Infection by Leishmania spp. in Free-Ranging Opossums ( Didelphis albiventris ) in an Environmentally Protected Area Inhabited by Humans in Southeastern Brazil

Introduction

Leishmaniasis became a growing public health concern in many countries around the world, since natural or anthropogenic environmental changes have increased the risk of this disease (WHO 2010). In Brazil, zoonotic transmission cycles commonly occur in areas where human dwellings are located close to the sylvatic transmission cycle of the parasite (WHO 2010).

Thus, discussions have increasingly focused on the participation of wildlife in leishmaniasis epidemiology, given that environmental changes can provide a greater degree of contact between these animals and humans and/or domestic animals. This is an important factor to be considered in leishmaniasis control programs, considering that a variety of mammal host species have been described infected by Leishmania in the Americas (Roque and Jansen 2014).

The municipality of Campinas, State of São Paulo, southeastern Brazil, has an environmentally protected area (EPA) containing Atlantic forest fragments, which has undergone severe environmental changes and urbanization. Outbreaks of human cutaneous leishmaniasis (CL) were reported in the 1990s (Corte et al. 1996) in this area, and currently, the sole focus of autochthonous transmission of visceral leishmaniasis (VL) in Campinas occurs in the EPA (Von Zuben et al. 2014). Based on these facts, we conducted an investigation of a possible cycle of transmission of Leishmania spp. among free-ranging wild mammals from this area.

Materials and Methods

Monthly captures of wild mammals were conducted between April 2014 and March 2015 at 18 points in the EPA of Campinas, located between the latitudes 22°45′00″ and 22°56′00″ S and longitudes 46°52′30″ and 47°00′00″ W. Sixty tomahawk traps (30 × 21 × 20 cm and 55 × 20 × 20 cm) were used for three consecutive nights/month, baited with banana and chicken.

The captured mammals were identified with microchip implants (Microchips Brasil^®, Brazil), and blood samples were collected by venipuncture. DNA extraction of total blood was performed within 48 h after the end of each capture, using a QIAamp DNA mini kit in the QIAcube^® DNA extractor (Qiagen^®, Netherlands).

PCR was performed using 1 μL of extracted DNA (10 ng) and 11 μL of PCR mix containing 1.3 μL buffer (50 mM KCl, 20 mM Tris-HCl pH 8.4), 0.4 μL MgCl₂ (1.6 μM), 0.25 μL of each oligonucleotide (0.2 μM), 0.25 μL dNTP (0.2 mM), 0.25 μL of Platinum^® Taq DNA polymerase (Invitrogen, Brazil), and 8.3 μL of ultrapure water.

To evaluate the endogenous quality, DNA integrity, and presence of inhibitors, we used primers for the conserved gene of the interphotoreceptor retinoid-binding protein (IRBP), IRBP-CF-FWD (5′-TCCAACACCACCACTGAG ATCTGGAC-3′) and IRBP-CF-REV (5′-GTGAGGAAGA AATCGGACTGGCC-3′) (Ferreira et al. 2010), or for β1 (5′-ACCACCAACTTCATCCACGTTCACC-3′) and β2 (5′-CTTCTGACACAACTGTGTTCACTAGC-3′) (Lee et al. 2001).

For Leishmania spp. DNA research, we used primers for the internal transcribed spacer region-1 (ITS-1), LITSR (5′-CTGGATCATTTTCCGATG-3′) and L5.8S (5′-TGATA CCACTTATCGCACTT-3′) (El Tai et al. 2000). As positive controls we used 10 ng of DNA extracted from an in vitro culture of L. (L.) infantum (MHOM/BR/2002/LPC-RPV), L. (V.) braziliensis (MHOM/BR/1975/M2903), L. (L.) major (MHOM/IL/1980/FRIEDLIN), and ultrapure water as negative control.

The amplified products were identified by agarose gel electrophoresis 1.5% containing 1.0 μL/10 mL of SYBR^® safe DNA gel stain (Invitrogen^®; Life Technologies) and purified with the Illustra GFX^™ PCR DNA and Gel Band Purification kit (GE Healthcare^®, United Kingdom). Genetic sequencing by the Sanger method was performed in the Genetic Analyzer 3500 automated sequencer using the BigDye Terminator v 3.1 Cycle Sequencing kit (Applied Biosystems^®; Life Technologies).

The sense and antisense sequences were visualized using Chromas v 2.1.1 software (Technelysium Pty Ltd., Australia), subjected to global alignment using MEGA5 software (Tamura et al. 2011) and compared with those sequences deposited in GenBank, using the nucleotide basic local alignment search tool (BLASTn, www.ncbi.nlm.nih.gov/BLAST).

This research was approved by the Ethics Committee on Animal Use of the State University of Campinas (No. 3296-1) and by the Brazilian Institute of Environment and Renewable Natural Resources (IBAMA), through the Biodiversity Authorization and Information System (SISBIO, No. 42926-1/2).

Results

Eighty-two wild mammals of six species were sampled, as follows: 1/82 (1.2%) Mazama gouazoubira (brocket deer), 1/82 (1.2%) Sciurus (Guerlinguetus) aestuans (squirrel), 8/82 (9.8%) Callithrix jacchus (white-tufted-ear marmoset), 11/82 (13.4%) Didelphis aurita (black-eared opossum), 18/82 (22.0%) Callithrix penicillata (black-tufted-ear marmoset), and 43/82 (52.4%) Didelphis albiventris (white-eared opossum). Six D. albiventris (6/82, 7.32%; 95% CI 1.68–12.95%) were sampled again in a second capture.

The molecular identities of amplicons obtained from samples of two D. albiventris (2/82, 2.4%, 95% CI 0.3–8.5%) were confirmed by genetic sequencing. The sequence of one amplicon (GenBank accession number KX580706) showed 100% similarity with sequences of Leishmania (Leishmania) infantum (query cover 100%; value E = 2e-135, access KR081265.1 and others) deposited in GenBank, and the sequence of the other amplicon (GenBank accession number KX580707) showed 100% similarity with sequence of Leishmania (Viannia) guyanensis (query cover 100%; E value = 5e-100, access FJ753388.1 and others) and 99% similarity with sequences of L. (V.) peruviana (query cover 100%; E value = 2e-98; access HG512896.1 and others), L. (V.) braziliensis (query cover 100%; E value = 2e-98, access FJ753382.1 and others), and L. (V.) panamensis (query cover = 100%; E value = 3e-97; access CP009396.1 and others). This is the first report of infection by Leishmania spp. in wild animals in the municipality of Campinas.

Discussion

In 1996, Corte et al. associated cases of human CL that occurred in the EPA of Campinas to environmental changes due to urban expansion in the 1970s and 1980s, suggesting the importance of identifying possible wild reservoirs involved. Also in this area, the finding of Lutzomyia longipalpis infected by L. (L.) infantum and the VL transmission among dogs, which has been restricted to this geographical region of the municipality since 2009 (Von Zuben et al. 2014), have aroused interest of local health institutions.

The species D. albiventris possibly acts as an L. (L.) infantum reservoir in Brazilian zoonotic VL, besides zoonotic CL and mucocutaneous leishmaniasis in Peru (WHO 2010). In Brazil, L. (L.) infantum was first isolated from this species in Bahia (Sherlock et al. 1984) and their infectivity to vectors was demonstrated by xenodiagnosis (Sherlock 1996).

The first report of free-ranging opossums with parasitemia by pathogenic species of Leishmania spp. in the EPA of Campinas, in addition to previous epidemiological data and the area's environmental and historical characteristics, indicates a possible role of wildlife in the introduction and/or maintenance of transmission cycles of causative agents of leishmaniasis.

Although genetic sequencing enabled the identification of the parasite only in two animals, the occurrence of Leishmania infection in wild species in the region was previously unknown. Furthermore, sequencing of one sample made it possible to confirm the presence of L. (L.) infantum, but in another sample a sequence was obtained that is common to sequences of different species from the subgenus Viannia. However, based on similarity and e values obtained, it is possible to infer that the Leishmania species detected in this blood sample is probably L. (V.) guyanensis (greater similarity and lower e value), whose circulation has never been reported in the study area.

These findings are especially important from a public health perspective, considering that opossums are reservoirs of the parasite (WHO 2010) and that the EPA is inhabited by humans.

It must be noted, however, that Leishmania spp. may be transmitted not only from wildlife but also for those animals from infected pets or even from infected humans living in wild areas, if there is the presence of vectors.

The measures proposed by the Brazilian Visceral Leishmaniasis Control Program have been insufficient to prevent the spread and to reduce the prevalence of the disease. The low effectiveness is probably due to several factors, but the main pillar of this program is the elimination of the canine reservoir (Roque and Jansen 2014). The results of this investigation suggest that eliminating dogs with positive serology may not be sufficient to prevent the maintenance and/or expansion of VL foci in places where there is involvement of infected wild reservoirs.

Conclusions

This work reports the circulation of pathogenic species of Leishmania spp. in wild animals considered reservoirs of the parasite (Didelphis albiventris) in an EPA inhabited by humans. The results suggest the need for periodic reviews and flexibility in leishmaniasis control and surveillance strategies, considering the specificities of each locality.