Didelphis, it has already been isolated in different species of mammals and triatomines, and has been found in all forest strata. This DTU is responsible for CD cases in the northern countries of South America, as well as in the Amazon region in Brazil. Molecular studies using different markers demonstrated the high heterogeneity of DTU TcI. In samples obtained in Colombia, DTU TcI was divided into four groups or haplotypes (Ia to Id), which correspond to haplotypes found in human infection, in triatomines of the genus Rhodnius in the wild, in the transmission cycle of T. cruzi in the peridomiciliary environment and in the species of Triatomadimidiatans in sylvatic transmission cycles. In Chile, a new haplotype (Ie) of TcI was described, associated with the domestic transmission cycle. Phylogenetic studies using nuclear and mitochondrial markers have demonstrated the existence of a population of TcI: TcIdom occurring in Central America to South America and associated with the domestic transmission cycle. Currently, two TcI genotypes are described: i) TcIdom associated with the domestic transmission cycle and ii) sylvatic TcI associated with the sylvatic transmission cycle.
The DTU TcII, considered to be the third most frequent DTU in the American continent, in Brazil this profile is not observed, since it represents the second most isolated DTU in the country. Although this DTU is considered to be associated with the domestic transmission cycle of the T. cruzi, in the central and southern regions of South America, being responsible for severe cases of CD, TcII has already been described in the Brazilian Amazon region, Colombia, Mexico and the United States. Despite the supposed association with the domestic transmission cycle, TcII has been found in the wild in mammals and vectors, having already been described in primates, rodents, marsupials, bats, carnivores and in triatomines of the genera Triatoma and Rhodnius.
The TcIII and TcIV DTUs, previously classified as a single Z3 group, have been described as almost exclusively associated with wild hosts, in more restricted and focal transmission cycles. DTU TcIII is widely distributed in nature, being found in Mexico, from Venezuela to Argentina. It has been proposed that this DTU is associated with the terrestrial transmission cycle and the species Dasypusnovemcinctus. In Brazil, this DTU has already been described in the Atlantic Forest, Caatinga, Amazon and Pantanal biomes and in different species of mammals, including arboreal mammals. It has already been isolated from domestic dogs and from triatomines of the genera Panstrogylus, Rhodnius and Triatoma. The DTU TcIV, described in the United States of America, Guatemala and South America, in Brazil was found in the Atlantic Forest of southeastern Brazil, circulating in triatomines of the species Triatomavitticeps and in the Amazon region. According to some authors, TcIV can be subdivided into two genotypes: one related to isolates from North America and the other to isolates from South America. It has been proposed that TcIV is associated with the tree transmission cycle, in primates and triatomines of the genus Rhodnius. In fact, little is known about the distribution of these DTUs (TcIII and TcIV) in Brazil, but it can be said that they are less frequent than the TcI and DTU TcIIs and that they were described as of little importance in human infection, however, they have recently oral ACD outbreaks caused by DTUs TcIII and TcIV have been observed. Outbreaks of CD caused by mixed infection by TcI and TcIII or TcIV DTUs have also been described in Amapá in 1996. In addition, in areas of recorded outbreaks in the Brazilian Amazon, 50% of triatomines collected in the vicinity of houses and villages are infected with TcI and TcIII or TcIV DTUs. Regarding the origin of the DTUs TcIII and TcIV, these genotypes are considered to have arisen from hybridization events between the DTUs TcI and TcIIs, and that the DTU TcIII is an ancestral lineage, however the matter is quite controversial.
DTUs TcV and TcVI have been proposed to be associated with CD cases in the south-central region of South America and described mainly in Argentina, Bolivia, Chile, and Paraguay. They have already been found in Mexico, Colombia, Brazil, Ecuador, and Peru, being isolated in the wild, from marsupials and rodents. These DTUs are considered hybrids, due to the observation of heterozygosity in their isolates. Hybridization events, in which there is a genetic exchange between TcII and TcIII, are proposed to explain this heterogeneity.
The DTU TcBat is the most recent and was described for the first time in bats of the genera Myotis and Noctilio in the states of São Paulo and Mato Grosso do Sul. It is known that its distribution is wider, with reports of its finding in other Brazilian states, as well as in Panama. Previously restricted to bats, this DTU has already been described in humans, in human mummy and its occurrence has also been described in triatomine species T. sordida. Phylogenetic analyzes demonstrated TcBat as an independent DTU, being the same genetically closer to the TcI DTU.
Much has been discussed and studied about the origin of the T. cruzi clade. The first studies related to this subject showed that this clade would have originated from the theory of the southern supercontinents. This theory suggests that the trypanosomatid species of this clade appeared from a species of Trypanosoma present in marsupials, when South America, Australia, and Antarctica formed a single continent. The theory gained strength, once Australian marsupial trypanosomatid species were included in the T. cruzi clade, grouping together in its basal region. The description of trypanosomatid species in African mammals and new species of trypanosomatids in African and American bats, within the T. cruzi clade, resulted in the proposal that the species of the T. cruzi clade originated from a bat trypanosomatid and that it was adapting to other species of mammals in several different events. This theory is known as the bat-seeding hypothesis.Figure 2 shows the spatial distribution of the six DTUS (TcI to TcVI) of T. cruzi obtained from wild and domestic mammals and vectors examined in Brazilian biomes with different phytogeographic characteristics.
The parental DTUs are represented by the purple (DTU TcI) and light blue (DTU TcII) dots, and it can be observed that the DTU TcI predominates throughout the Brazilian territory, corresponding to 58% of the T. cruzi isolates and that the DTU TcII , despite being significantly less frequent (17%), is also widely distributed. The DTU TcII, previously exclusively associated with human infection and disease and later associated with armadillos, shows the second largest distribution, in addition to a wide range of wild hosts, being observed in rodents, marsupials, primates and carnivores in all observed biomes. The cross distribution of the two DTUs in nature shows that there is no association with biome or habitat. Concomitant infection by DTUs TcI and TcIIs, in addition to being the most common and widely dispersed, 16% of infected mammals from all biomes, including arboreal and terrestrial species, as well as bats, was especially common in coatis and in Didelphimorphia. The most common pattern of concomitant infection was observed between DTUs TcI and TcIV, observed in Chiroptera, Didelphimorphia and Primates. DTUs TcIII and TcIV were identified in single and mixed infections of wide dispersion in the five Brazilian biomes, with the DTU TcIV being the most common. The data indicate that the DTUs TcIII and TcIV are more widespread and infect a greater number of mammalian species than previously thought. In addition, they are transmitted in restricted foci and cycles, but in different micro habitats and areas with different ecological profiles.
Regarding the biomes, the Pantanal presented the highest number of isolates characterized as DTUs TcIII and TcIV in single and mixed infections, followed by the Atlantic Forest and Amazon Forest biomes.
Species of the order Didelphimorphia showed the highest frequency of infection and were found in all five biomes. The infection of a species of the order Artiodactyla by the DTU TcIII was reported for the first time, in addition to new host species: five mammals (marsupials and rodents) and two genera of Hemiptera.
Taken together, the results presented in Figure 2 demonstrate the complexity of the T. cruzi reservoir system and its transmission strategies, indicating that there is considerably more knowledge regarding the ecology of T. cruzi. It also emphasizes that there are large areas that have not yet been sampled and that the distribution of the different DTUs of T. cruziis dynamic and is still far from being fully known.
Cartography & Health
Use of geotechnologies in the context of single health: human, animal and environment health
Samanta Cristina das Chagas Xavier
Laboratory of Biology of Trypanosomatids, Instituto Oswaldo Cruz/Fiocruz
Luiz Felipe Coutinho Ferreira da Silva
Department of Cartographic Engineering, Military Institute of Engineering, IME, Rio de Janeiro
The current distribution of organisms does not occur by chance. It has patterns that are repeated, obeying rules that are, in most cases, determined by the environment. To understand why a species has a wide distribution and others are restricted to only one type of environment, it is necessary to study the evolutionary processes of each organism, since it is important to determine which factors modulate the distribution of an organism in space and in time. The study of Biogeography, more specifically Terrestrial Biogeography, which involves the study of ecoregions and ecosystems, is the most used in research on zoonoses as it facilitates the study of hosts and their relationship with the environment. Therefore, Biogeography is an important science for the spatial-temporal understanding of life on earth, thus contributing to the study of the global environment and, consequently, collaborating for the studies of emerging and re-emerging zoonoses.
Historically, it was up to John Snow, in the 19th century, to realize that the cholera epidemic in London at the time could be being propagated through the surface runoff of contaminated water. Using maps showing the geographic distribution of deaths from cholera as well as the water distribution infrastructure, in 1854, this researcher proved the association between mortality and regions drained by contaminated water.
One cannot think about the ecology of Trypanosoma cruzi and Chagas’ disease, a zoonosis maintained among hundreds of mammal species and wild vectors that eventually expands or retracts without taking a comprehensive look. This becomes even more relevant if the objective is to adopt measures for the prevention and/or control of Chagas’ disease, considered reemerging and which results in about 200-250 new cases per year in Brazil, most of the cases in Pará. (87.1%) and in Amapá (5.5%) especially by oral vector (MS, 2017). The cycle of this parasite in nature is complex. It is a large web, a network of transmission of the parasite through contaminative and oral vectors that involves triatomines and mammals that use all forest plant extracts with local and temporal characteristics and particularities. Thus, it is essential that studies on Chagas’ disease are carried out within the One Health vision. This “One Health” approach is represented by the triad between human and animal health and the characteristics of the environment where they are inserted (Figure 1). The integrated study means considering the rates and profile of infection in domestic and wild animals and the local environmental characteristics. It was with this dataset and applying an interpolation analysis and map algebra that Xavier et al., (2012) confirmed the proposal to use dogs as sentinels of risk of human infection in areas of enzootic transmission. It was also evaluating the cultural and economic scenario, in addition to the enzootic scenario, that Xavier et al., (2014) explained how human cases were happening in Belém in the State of Pará, resulting from infected insects that are transported from nearby islands to the two locations. that most report cases in the city.Considering that biological phenomena do not obey a linear logic, the “one health” approach, associated with predictive modeling, will allow the identification of environmental factors that favor or not the occurrence of cases, and mainly result in risk maps.
Although it is not possible to make direct causal inferences from the etiological point of view, knowledge of the spatial and temporal variations in the incidence of diseases, concomitantly with specified environmental situations, is important for planning prevention and control actions. Chagas’ disease, yellow fever, dengue fever, Zika, chikungunya, malaria, Leishmaniasis and others, when neglected, can become urban diseases that are difficult to control. The monitoring of these diseases through geoprocessing tools allows to know the potential of appearing in a new area or persisting in a region.
The development of digital mapping and spatial analysis technologies, particularly in the context of Geographic Information Systems (GIS), has opened a wide field for the study of problems related to Biogeography and the occurrence of diseases in animal and human populations.
Geographic mapping is essential for epidemiological surveillance because, in addition to generating information on the punctual distribution of diseases, it makes it possible to monitor the spatial-temporal distribution of environmental factors that modulate their occurrence. Knowledge of the geographic pattern of diseases can provide information about the etiology and pathophysiology of certain morbid events. The virulence of certain infectious agents can be influenced by physical and climatic factors.
The life cycle of vectors, as well as reservoirs and hosts that participate in the transmission chain of parasites, are strongly related to the environmental dynamics of the ecosystems where they live, being limited by environmental variables such as temperature, precipitation, humidity, patterns of use and soil coverage. The influence of climate change on the dynamics of transmission cycles can also geographically expand the distribution area of triatomines that does not occur in certain locations due to the temperate climate, which prevents the proliferation of vectors. As an example, the study of the potential area of occurrence of Chagas’ disease in Texas, where a potential shift to the north in the distribution of T. gerstaeckeri and a distributional shift to the north and south of T. sanguisuga in its current range are predicted due to the climate change.
GIS allow the detailed description of environmental processes and the establishment of a complex network of facts with geographical expression. They also allow global analyzes of parasites, their hosts and vectors, the disease and the environment, in addition to a spatial and temporal assessment of ecological and epidemiological risks. The construction of a GIS focused on the study of the enzootic cycles of transmission of trypanosomatids may contribute with early information on the occurrence of the circulation of parasites in wild animals and predict the risk for domestic animals and humans. It will be possible to produce diagnoses of the areas as an alert of the occurrence of risk factors for the surrounding population arising from the local biodiversity and for the validation of models for predicting ecological opportunities for the emergence of diseases. An advantage of this tool is that it allows the creation and visualization of a great diversity of environmental transmission contexts (due to the existence of different species of vectors, reservoirs and parasites associated with the action of man on the environment), quickly, facilitating the analysis and directing strategies for control that must be specific. Hence the need to consider the regional epidemiological pattern and the transmission behavior in each location.
Remote sensing is one of the geoprocessing techniques with potential application to map parasite vectors and contribute to the analysis of trypanosomatid transmission cycles. Vegetation responds quickly to changes in other environmental variables such as precipitation, temperature, and humidity. Using the spatial, temporal and spectral characteristics of the sensors, it is possible to monitor, in a systematic and regular way, the terrestrial conditions, providing large amounts of climatic data and about the vegetation cover and land use. Among the environmental factors most related to endemics caused by vectors and which can be observed from space platforms, the following stand out: temperature, water, soil moisture, vegetation cover conditions, deforestation, urban characteristics, ocean color, and topography. This information can be used for studies on parasites with focal transmission, from the use of images obtained by satellites of higher spatial resolution. The simple visualization of the images can already be informative for the identification of patterns relevant to the occurrence of diseases. However, some techniques applied to the image, based on spectral responses, can generate new information and contribute to enhance environmental characteristics. The NDVI “Normalized Difference Vegetation Index” operation generates information about the vegetation, which can be related to the presence of parasite vectors. The presence or absence of vegetation cover is essential in maintaining the biological cycle of vectors and infectious agents.
Ecological Niche Modeling (ENM) is a tool used for potential distribution models that have been widely used in various situations, helping to indicate priority areas for biodiversity conservation, to assess the potential for invasion of exotic species, to study the impacts of climate change in biodiversity, and monitoring of infectious disease vectors, for example. ENM identifies areas in the landscape that have environments similar to those in which the species was observed. This information can be extremely useful in a very wide range of applications in spatially explicit studies.
Systems of this nature are increasingly present in areas of knowledge that have problems of a spatial nature, such as, for example, to support health actions, whether for prevention or for a more effective action in the control of diseases and endemic diseases. The geographical approach refines and makes the analysis of the factors that guide and condition the definition of public policies in the health area more precise. In epidemiology, it can contribute to improving the possibilities of description and spatial analysis of diseases in large sets of geographically referenced data, for the evaluation of environmental variables and for the planning of prevention and control actions.
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Boletim Epidemiológico Secretaria de Vigilância em Saúde − Ministério da Saúde Volume 48 N° 4 – 2017 ISSN 2358-945.
Paulo Sergio D’Andrea
Department of Tropical Medicine, Instituto Oswaldo Cruz/Fiocruz
Bernardo Rodrigues Teixeira and Marconny Gerhardt da Rocha
Laboratory of Biology and Parasitology of Wild Mammal Reservoirs, Instituto Oswaldo Cruz/Fiocruz
Email: firstname.lastname@example.org, email@example.com
Disturbances in natural habitats caused essentially by human activities have directly affected the structure of animal and plant communities. These environmental changes may allow the contact of human populations with epizootics previously restricted to the sylvatic cycle. In these situations, many species of mammals have been identified as natural reservoirs of parasites that affect humans.
Several studies on mammal reservoirs of zoonoses have been carried out in Brazil. However, considering that the diversity of the Brazilian mammal fauna is still not completely known, and that the emergence and re-emergence of zoonoses is a current public health problem, studies on ecology, taxonomy and parasitology of wild mammals are extremely important. need and must be continually developed.
In order to carry out these studies, the region to be investigated must initially be determined. Regions with recent occurrences of Chagas’ disease outbreaks, disease monitoring regions or regions where investigations of an investigative nature are desired are chosen.
In these regions, places are determined for the capture of animals in a wild environment, peridomiciliary (chicken coops, warehouses, plantations, etc.) and intra-domiciliary environment (inside homes). In the wild environment, linear transects with 20 capture points each are established. All transects are geo-referenced using a GPS device. At these points, live-trap traps (which capture the live animal), Tomahawk® (braided wire, galvanized) and Sherman® (aluminum traps) models, suitable for capturing small mammals with up to 3 kg. It is important to always place the traps in places protected from the sun.
The collections generally last for five consecutive nights and the bait used is a mixture of bacon, oatmeal, banana and peanut butter, whose purpose is to be generalist and attract rodents and marsupials with different eating habits (granivores, frugivores, insectivores, omnivores, and carnivores).
The transects are walked daily in the morning (between 6 and 8 AM) to check the traps for the presence of animals. Animal traps are removed and later replaced with others. The captured animals are then removed to a laboratory base, being transported in pick-up cars, with a separate cabin for transporting people. The laboratory base is established at a central point between the collection areas, since the animals begin to be processed on the same morning as they are captured.
At the field laboratory base, after the entire team is outfitted with individual bio-protection equipment (level III PPE), the animals are removed from the traps and contained in cloth bags to be anesthetized.
At the time of capture, the following data are observed and recorded: date and place of collection, point of capture, species, weight, sex, reproductive condition, degree of eruption and functionality of marsupial teeth, body measurements and individual observations.
Two types of work methodologies can be performed: capture-mark-release or removal.
This technique is used with the objective of recapturing the marked individuals and, then, carrying out studies on the population dynamics of the species involved, movements and the variation in the infection rates of zoonoses. Animals are individually marked with numbered earrings and blood samples are collected for diagnosis of Trypanosoma cruzi infection. The animals are weighed and after observation of their reproductive conditions and complete recovery from anesthesia, they are released at their respective capture points. For this type of study, it is recommended that capture expeditions be regular, preferably bimonthly, due to the short average longevity of the animals being investigated.
Captured animals are collected and necropsied according to the species and quantities authorized in licenses granted by IBAMA. For this type of study, it is recommended that capture expeditions are frequent, preferably in the wet and dry seasons. The necropsy of animals is necessary to:
- collection of tissue and organ samples for diagnosis of parasite infections. A significant amount of samples is needed to detect the circulation of parasites among natural populations, isolate and characterize trypanosomatid ancestries/strains;
- specific level identification of rodents by cytogenetic techniques. In the case of most species it is not possible to identify at a specific level only by analyzing the external morphology. The cytogenetic technique used (karyotyping) is based on cell culture of the femoral marrow and subsequent analysis of the number and morphology of chromosomes;
- identification at specific level by morphometric analysis (rodents and marsupials). Morphometry is based on dental characters and analysis of measurements of cranial bones and is one more tool for the correct identification of species;
- preparation of witness material for deposit in scientific zoological collections of reference. After performing the procedures, the skin and skeleton of the individuals are prepared and deposited in the mammal collection of the Museu Nacional/UFRJ. The zoological collections of Brazilian mammals are insufficient for all species and their distribution areas, and there is a need to increase witness material for the knowledge of the diversity of our fauna. In this way, a more in-depth study of this diversity is possible, which will provide subsidies for species conservation strategies.
All the information obtained will support studies related to the transmission dynamics of T. cruzi. Information on the animals’ habitat is obtained; their population sizes; survival and recruitment rates; diversity, equitability and abundance indices; in addition to the determination of age classes according to weight and reproductive condition to analyze the age structure of each population. These data are related to climatic data from the region to support the understanding of the population dynamics of the species.
The parasitological parameters (prevalence and incidence of parasites) are analyzed in relation to these population, climatic and habitat parameters to understand the dynamics of parasite transmission in the study area.