Antibodies to Toxoplasma gondii, Leishmania spp., and Leptospira spp. in Free-Ranging Six-Banded Armadillos (Euphractus sexcinctus) from Northeastern Brazil

Armadillos can carry a great diversity of pathogens. For instance, the nine-banded armadillo (Dasypus novemcinctus) is considered an important reservoir of Mycobacterium leprae, the agent of leprosy (Da Silva et al. 2018). Other important organisms infecting these animals are Toxoplasma gondii, Trypanosoma cruzi, Sarcocystis spp., Leptospira spp., and Paracoccidioides spp. (Antunes et al. 2012; Morocoima et al. 2012; Vitaliano et al. 2014; Fornazari et al. 2018; Hrycyk et al. 2018). The investigation of pathogens infecting armadillos can provide valuable information on the epidemiology of different diseases, the health status of these animals, and their possible roles as reservoirs of zoonoses. Hence, we aimed to investigate antibodies to T. gondii, Leishmania spp., and Leptospira spp. in six-banded armadillos (Euphractus sexcinctus).

The study was conducted in the municipality of Mossoró (5°11′16″S, 37°20′38″W), in the northwest region of Rio Grande do Norte State, northeast Brazil. The city has approximately 295,000 inhabitants and is situated in the Caatinga biome, which occurs only in Brazil. Despite the high level of urbanization in Mossoró, approximately 9% of its population lives in rural areas (Brazilian Institute of Geography and Statistics 2010), where they have close contact with wildlife and occasionally use them as food, especially six-banded armadillos (Barboza et al. 2016).

Blood samples from 33 free-ranging six-banded armadillos were tested. Six animals were hunted by Mossoró residents, who kept them alive prior to consumption and consented to the extraction of blood samples. The remaining 27 armadillos were rescued by the local authorities from rural areas of Mossoró and sent to the Federal University of the Semi-Arid Region (UFERSA); details on the exact origin of these animals were not provided. The armadillos were chemically immobilized and blood was collected from the femoral vein. All the procedures were approved by the Chico Mendes Institute for Biodiversity Conservation (42879-1) and the Ethics Committee of UFERSA (23091. 002959/2016-43).

Antibodies to T. gondii were detected through the modified agglutination test (MAT-Toxo) according to Desmonts and Remington (1980), using 1:25 as cut-off point. A standard RH strain was used as antigen according to the protocol previously described by Fornazari et al. (2011). Antibodies to Leishmania spp. were assessed through the direct agglutination test (DAT) using a liquid antigen produced from Leishmania (Leishmania) infantum promastigotes, following the methodology described by Paiz et al. (2015). A dilution of 1:320 was adopted as the cut-off point. To detect anti–Leptospira spp. antibodies, the microscopic agglutination test (MAT-Lepto) was employed. A collection of 28 antigens previously described by Fornazari et al. (2018) was used, considering 1:100 as cut-off point. Details on the MAT-Lepto protocol were described by Fornazari et al. (2012).

Two animals were positive for antibodies to T. gondii (6.0%; 95% confidence interval [CI] 0.7–20.2%), both showing a titer of 400. Regarding Leishmania spp., three animals were positive (9.0%; 95% CI 1.9–24.3%), all with a titer of 320. Antibodies to Leptospira spp. were detected in two armadillos (6.0%; 95% CI 0.7–20.2%), both with a titer of 200. One was positive for the Canicola serogroup and the other for the Andamana serogroup. No animals were positive for more than one test.

Toxoplasma gondii is one of the most cosmopolitan parasites worldwide and infects a great diversity of warm-blooded animals. Although both armadillos presented serologic titers considerably above the cut-off value, inferences such as the stage of infection or the reservoir status cannot be made since MAT-Toxo has never been standardized for this animal species. This statement is also valid for the DAT and MAT-Lepto. Nevertheless, previous investigations in southeastern Brazil have isolated viable T. gondii from six-banded armadillo tissues, indicating that this animal can act as a reservoir of this parasite (Vitaliano et al. 2014). Given the habit of hunting and eating six-banded armadillos in the Caatinga region (Barboza et al. 2016), the risks of human infection should be further investigated, since the ingestion of undercooked tissues is an important transmission route for T. gondii.

Mammals belonging to the Cingulata order have a long coevolutionary history with trypanosomatids. However, studies on leishmaniasis in armadillos are scarce, and our report of exposure of six-banded armadillos to Leishmania is unique. Although the DAT was performed using promastigotes of Leishmania (Leishmania) infantum, the agent of visceral leishmaniasis, serologic cross-reactivity may occur with other species of the genus Leishmania (Paiz et al. 2015).

Although domestic dogs are the main reservoir of Leishmania (Leishmania) infantum, the role of other animal species has been investigated in Brazil, including foxes, rodents, primates, and opossums (Roque and Jansen 2014). However, the importance of wildlife in the maintenance and transmission of Leishmania still requires further study. There have been 28 human cases of visceral leishmaniasis between 2015 and 2017 in Mossoró municipality (Notifiable Diseases Information System 2019), which emphasizes the need to find other reservoirs (besides dogs) and elucidate their epidemiologic relevance.

Leptospirosis is endemic in Brazil and infects several wild species (Fornazari et al. 2018). Our results showed that six-banded armadillos have been exposed to Andamana and Canicola serogroups, indicating the circulation of these bacteria in the environment. Neither of these serogroups is considered of major relevance to public health in Brazil, and the lack of studies in the Caatinga biome hinders further conclusions on this topic. Surprisingly, one of the few studies concerns the isolation of leptospires from the urine of two six-banded armadillos captured in the Caatinga, thus proving that this animal species can act as a reservoir and shed this bacterium into the environment (Silva et al. 2015).

In conclusion, we found evidence of exposure to T. gondii, Leishmania spp., and Leptospira spp. in free-ranging six-banded armadillos in the Caatinga biome of Mossoró municipality, Brazil. Further studies are needed to understand the outcome of these events and identify if these animals pose risks to public health. We highlight the importance of such studies given the widespread habit of eating armadillos, especially in the northeast of Brazil. This practice can lead to infections while handling the living animal, when the carcass is being cleaned, or through meat consumption.