Prevalence of Trypanosoma cruzi in Dogs ( Canis familiaris ) and Triatomines During 2008 in a Sanitary Region of the State of Mexico,Mexico

Abstract

American trypanosomiasis is a public health problem in Latin America and southern parts of the United States. Infection in triatomines (vector) and domestic dogs (reservoir host) is a good indicator of Trypanosoma cruzi circulation and human risk of infection. The State of Mexico, Mexico, has been considered free of T. cruzi, and no detailed epidemiologic study has been conducted to assess the intricacies of the transmission cycle of the parasite in the region. Such studies would enhance our understanding of the epidemiology of T. cruzi infection in this geographic region and provide regional sanitary authorities with stronger fundamental knowledge for making decisions and allocating funds for Chagas disease control programs in the State of Mexico. The objective of this study was to determine the prevalence of T. cruzi infection in dogs (seroprevalence) and triatomines (fecal parasites) in a previously identified, discrete endemic region of parasite circulation and to widen our studies in the Tejupilco Sanitary Region located in the southern part of the State of Mexico. Dog blood samples (n = 102) were analyzed for the presence of anti-T. cruzi antibodies by two assays, namely indirect hemagglutination assay and enzyme-linked immunosorbent assay. Triatomines (n = 88) were collected and fecal aliquots were analyzed for the presence of parasites by light microscopy. Average seroprevalence in dogs in the Tejupilco Sanitary region was 24.5%, and the overall triatomine infection rate was 34.01%. Triatoma pallidipennis was the only triatomine species found in this region. Our data demonstrate that T. cruzi is actively circulating in the Tejupilco Sanitary Region and emphasize the requirement for epidemiologic surveillance programs throughout the putative endemic areas of the State of Mexico.

Introduction

I t has been a century since Carlos Chagas first described American trypanosomiasis and approximately half a century since it was first recognized as a public health problem in Mexico (Velasco-Castrejón et al. 1992). Chagas disease is caused by a flagellate protozoan parasite, Trypanosoma cruzi, and transmitted by a hematophagous insect vector (the “kissing bug”) of the Triatominae subfamily. Approximately 9 million people are infected with T. cruzi in 19 countries in Latin America and ∼40,000,000 people live at risk of getting infected (Schofield et al. 2006). In Mexico, health authorities suggest that ∼1.6 million people may be infected with T. cruzi (Velasco-Castrejón and Guzman-Bracho 1986, Velasco-Castrejón et al. 1992, Guzman-Bracho 2001), although this number could be underestimated according to Cruz-Reyes and Pickering-Lopez (2006). Most critical transmission areas are located in the 23 states south of the Tropic of Cancer (Guzman-Bracho 2001, Cruz-Reyes and Pickering-Lopez 2006). The State of Mexico was considered free of T. cruzi infection until 2006, when our group documented T. cruzi-specific antibodies in 7.1% humans and 21% dogs from rural areas of the State of Mexico (Estrada-Franco et al. 2006). In the published report, we demonstrated that the seroanalysis of dogs may help to predict human risk of vectorial T. cruzi infection in the southern areas of the province (Estrada-Franco et al. 2006). Yet, a major drawback of this study was that we did not address whether T. cruzi transmission from vectors occur in this region or if seropositive humans and dogs migrated from other endemic areas to the studied region in the State of Mexico.

In this study, we aimed to investigate T. cruzi infectivity in dogs and triatomines in the southern areas of the State of Mexico. We have employed a random sampling approach (using “village” as the study unit) to determine if T. cruzi distribution is widespread throughout the sanitary region of the Tejupilco municipality and to test the hypothesis that the geographical range of the parasite is not restricted to a few small foci. We used seroprevalence in dogs as an indicator to assess the human risk of infection in the study area and determined T. cruzi infection prevalence in triatomines living in the human dwellings to evaluate if active transmission of T. cruzi could be occurring in the State of Mexico.

Materials and Methods

Study area and sample collection

The study was conducted in 16 villages from the Tejupilco Sanitary Region of the State of Mexico, which consists of six municipalities: Temascaltepec, San Simón de Guerrero, Tejupilco, Luvianos, Amatepec, and Tlatlaya. Names of villages and municipalities with their geographical coordinates and number of inhabitants per village are listed according to the last census from INEGI (2005) (Table 1 and Fig. 1). The area has seasonal climate variations (dry season from November through May and rainy season from June through October). The main occupations of the population are agriculture and livestock production. Large areas of the municipalities share common climatic and ecological conditions. Altitude ranges from 600 to 2000 m, with a mean rain precipitation of 795.5 mm per year, and a mean year temperature of 25.2°C with a minimum value of 18.6°C and maximal of 29.4°C (INEGI 2005).

FIG. 1.

Sixteen villages from the State of Mexico surveyed for triatomine infectivity and seroprevalence in dogs are highlighted with black dots.

Table 1.

Seroprevalence in Dogs and Infected Triatomines from 16 Villages of the Tejupilco Sanitary Region, State of Mexico, During 2008

Municipalities	Village name and geographical coordinates ^a	Village human population ^a	No. of houses ^a	Altitude (meters) ^a	Dogs prevalence seropositive/total	Triatomine prevalence infected/total
Tlatlaya	Revelado 18,33,38 N 100,04,26 W	149	31	960	0/6	0/4
Tlatlaya	Corral de Piedras 18,34,13 N 100,14,13 W	154	23	1000	2/6	1/6
Tlatlaya	Cuadrilla de Flores 18,38,00 N 100,14,13 W	142	31	1300	0/1	0/4
Tlatlaya	La Parota 18,26,54 N 100,14,50 W	35	5	700	0/4	4/9
Tlatlaya	Pueblo Nuevo 18,38,35 N 100,07,22 W	380	73	730	2/4	4/8
Tlatlaya	Miraveles 18,39,15 N 100,12,38 W	67	13	1380	2/9	3/7
Amatepec	Cerro de los Cacahuates 18,44,00 N 100,21,27 W	35	5	900	2/4	1/4
Amatepec	Mango Matuz 18,14,13 N 100,26,07 W	136	24	700	5/8	2/7
Amatepec	San José del Palmar 18,39,53 N 100,22,12 W	212	41	920	4/8	6/14
Amatepec	Las Ciebas 18,39,56 N 100,26,22 W	139	25	800	0/10	1/5
Luvianos	Hormigueros 18,52,37 N 100,22,49 W	79	15	1700	0/4	2/7
Luvianos	Palos Prietos 18,51,50 N 100,25,32 W	76	17	1740	3/9	1/4
Tejupilco	Plaza de Gallos 18,50,46 N 100,14,15 W	149	24	970	2/9	4/7
Tejupilco	Ojo de Agua 18,56,44 N 100,07,51 W	197	39	1640	1/4	1/2
San Simón de Guerrero	Mina de Agua 19,01,20 N 100,01,49 W	120	25	1980	1/11	0
Temascaltepec	Carboneras 19,03,22 N 100,00,29 W	1201	224	2000	1/5	0
Total	16	3271	615	1213.7	25/102 (24.5%)	30/88 (34.01%)

Taken from INEGI (2005).

For the dog population, a sample size of 97 was estimated by using a 90% confidence interval, 5% absolute precision, and an expected T. cruzi dog prevalence of 10% according to previous studies (Estrada-Franco et al. 2006). For the triatomines, the sample size was also estimated to be 97 by using a 90% confidence interval, 5% absolute precision, and an expected prevalence of, at least, 10% according to previous studies (Martínez-Pérez et al. 2002). The sampling model is based on an infinite population according to a sample size model (Roasoft 2004). All localities from the Tejupilco health jurisdiction were numbered, and localities where samples were collected were chosen randomly by using a random numbers program (Haahr 2009). Exclusion factors were altitude above 2000 m and population above 2500 people.

Dog (n = 102) and triatomine (n = 88) samples were collected from 16 randomly selected villages from the 1246 villages in the Tejupilco Sanitary Region. Dog blood samples were taken after informed consent was obtained from the owners. Only dogs older than 2 months were included in the study. Blood (7 mL) was obtained by puncture of the cephalic vein, through a vaccutainer system. Negative control samples were collected from 30 healthy dogs from Toluca, the capital city of the State of Mexico (altitude 2640 m; average temperature 15°C, range: 5–24°C). Toluca is considered free of vectorial T. cruzi transmission, as triatomines have never been documented in the area, and triatomines are believed to not proliferate in most localities at altitudes >2500 m (Velasco-Castrejón and Guzman-Bracho 1986, Guzman-Bracho 2001). The animal welfare committee of the University of the State of Mexico (UAEM) reviewed and approved all animal studies. The site of collection was normally in villages located several hours away from the laboratory. Therefore, blood samples were protected from light and extreme temperatures in coolers and transported to the laboratory within 24 h of collection time. Blood samples were centrifuged at 1800 g for 10 min at room temperature, and serum (supernatant) was collected by aseptic procedure, aliquoted, and stored at −20°C, until analyzed.

Triatomines were collected by the people living in the localities visited, by using a collection protocol reported by Bautista et al. (1999). Collected triatomines were classified according to Lent and Wygodzinsky (1979). Feces were collected by abdominal pressure and then diluted with 200 μL phosphate-buffered saline (pH 7.2). Metacyclic trypomastigotes were identified by the characteristic trypomastigote motility by light microscopy (magnification: dry 20 × and 40 ×, and immersion 100 ×).

The quality of the building material in houses was evaluated to stratify risk of transmission with respect to the building material. Roofing, wall, and floors were categorized and recorded as follows: (i) thatched, asbestos, or concrete roofing; (ii) adobe brick wall/plastered wall houses; and (iii) dirt or concrete flooring.

Serology

Two commercial serologic kits were used to detect anti-T. cruzi antibodies: indirect hemagglutination assay (IHA) (Polichaco^®; Laboratorio Lemos SRL, Buenos Aires, Argentina) (with 98% sensitivity and 99% specificity, according to the manufacturer's specifications) and enzyme-linked immunosorbent assay (ELISA) (Laboratorio Lemos SRL) (with 100% sensitivity and 100% specificity, according to the manufacturer's specifications). All assays were performed according to manufacturer's instructions, except that the horseradish peroxidase-labeled, anti-human IgG antibody in the ELISA kit was replaced with horseradish peroxidase-labeled anti-dog IgG (Koma Biotech, Seoul, Korea) as a secondary antibody (Barbabosa-Pliego et al. 2009). The cutoff value for IHA was set at ≥1:16 sera dilutions. As ELISA was modified from the manufacturer's instructions, it was standardized by using the sera from healthy dogs. Sera samples from healthy dogs (n = 30, 1:100 dilution) were analyzed in triplicate, and a cutoff value was set at 0.129 OD_450nm ± 0.1 (or +2 SD). Samples were considered positive when reactive for both assays.

Results

Before sampling the dogs' sera and triatomine collection, 117 of the householders in 16 villages were interviewed from 615 total houses. People were interviewed either because they had found a triatomine in their house or because they accepted to have their dogs sampled. A total of 102 dog sera samples and 88 triatomines were collected (Table 1). The population size of these villages ranged between 35 and 1201 people and the number of houses from 5 to 224. Most people in these villages had low income. This statement is supported by the following socioeconomic data: 51% houses were made of adobe, the roofing was made of tiles (63.2%) and asbestos laminate (11.9%), and 34% houses had dirt floor. Additionally, more than half of the houses (56.4%) were not fumigated for pest control at least during a 1-year period.

Triatomines (n = 88) were collected from 14 of the 16 villages and from four of the six municipalities included in the study. Of the 88 triatomines collected from these villages, 30 (34%) were infected with T. cruzi, as determined by light microscopic examination of metacyclic trypomastigotes in fecal material. Most triatomines (81/88) were found outdoors in close vicinity to the houses. All triatomines belonged to the species Triatoma pallidipennis according to Lent and Wygodzinsky (1979).

Dogs sampled represented all 16 communities. Twenty-five (24.5%) dogs were seropositive for T. cruzi-specific antibodies by two serodiagnostic assays (ELISA and IHA). Anti-T. cruzi seroreactivity in dogs was found in 11 of the 16 villages (69%) from all six municipalities included in the study (Table 1). Seroprevalence in dogs was highest in Amatepec (36.6%), followed by Tejupilco and Luvianos (23.07%), Temascaltepec (20%), Tlatlaya (19.35%), and San Simon de Guerrero (9.09%).

Discussion

In Mexico, Triatoma pallidipennis has been reported as the dominant triatomine species in the states of Morelos, Michoacan, and the State of Mexico, and it is observed at low frequency in Oaxaca, Veracruz, Colima, and San Luis Potosi. Conversely, Triatoma dimidiata has been reported as the dominating species in the states of Oaxaca, Hidalgo, the Yucatan peninsula, and Veracruz, and it is observed at low frequency in the states of Hidalgo and Mexico. In the State of Mexico, Triatoma pallidipennis has been regularly found at low altitudes (<2000 m) that mainly comprise the southern region of the state (reviewed in Bautista et al. 1999, Vidal-Acosta et al. 2000, Martínez-Pérez et al. 2002). In this study, all specimens collected were Triatoma pallidipennis, and we found no Triatoma dimidiata in this region. Recently, as part of the surveillance activities from the health authorities of the State of Mexico and our own observations, only a few Triatoma dimidiata specimens were found in the region of our study (Estrada-Franco, pers. comm. 2008; Ochoa-Garcia, pers. comm. 2009). Therefore, we can surmise that Triatoma pallidipennis is the dominating species in the southern State of Mexico.

In a previous study conducted in the state of Morelos, a southern neighbor of the State of Mexico, the effects of pesticide on triatomine populations in human dwellings were estimated. Before pesticides were sprayed, T. pallidipennis infestation indices were noted to be 38% and 44.7% in peridomicile and domicile habitats, respectively (Bautista et al. 1999). Shortly after pesticide spraying, infestation of triatomines in human dwellings was reduced significantly (0–0.6%). However, 1 year after spraying, the triatomine population reached to about 50% of the population found before pesticide spraying. All insects (Triatoma pallidipennis) collected in this study were distributed in 75% of the communities studied, and a majority (81/88) were found outdoors in close vicinity to houses. Finding triatomines in the selected communities was not surprising, because infestation of Triatoma pallidipennis has been reported in the neighboring state of Morelos and in five communities belonging to the Tejupilco municipality of the State of Mexico (Martínez-Pérez et al. 2002, Cohen et al. 2006). These regions, Morelos and the southern State of Mexico are located in the same geographical gradient sharing many ecological characteristics. Accordingly, we reasoned that the triatomine population distribution would follow a similar pattern and essentially would be the same along the two regions. The pesticide spray program established by local health authorities has limited financial and personnel resources. For local health authorities, the main vector-borne disease targets are those transmitted by mosquitoes, and therefore, pesticide spray is executed, primarily linked to the time and places where dengue outbreaks occur, and it may not be delivered in all communities every year. In fact, 56.4% of the houses included in the study had not been sprayed for over a year. This irregular pesticide spraying could place selective pressure on triatomines resistant to pesticides in the region, a situation that needs further study.

It has been demonstrated that the presence of triatomines is associated with some types of housing material and animals in the peridomicile and domicile and represents risk factors for T. cruzi transmission (Cohen and Gurtler 2001, Cuba et al. 2002). We surveyed the type of materials and animals present in the houses from which samples were collected and found that at least 63% houses were composed of rustic materials (adobe brick walls with thatched, asbestos tile, or cardboard laminate roofs). We also found that in the peridomicile, a high proportion of the yards surrounding houses had piles of wood and/or stones, and animals (chicken coops and dogs among others). We did not find any correlation between these factors and the presence of triatomines within the houses. However, this study was not designed to look into this topic in depth, and further research should be conducted to evaluate this aspect of the epidemiology of Chagas disease in the southern region of the State of Mexico.

T. cruzi-infected triatomines have been reported to maintain the vectorial mode of parasite transmission to mammals, including humans (Sanmartino and Crocco 2000, Vásquez et al. 2004). Thus, we examined all triatomines collected for the presence of fecal epimastigotes and/or metacyclic trypomastigotes. In a previous study, Bautista et al. (1999) reported 33.3% infectivity of the triatomines captured in the neighboring state of Morelos, Mexico. In this study, 34% of the triatomines captured in the southern State of Mexico were infected with T. cruzi, as determined by direct observation of phosphate-buffered saline-diluted feces under light microscopy. We have found, however, that this diagnostic method resulted in an underestimation of the proportion of infected triatomines, as we found in experimentally infected triatomines that parasitic load in feces varies between one examination and the next, few days apart. Accordingly, multiple fecal examinations are required to validate the absence of T. cruzi infection. Further studies employing sensitive diagnostic techniques, such as polymerase chain reaction, would give an accurate estimate of infection prevalence in triatomines. Yet, our observation of a 34% infectivity index in triatomines leads us to suggest that dogs and humans are exposed to the risk of vector-mediated T. cruzi infection in the area of study.

Dogs are an important reservoir host of T. cruzi and considered to provide a risk factor for human Chagas disease (Moncayo 1992). In natural conditions, dogs are infected with T. cruzi more frequently than humans (Gurtler et al. 1996), and dogs maintain acute parasitemia for longer periods (Gurtler et al. 1986). Dogs are a major source of transmission of T. cruzi to triatomines in domiciliary/peridomiciliary surroundings (Montenegro et al. 2002, Gurtler et al. 2007). In these scenarios, a presumably large but undetermined proportion of animals are able to move freely among the different microecological niches of the disease, getting exposed to infected triatomines, eating from infected mammals, and thus acting as reservoirs of the domestic cycle, thereby threatening the health of the human population surrounding them. The prevalence rate of infected triatomines in a household increases with the number of infected dogs in the vicinity (Gurtler et al. 1996), and the prevalence of adult human infection is increased in households with two or more infected dogs (Gurtler et al. 1998). In South America, in Triatoma infestans, Triatoma dimidiata, and Triatoma sordida circulation areas, domesticated dogs have been recognized to present a risk factor associated with human infection (Guzman Bracho et al. 1998). Few studies have been conducted to suggest an association between dogs' seropositivity and parasite vectorial transmission in the states of Puebla and Yucatan in the Mexican Republic (Sosa-Jurado et al. 2004, Jimenez-Coello et al. 2008). Sosa-Jurado et al. (2004) have shown T. cruzi-specific antibodies in dogs (10%) in rural villages of Puebla, where active vectorial transmission was also observed. Studies carried out in stray dogs from the Yucatan State showed seroprevalences fluctuating between 9.8% in rural settings versus 14.4% in urban premises (Jimenez-Coello et al. 2008). It was assumed that the vector involved in these states was Triatoma dimidiata. Recently, we have reported 21% seroprevalence for T. cruzi-specific antibodies in dogs in five localities of the Tejupilco sanitary region of the State of Mexico (Estrada-Franco et al. 2006). In this study, our finding of 24.5% seroprevalence in dogs and 34% infectivity of triatomines indicates that dogs are exposed to active infection from a vector in the State of Mexico. Our seroprevalence values are somewhat higher than those reported by Sosa-Jurado et al. in Puebla (10%) and Jimenez-Coello et al. in Yucatan (9.8–14.4%), findings which may point to behavioral feeding preferences of the circulating vector in the respective areas. Triatoma barberi is reported to be the most abundant vector in the Puebla state, and Triatoma dimidiata in the Yucatan state. We have found only Triatoma pallidipennis in the State of Mexico.

We surmise that the active transmission of T. cruzi occurs across most of the studied area in the southern State of Mexico. T. cruzi strains isolated from triatomines from this region were pathogenic in dogs and resulted in chronic cardiomyopathy (Barbabosa-Pliego et al. 2009). T. cruzi infection in triatomines and seropositive dogs has also been detected in other sanitary regions of the southern area of the State of Mexico by the local health authorities and us. Our observations emphasize the importance of active epidemiologic surveillance programs throughout the State of Mexico and implementation of vector control strategies in endemic areas.

Footnotes

Acknowledgments

This work was supported, in part, by grants from the Universidad Autónoma del Estado de México (UAEM) (2381/2006U to J.C.V.C.) and from CONACYT Mexico (84863/2008 to J.G.E.F). The authors are indebted to Dr. Miguel Angel Estrada Botello from the Sanitary Tejupilco Region for the support in collecting samples.

Disclosure Statement

No competing financial interests exist.

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