Trypanosoma cruzi is a protozoan parasite that is transmitted by triatomine vectors to mammals. It is classified in six discrete typing units (DTUs). In Chile, domestic vectorial transmission has been interrupted; however, the parasite is maintained in non-domestic foci. The aim of this study was to describe T. cruzi infection and DTU composition in mammals and triatomines from several non-domestic populations of North-Central Chile and to evaluate their spatio-temporal variations.
A total of 710 small mammals and 1140 triatomines captured in six localities during two study periods (summer/winter) of the same year were analyzed by conventional PCR to detect kDNA of T. cruzi. Positive samples were DNA blotted and hybridized with specific probes for detection of DTUs TcI, TcII, TcV, and TcVI. Infection status was modeled, and cluster analysis was performed in each locality. We detected 30.1% of overall infection in small mammals and 34.1% in triatomines, with higher rates in synanthropic mammals and in M. spinolai. We identified infecting DTUs in 45 mammals and 110 triatomines, present more commonly as single infections; the most frequent DTU detected was TcI. Differences in infection rates among species, localities and study periods were detected in small mammals, and between triatomine species; temporally, infection presented opposite patterns between mammals and triatomines. Infection clustering was frequent in vectors, and one locality exhibited half of the 21 clusters found.
We determined T. cruzi infection in natural host and vector populations simultaneously in a spatially widespread manner during two study periods. All captured species presented T. cruzi infection, showing spatial and temporal variations. Trypanosoma cruzi distribution can be clustered in space and time. These clusters may represent different spatial and temporal risks of transmission.
Trypanosoma cruzi is a parasite that infects mammals, transmitted by triatomine insect vectors in America, causing Chagas disease in humans. There are six T. cruzi discrete typing units (DTUs). Our goal was to estimate T. cruzi infection rates and describe the DTUs present in mammals and triatomines of Chile, evaluating spatial and temporal variation. We captured nine small mammal and two triatomine species in six localities during two periods (summer/winter) of the same year. We detected T. cruzi DNA and some DTUs were identified. We report one mammal species infected for the first time. Infection presented significant variation among species. The endemic vector had higher infection rates than Triatoma infestans. The DTUs TcI, TcII, TcV and TcVI were present, with predominance of TcI. Temporally, we detected higher rates of infection during summer in small mammals and during winter in triatomines. Infection was spatially and temporally aggregated in small mammals and vectors. Some species might have higher risk of infection, and this may be different between localities or periods, or even within the same locality.
Citation: Ihle-Soto C, Costoya E, Correa JP, Bacigalupo A, Cornejo-Villar B, Estadella V, et al. (2019) Spatio-temporal characterization of Trypanosoma cruzi infection and discrete typing units infecting hosts and vectors from non-domestic foci of Chile. PLoS Negl Trop Dis 13(2): e0007170. https://doi.org/10.1371/journal.pntd.0007170
Editor: Ricardo E. Gürtler, Universidad de Buenos Aires, ARGENTINA
Received: June 4, 2018; Accepted: January 17, 2019; Published: February 15, 2019
Copyright: © 2019 Ihle-Soto et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: Data of each individual analyzed are available from the Harvard Dataverse site: https://dataverse.harvard.edu/dataverse.xhtml Bacigalupo, Antonella; Ihle-Soto, Camila; Costoya, Eduardo; Correa, Juana P.; Cornejo-Villar, Berenice; Estadella, Viviana; Solari, Aldo; Ortiz, Sylvia; Hernández, Héctor J.; Botto-Mahan, Carezza; Gorla, David E.; Cattan, Pedro E., 2019, "Replication Data for: Ihle-Soto, Costoya et al.", https://doi.org/10.7910/DVN/ESN21F, Harvard Dataverse, V1, UNF:6:IFlAKUfeSnp07ID8Z67Y+g==[fileUNF]
Funding: This study was funded by Fondo Nacional de Desarrollo Científico y Tecnológico (http://www.conicyt.cl/fondecyt/), which provided the following Research Funds: CONICYT-FONDECYT 1100339 (PEC, HJH, AB, BCV, VE, CIS, and EC); CONICYT-FONDECYT 1120122 (AS, PEC, SO, CIS, and EC); CONICYT-FONDECYT 1140650 (PEC, AB, JPC, and DEG); CONICYT-FONDECYT 1180940 (PEC, and AB); CONICYT-FONDECYT 1170367 (CBM, and JPC); CONICYT-FONDECYT 3140543 (JPC). The funder, Fondo Nacional de Desarrollo Científico y Tecnológico, had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Chagas disease is a zoonotic parasitic disease, endemic in 22 countries of America, caused by the flagellated protozoa Trypanosoma cruzi. This disease affects approximately 7 million people in the world and represents the third parasitic disease of major world impact . The parasite is transmitted through contact of contaminated feces of hematophagous insects from the Triatominae subfamily with wounds or mucosae of mammals, by blood transfusions, congenital transmission, organ transplants, laboratory accidents, and oral transmission . Vectorial transmission occurs from southern United States to Patagonia (40°N to 45°S) [3, 4]; however, in the last decades, Chagas disease has spread to other continents due to alternative infection routes and migration . In Chile, the disease is endemic in rural and suburban areas from latitudes 18°30’ to 34°36’ S .
Trypanosoma cruzi is a mono-flagellar protist (Kinetoplastida). Its kinetoplastidic DNA (kDNA) displays like a concatenated discal web of maxicircles (20–40 kb; 20–25 copies/cell) and minicircles (0.5–10 kb; 20000 copies/cell) . Minicircles are organized in four conservative regions (conserved sequence blocks, CSB) separated by four variable regions . Due to the high number of minicircle copies and conserved sequences, they are used in the diagnosis of infection through polymerase chain reaction (PCR), using primers that bind to the CSB . Minicircle DNA amplification creates a very polymorphic product of the variable region, useful for T. cruzi genotyping through hybridization methods using characterized probes . Six genetically related lineages of T. cruzi have been described, identifiable by markers, called discrete typing units (DTUs): TcI, TcII, TcIII, TcIV, TcV, TcVI . A new genotype called TcBat has been discovered in bats from Brazil . Several approaches have been used to evaluate the biochemical and genetic diversity of T. cruzi isolates, but there is no unique genetic target that allows complete DTU resolution .
TcI exhibits the wider distribution, from Southern USA to Central Chile and Argentina . TcII is found primarily in the domestic cycle from the South-Central region of South America. TcIII ranges from western Venezuela to the Argentine Chaco, mainly linked to the wild cycle in Brazil . TcIV possess a similar distribution to TcIII but is absent in the Gran Chaco area. Finally, TcV and TcVI are found in Central and Southern South America . So far, no clear association between the parasite genotype and the manifestation of the disease or drug resistance has been detected , but there is evidence that suggests a selective role of hosts and vectors on the different DTUs [15–17].
More than 150 wild, synanthropic, and domestic mammal species have been found infected with T. cruzi in America, including most of the terrestrial mammal orders present [3, 18], playing a relevant role in the maintenance and interplay among wild, peridomestic and domestic cycles . Small mammals are common feeding sources for triatomines in the sylvatic cycle of the endemic zone of Chile , presenting smaller home ranges than larger mammal species . Since the home range of triatomines is also small , these mammals can act as important T. cruzi hosts, acquiring and maintaining the infection [23, 24]. Infected species in Chile are the rodents Octodon degus, Phyllotis darwini, Abrothrix olivaceus, Rattus rattus, the lagomorph Oryctolagus cuniculus, and the marsupial Thylamys elegans, ranging from 32% to 83.6% [18, 23, 25–27]. North-Central Chile is a Mediterranean climatic influenced area characterized by lower richness of terrestrial mammals than other Mediterranean areas of the world , and over 40% of the 30 wild or synanthropic mammal species present are small mammals, exhibiting relatively high abundances .
Triatomines can get infected with T. cruzi at any stage posterior to hatching, by consumption of contaminated mammal blood, cannibalism or coprophagy . In Chile there are four triatomine species: Triatoma infestans, Mepraia spinolai, M. parapatrica, and M. gajardoi [30, 31], where T. infestans has been found in domiciliary and wild habitats [32, 33], while M. spinolai in domestic, peridomestic but mainly wild habitats . Mepraia gajardoi and M. parapatrica are present in wild coastal areas . Mepraia spinolai and T. infestans are distributed sympatrically in part of the endemic area . Infection rates of T. cruzi detected by conventional PCR in sylvatic T. infestans and M. spinolai from Chile vary spatially and temporally, ranging from 36.5% to 68.6% and 14.9% to 76.1%, respectively [32, 33, 36–38]. TcI is the most frequently circulating DTU in T. infestans , and M. spinolai , as well as in Chilean small mammals, present as single and mixed infections [16, 27]. However, there are differences between species regarding their infecting DTU [16, 25, 27, 36, 37].
Infection events can have one of three different spatial configurations: regular or uniform, random, or aggregated (clustered); however, to our knowledge the spatial configuration of T. cruzi infection in hosts and vectors has not been previously evaluated. To understand transmission cycles, it is important to establish whether cases of an infection–i.e., the infected individuals—have the tendency to cluster together more than it would be expected by the natural clustering of the population affected . In the present study, we aimed to assess spatial and temporal variations of T. cruzi infection, detecting the DTUs, by sampling triatomines and small mammals of the same areas in two contrasting seasons of the same year, using conventional and spatially-explicit statistical techniques.
Trapping sites and dates
Small mammals and triatomines were captured from January to February (austral summer season) and from July to August (austral winter season) of 2011. The six study sites—Localities 1 to 6—were in North-Central Chile, from 30º49’S to 33º39’S, encompassing around 300 km from the northernmost to the southernmost study site (S1 Fig). Most of the rainfall in all study sites concentrates between May and August, which are also the colder months  Details of each locality are shown in S1 Table. Base layers (shapefiles) of administrative boundaries, rivers and elevation were obtained from freely available sources for academic use and other non-commercial use [41, 42]; point shapefiles of trapping sites and maps were produced specifically for this investigation, in QGIS Desktop 2.18.2 software, a free and open source Geographic Information System .
Small mammal sampling
Small mammals were captured using live traps (Rodentrap Special Forma and Rodentrap Berlin Forma, Santiago, Chile, and Tomahawk traps, Wisconsin, USA) with rolled oat as bait and cotton as shelter for the captured animals. Traps were placed in linear patterns separated by approximately 10 m, labeled and georeferenced. Each captured mammal was anesthetized with isoflurane and blood sampled in a field laboratory. Detailed procedure is available at dx.doi.org/10.17504/protocols.io.wnxfdfn.
Sampling procedures were authorized by the Servicio Agrícola y Ganadero (SAG Resolution N° 6853) and by the Bioethics Committee of the Facultad de Ciencias Veterinarias y Pecuarias, Universidad de Chile (Certificate Nº 17), regulated in Chile according to Ley 19.473 about wildlife hunt and capture , and Ley 20.380 about animal protection . Live capture, marking, holding, maintenance, and blood withdrawal followed international guidelines for wild and laboratory mammals [46, 47].
Triatomines were captured using baited traps. A total of 90 yeast baited traps per day were set during summer, and 72 mouse baited traps during winter. Traps were placed following linear patterns, separated by 10 m in rocky outcrops and rock piles; and assorted according to the availability of terrestrial bromeliads if present. Each trap was georeferenced in UTM WGS84 19S coordinate system. Traps were activated at sunset and collected the next morning. Detailed capture procedure is available at dx.doi.org/10.17504/protocols.io.wnpfddn. Captured triatomines were transferred to individual flasks for transportation. Triatomine species were identified based on its morphological description [30, 48]. Insects were euthanized with ether overdose, and their abdomens were dissected using individual scalpels.
DNA was extracted from small mammals’ blood samples (100 μl) using the Quick-gDNA Blood MiniPrep kit. Triatomines’ abdomens were macerated with 190 μl Guanidine-HCl 6 M—EDTA 0.2 M solution and incubated with 10 μl of proteinase K solution (20 mg/ml) during 3 hours at 54°C. Samples were then centrifuged for 1 min at 10.000 x g; the supernatant was transferred to another microcentrifuge tube and its DNA was extracted using the Quick-gDNA MiniPrep (Zymo Research) kit. Both blood and triatomine eluates were resuspended in 100 μl nuclease free water.
Trypanosoma cruzi infection
Trypanosoma cruzi infection status of each DNA sample was determined by conventional PCR using a master mix containing 5 μl of DNA sample, buffer solution 1x; dATP, dCTP, dGTP y dTTP 0.38 mM each; MgCl2 1.37 mM; 1.3 units of Paq DNA Polimerase (Agilent); 0.4 μM of each oligonucleotide: 121 and 122, which anneal to CSB2 and CSB3 of T. cruzi’s kinetoplast minicircles, respectively ; and nuclease free water to complete 32 μl. Each run included a positive (purified T. cruzi kDNA) and a template free control (nuclease free water). Amplification was performed with a cycling protocol of: 1 min at 98°C and 2 min at 64°C; followed by 33 cycles at: 94°C for 1 min and 64°C for 1 min; ending with a 10 min cycle at 72°C. Ten μl of amplified samples were run in a 2% Tris-Borate-EDTA agarose gel with GelRed nucleic acid stain for 60 min at 90 volts. Samples were considered positive to T. cruzi infection when a 330 pair base band was observed by ultraviolet transillumination after electrophoresis.
PCR positive samples were genotyped with a DNA blot technique. In this procedure it is expected that two minicircle sequences will cross-react if hybridized under high stringency conditions only if they belong to the same sequence class; that is, if they present homologies in the divergent region . Minicircle hybridization is a complex technique that has the advantage of working with low parasite amounts, and may be used for direct genotyping without the bias of parasite isolation and culture, which may favor the selection of some T. cruzi clones from a mixture [14, 51]. Further details are specified in dx.doi.org/10.17504/protocols.io.sz2ef8e.
R software version 3.5.1, with packages rcompanion, RVAideMemoire, Lme4 and epiDisplay, were used for statistical analyses. Descriptive statistics of the infection status were included for species, localities, and study periods. Differences in the frequencies of infection were analyzed by species using Fisher’s exact test, testing a posteriori differences between mammal species using a pairwise test of independence for nominal data, with a significance level of α = 0.05. The infection status of vectors was modeled using the locality (1–6), study period (summer vs. winter) and the species (T. infestans and M. spinolai) as predictors in a factorial logistic regression. A separate model was generated for the infection status of small mammals, using three categories for the species variable: Octodon sp., P. darwini, and all other small mammals combined, along with the variables locality and study period. Bar charts were created in Microsoft Excel (Microsoft Office Professional Plus 2010, version 14.0.7208.5000).
Cluster analysis was performed using SaTScan v9.4.4 64-bit software (Kulldorff M. and Information Management Services, Inc. SaTScan v8.0: Software for the spatial and space-time scan statistics. http://www.satscan.org/, 2009. “SaTScan is a trademark of Martin Kulldorff. The SaTScan software was developed under the joint auspices of (i) Martin Kulldorff, (ii) the National Cancer Institute, and (iii) Farzad Mostashari of the New York City Department of Health and Mental Hygiene”). Spatial, temporal and space-time cluster detection were performed in each locality for small mammals and triatomines, separated and combined. We used the default software settings except by using an elliptical scanning window and 9999 iterations of Standard Monte Carlo procedure for calculations [52, 53]. To determine if there was clustering of the infection status, we used the Bernoulli model , where each individual (triatomine or small mammal) was either a case (1)—which corresponded to an infected vector or host—or a control (0)–an uninfected individual.
A total of 710 small mammals and 1140 triatomines were captured. Small mammals belonged to two rodent Suborders: Hystricomorpha and Myomorpha, and to one marsupial Order: Didelphimorphia . Almost 76.5% of the captured mammals were Octodon sp. (n = 356) and P. darwini (n = 187). Mepraia spinolai (n = 595) and T. infestans (n = 545) were collected, found in sympatry only in Locality 4 (Fig 1). Detailed number of small mammals and triatomines captured by locality and study period is available in S2 Table.
All infection results are presented indicating the average and the 95% confidence interval, in Tables 1 and 2. We detected 215 small mammals infected with T. cruzi (30.3% of infection; 95% CI 27.0–33.7%), presenting different infection rates among mammal species, without considering locality or study period (Fisher’s exact test, p<0.001). A posteriori pairwise comparisons showed that only Octodon sp. and P. darwini were statistically different (adjusted p = 0.0287), with P. darwini showing higher rates of infection (39.0%) than Octodon sp. (25.0%). Rattus norvegicus and A. longipilis showed the highest and lowest infection rates, respectively. Locality 3 showed the highest, and Locality 1 showed the lowest infection rate when considering all mammals combined. During the summer, infection of small mammals was 35.6% and in the winter was 25.6%. In summer, the most and the less infected species were A. olivaceus and O. longicaudatus, respectively. During winter, R. norvegicus and A. longipilis presented the highest rate and lowest infection rates, respectively. Detailed results of infection are presented in Tables 1 and 2, and in S2 Table. In the factorial logistic regression, all the tested variables were retained as predictors for infection status of small mammals. Phyllotis darwini and other small mammals presented greater odds of infection than Octodon sp; Locality 1 presented lower odds than all the rest localities; finally, small mammals exhibited lower odds of being infected in winter versus summer (Table 3).
We detected 389 triatomines infected with T. cruzi (34.1% of infection; 95% CI 31.4–36.9%), with higher infection rates in M. spinolai (39.7%) than T. infestans (28.1%) when comparing both species without considering locality or study period (Fisher's exact test, p<0.001). Locality 5 presented the highest triatomine infection rates (M. spinolai: 43.5%) and Locality 4 the lowest (both triatomine species combined: 26.5%; M. spinolai: 27.1%; T. infestans: 25.0%). Disregarding triatomine species and locality, higher infection rates were detected during winter (41.2%, n = 170) compared to summer (32.9%, n = 970). During summer, Mepraia spinolai presented 38.9% of infection, and T. infestans 28.0%, combining all localities, and in winter, M. spinolai showed 41.6%, and T. infestans 33.3%. During summer, Locality 2 had the highest rate of infection, and Locality 3 the lowest. Meanwhile, during winter, Locality 3 presented the highest infection rates, and Locality 1 the lowest. Detailed results of infection are presented in Tables 1 and 2, and in S2 Table. The model selected for triatomines retained only the species as predictor, showing that M. spinolai individuals were more frequently infected than T. infestans (p<0.001; Table 3).
In small mammals, 45 out of 215 positive PCR samples hybridized with at least one probe tested (45 effective hybridizations). Only 110 out of 389 triatomine positive samples corresponded to effective hybridizations. We detected, in decreasing frequency, TcI, TcII, TcVI and TcV in small mammals (Table 4). Positive samples from A. bennetti, A. olivaceus and R. norvegicus did not bind to any probe. Only one positive sample of A. longipilis and R. rattus hybridized with TcI and TcII as single infections, respectively. Only Octodon sp. and P. darwini presented all four DTUs tested (S3 Table). The DTUs detected in triatomines were TcI, TcII, TcV and TcVI, in decreasing frequency. In M. spinolai TcV was not detected, and TcVI was detected in only one sample (Table 4).
One individual may be infected with more than one DTU. M = Mammals; T = Triatomines.
We detected the four DTUs in all localities, except in Localities 2 and 4 where TcV was not detected. Locality 6 was the only study site with all four DTUs detected both in triatomines and small mammals. Disregarding locality, during summer in Octodon sp. and P. darwini only TcI and TcII were detected, as single infections, but during winter, all four DTUs were found. We observed the opposite pattern in triatomines, in which we detected all four DTUs during summer and just TcI and TcII in winter. Detailed results of DTUs are shown in S3 Table.
In small mammals, we detected 62.2% single infections (hybridization with just one DTU) and 37.8% mixed infections (hybridization with more than one DTU) (Table 5). When analyzing the two most abundant species, Octodon sp. showed more single (72.7%) than mixed infections (27.3%), while P. darwini presented a similar proportion of single (55.6%) and mixed (44.0%) infections. In triatomines, we detected 66.4% of single and 33.6% mixed infections, with T. infestans showing more mixed infections than M. spinolai (43.9% v/s 18.2%, respectively) (Table 5). We detected a mixed infection in one O. longicaudatus with TcI+TcVI, and a single infection in the same rodent species with TcV. The marsupial species T. elegans presented a mixed infection with TcI+TcII, and the other two with DTU determined were single infections with TcI.
Only effective hybridizations are included. A positive individual was categorized as having either single or mixed infections.
When evaluating single and mixed infections by locality, there is not a clear pattern, but it seems that single infections were more frequent in both small mammals and triatomines. We did not detect mixed infections in small mammal species during summer. In triatomines we detected similar proportions of single and mixed infections in both study periods.
We detected a total of 21 significant spatial, temporal and spatio-temporal clusters in five localities (S4 Table). In general terms, T. cruzi clustering was more common in vectors than in hosts, with a total of 10 purely spatial and spatio-temporal clusters detected in triatomines in three localities; when combining vectors and hosts, we found 9 clusters. Most clusters were detected in Locality 6 (11 out of 21). We mapped only purely spatial clusters of infection (Fig 2).
In this study, we analyzed spatio-temporal infection and DTUs detected in populations of small mammals and triatomines from an endemic region of Chile. Here, the rodents Octodon sp. and P. darwini, and the triatomines M. spinolai and T. infestans appear to be particularly important wild hosts and vectors of T. cruzi, respectively. All tested DTUs were detected, with predominance of TcI. Infection varied among species, localities and study periods in small mammals, and between triatomines. Significant spatial, temporal and spatio-temporal clusters for infection were detected, mainly in vectors from the southernmost localities.
Octodon sp. and P. darwini were the most frequent and ubiquitously captured mammal species in this study. Octodontids’ specimens were not identified at species level; however, we assumed that they were Octodon degus. The infection rates of both rodent species might be related to their higher relative abundances and life history traits, increasing their probability to become a feeding source for triatomines due to higher contact rates [20, 55]. These two rodent species have partially overlapping distributions and home ranges . Octodon sp. has been found associated to Puya sp., a terrestrial bromeliad of semiarid Chile , described as refuge for T. infestans and M. spinolai . Phyllotis darwini’s nests are commonly found within abandoned Octodon sp. burrows . Despite these species’ ecological proximity, P. darwini exhibited significantly higher infection rates than Octodon sp. This difference might be explained by their behavior: P. darwini is nocturnal and M. spinolai diurnal, making this host easily available for the triatomine during the day, when P. darwini rests. On the contrary, the nocturnal T. infestans would feed on the diurnal O. degus during the night [27, 33]. This different infection rate is relevant, given that infected P. darwini are reported to travel more than the uninfected, dispersing the parasite, while infected O. degus move less than uninfected specimens .
Synanthropic species—R. norvegicus and R. rattus—were less abundant in this study, but had high infection rates, as previously reported in Chile [23, 27]. These rodents could have an important role in Chagas disease epidemiology, since they circulate in sylvatic and domestic areas [23, 60]. Some small mammal species captured were not abundant but were nonetheless infected with T. cruzi. To our knowledge, this is the first report of infection in O. longicaudatus, with eight out of 45 infected. Thylamys elegans, A. bennetti and A. longipilis were also infected, with 42,9%, 22.2%, and 9.5% of infection, respectively. Comparing our findings with the infection rates found previously in small mammals with molecular techniques, they are similar to those found in Chile [19, 27, 61] and in other countries [3, 62, 63]; however, they are quite different to previous reports from Bolivia and Argentina [64, 65].
As mentioned, the infection status of small mammals was explained by the host species, locality and study period. It is possible that the particular geo-climatic conditions–temperature, precipitation and elevation–could influence T. cruzi transmission among vectors and mammals, as reported in mice inoculated with T. cruzi isolates from higher elevation, which showed the lowest parasitemia . However, differential availability of vertebrates could also explain these differences in small mammals’ infection between localities, since the localities with higher odds of infection for mammals presented also lower numbers of mammals captured. It is possible that when there are fewer individuals available, their probability of becoming the vectors’ blood-meal, and therefore, their chance of becoming infected, increase. Regarding the study period, during winter mammals showed lower odds of infection than during summer. Hosts seem to control infection after the acute period, reducing their parasitemia [24, 67]; therefore, this control could occur during the winter in our study system.
In triatomines we detected that T. cruzi infection rate was higher in M. spinolai than T. infestans. Mepraia spinolai has been described as the most relevant vector in the sylvatic transmission cycle of T. cruzi in Chile ; however, T. infestans has been traditionally considered the vector to humans , given its domestic and peridomestic habitat preferences and higher transmission efficiency by a faster post-feeding defecation than M. spinolai . Although the lower T. cruzi infection detected in T. infestans could be an optimistic result, T. cruzi infection modifies M. spinolai’s behavior, reducing its defecation time , improving its ability as vector. Thus, M. spinolai’s relevance should not be neglected, considering its high infection rates and widespread distribution in North-Central Chile. Previous studies in sylvatic areas of Chile showed variable T. cruzi infection rates, ranging from 36.5% to 57.7% in T. infestans and 29.9%-76.1% in M. spinolai [32, 33, 36–38, 71]. Small mammal species composition at the localities where T. infestans and M. spinolai were found was slightly different, but their availability of larger mammals was probably very different. This may be explaining why in Locality 4, where both triatomine species were present, they showed similar infection rates, so different mammals’ availability may influence triatomines’ infection [37, 61]. Unfortunately, our design precluded the study of larger mammals.
Triatomine species was the only variable retained as predictor of triatomine infection status. Locality and study period seemed to be less relevant for triatomines’ infection status than to hosts. Previous studies have shown temporal variations in density and infection rates of hosts and vectors, comparing different years [33, 68, 72]. Here we analyzed two study periods within one year, and small mammals were significantly more infected during summer than winter. On the contrary, triatomines’ infection was higher during winter than summer. Previous studies have shown that abundance and home range of hosts and triatomines increase during summer in the North-Central Chile and Argentina [22, 57, 73–75]. Additionally, the maximum overall densities of M. spinolai occurred in summer months ; accordingly, a study of T. infestans in Argentina showed higher densities in houses between spring and autumn and a decrease in winter, and also that the number of parasites in triatomines’ rectal contents showed seasonal changes, with higher values in late spring . In Triatoma protracta, lower environmental temperatures retarded and higher temperatures increased the number of metacyclic trypomastigotes released in its dejections . During warm weather there was a larger diversity of alimentary sources than in cold weather in M. spinolai , supporting the idea of higher T. cruzi transmission risk to small mammals during warmer months in Chile that could have led to high T. cruzi’s parasitemia and higher detection of infection in their blood samples.
Vector population composition varied between study periods for M. spinolai, with concomitant higher infection rates in winter . Also, it is possible that infection in triatomines is more easily detected after some time since parasite ingestion, allowing T. cruzi to multiply . Sylvatic Triatoma brasiliensis showed higher infection when its nutritional status was better . Long fasting periods can eliminate 99.5% of T. cruzi flagellates in the triatomines’ rectum , explaining why M. spinolai increased its infection rate in dejections with supplementary feeding . We expected that during winter the availability of hosts were lower, but we captured lower numbers of small mammals during winter only in two localities, so this may suggest that triatomines captured during winter could have maintained their T. cruzi populations. Unfortunately, nutritional status of the captured triatomines was not evaluated.
In this study, the number of triatomines caught during summer was almost six times the number of triatomines found during winter; it is possible that a differential bait attraction could account for this difference. However, previous studies have shown that M. spinolai and T. infestans are not active when temperature is below 15 ºC , which is frequent during the cold season in the sampled localities. The higher infection rates detected in winter may have been related to this lower sample size, but in laboratory, T. cruzi-infected M. spinolai showed reduced time to detect potential hosts in comparison to uninfected insects , so they might have been able to find baited traps more easily in adverse climatic conditions.
A high number of positive samples did not hybridize with any of the probes used in our study. It is possible that some samples corresponded to DTUs not tested in this study (TcIII or TcIV) or to the genotype TcBat. It is also possible that the unidentified samples corresponded to one of the tested DTUs, but with slight genetic differences that prevented hybridization, as reported in other endemic areas . This is particularly relevant for TcI, with greater internal diversity than the other DTUs . Another possibility explaining our low efficiency in the hybridization tests is a reduced amount of kDNA transferred to the nylon membranes.
TcI was the most frequently detected DTU in small mammals and triatomines, agreeing with previous reports in small mammals [16, 25, 27], triatomines [36, 37, 51, 87], and humans in Chile [17, 51], and in sylvatic and domestic cycles of America . Regarding the type of infection, this study agrees with others where single infections were more common than mixed ones in triatomines and small mammals [25, 27, 36, 37]. However, given our low number of positive samples effectively hybridized, we cannot be confident that this tendency would have remained the same under complete DTUs detection. We were able to detect mixed infections in four small mammal species. In triatomines, we detected higher number of mixed infections in T. infestans.
We found more infection clusters in triatomines than in small mammals. We also found clustering when combining triatomines and small mammals, agreeing with a report relating host probability of infection with their distance to M. spinolai colonies . As supported here, there are spots in space where infected individuals aggregate. Mepraia spinolai exhibits a sit–and-wait strategy for finding hosts , and seems to feed on species according to availability [20, 22], same as T. infestans . Therefore, triatomines’ cohorts from eggs laid in the same microsite would feed on the nearest available host. If these hosts had been infected, triatomines would become infected and later infect other small mammals, producing clustered spatial patterns of higher rates of infection. Moreover, infection among triatomines could be enhanced by coprophagy and cannibalism . In mammals, potential congenital transmission [89, 90] could also perpetuate infection on site.
Locality 6 had most of the purely spatial clusters of infection, followed by Locality 5, where the ecotope providing shelter for both triatomines and small mammals were terrestrial bromeliads, presenting a spatially aggregated distribution [27, 32, 91]. Dispersion from these patches may be more difficult than from a continuous ecotope, as rocky outcrops, enhancing transmission in case they were infected. Future studies should evaluate the variables that differentiate cluster areas from the rest, which could be related with biotic conditions, as reported for sylvatic Rhodnius spp. inhabiting palms . Locality 6 clusters were near human dwellings, so extra precautions should be taken to avoid exposure to vectors. Sporadic dwelling invasion of wild triatomines has been reported as the main vectorial risk in Chile .
We found purely temporal clusters of infection, with higher infection rates during summer in Localities 1 and 2. The temporal differences of infection in these localities might be related with the changes in density and composition of small mammals’ community, mainly due to differences in density and infection of the two most abundant small mammals, Octodon sp. and P. darwini.
The detected spatio-temporal clustering of infection shows sites presenting different rates between periods. Site’s microclimatic conditions may vary transmission, or individuals could aggregate in these sites during some periods.
In sum, our study evaluated T. cruzi infection, described DTUs and clustering from locations in a vast geographical extension during two contrasting seasons, determining in a widespread manner T. cruzi infection distribution in host and vector populations simultaneously, unveiling some of the eco-epidemiological complexity of T. cruzi wild cycle in Chile.
This study describes Trypanosoma cruzi infection status, infecting DTUs, and determines the spatial and temporal variations of infection in small mammals and triatomines of the endemic zone of Chile. Octodon sp. and Phyllotis darwini were the most represented small mammals, and they showed high infection rates, thus representing important wild hosts. Mepraia spinolai presented higher infection rate than Triatoma infestans; however, non-domestic populations of both vectors were infected in all localities and study periods evaluated, emphasizing the need for sustaining prevention measures even if domestic vectorial transmission has been interrupted. We detected the four tested DTUs in triatomines and small mammals, with an overall predominance of TcI, following the trend of Chile and America. Significant spatial, temporal and spatio-temporal clusters for infection were detected within localities, mainly in triatomines. Finally, we can conclude that T. cruzi infection varies between host and vector species, localities and study periods in North-Central endemic zone of Chile.
S1 Table. Geographic and bioclimatic description of the study sites.
S2 Table. Trypanosoma cruzi infection in small mammals and triatomines.
Number of captured individuals by species, locality and study period. Percentage of infection with T. cruzi between parentheses.
S3 Table. Number of individuals with detected DTU by species, locality and study period.
One individual might be infected with more than one DTU.
S4 Table. Description of statistically significant spatial, temporal or spatio-temporal clusters of infection status detected in small mammals, triatomines or both.
To undergraduate students (Raúl Mena, Felipe Rojas, Ricardo González), technicians (Natalia Lártiga, Patricio Arroyo, Mariela Puebla) and to many volunteers, for their field and laboratory work. To MINSAL, and MINEDUC for logistic support in the field. To CONAF for authorizing the research in Reserva Nacional Las Chinchillas.
- 1. Coura JR, Vinas PA. Chagas disease: a new worldwide challenge. Nature. 2010;465(7301):S6–7. pmid:20571554
- 2. Rassi A Jr, Rassi A, Marin-Neto JA. Chagas disease. Lancet. 2010;375(9723):1388–402. pmid:20399979
- 3. Noireau F, Diosque P, Jansen AM. Trypanosoma cruzi: adaptation to its vectors and its hosts. Vet Res. 2009;40(2):26. pmid:19250627
- 4. Marinkelle CJ. Direct transmission of Trypanosoma cruzi between individuals of Rhodnius prolixus Stal. Rev Biol Trop. 1965;13:55–8.
- 5. Canals M, Gonzalez C, Canals L, Canals A, Caceres D, Alvarado S, et al. [What do the numbers tell us about the temporal evolution of Chagas' disease?]. Rev Chil Infectol. 2017;34(2):120–7.
- 6. Lukes J, Guilbride DL, Votypka J, Zikova A, Benne R, Englund PT. Kinetoplast DNA network: evolution of an improbable structure. Eukaryot Cell. 2002;1(4):495–502. pmid:12455998
- 7. Degrave W, Fragoso SP, Britto C, van Heuverswyn H, Kidane GZ, Cardoso MA, et al. Peculiar sequence organization of kinetoplast DNA minicircles from Trypanosoma cruzi. Mol Biochem Parasitol. 1988;27(1):63–70. pmid:2830509
- 8. Avila HA, Sigman DS, Cohen LM, Millikan RC, Simpson L. Polymerase chain reaction amplification of Trypanosoma cruzi kinetoplast minicircle DNA isolated from whole blood lysates: diagnosis of chronic Chagas' disease. Mol Biochem Parasitol. 1991;48(2):211–21. pmid:1662334
- 9. Veas F, Breniere SF, Cuny G, Brengues C, Solari A, Tibayrenc M. General procedure to construct highly specific kDNA probes for clones of Trypanosoma cruzi for sensitive detection by polymerase chain reaction. Cell Mol Biol. 1991;37(1):73–84. pmid:2059987
- 10. Zingales B, Andrade SG, Briones MRS, Campbell Da, Chiari E, Fernandes O, et al. A new consensus for Trypanosoma cruzi intraspecific nomenclature: second revision meeting recommends TcI to TcVI. Mem Inst Oswaldo Cruz. 2009;104(7):1051–4. pmid:20027478
- 11. Marcili A, Lima L, Cavazzana M, Junqueira AC, Veludo HH, Maia Da Silva F, et al. A new genotype of Trypanosoma cruzi associated with bats evidenced by phylogenetic analyses using SSU rDNA, cytochrome b and Histone H2B genes and genotyping based on ITS1 rDNA. Parasitology. 2009;136(6):641–55. pmid:19368741
- 12. Zingales B, Miles Ma, Campbell Da, Tibayrenc M, Macedo AM, Teixeira MMG, et al. The revised Trypanosoma cruzi subspecific nomenclature: rationale, epidemiological relevance and research applications. Infect Genet Evol. 2012;12(2):240–53. pmid:22226704
- 13. Llewellyn MS, Lewis MD, Acosta N, Yeo M, Carrasco HJ, Segovia M, et al. Trypanosoma cruzi IIc: phylogenetic and phylogeographic insights from sequence and microsatellite analysis and potential impact on emergent Chagas disease. PLoS Negl Trop Dis. 2009;3(9):e510. pmid:19721699
- 14. Zingales B. Trypanosoma cruzi genetic diversity: Something new for something known about Chagas disease manifestations, serodiagnosis and drug sensitivity. Acta Trop. 2018;184:38–52. pmid:28941731
- 15. Schaub GA. Trypanosoma cruzi: Quantitative studies of development of two strains in small intestine and rectum of the vector Triatoma infestans. Exp Parasitol. 1989;68(3):260–73. pmid:2649388
- 16. Campos R, Acuna-Retamar M, Botto-Mahan C, Ortiz S, Cattan PE, Solari A. Susceptibility of Mepraia spinolai and Triatoma infestans to different Trypanosoma cruzi strains from naturally infected rodent hosts. Acta Trop. 2007;104(1):25–9. pmid:17904090
- 17. Coronado X, Zulantay I, Albrecht H, Rozas M, Apt W, Ortiz S, et al. Variation in Trypanosoma cruzi clonal composition detected in blood patients and xenodiagnosis triatomines: Implications in the molecular epidemiology of Chile. Am J Trop Med Hyg. 2006;74(6):1008–12. pmid:16760511
- 18. Wilson DE, Reeder DM. Mammal species of the world: a taxonomic and geographic reference. 3rd ed. Baltimore: Johns Hopkins University Press; 2005. 2142 p.
- 19. Rozas M, Botto-Mahan C, Coronado X, Ortiz S, Cattan PE, Solari A. Short report: Trypanosoma cruzi infection in wild mammals from a chagasic area of Chile. Am J Trop Med Hyg. 2005;73(3):517–9. pmid:16172474
- 20. Chacon F, Bacigalupo A, Quiroga JF, Ferreira A, Cattan PE, Ramirez-Toloza G. Feeding profile of Mepraia spinolai, a sylvatic vector of Chagas disease in Chile. Acta Trop. 2016;162:171–3. pmid:27349188
- 21. Muñoz-Pedreros A, Yáñez J. Mamíferos de Chile. Valdivia, Chile: CEA Ediciones; 2000. 464 p.
- 22. Botto-Mahan C, Cattan PE, Canals M, Acuna M. Seasonal variation in the home range and host availability of the blood-sucking insect Mepraia spinolai in wild environment. Acta Trop. 2005;95(2):160–3. pmid:15949784
- 23. Yefi-Quinteros E, Munoz-San Martin C, Bacigalupo A, Correa JP, Cattan PE. Trypanosoma cruzi load in synanthropic rodents from rural areas in Chile. Parasit Vectors. 2018;11(1):171. pmid:29530074
- 24. Rojo G, Sandoval-Rodríguez A, López A, Ortiz S, Saavedra M, Botto-Mahan C, et al. Within-host temporal fluctuations of Trypanosoma cruzi discrete typing units: the case of the wild reservoir rodent Octodon degus. Parasit Vectors. 2017;10:380. pmid:28784152
- 25. Rozas M, Botto-Mahan C, Coronado X, Ortiz S, Cattan PE, Solari A. Coexistence of Trypanosoma cruzi genotypes in wild and periodomestic mammals in Chile. Am J Trop Med Hyg. 2007;77(4):647–53. pmid:17978065
- 26. Botto-Mahan C, Acuna-Retamar M, Campos R, Cattan PE, Solari A. European rabbits (Oryctolagus cuniculus) are naturally infected with different Trypanosoma cruzi genotypes. Am J Trop Med Hyg. 2009;80(6):944–6. pmid:19478255
- 27. Galuppo S, Bacigalupo A, Garcia A, Ortiz S, Coronado X, Cattan PE, et al. Predominance of Trypanosoma cruzi genotypes in two reservoirs infected by sylvatic Triatoma infestans of an endemic area of Chile. Acta Trop. 2009;111(1):90–3. pmid:19426670
- 28. Lacher TEJ, Mares MA. The structure of Neotropical mammal communities: an appraisal of current knowledge. Rev Chil Hist Nat. 1986;59(2):121–34.
- 29. Iriarte A. Mamíferos de Chile. Barcelona, Spain: Editorial LYNX; 2008. 420 p.
- 30. Lent H, Wygodzinsky PW. Revision of the Triatominae (Hemiptera, Reduviidae), and their significance as vectors of Chagas' disease. B Am Mus Nat Hist. 1979;163:125–520.
- 31. Frias-Lasserre D. A new species and karyotype variation in the bordering distribution of Mepraia spinolai (Porter) and Mepraia gajardoi Frias et al (Hemiptera: Reduviidae: Triatominae) in Chile and its parapatric model of speciation. Neotrop Entomol. 2010;39(4):572–83. pmid:20877994
- 32. Bacigalupo A, Segura JA, Garcia A, Hidalgo J, Galuppo S, Cattan PE. [First finding of Chagas disease vectors associated with wild bushes in the Metropolitan Region of Chile]. Rev Med Chil. 2006;134(10):1230–6. pmid:17186091
- 33. Bacigalupo A, Torres-Perez F, Segovia V, Garcia A, Correa JP, Moreno L, et al. Sylvatic foci of the Chagas disease vector Triatoma infestans in Chile: description of a new focus and challenges for control programs. Mem Inst Oswaldo Cruz. 2010;105(5):633–41. pmid:20835609
- 34. Frias-Lasserre D, Gonzalez CR, Valenzuela CR, de Carvalho DB, Oliveira J, Canals M, et al. Wing polymorphism and Trypanosoma cruzi infection in wild, peridomestic, and domestic collections of Mepraia spinolai (Hemiptera: Reduviidae) from Chile. J Med Entomol. 2017;54:1061–6. pmid:28399301
- 35. Canals M, Solis R, Valderas J, Ehrenfeld M, Cattan PE. Preliminary studies on temperature selection and activity cycles of Triatoma infestans and T. spinolai (Heteroptera: Reduviidae), Chilean vectors of Chagas' disease. J Med Entomol. 1997;34(1):11–7. pmid:9086704
- 36. Bacigalupo A, Segovia V, Garcia A, Botto-Mahan C, Ortiz S, Solari A, et al. Differential pattern of infection of sylvatic nymphs and domiciliary adults of Triatoma infestans with Trypanosoma cruzi genotypes in Chile. Am J Trop Med Hyg. 2012;87(3):473–80. pmid:22802439
- 37. Coronado X, Rozas M, Botto-Mahan C, Ortiz S, Cattan PE, Solari A. Molecular epidemiology of Chagas disease in the wild transmission cycle: the evaluation in the sylvatic vector Mepraia spinolai from an endemic area of Chile. Am J Trop Med Hyg. 2009;81(4):656–9. pmid:19815882
- 38. Correa JP, Bacigalupo A, Fonturbel FE, Oda E, Cattan PE, Solari A, et al. Spatial distribution of an infectious disease in a small mammal community. Naturwissenschaften. 2015;102(9–10):51. pmid:26289933
- 39. Chetwynd AG, Diggle PJ, Marshall A, Parslow R. Investigation of spatial clustering from individually matched case-control studies. Biostatistics. 2001;2(3):277–93. pmid:12933539
- 40. Climate-Data.org. Oedheim: AM Online Projects: Alexander Merkel; [cited 2018 May 2]. Available from: https://es.climate-data.org/.
- 41. Coberturas SIG para la enseñanza de la Geografía en Chile [Internet]. Universidad de La Frontera. Temuco. 2012. Available from: www.rulamahue.cl/mapoteca.
- 42. GADM database of Global Administrative Areas version 1.0 [Internet]. 2009 [cited March 2009]. Available from: www.gadm.org.
- 43. QGIS Geographic Information System [Internet]. Open Source Geospatial Foundation Project. [cited 2017]. Available from: http://qgis.osgeo.org.
- 44. Ley Nº 19.473 [Internet]. 1996. Available from: http://bcn.cl/1v8wm.
- 45. Ley Nº 20.380 [Internet]. 2009. Available from: http://bcn.cl/1v48z.
- 46. Removal of blood from laboratory mammals and birds. First report of the BVA/FRAME/RSPCA/UFAW Joint Working Group on Refinement. Laboratory animals. 1993;27(1):1–22. pmid:8437430
- 47. Gannon WL, Sikes RS, Mammalogists AcauCotASo. Guidelines of the American Society of Mammalogists for the use of wild mammals in research. J Mammal. 2007;88(3):809–23.
- 48. Frias D, Martinez H, Wallace A. [Some taxonomic features of Triatoma spinolai Porter (Hemiptera: Reduviidae)]. Acta Entomol Chil. 1987;14:155–70.
- 49. Wincker P, Britto C, Pereira JB, Cardoso MA, Oelemann W, Morel CM. Use of a simplified polymerase chain reaction procedure to detect Trypanosoma cruzi in blood samples from chronic chagasic patients in a rural endemic area. Am J Trop Med Hyg. 1994;51(6):771–7. pmid:7810810
- 50. Macina RA, Arauzo S, Reyes MB, Sanchez DO, Basombrio MA, Montamat EE, et al. Trypanosoma cruzi isolates from Argentina and Chile grouped with the aid of DNA probes. Mol Biochem Parasitol. 1987;25(1):45–53. pmid:2823134
- 51. Arenas M, Campos R, Coronado X, Ortiz S, Solari A. Trypanosoma cruzi genotypes of insect vectors and patients with Chagas of Chile studied by means of cytochrome b gene sequencing, minicircle hybridization, and nuclear gene polymorphisms. Vector Borne Zoonotic Dis. 2012;12(3):196–205. pmid:22022808
- 52. Carpenter TE. Methods to investigate spatial and temporal clustering in veterinary epidemiology. Prev Vet Med. 2001;48(4):303–20. pmid:11259822
- 53. Kulldorff M, Heffernan R, Hartman J, Assuncao R, Mostashari F. A space-time permutation scan statistic for disease outbreak detection. PLoS Med. 2005;2(3):e59. pmid:15719066
- 54. Kulldorff M. A spatial scan statistic. Commun Stat-Theor M. 1997;26(6):1481–96.
- 55. Alzamora A, Correa P, Gaggero E, Acuña-Retamar M, Cattan PE. [Feeding behaviour of Mepraia spinolai in two frequent hosts from its habitat]. Parasitol Latinoam. 2007;62:112–7.
- 56. Jimenez J, Lorca M. American trypanosomiasis in wild vertebrates and its relation to the vector Triatoma spinolai. Arch Med Vet. 1990;22(2):179–83.
- 57. Meserve PL, Martin RE, Rodriguez J. Comparative ecology of the caviomorph rodent Octodon degus in two Chilean Mediterranean-type communities. Rev Chil Hist Nat. 1984;57:79–89.
- 58. Bozinovic F, Rosenmann M, Veloso C. [Behavioral thermoregulation in Phyllotis darwini (Rodentia: Cricetidae): effects of ambient temperature, nest use, and huddling on energy expenditure. Rev Chil Hist Nat. 1988;61(1):81–6.
- 59. Jiménez C, Fontúrbel FE, Oda E, Ramírez PA, Botto-Mahan C. Parasitic infection alters rodent movement in a semiarid ecosystem. Mamm Biol. 2015;80(4):255–9.
- 60. Lobos G, Ferres M, Palma RE. [Presence of the invasive genera Mus and Rattus in natural areas in Chile: an environmental and epidemiological risk]. Rev Chil Hist Nat. 2005;78(1):113–24.
- 61. Oda E, Solari A, Botto-Mahan C. Effects of mammal host diversity and density on the infection level of Trypanosoma cruzi in sylvatic kissing bugs. Med Vet Entomol. 2014;28(4):384–90. pmid:24844934
- 62. Mejía-Jaramillo AM, Agudelo-Uribe LA, Dib JC, Ortiz S, Solari A, Triana-Chavez O. Genotyping of Trypanosoma cruzi in a hyper-endemic area of Colombia reveals an overlap among domestic and sylvatic cycles of Chagas disease. Parasit Vectors. 2014;7:108. pmid:24656115
- 63. Orozco MM, Piccinali RV, Mora MS, Enriquez GF, Cardinal MV, Gurtler RE. The role of sigmodontine rodents as sylvatic hosts of Trypanosoma cruzi in the Argentinean Chaco. Infect Genet Evol. 2014;22:12–22. pmid:24394448
- 64. Torrico MC, Téllez T, Tenorio O, Rojas L, Huaranca JC, De La Barra A, et al. Tripanosomátidos aislados de mamíferos silvestres en tres departamentos de Bolivia (Cochabamba, Potosí y Santa Cruz de la Sierra). Gac Med Bol. 2013;36(1):6–10.
- 65. Alvarado-Otegui JA, Ceballos LA, Orozco MM, Enriquez GF, Cardinal MV, Cura C, et al. The sylvatic transmission cycle of Trypanosoma cruzi in a rural area in the humid Chaco of Argentina. Acta Trop. 2012;124(1):79–86. pmid:22771688
- 66. De Fuentes-Vicente JA, Cabrera-Bravo M, Enriquez-Vara JN, Bucio-Torres MI, Gutierrez-Cabrera AE, Vidal-Lopez DG, et al. Relationships between altitude, triatomine (Triatoma dimidiata) immune response and virulence of Trypanosoma cruzi, the causal agent of Chagas' disease. Med Vet Entomol. 2017;31(1):63–71. pmid:27753118
- 67. Martinez-Hernandez F, Rendon-Franco E, Gama-Campillo LM, Villanueva-Garcia C, Romero-Valdovinos M, Maravilla P, et al. Follow up of natural infection with Trypanosoma cruzi in two mammals species, Nasua narica and Procyon lotor (Carnivora: Procyonidae): evidence of infection control? Parasit Vectors. 2014;7:405. pmid:25174672
- 68. Botto-Mahan C, Campos R, Acuna-Retamar M, Coronado X, Cattan PE, Solari A. Temporal variation of Trypanosoma cruzi infection in native mammals in Chile. Vector Borne Zoonotic Dis. 2010;10(3):317–9. pmid:19505255
- 69. Canals M, Ehrenfeld M, Solís R, Cruzat L, Pinochet A, Tapia C, et al. [Comparative biology of Mepraia spinolai in laboratory and field conditions: five years study]. Parasitol Dia. 1998;22:72–8.
- 70. Botto-Mahan C, Cattan PE, Medel R. Chagas disease parasite induces behavioural changes in the kissing bug Mepraia spinolai. Acta Trop. 2006;98(3):219–23. pmid:16780784
- 71. Botto-Mahan C, Ortiz S, Rozas M, Cattan PE, Solari A. DNA evidence of Trypanosoma cruzi in the Chilean wild vector Mepraia spinolai (Hemiptera: Reduviidae). Mem Inst Oswaldo Cruz. 2005;100(3):237–9. pmid:16113860
- 72. Botto-Mahan C, Rojo G, Sandoval-Rodriguez A, Pena F, Ortiz S, Solari A. Temporal variation in Trypanosoma cruzi lineages from the native rodent Octodon degus in semiarid Chile. Acta Trop. 2015;151:178–81. pmid:26086950
- 73. Quirici V, Castro RA, Ortiz-Tolhuysen L, Chesh AS, Burger JR, Miranda E, et al. Seasonal variation in the range areas of the diurnal rodent Octodon degus. J Mammal. 2010;91(2):458–66. pmid:22328788
- 74. Lima M, Julliard R, Stenseth NC, Jaksic FM. Demographic dynamics of a neotropical small rodent (Phyllotis darwini): feedback structure, predation and climatic factors. J Anim Ecol. 2001;70:761–75.
- 75. Vazquez-Prokopec GM, Ceballos LA, Marcet PL, Cecere MC, Cardinal MV, Kitron U, et al. Seasonal variations in active dispersal of natural populations of Triatoma infestans in rural north-western Argentina. Med Vet Entomol. 2006;20(3):273–9. pmid:17044877
- 76. Cattan PE, Pinochet A, Botto-Mahan C, Acuna MI, Canals M. Abundance of Mepraia spinolai in a periurban zone of Chile. Mem Inst Oswaldo Cruz. 2002;97(3):285–7. pmid:12048552
- 77. Giojalas LC, Catala SS, Asin SN, Gorla DE. Seasonal changes in infectivity of domestic populations of Triatoma infestans. Trans R Soc Trop Med Hyg. 1990;84(3):439–42. pmid:2124395
- 78. Wood SF. Environmental temperature as a factor in development of Trypanosoma cruzi in Triatoma protracta. Exp Parasitol. 1954;3(3):227–33. pmid:13161964
- 79. Canals M, Cruzat L, Molina MC, Ferreira A, Cattan PE. Blood host sources of Mepraia spinolai (Heteroptera: Reduviidae), wild vector of Chagas disease in Chile. J Med Entomol. 2001;38(2):303–7. pmid:11296839
- 80. Ordenes H, Ehrenfeld M, Cattan PE, Canals M. [Tripano-triatomine infection index of Triatoma spinolai in a zone with epidemiological risk for Chagas disease]. Rev Med Chil. 1996;124(9):1053–7. pmid:9197018
- 81. Garcia ES, Ratcliffe NA, Whitten MM, Gonzalez MS, Azambuja P. Exploring the role of insect host factors in the dynamics of Trypanosoma cruzi-Rhodnius prolixus interactions. J Insect Physiol. 2007;53(1):11–21. pmid:17141801
- 82. Sarquis O, Carvalho-Costa FA, Oliveira LS, Duarte R, PS DA, de Oliveira TG, et al. Ecology of Triatoma brasiliensis in northeastern Brazil: seasonal distribution, feeding resources, and Trypanosoma cruzi infection in a sylvatic population. J Vector Ecol. 2010;35(2):385–94. pmid:21175946
- 83. Kollien AH, Schaub GA. The development of Trypanosoma cruzi in Triatominae. Parasitol Today. 2000;16(9):381–7. pmid:10951597
- 84. Egana C, Vergara F, Campos R, Ortiz S, Botto-Mahan C, Solari A. Trypanosoma cruzi infection in Mepraia gajardoi and Mepraia spinolai: the effect of feeding nymphs from the field. Am J Trop Med Hyg. 2014;91(3):534–6. pmid:24935951
- 85. Bosseno MF, Espinoza B, Sanchez B, Breniere SF. Mexican Trypanosoma cruzi stocks: analysis of minicircle kDNA homologies by cross-hybridization. Mem Inst Oswaldo Cruz. 2000;95(4):473–6. pmid:10904401
- 86. Tibayrenc M, Ayala FJ. Isozyme variability in Trypanosoma cruzi, the agent of Chagas disease: genetical, taxonomical, and epidemiological significance. Evolution. 1988;42:277–92. pmid:28567853
- 87. Torres JP, Ortiz S, Munoz S, Solari A. Trypanosoma cruzi isolates from Chile are heterogeneous and composed of mixed populations when characterized by schizodeme and Southern analyses. Parasitology. 2004;128(Pt 2):161–8. pmid:15030003
- 88. Breniere SF, Waleckx E, Barnabe C. Over six thousand Trypanosoma cruzi strains classified into discrete typing units (DTUs): Attempt at an inventory. PLoS Negl Trop Dis. 2016;10(8):e0004792. pmid:27571035
- 89. Torres-Vargas J, Jimenez-Coello M, Guzman-Marin E, Acosta-Viana KY, Yadon ZE, Gutierrez-Blanco E, et al. Quantitative and histological assessment of maternal-fetal transmission of Trypanosoma cruzi in guinea pigs: An experimental model of congenital Chagas disease. PLoS Negl Trop Dis. 2018;12(1):e0006222. pmid:29364882
- 90. Anez N, Crisante G, Soriano PJ. Trypanosoma cruzi congenital transmission in wild bats. Acta Trop. 2009;109(1):78–80. pmid:18823929
- 91. Wisnivesky-Colli C, Schweigmann NJ, Pietrokovsky S, Bottazzi V, Rabinovich JE. Spatial distribution of Triatoma guasayana (Hemiptera:Reduviidae) in hardwood forest biotopes in Santiago del Estero, Argentina. J Med Entomol. 1997;34(2):102–9. pmid:9103752
- 92. Abad-Franch F, Ferraz G, Campos C, Palomeque FS, Grijalva MJ, Aguilar HM, et al. Modeling disease vector occurrence when detection is imperfect: infestation of Amazonian palm trees by triatomine bugs at three spatial scales. PLoS Negl Trop Dis. 2010;4(3):e620. pmid:20209149