Trypanosoma cruzi diversity in infected dogs from

Veterinary Parasitology: Regional Studies and Reports 5 (2016) 42–47

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Trypanosoma cruzi diversity in infected dogs from areas of the north coast of Chile S. Ortiz a,1, M.J. Ceballos b,1, C.R. González c,d, C. Reyes d, V. Gómez e, A. García e, A. Solari a,⁎ a

Programa de Biología Celular y Molecular, ICBM, Facultad de Medicina, Universidad de Chile, Santiago, Chile Escuela de Medicina Veterinaria, Facultad de Ciencias Agropecuarias, Universidad Pedro de Valdivia, La Serena, Chile c Instituto de Entomología, Facultad de Ciencias Básicas, Universidad, Metropolitana de Ciencias de la Educación, Santiago, Chile d Laboratorio de Entomología Médica, Sección Parasitología, Instituto de Salud, Pública de, Chile e Facultad de Medicina, Universidad Pedro de Valdivia, La Serena, Chile b

a r t i c l e

i n f o

Article history: Received 15 June 2016 Received in revised form 13 September 2016 Accepted 19 September 2016 Available online 22 September 2016 Keywords: Trypanosoma cruzi diversity Infected dogs Mepraia North coast of Chile

a b s t r a c t As part of a multi-site research program on the eco-epidemiology and control of Chagas disease in northern Chile, we sought to identify the Trypanosoma cruzi discrete typing units (DTUs) infecting rural and peridomestic dogs, using direct methods without grown of the parasite in the laboratory and thus to assess the use of this species as a sentinel of the disease in well-defined endemic areas of T. cruzi in Chile. Infected dogs (35) from three villages were included in the study. The studied villages were Caleta Río Seco and Caleta San Marcos, both in the Tarapacá Region, and La Serena in the Coquimbo Region. These villages were selected based on previous evidence of Mepraia infection reports of the Chilean Ministry of Health. Amplicons from nested-PCR positive samples were used as targets to determine the infective T. cruzi DTUs circulating in blood using PCR-DNA blotting and hybridization assays with five specific DNA probes (TcI, TcII, TcIII, TcV and TcVI). Results of hybridization with dog samples from Caleta Rio Seco showed single infections in 2 out of 16 and mixed infections in 14 out of 16. TcVI was the most frequent DTU found in this area. A highlight is that for the first time the presence of TcIII is reported in this area. Samples from Caleta San Marcos showed single infections in 5 out of 9 and mixed infections in 4 out of 9. TcVI was the most frequent DTU found in this area. Samples from La Serena showed single infections in 5 out of 10 and mixed infections in 2 out of 10; we were unable to genotype the other 3 samples. Our results indicate that infection by T. cruzi DTUs in dogs is not homogeneously distributed but rather specific to each region of our country, as demonstrated by the differences in the T. cruzi DTU distribution in some localities. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Trypanosoma cruzi, the etiological agent of Chagas disease, is a multi-host parasite infecting approximately 180 species of mammals (WHO, 2014). T. cruzi has a large genetic diversity classified into six DTUs, T. cruzi I–VI (Zingales et al. 2009); a new entity only found in bats (Tcbat) has been described (Marcili et al. 2009) T. cruzi DTUs are distributed differentially among vectors, mammalian hosts and geographic regions; TcIII and TcIV usually occur in sylvatic transmission cycles; TcII, TcV and TcVI circulate in domestic cycles and TcI in both cycles (Miles et al. 2009). In Chile, Triatoma infestans, the main vector of Chagas disease has been controlled, but three other endemic species of sylvatic triatomines, Mepraia spinolai, M. gajardoi and M. parapatrica occupy different habitats. M. gajardoi, distributed in the coastal zone from 18° to 26°S, presents occurrence consistent with permanent ⁎ Corresponding author at: Programa de Biología Celular y Molecular, ICBM, Facultad de Medicina, Universidad de Chile, 8380453 Santiago, Chile. E-mail address: [email protected] (A. Solari). 1 Both contributed as first author.

http://dx.doi.org/10.1016/j.vprsr.2016.09.004 2405-9390/© 2016 Elsevier B.V. All rights reserved.

human settlements and transient summer occupation in rocky habitats of northern Chile (Toledo et al., 2013). Domestic dogs (Canis lupus familiaris) and cats (Felis silvestris catus) are considered major reservoir hosts of T. cruzi in the domestic environment, and a risk factor for human infection (Beard et al. 2003; Cardinal et al. 2008; Gürtler et al. 2005, 2007). The importance of each mammalian species in the maintenance and dispersion of a multi-host parasite such as T. cruzi depends mainly on the ability of the parasite to persist in the mammalian host and be transmitted to the vector (blood-sucking insects), as well as the relative abundance of the host. A mammalian host responsible for the long-term maintenance of a population of infectious agents is considered a reservoir host (Ashford, 1996). Dogs and cats, with an average life span of 7 years, play a significant part in the dynamics of transmission in the human environment. The importance of domestic dogs as reservoirs of T. cruzi varies throughout Latin America. Dogs acquire the infection in several ways: ingestion of triatomines or infected prey, which represents infections with high T. cruzi parasitaemia, or by means of triatomine bites and subsequent defecation near the wound site, which may represent an infection with a low parasitaemia. In northwestern Argentina dogs display high parasite burden and

S. Ortiz et al. / Veterinary Parasitology: Regional Studies and Reports 5 (2016) 42–47

infectiousness to vectors for long periods (Gürtler et al., 2007, Enriquez et al., 2014), whereas in most countries, including Brazil, dogs display high seroprevalence but rarely present a high parasite burden (Xavier et al. 2012). A recent entomological and serological study of Chagas disease in two localities of the northern Chilean coast found 10.3% of canids seropositive and 5.8% M. gajardoi infected the vector associated with the sylvatic cycle in this area (González et al., 2015). In humans, Chagas disease is characterized by a lifelong chronic phase often associated with cardiac and digestive lesions. This parasite is capable of infecting almost all cell types (Noireau et al. 2009). In about 60–70% of infected humans the acute disease resolves spontaneously and patients remain asymptomatic. However, 30–40% of patients will develop cardiac, digestive or cardiodigestive problems 10–30 years after the initial infection. Pathological alterations found in naturally and experimentally infected dogs are similar to those found in human Chagas disease (Guedes et al. 2009; Kjos et al. 2008). T. cruzi DTUs identified in humans have been found in domestic dogs from Argentina, Colombia and elsewhere in Latin America (Diosque et al. 2003; Cardinal et al. 2008; Cura et al. 2012; Enriquez et al. 2013; Ramírez et al., 2013). Dogs usually present with high and persistent levels of parasitaemia, acting as ideal reservoirs (Gürtler et al. 2007). In a hyper-endemic area of Colombia with a T. cruzi infection prevalence of 50% in humans, some of the infected dogs showed signs of the disease (hair loss, ulcers, nail overgrowth, nasal depigmentation, weight loss and inactivity) (Mejía-Jaramillo et al. 2014). Such domestic reservoirs are a risk factor for humans living in the same dwelling. Dogs may also serve as natural sentinels in the surveillance phase of the disease, providing information on the spread of T. cruzi into the domestic cycle. It is known that the household presence of dogs seropositive for T. cruzi increases the risk of parasite transmission to local vectors and humans (Catalá et al. 2004). Dogs had a 17 times greater force of infection than children (Gürtler et al. 2005). In the southern cone countries of South America, the main DTUs identified from humans, domestic dogs and triatomine bugs were TcI, TcII, TcIII, TcV and TcVI (Diosque et al. 2003; Cardinal et al. 2008; Barnabé et al. 2011; Yeo et al. 2011; Cura et al. 2012). In the northern Argentinian provinces Santiago del Estero and El Chaco, domestic Triatoma infestans and dogs are frequently infected with TcVI (Diosque et al. 2003; Cardinal et al. 2008). Despite the large contribution of this reservoir host to the domestic transmission of T. cruzi, only a few studies have identified the DTUs infecting dogs. T. cruzi isolates were obtained to genotype T. cruzi in two infested rural villages of the humid Argentine Chaco. TcVI was identified in 37 dogs, TcV was identified in five dogs and TcIII in two dogs. No mixed infections were detected with this indirect method (Enriquez et al., 2013). In another study with direct analysis of DTUs performed in the Gran Chaco, T. cruzi DNA was reported in 53% (35/66) of dog samples. Among the single infections TcVI (42.8%) was the highest and TcI/TcVI (14.3%) was the most prevalent mixed infection in dogs (Monje-Rumi et al., 2015). As part of a multi-site research program on the eco-epidemiology and control of Chagas disease in northern Chile, we sought to identify the DTUs infecting rural and peridomestic dogs, using a direct method. This method avoids the need for parasite culture to characterize the structure and transmission cycles of T. cruzi. Another aim of this study is to assess the use of this species as a sentinel of the disease in well-defined endemic areas of Chile. 2. Material and methods 2.1. Study areas Dogs from three villages were included in the study. The study villages were Caleta Río Seco and Caleta San Marcos, both in the Tarapacá Region, and La Serena in the Coquimbo Region (Fig. 1). These villages were selected based on previous evidence of Mepraia infection reports of the Chilean Ministry of Health. The samples from Caleta Río Seco (20°59′46.02′′S/70°09′37.57′′W) and Caleta San Marcos (21°06′46.90′′S/70°07′12.62′′W), fishing villages

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Fig. 1. Map of localities studied in north coast of Chile.

located approximately 80 km south of the city of Iquique, were collected during 2012. The study sites have a coastal desert climate with b2 mm annual precipitation (Muñoz-Schick et al., 2001). These sites are extremely arid, have low plant cover and include beaches with a mixture of rocks, pebbles, cobblestones and sand. Lizards, sea birds and wild rodents inhabit the collecting sites. According to information provided by staff of the SEREMI de Salud (Regional Health Department) from a survey prior to the study, some homes are built with lightweight material and are situated on or near rocky bluffs and guano (seabird excrement) deposits; the remaining are of solid material, situated on a road a few meters from the coastline. The number of pet animals in the fishing villages is variable. Stray dogs are common, and there are

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no programs to suppress breeding of strays, therefore their age is unknown. The number of dogs counted in the two settlements was 56. The other area studied was La Serena, a city in north-central Chile, capital of the Coquimbo Region, 29°54′S 71°15′W. The city is located on ocean terraces, which are clearly noticeable from the coastal area; the rest of the urban area is located on several small hills, valleys and plains. La Serena has a cool desert climate, clearly seasonal; in summer there is no precipitation, but with abundant morning cloudiness and drizzles. These dissipate around noon, giving way to clear skies and warm 22 °C days. To the west of the city are mainly areas for growing vegetables, and there are a large number of plantations. The sample was 52 dogs and the average age of La Serena dogs was 2.9 years reported by owners. 2.2. Sampling The dogs tested (38 in Caleta San Marcos and 18 in Caleta Río Seco) were taken by their owners to places prepared for sampling, and approximately 3 mL of venous blood was extracted from the cephalic vein. Each sample was then transferred to a Khan tube and maintained at room temperature for 24 h to remove the serum. The serum was stored at −20 °C until use. Dog surveys in La Serena were a house-to-house census of dogs in endemic neighborhoods. Blood samples were taken from 52 dogs. Six milliliters of blood samples were collected by puncture of the cephalic vein using a Vacutainer® system. Our sample included dogs ranging from age 3 months to 14 years. The majority of adult dogs (71%) were used in cattle herding and slept outside the house. Owners also reported that their dogs hunt and that they go out by themselves for several consecutive days. Sampling was performed with owner approval and informed consent, in accordance with the international guiding principles for biomedical research involving animals and this project obtained ethical approval of each local health department. 2.3. Blood and serum samples Circulating antibodies for antigens of T. cruzi in the sera obtained from dogs of Caleta Rio Seco and San Marcos were analyzed as described (González et al., 2015).

DNA amplified bands, using oligonucleotides CV1 and CV2, CV1 (5′-GATTGGGGTTGGAGTACTAT-3′) and CV2 (5′-TTGAACGGCCCTCCGA AAAC-3′), which anneal to the constant regions of the minicircle (Wincker et al., 1994). Samples subjected to electrophoresis were transferred onto five identical Hybond N+ nylon membranes (Amersham, Little Chalfont, United Kingdom) and cross-linked with ultraviolet light to fix the DNA. The minimum amount of amplified DNA to perform hybridization tests is 30 ng, and under these conditions any probe used should cross-react with the immobilized DNA in the membranes, unless they are heterologous DNAs. 2.6. DNA blot analysis, hybridization assays and DNA probe construction Different T. cruzi DTUs (TcI: sp104 cl1, TcII: CBB cl3, TcIII: M5631cl 5, TcV: NR cl3 and TcVI: V195cl1) were used as DNA templates to generate DNA probes for to determine by hybridization the parasite DTUs infecting each dog. The next set of stocks and reference strains representing the known T. cruzi DTUs: Tcbat 4 TCC793 (Tcbat); Tcbat 5 TCC1122 (Tcbat); Cuica cl1 (TcI); sp104 cl1 (TcI); CBB cl3 (TcII); Esm cl3 (TcII); M5631 cl5 (TcIII); ARMA18 cl3 (TcIII); CanIII cl1 (TcIV); Dog Theis cl1 (TcIV); 92.80 cl2 (TcV); NR cl1 (TcV); V195cl1 (TcVI); CH2 cl1 (TcVI), were incorporated as negative and positive probes control lanes (Fig. 2). The primers for probe generation were CV1 and CV2, which produced a 270-bp fragment. These fragments were further digested with restriction endonucleases Sau96I and ScaI (Fermentas, Ontario, Canada) to obtain a 250-bp band containing DNA sequences from the minicircle variable regions and remove all sequences from the minicircle constant regions (Veas et al.1991). Finally, the probes were labeled using the random primer method with [α-32P] dATP. The five membranes were prehybridized in a solution of 6 × SSC and 1 mM EDTA, pH 7.4, with salmon sperm DNA at 55 °C. The membranes were hybridized overnight with the labeled probe at 55 °C in the same solution and then washed in high-stringency conditions (0.1 × SSC and 0.1 × SDS at 55 °C). The membranes were exposed for 1–4 h in a Molecular Imager (FX Bio-Rad Laboratories, Hercules, CA). This method has been validated by hybridization with the probes constructed by PCR amplification of T. cruzi DNA of the different DTUs (Brenière et al., 1998; Monje-Rumi et al., 2015). The hybridization profiles were analyzed to

2.4. DNA extraction The samples of peripheral blood of the La Serena dogs were preserved in (6 M) Guanidine-(0.2 M) EDTA and boiled for 15 min at 98 °C before extraction and purification of DNA using the EZNA kit (Omega Bioteck, Doraville, GA) according to the manufacturer's instructions, and maintained at − 20 °C until use. Whole genomic DNA was isolated from 0.2-mL of the Guanidine-EDTA samples. The procedure of extraction and purification of DNA was applied to the serum samples from dogs of Caleta Rio Seco and San Marcos after phenolic extraction. 2.5. Minicircle PCR The amplification reaction was performed with oligonucleotides 121 (5′-AAATAATGTACGGGT/GGAGATGCATGA-3′) and 122 (5′-GGTTCGAT TGGGGTTGGTGTAATATA-3′) which anneal to the conserved sequences present in the constant regions of each minicircle. The amplicon mainly contains DNA sequences of the minicircle variable regions. The reaction mixture for the standard PCR was composed of 5 μL of the DNA sample in a volume of 50 μL with 33 cycles and conditions already described (Wincker et al. 1994). Each experiment included a control that contained water instead of DNA and a positive control that contained purified kinetoplast (kDNA) of T. cruzi. The 330-bp PCR product was analyzed by electrophoresis in a 2% agarose gel and visualized by staining with ethidium bromide. Then all samples were amplified by PCR and subsequently subjected to nested PCR (N-PCR) to improve

Fig. 2. Hybridization patterns of different Trypanosoma cruzi stocks belonging to different DTUs as negative and positive probe controls. Different T. cruzi DTUs probes (TcI- sp 104 cl1; TcII- CBB cl3, TcIII- M5631 cl5; TcV- NR cl1; TcVI- V195 cl1. Minicircle PCR amplicons stained with ethidium bromide Lane M, 100-base pair (bp) DNA ladder. A. Hybridization with probe TcI; B. Hybridization with probe TcII; C. Hybridization with probe TcIII; D. Hybridization with probe TcV; E. Hybridization with probe TcVI.


compare the intensity of the ethidium bromide-stained bands in each membrane with the presence of the radioactive bands obtained with each probe. The specificity of the probes was tested in hybridization controls against DNA of the probes themselves. The profiles of radioactive hybridization signals with multiple probes are indicative of mixed infections with two or more T. cruzi DTUs. 2.7. Statistical analyses We compared the T. cruzi DTU composition of three villages using the chi-square tests with α = 0.05. 3. Results 3.1. T. cruzi detection by PCR in dogs The number of dogs tested in the Tarapacá Region accounted for 50% of the dogs registered (25/51). The presence of T. cruzi DTUs circulating was studied in 16 dogs from Caleta Río Seco and 9 dogs from Caleta San Marcos. The number of dogs infested in La Serena included 13 out of the 52 dogs registered. 3.2. Distribution of T. cruzi DTUs in dogs Amplicons from PCR positive samples were used as targets to determine the infective T. cruzi DTUs using PCR-DNA blotting and hybridization assays with the five specific DNA probes (Campos-Soto et al., 2016). Infecting DTUs were identified in 9 out of 9 samples from PCR-positive dogs of Caleta San Marcos, and 16 out of 16 samples from PCR-positive dogs of Caleta Rio Seco. Infecting DTUs were identified in 7 out of 10 samples from N-PCR positive dogs of La Serena. Fig. 3 summarizes the results of the T. cruzi characterizations obtained in the samples. The results of hybridization with samples from Caleta Rio Seco showed single infections in 2 out of 16, and mixed infections in 14 out of 16 samples. TcVI was the most frequently found DTU in this area. A highlight is that for the first time the presence of TcIII is reported in this area. Results of hybridization with samples from Caleta San Marcos showed single infections in 5 out of 9 and mixed infections in 4 out of 9. TcVI was also the most frequent DTU found in this area. Results of hybridization with samples from La Serena showed single infections in 5 out of 10 samples and mixed infections in 2 out of 10. Three samples were not recognized by any probe used. TcV was the most frequent DTU found in this area. The genotype distribution of TcI, TcIII and TcVI T. cruzi DTUs between the three villages was significantly different (χ2 = 15.23, χ2 = 8.7, χ2 = 15.92 d.f. = 2, P b 0.05, respectively). The genotype distribution of TcI, TcIII and TcVI was

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significantly different between Caleta Rio Seco and Caleta San Marcos (χ2 = 5.39, χ2 = 4.58, χ2 = 4.63 d.f. = 1, P b 0.05, respectively). The genotype distribution of TcI, TcIII and TcVI was significantly different between Caleta Rio Seco and La Serena (χ2 = 15.35, χ2 = 4.85, χ2 = 18.64 d.f. = 1, P b 0.05, respectively). The genotype distribution of TcVI was significantly different between Caleta San Marcos and La Serena (χ2 = 8.87 d.f. = 1, P b 0.05). Some samples showed a complex hybridization pattern with more than one probe, suggesting that those cases represent mixed infections with different T. cruzi DTUs (Fig. 3). 4. Discussion Our results indicate that T. cruzi infection in dogs is not homogeneously distributed but is rather different in each region of Chile, as demonstrated by the differences in T. cruzi DTU distribution in some localities. Thus we can highlight TcVI as prevalent in Caleta Rio Seco and Caleta San Marcos and TcV in La Serena. Although the Pacific coast of Chile has been considered an area without active transmission of Chagas disease, the coastal populations of M. gajardoi are infected with T. cruzi and represent a threat for humans, such as fisherman and alga collectors living in those areas. The same T. cruzi DTU is also the most prevalent circulating in M. gajardoi of this sector (Toledo et al., 2013; Campos-Soto et al., 2016). Also, notoriously there are multiple and varied combinations of mixed infections in Caleta Rio Seco. However TcV prevails in La Serena and mixed infections are sharply reduced compared to the northerly areas with higher infection rates. These differences may be due to ecological features and mammal host species carrying different T. cruzi DTUs in those endemic areas 1200 km apart. T. cruzi is a multi-host parasite that displays a huge intraspecific heterogeneity and a complex transmission cycle that may exhibit local peculiarities. The distribution observed in dogs may be associated with their proximity to the coast, rural areas and with the loss of richness and abundance of flora and rates of infection with T. cruzi in the small wild mammal fauna. However the distribution of different and mixtures of T. cruzi DTUs could be explained by coinfection or poly-parasitism with helminthes as reported by Enriquez et al. (2016). The importance of a host species as a reservoir of a vectorborne parasite mainly depends on its prevalence of infection, capacity to infect the vector and the rate of host-vector contact. The small wild mammalian fauna diversity plays an important role in the profile of the enzootic infection patterns in a given area, as shown by the notoriously high transmission described in a previous study (Herrera et al., 2005). The current evidence indicates that preserving ecosystems and their endemic biodiversity intact should generally reduce the prevalence of infectious diseases (Keesing et al., 2010). However, the global trend and particularly in Chile is the fragmentation of ecosystems by

Fig. 3. Trypanosoma cruzi amplicons of dog infected samples stained with ethidium bromide. Hybridization profiles obtained with genotype-specific probes corresponding to TcI, TcII, TcIII, TcV, and TcVI. Lane M = molecular mass marker. A 330-base pair (bp) product indicates a positive. A. Samples of dogs infected with T. cruzi from Caleta Rio Seco. B. Samples of dogs infected with T. cruzi from Caleta San Marcos. C. Samples of dogs infected with T. cruzi from La Serena.

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the incursion of people in wild areas, transforming them into areas for housing and the introduction of agricultural species, and also by overexploitation of existing resources (Loreau, 2010; Jorquera-Jaramillo et al., 2012). The determination of the spatial distribution of the elements that compose the epidemiological chain of a parasitic disease is of pivotal importance for the determination of trends and risk evaluation. Moreover, it is worth mentioning that attempts to control a given multi-host parasite based on the control of one single vector or host species will always be insufficient, because parasite transmission very rarely relies on a single system. The simplification of the mammalian host diversity associated with an increase in the abundance of competent reservoir host species as described here is certainly one of the risk factors involved in the reemergence of Chagas disease (Xavier et al., 2007). Northern Chile shows the presence of a high density of naturally infected T. cruzi Triatomines of the genus Mepraia, which are the main vectors in both regions studied here (Campos-Soto et al., 2016). Our results point to an active T. cruzi transmission by these vectors to the dogs in the northern coastal area of Chile. However, a noticeable rise in the prevalence of Chagas disease in the human population of the region has not been detected in the last decades (Lorca et al. 2001). This may reflect the effectiveness of the epidemiological surveillance campaigns against the domestic Triatoma infestans exerted in these areas. Local people are aware of the risk of disease and adopt local measures to avoid infection risk. The presence of T. cruzi in dogs in these regions can be attributed to the elevated rate of contact among these domestic animals and the wild environment because the houses overlap the wild areas, especially in summer when the home range of Mepraia increases (Botto-Mahan et al., 2005). In these areas it is difficult to delimit the peridomestic and wild areas; many local inhabitants and dogs are involved in hunting activities and there is potential exposure of dogs to infected vectors or other animals when they are permanently out in the interface between the peridomestic and wild environments. Surveillance for canine Chagas disease should be a useful tool for the design of suitable epidemiological control programs in areas where sylvatic triatomines are responsible for human infection, as in many rural endemic areas (Pineda et al., 2011). The distribution of different T. cruzi DTUs found in this study is consistent with results reported by other authors in Latin America (Diosque et al. 2003; Cardinal et al. 2008; Cura et al. 2012; Enriquez et al. 2013; Ramírez et al., 2013). The high prevalence of TcVI in the two study areas located further north in Chile coincides with other reports in Argentina at the same latitude east of the Cordillera Los Andes (Diosque et al. 2003; Cardinal et al. 2008, Enriquez et al., 2013; Monje-Rumi et al., 2015). Also, having detected the presence of TcIII in Rio Seco coincides with reports of Tc III in Argentina (Enriquez et al., 2013, Monje-Rumi et al., 2015). In the area of La Serena mainly TcV and few TcI, TcII and TcVI DTUs were detected. However it was not possible to identify the T. cruzi DTUs present in three samples. One possible explanation would be that they correspond to the presence of TcI or TcII variants or TcIV which were not tested in this opportunity. To conclude, this study gathered information on the T. cruzi DTU composition in dogs and recommends paying attention to dogs as sentinels of Chagas disease. The results obtained are useful to determine T. cruzi variability in coastal areas of distribution of this parasite in northern Chile, and to prevent the risk that this parasite variability be transmitted to the human populations. Acknowledgements This work was supported by FONDECYT-Chile 1120122 to A. Solari. References Ashford, R.W., 1996. Leishmaniasis reservoirs and their significance in control. Clin. Dermatol. 14, 523–532.

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