Therya_notes_NE11

THERYA NOTES 2024, Vol. 5 : 162-166 DOI: 10.12933/therya_notes-24-165 ISSN 2954-3614

Molecular detection of Trypanosoma cruzi in Mus musculus in a rural town in Mérida, Yucatán, México

Detección molecular de Trypanosoma cruzi en Mus musculus en una localidad rural de Mérida, Yucatán, México

Jesús Alonso Panti-May1, Marco Torres-Castro1, Javier Escobedo-Ortegón1, Elsy B. Canché-Pool1, and Hugo A. Ruiz-Piña1*

1Centro de Investigaciones Regionales “Dr. Hideyo Noguchi”, Universidad Autónoma de Yucatán. Avenida Itzáes 490, C. P. 9700, Mérida. Yucatán, México. E-mail: alonso.panti@correo.uady.mx (JAP-M); antonio.torres@correo.uady.mx (MT-C); eortegon@correo.uady.mx (JE-O); elsy.canche@correo.uady.mx (EBC-P); rpina@correo.uady.mx (HAR-P).

*Corresponding author

American trypanosomiasis is a disease caused by the protozoan Trypanosoma cruzi and affects approximately 6 million people in the Americas. Commensal rodents are a food source for the vector and potential reservoirs of T. cruzi. The objective of this study was to estimate the prevalence of T. cruzi in commensal rodents that inhabit the rural town of Molas, Yucatán, México. Rodents were captured in households using Sherman traps. DNA extracted from heart samples from the captured rodents was used to detect T. cruzi using a PCR test. The prevalence of T. cruzi was compared by considering the sex and age of the rodents with the Chi-square test. A total of 114 Mus musculus mice were captured. The prevalence of T. cruzi was 21.1 % (n = 24) in the individuals examined. The comparison of the prevalence of T. cruzi between males and females and between adults and juveniles of M. musculus did not show statistically significant differences (P > 0.05). The prevalence of T. cruzi in M. musculus was high compared to other previous studies in México, and infection was observed regardless of rodent sex and age. These results show that M. musculus participates in the biological cycle of T. cruzi at Molas. Further studies are needed to understand the type of involvement (e.g., reservoir) of these rodents in the transmission dynamics of this parasite.

Key words: American trypanosomiasis; commensal rodents; molecular biology; southeastern México; tropical.

La tripanosomiasis americana es una enfermedad causada por el protozoario Trypanosoma cruzi y afecta aproximadamente a 6 millones de personas en las Américas. Los roedores comensales son una fuente de alimento para el vector y potenciales reservorios de T. cruzi. El objetivo del presente estudio fue estimar la prevalencia de T. cruzi en roedores comensales de la localidad rural de Molas, Yucatán, México. Se capturaron roedores en viviendas usando trampas Sherman. El DNA extraído de muestras de corazón de los roedores capturados fue utilizado para la detección de T. cruzi por medio de la técnica de PCR. Se comparó la prevalencia de T. cruzi considerando el sexo y edad de los roedores con la prueba de Chi-cuadrada. Un total de 114 ratones Mus musculus fueron atrapados. La prevalencia de T. cruzi fue 21.1 % (n = 24) en los individuos examinados. La comparación de las prevalencias de T. cruzi entre machos y hembras y entre los adultos y juveniles de M. musculus no arrojó diferencias estadísticas significativas (P > 0.05). La prevalencia de T. cruzi en M. musculus fue alta, en comparación con otros estudios previos en México, y la infección se presentó independientemente del sexo y la edad de los roedores. Estos resultados demuestran que M. musculus participa en el ciclo biológico de T. cruzi en Molas. Es necesario realizar mayores estudios para entender el tipo de participación (e.g., reservorio) de estos roedores en la dinámica de transmisión de este parásito.

Palabras clave: Biología molecular; roedores comensales; sureste de México; tripanosomiasis americana; tropical.

American trypanosomiasis, known as Chagas disease, is caused by the flagellated protozoan Trypanosoma cruzi. It is a life-threatening disease that has been neglected, which affects approximately 5,742,167 people and puts 70,199,360 people at risk of infection in Latin America (WHO 2015). Trypanosoma cruzi is transmitted through hematophagous hemipteran insects (vectors), such as Rhodnius prolixus, Triatoma dimidiata, and Triatoma infestans, accounting for 80 % of reported cases (OPS 2018). Vector transmission occurs when an infected vector feeds on a mammal and, after feeding, defecates on it, contaminating the feeding site or adjacent mucous membranes with parasites (WHO 2015). Other modes of transmission are transfusion of contaminated blood, congenital (vertical), transplantation of infected organs, and consumption of food contaminated with the parasite (Coura 2014).

Trypanosoma cruzi has been reported in more than 100 species of wild mammals, including marsupials, rodents, bats, armadillos, and primates (Jansen et al. 2017). In wild environments, rodents have been suggested to play a secondary role as reservoirs for this parasite due to the low infection frequencies reported (usually 0.4 %–5 %) compared to other mammals such as marsupials (11 %–90 %; Jansen et al. 2017). In anthropized environments, there are rodents known as commensals, such as the house mouse Mus musculus and the black rat Rattus rattus, which take advantage of food sources and shelter that favor large populations and infestations in households throughout the year (Battersby et al. 2008). This has suggested that commensal rodents may play an important role in domestic and peridomestic transmission of T. cruzi (Pinto et al. 2006; Ayaqui and Ruelas 2020). There are reports of T. cruzi infection in M. musculus and R. rattus in these environments in Ecuador and Chile, with contrasting results. Although M. musculus was the rodent most frequently captured (n = 79) in households in Guayas, Ecuador, all specimens tested negative, while in R. rattus (n = 61), the second most captured species, the infection rate was 11.5 %. In Coquimbo, Chile, R. rattus (n = 46) was captured more frequently than M. musculus (n = 5), but both species showed similar infection rates, 83.6 % and 83.3 %, respectively.

In the city of Mérida, Yucatán, México, and its rural surroundings, people positive for T. cruzi, have been reported, as well as vectors (Guzmán-Tapia et al. 2007; García-Montalvo 2011) and the rodents M. musculus and R. rattus (Panti-May et al. 2017) also positive for T. cruzi. Although experimental infections with this protozoan in M. musculus are abundant, few studies have reported the natural infection rate in this rodent compared to R. rattus (Jansen et al. 2017). Therefore, the present study aimed to determine the prevalence of T. cruzi infection in M. musculus living in a rural town in Yucatán, México.

As part of a multidisciplinary study on zoonotic pathogens (Pacheco-Castro et al. 2013), commensal rodents were sampled in the rural town of Molas (20° 48' 55.42" N, 89° 37' 48.40" W), ca. 8 km south of the city of Mérida, in the municipality of Mérida, Yucatán, México (Figure 1). During 2009–2010, 200 Sherman traps were placed monthly in 20 households for 3 consecutive nights. The bait used was a mixture of oat flakes and artificial vanilla essence. The captured rodents were euthanized with sodium pentobarbital administered intraperitoneally (Leary et al. 2020). Subsequently, a mid-thoracic incision was performed to remove the heart, which was preserved in 10 % buffered formalin for subsequent molecular testing and histopathology analyses. The species, sex, and age of each rodent specimen were recorded; age was determined according to Panti-May et al. (2012). All field and laboratory protocols were approved by the Bioethics Committee of the Campus of Biological and Agricultural Sciences, Autonomous University of Yucatán (protocol no. CB-CCBA-L-2009-001).

From each heart sample, a 0.5 g section was obtained and placed in 200 μL of phosphate buffer (PBS) and incubated at room temperature for 24 hr. Afterward, the PBS was discarded, and the DNA was purified with the InstaGene™ Matrix resin (Bio-Rad®, Hercules, California) following the manufacturer's protocol. For the detection of T. cruzi, primers 121 and 122 were used, which amplify a region of the kinetoplast DNA (kDNA) of T. cruzi of approximately 330 base pairs (Wincker et al. 1994). Each PCR reaction included 50-100 ng of DNA, 10 μL of GoTaq Green Master Mix (Promega, Madison, Wisconsin), 200 ng of each primer and nuclease-free water to a final volume of 20 μL. The amplification conditions were 5 min at 94 °C; 35 cycles of 94 °C for 60 seconds, 57 °C for 60 seconds, and 72 °C for 60 seconds; and a final extension at 72 °C for 10 minutes. Positive controls (T. cruzi DNA) and negative controls (molecular-grade water) were included in each reaction. Electrophoresis of PCR products was performed on 1.5 % agarose gels stained with ethidium bromide. The results were visualized and recorded on a photo-documenter (Bio-Rad®, Hercules, California).

The prevalence of T. cruzi infection and its 95 % confidence intervals (CI) were estimated with the Quantitative Parasitology 3.0 program (Rózsa et al. 2000). Likewise, the prevalence of infection was compared between host sexes and between age groups using the Chi-square test and the program mentioned above.

A total of 114 individuals of M. musculus were captured, including 47 females and 67 males; of these, 89 were adults and 25 were juveniles. Twenty-four samples of M. musculus (21.1 %, CI = 14.4 %–29.7 %) tested positive for T. cruzi (Figure 2). The comparison of the prevalence of T. cruzi between males (25.4 %, CI = 16.3 %–37.2 %) and females (14.9 %, CI = 7.1 %–28.5 %), was not significantly different (ꭓ2 = 1.8, d.f. = 1, P = 0.18). In addition, no significant differences were found (ꭓ2 = 3.3, d.f. = 1, P = 0.07) in the infection rate with T. cruzi between adults (27.7 %, CI = 16.7 %–34.8 %) and juveniles (8 %, CI = 1.5 %–25.6 %).

The presence of T. cruzi in M. musculus through molecular methods has been reported in some regions of the Americas. The prevalence of T. cruzi found in M. musculus in Molas (21.1 %) is higher than the 6.8 % reported in the United States of America (Herrera et al. 2015), but lower than 83.3 % in Chile (Yefi-Quinteros et al. 2018). In México, a 7.9 % prevalence of T. cruzi in this rodent was reported in the state of Morelos (Ramsey et al. 2012). Particularly in the city of Mérida, Panti-May et al. (2017) determined the infection of M. musculus with T. cruzi in an urban and a suburban area located ca. 11 km and 8 km north of Molas, respectively. In the urban area of Mérida, no specimens of M. musculus were positive for T. cruzi, while in the suburban area, the infection rate was 15.7 %. Another study conducted in Yucatán investigated T. cruzi infection in M. musculus and R. rattus living in a rural community in the municipality of Cenotillo; no specimens of M. musculus were positive (Hernández-Cortazar et al. 2018). This shows the wide variability of infection with T. cruzi in commensal rodents, even within the same region.

The present study recorded infection with T. cruzi in M. musculus regardless of rodent sex or age. This involves the exposure of this rodent to the parasite through several transmission routes, such as vectorial, oral, vertical, and sexual. Blood from M. musculus has been found in the gut of vectors such as T. dimidiata in Guatemala (Bustamante et al. 2014) and México (Torres-Montero et al. 2012) and Panstrongylus geniculatus in Venezuela (Segovia et al. 2023). The foregoing shows that M. musculus is a food source for vectors, which can promote vector-borne transmission. Experimental studies have shown that M. musculus can be infected through oral inoculum with blood and metacyclic trypomastigotes of T. cruzi (Dias et al. 2013), which may favor the maintenance of the parasite in rodents through the consumption of vectors that carry the metacyclic forms and contact with the blood of mice infected with trypomastigotes during fights. Vertical and sexual transmission routes have been experimentally demonstrated in M. musculus, but it has been concluded that they do not contribute significantly to the transmission and maintenance of the parasite (Rios et al. 2018; Faral-Tello et al. 2023).

This study used heart tissue to detect T. cruzi DNA in naturally infected mice. Infection with T. cruzi is characterized by an acute phase in which the parasite is found in the blood and various tissues, including the heart, and a chronic phase in which the parasite infects several tissues, such as the heart, skeletal muscle, kidney, liver, and spleen (Cencig et al. 2011; Caldas et al. 2012). Although T. cruzi can parasitize multiple tissues, the parasite load can vary in the different infection stages, depending on the parasite strain, the inoculum, and reinfections (Andrade et al. 1999; Cummings and Tarleton 2003; Cencig et al. 2011).

Commensal rodents have been identified as potential sentinels of vector transmission of T. cruzi because they are susceptible to infection, can be a source of food for vectors, are abundant, easy to capture, and live in proximity with the local inhabitants (Pinto et al. 2006; Bustamante et al. 2014; Panti-May et al. 2017; Ayaqui and Ruelas 2020). Mus musculus is the most frequently reported commensal rodent species in the city of Mérida (Panti-May et al. 2012, 2016). However, R. rattus may be more abundant in areas with tree vegetation due to its semi-arboreal behavior (Battersby et al. 2008). Therefore, R. rattus should be considered in studies addressing pathogens in commensal rodents. The results obtained in the present work highlight the need to study the participation of M. musculus in the biological cycle of T. cruzi in the city of Mérida and the rest of the Yucatán state.

Acknowledgements

The authors wish to thank W. I. Moguel-Chin for his assistance in the preparation of Figure 1. Thanks also to the Secretariat of Public Education for financing through the project "Estudio Multidisciplinario para la identificación de variables asociadas a la transmisión de enfermedades zoonóticas y ETVs en Yucatán” (Multidisciplinary study for the identification of variables associated with the transmission of zoonotic diseases and VBDs in Yucatán), project no. 103.5/09/1258. The comments of two anonymous reviewers improved the first version of this note. M. E. Sánchez-Salazar translated the manuscript into English.

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Associated editor: Beatríz Bolívar Cimé.

Submitted: February 28, 2024; Reviewed: May 22, 2024.

Accepted: June 12, 2024; Published on line: June 19, 2024.