Figures
Abstract
Background
Visceral leishmaniasis (VL) is a complex zoonotic disease transmitted to humans through the bites of Leishmania-infected phlebotomine sand flies (Diptera:Psychodidae:Phlebotominae). Its transmission dynamics are shaped by environmental, ecological, and anthropogenic factors. Rapid urbanization, landscape modification, and the rising incidence of human and canine VL in endemic cities raise concerns about parasite circulation at the human-wildlife interface, including within captive animal populations housed in urban zoological institutions.
Methodology/principal findings
This study was conducted at the Belo Horizonte Zoo (Minas Gerais, Brazil), located in one of city’s most VL-affected districts. We collected 31 skin biopsies and 40 blood samples from 13 captive mammal species—11 primates (Alouatta guariba, Aotus nigriceps, Ateles marginatus, Gorilla gorilla, Lagothrix lagotricha, Leontopithecus rosalia, Leontopithecus chrysopygus, Pan troglodytes, Pithecia irrorata, Saguinus imperator, Sapajus xanthosternos) and 2 carnivores (Chrysocyon brachyurus and Leopardus colocolo). Samples were screened using a complementary molecular diagnostic panel: Leishmania nested PCR (LnPCR), real time PCR (qPCR), and K26 loop-mediated isothermal amplification (K26-LAMP). Leishmania DNA was detected in the blood of a clinically healthy adult maned wolf (Chrysocyon brachyurus). Sequencing and phylogenetic analysis of the LnPCR amplicon confirmed the presence of Leishmania infantum. The animal was housed in an enclosure with documented high densities of Lutzomyia longipalpis, the primary VL vector in the region.
Conclusions/significance
The detection of Leishmania infantum DNA in a zoo-housed wild canid within an VL-endemic urban area highlights the potential exposure of captive wildlife to local transmission cycles. Although infectivity and reservoir competence were not assessed, these findings underscore the importance of integrating minimally invasive molecular surveillance with entomological monitoring in zoological institutions. Such an integrated approach can strengthen One Health surveillance frameworks by enabling early detection of parasite circulation, informing risk-based management strategies, and supporting VL prevention efforts in complex urban environments.
Author summary
Visceral leishmaniasis (VL) is a serious disease transmitted to humans and animals by infected sand flies. Urban expansion and the increasing incidence of VL raises concerns about parasite circulation at the interface of people, domestic and wild animals, and the environment—including wildlife in urban zoos. In this study, we analyzed 31 skin and 40 blood samples from 13 species of captive primates and carnivores at the Belo Horizonte Zoo, located in a highly VL-endemic area of Brazil. Using molecular diagnostic methods, we detected Leishmania infantum DNA in the blood of a maned wolf (Chrysocyon brachyurus), providing evidence that zoo-housed wild animals can become infected in urban endemic settings. Notably, the enclosure housing this animal exhibited a high density of Lutzomyia longipalpis, the primary vector of VL in Brazil. Although our study did not assess the infectivity of the detected parasite or the animal’s capacity to transmit it, our findings underscore the value of integrating molecular surveillance with vector monitoring in zoological institutions. Such an integrated approach aligns with the One Health framework, enabling early detection of parasite circulation at the wildlife–vector–human interface and informing both public health wildlife conservation strategies.
Citation: Pereira NCL, Michalsky ÉM, de Avelar DM, Saraiva L, Antunes Nobi RC, Coelho CM, et al. (2026) Detection of Leishmania infantum DNA in captive wild mammals from an urban zoo in a visceral leishmaniasis–endemic area of Brazil. PLoS Negl Trop Dis 20(3): e0014098. https://doi.org/10.1371/journal.pntd.0014098
Editor: Mehmet Aykur, Tokat GaziosmanpaÅŸa University Faculty of Medicine: Tokat Gaziosmanpasa Universitesi Saglik Bilimleri Fakultesi, TÃœRKIYE
Received: September 19, 2025; Accepted: February 26, 2026; Published: March 20, 2026
Copyright: © 2026 Pereira et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All data underlying the findings are within the manuscript.
Funding: CAPES (Coordenação de Aperfeiçoamento de Pessoal de NÃvel Superior, Brazil) granted a Doctoral fellowship to NCLP (Financing code 001). ESD is a productivity fellow from the Conselho Nacional de Desenvolvimento CientÃfico e Tecnológico, Brazil (CNPq process no. 308394/2021-6). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Leishmaniasis is a neglected tropical and zoonotic disease caused by protozoa of the genus Leishmania (Ross, 1903) and transmitted in the New World primarily by infected phlebotomine sand flies of the genus Lutzomyia (Diptera: Psychodidae: Phlebotominae). In the Americas, leishmaniasis constitutes a major public health problem, characterized by diverse clinical manifestations, marked biological complexity, and substantial epidemiological heterogeneity [1,2].
Disease outcomes vary widely depending on the infecting Leishmania species. Visceral leishmaniasis (VL), also known as kala-azar, is the most severe form and is almost invariably fatal if left untreated. It typically presents with irregular fever, weight loss, hepatosplenomegaly, and anemia. Globally, an estimated 50,000–90,000 new VL cases occur annually; although only 25–45% are officially reported to the World Health Organization [3]. Brazil remains among the countries with the highest VL burden worldwide, and notification of human cases to the Ministry of Health is mandatory.
Leishmania parasites exhibit high genetic diversity and a broad host range, infecting a wide variety of domestic and wild mammals, and humans [4]. Numerous wildlife species have been identified as natural reservoirs, including representatives of Carnivora, Cingulata, Marsupialia, Pilosa, Primates, Rodentia, and Chiroptera [4,5]. These hosts play a fundamental role in maintaining parasite transmission cycle in nature [6]. Their presence in urban and peri-urban environments may contribute to sustained transmission, particularly in areas where Lutzomyia longipalpis—the primary vector of Leishmania infantum—is well established [4].
In Belo Horizonte, the capital of Minas Gerais state, VL was first reported in 1994 and has since undergone sustained urban expansion, with continuous detection of both human and canine cases. The municipality is officially classified as endemic by the Brazilian Ministry of Health. Between 2014 and 2024, 425 new human VL cases were confirmed [7] and 5,770 dogs tested seropositive through the municipal surveillance program [8]. Of the reported human cases, 23 occurred in the Pampulha district, where the municipal zoo (BH Zoo) is located. Pampulha borders districts with high VL incidence—Northeast, Northwest, and Venda Nova—which together accounted for 209 human cases during the same period [9]. Within this urban–peri-urban mosaic, captive wild animals may be exposed to infected vectors, due to favorable environmental conditions for sand fly proliferation and the high faunal diversity of the zoo, many species of which are susceptible to Leishmania infection.
Belo Horizonte has implemented a Municipal Canine Visceral Leishmaniasis Control Program aligned with national guidelines, encompassing entomological surveillance, canine surveys, vector control, environmental management, and health education actions. The primary objective of this program is to reduce transmission and prevent human and canine cases [9,10]. Nevertheless, despite sustained control efforts, VL elimination has not yet been achieved. The human VL case fatality rate increased from 5.3% in 2020 to 10.8% in 2021. In 2022, although the incidence reached its lowest recorded value (0.9 cases per 100,000 inhabitants), the highest fatality rate was observed (17.9%), remaining elevated in 2023 (17.1%) [11].
Comparable challenges have been reported in other endemic regions, including Eastern Africa, where expanded VL elimination strategies have been proposed, incorporating intensified diagnosis, optimized treatment, and integrated surveillance and vector control approaches [12]. These experiences highlight the importance of understanding local transmission dynamics, particularly in complex ecological interfaces such as zoological institutions.
The occurrence of VL in zoos represents an emerging challenge within the One Health framework, which integrates human, domestic animal, wildlife, and environmental health [13,14]. Infections in zoo wildlife may signal shifts in transmission dynamics and the early emergence of zoonotic diseases in urban settings [15]. Additionally, conservation programs, animal translocations, and reintroduction initiatives may inadvertently facilitate pathogen dispersal if infected individuals remain undetected [16].
The role of zoo-housed mammals in the maintenance of VL transmission cycles therefore remains poorly understood, representing a critical gap in One Health surveillance. From this perspective, the selection of biological samples for molecular diagnosis must balance animal welfare, feasibility across diverse taxa, and diagnostic sensitivity. Minimally invasive matrices—such as peripheral blood, buccal swabs, conjunctival swabs, and skin scrapings—have demonstrated good diagnostic performance for Leishmania infantum in domestic and wild mammals, making them particularly suitable for surveillance in zoological settings [17].
Despite increasing reports of Leishmania infection in captive wildlife worldwide, major knowledge gaps remain regarding the circulation of Leishmania infantum in zoological institutions located in urban endemic settings, particularly in South America. Systematic surveillance using minimally invasive sampling strategies in apparently healthy captive wildlife remains scarce. As a result, the epidemiological relevance of zoo-housed wild mammals within local transmission cycles is still poorly understood.
In this context, the present study aimed to screen captive wild mammals housed at a public urban zoo located in a visceral leishmaniasis–endemic area of Brazil for Leishmania spp. DNA using complementary molecular approaches, while prioritizing animal welfare and institutional biosafety constraints. By combining molecular detection and DNA sequencing, we sought to provide evidence-based data aiming to reinforce the relevance of sustained One Health surveillance in zoological settings.
Materials and methods
Ethical statements
The present study was approved by the Institutional Review Board of the Foundation of Municipal Parks and Zoobotany, Belo Horizonte, Minas Gerais, under the protocol no. FU010/2022.
Study site
The present study was conducted at the Belo Horizonte Zoo (BH Zoo), operated by the Foundation of Municipal Parks and Zoobotany (19°51′35″S; 44°00′38″W) in Belo Horizonte, Minas Gerais, Brazil. Located in the Pampulha district, the zoo houses approximately 3,500 animals representing over 250 species across the vertebrate classes: mammals, birds, reptiles, amphibians, and fish, with specimens from five continents. Animals are housed in 292 species-appropriate enclosures designed in accordance with Brazilian animal welfare regulations. The zoo also includes specialized facilities such the São Francisco River Aquarium, a Butterfly House, a Bird Immersion Enclosure, and an Environmental Education Center. On site veterinary care is provided through a dedicated hospital and a nationally and internationally recognized Animal Welfare Program [18].
Data on the animals’ date of arrival at the BH Zoo were not available for analysis because these records are maintained in an internal administrative database and are not accessible to external researchers, including the authors.
Sample collection
All animals were individually identified per institutional registry. Samples were collected opportunistically during veterinary procedures (e.g., health checks, clinical evaluations), according to established zoo protocols, from April 2022 to October 2023. Following a 12-hour fast, animals physically restrained and anesthetized via intramuscular injection of a tiletamine-zolazepam (Zoletil, Virbac at 5 mg/kg).
Whole blood (1–3 mL, depending on body weight) was collected via femoral venipuncture into 4.8 mL K₃EDTA Vacutainer tubes as part of routine clinical procedures at the BH Zoo and stored at −20°C until analysis. As EDTA-anticoagulant blood was obtained solely during standard veterinary care, serum was not available.
Skin samples were harvested from the inner hind limb or ear tip after shaving, followed by local disinfection with 2% chlorhexidine, 10% aqueous povidone-iodine solution and 70% ethanol, in this order. Asepsis was achieved by using concentric movements of sterile gauze. Local anesthesia with 2% lidocaine hydrochloride (0.1 mL/kg) was performed and a skin fragment of 0.5 × 1.0 cm was excised using sterile surgical scissors, forceps, and a sterile 2 mm biopsy punch. The biopsy sites were closed with 3.0 nylon (non-absorbable) in interrupted stitches. The skin samples were individually stored at -20°C in labeled 1.5 mL Eppendorf tubes.
In some cases, blood samples were collected from the same animal on two occasions, approximately one year apart, during routine clinical procedures at the BH Zoo. Both samples were included in the analysis because the interval between collections was sufficient to allow for potential changes in infection status over time.
DNA extraction
Blood DNA was isolated utilizing the GFX Genomic Blood DNA Purification Kit (GE Healthcare) and skin DNA was isolated utilizing the Tissue and Cell Extraction Kit (GE Healthcare) according to the manufacturer’s instructions. DNA purity and concentration were determined by the A260/A280nm ratio in a NanoDrop spectrophotometer (Thermo Scientific).
PCR of endogenous mammalian gene
To verify DNA integrity, absence of inhibitors and confirm mammalian origin, and endogenous control targeting a 227-bp fragment of the interphotoreceptor retinoid-binding protein (IRBP) [19] was amplified in all samples (Table 1). Canine skin DNA served as the positive control, while nuclease-free water replaced DNA in the negative control. The amplified bands were separated on 2% agarose gels to confirm the expected size, stained with ethidium bromide, and imaged under a UV light (ImageQuant LAS 4000 (GE HealthCare).
https://doi.org/10.1371/journal.pntd.0014098.t001
Molecular Detection of Leishmania DNA
Nested PCR (LnPCR).
The first reaction targeted the Leishmania SSU rRNA gene (Table 1). After 1:40 dilution, the product was submitted to a second round of amplification, using internal primers (R3 and R4) to the firstly amplified fragment. A ~ 358 bp product was expected for Leishmania sp. PCR amplifications were carried out on a Veriti thermocycler (Applied Biosystems). The procedure followed published protocols [20–22]. The positive control was Leishmania infantum DNA (strain MHOM/BR/PP75). In the negative control, DNA was replaced in volume by nuclease-free water.
Real-Time PCR (qPCR).
qPCR reactions (Table 1) were carried out according to [23], in 25 µL final volume with 12.5 µL SYBR Green Master Mix (Applied Biosystems), 0.3 pmol of each primer, 5 µL sample DNA, and 5.5 µL nuclease-free water. The melting curve was determined from 60°C to 95°C with 0.3°C/s with continuous fluorescence acquisition. The positive control was DNA extracted from a culture of Le. infantum (strain MHOM/BR/PP75). In the negative control DNA was replaced in volume by nuclease-free water.
Loop-Mediated isothermal amplification (K26-LAMP).
The loop-mediated isothermal amplification (K26-LAMP) assay (Table 1) was performed according to [24], using the WarmStart Colorimetric LAMP 2X Master Mix (New England Biolabs). The total volume was 25 µL, with 12.5 µL of Master Mix, 2.5 µL 10X primer mix (FIP, BIP: 1.6uM; Loop-F, Loop-B: 0.8uM; F3, B3: 0.2uM), 8 µL nuclease-free water, and 2 µL sample DNA. The positive control was prepared with 2µL of DNA extracted from a culture of Le. infantum (strain MHOM/BR/PP75). In the negative control any DNA sample was omitted and replaced with 2µL of nuclease-free water. The results were interpreted visually based on color change: pink (negative) to yellow (positive), reflecting pH shift due to amplification.
Specific identification of Leishmania spp
Species-level identification of Leishmania was performed by sequencing the 358-bp amplicon generated in the second round nested PCR (LnPCR). To minimize the risk of cross-contamination, sequencing was carried out using a fresh aliquot of the original DNA extract. The resulting sequence was identical to that obtained from the initial amplicon, confirming the reliability of the positive detection.
The target DNA band was excised from the agarose gel and purified using the QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany). Sequencing reactions were prepared in a final volume of 10 µL containing: 1 µL BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems), 1 µL 5x sequencing buffer, 1 µL each of the R3 and R4 primers (used in the second LnPCR round) at 3.2 pmol/µL each, 2 µL PCR product, and 5 µL nuclease-free water]. Reactions were run on an automated sequencer (ABI 3500 Genetic Analyzer), and both forward and reverse strands were sequenced to ensure accuracy.
The obtained sequences were edited and assembled using BioEdit software (version 7.2.5) and compared against reference sequences deposited in the GenBank database using the Basic Local Alignment Search Tool (BLAST). Species identification was based on the highest percent identity and query coverage with validated Leishmania reference sequences. Edited sequences were aligned and compared with the three main Leishmania species of epidemiological relevance in the study area—Leishmania braziliensis, Leishmania amazonensis, and Leishmania infantum—and subsequently deposited in GenBank.
Phylogenetic analysis
Multiple sequence alignment was performed using Molecular Evolutionary Genetics Analysis software (MEGA version 12), as described [25]. The 358-bp DNA sequence obtained from the maned wolf (Chrysocyon brachyurus; sample B19) was aligned with homologous Leishmania spp. sequences retrieved from GenBank (accession numbers: MN852437.1, MN852432.1, PP735724.1, PQ151612.1, MN852436.1, PQ635332.1, KX011481.1, KX011484.1, KF302746.1, JX030187.1, KF041799.1, KF041803.1, JX030136.1, KF302743.1).
Considering the polymorphism of Leishmania kDNA sequences, fragments were selected for phylogenetic inference using the Maximum Likelihood method under Jukes-Cantor substitution model [26], with uniform distribution, as selected by the Model Test function implemented in MEGA12. Initial trees for the heuristic search were generated automatically using the Neighbor-Joining and Maximum Parsimony algorithms. Branch support was assessed by bootstrap analysis with 1,000 replicates.
The final data set comprised 15 trypanosomatid sequences (241–250 bp in length) representing key Leishmania species circulating in the study region. Trypanosoma cruzi (GenBank accession number JF746736.1) was used as the outgroup.
All animal handling and sampling procedures were conducted in accordance with the institutional animal welfare, biosafety, and veterinary care protocols of the Belo Horizonte Zoo. According to these institutional guidelines, invasive procedures such as lymph node aspiration or bone marrow sampling are restricted to clinically ill animals and are not permitted for routine surveillance of apparently healthy individuals. Therefore, sample collection in the present study was limited to minimally invasive matrices, including peripheral blood and skin biopsies, which are considered ethically acceptable and operationally feasible for surveillance purposes in captive wildlife.
Results
Confirmation of the origin of the extracted DNA
The mammalian constitutive interphotoreceptor retinoid-binding protein (IRBP) gene was successfully amplified in all collected samples (Fig 1), yielding the expected 227-bp fragment. This result confirms the mammalian origin of the extracted DNA, its suitability for PCR amplification, and the absence of significant PCR inhibitors.
[A]. Mammalian IRBP gene amplification by conventional PCR (cPCR). Sample numbers are indicated on top. Positive amplification is indicated by the presence of a 227-bp band (indicated by the arrow). PC – Positive control (DNA isolated from dog skin); NC – Negative control (no DNA); MM – 120-bp molecular marker. [B]. DNA amplification with primers for the SSU rRNA gene (round 2 of LnPCR) of Leishmania sp. Samples are identified on top. Positive amplification is indicated by the presence of a 358-bp band (indicated by the arrow). PC – positive control (DNA from Leishmania infantum reference strain MHOM/BR/PP75); NC – negative control (no DNA); MM – 120 bp molecular marker. The positive sample for Leishmania DNA (B19 from maned wolf) is boxed in [A] and [B]. Photo credits: NLCP.
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Molecular detection of Leishmania DNA
A total of 31 skin biopsies and 41 blood samples from wild mammals (orders Primates and Carnivora) were screened for Leishmania DNA using three complementary molecular methods: nested PCR (LnPCR), real time PCR (qPCR), and K26-loop-mediated isothermal amplification (K-26 LAMP) (Table 2), following established protocols.
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All skin samples tested negative by all three assays. Among blood samples, only one—collected from a maned wolf (Chrysocyon brachyurus; sample no. 19, ID 245565)—yielded a positive result. This sample produced the expected 358-bp amplicon in the second round of LnPCR (Fig 1B) and was also positive by K26-LAMP, as indicated by a color change from pink to yellow (Fig 2). In contrast, this test sample tested negative by qPCR. All remaining blood samples (n = 39) were negative across all three molecular assays.
PC – Positive control (DNA from Leishmania infantum reference strain MHOM/BR/PP75); NC – Negative control (no DNA); B19 – Positive blood sample from Chrysocyon brachyurus. Photo credits: NLCP.
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Species identification by DNA sequencing
The 358-bp LnPCR amplicon obtained from the maned wolf (Chrysocyon brachyurus, sample B19) was sequenced and compared with reference sequences available in the GenBank database. BLAST analysis revealed 100% nucleotide identity with Leishmania infantum. The sequence was deposited in Gene Bank accession number PV797395).
Phylogenetic tree
A Maximum Likelihood phylogenetic tree based on small subunit ribosomal RNA (SSU rRNA) sequences corroborated the taxonomic assignment. The sequence obtained from the maned wolf (GenBank accession PV797395) clustered robustly within the Le. infantum clade, with 100% bootstrap support (Fig 3). The analysis included 14 reference Leishmania sequences, and Trypanosoma cruzi (JF746736.1) as the outgroup.
The tree was constructed using the Juke-Cantor substitution model with 1,000 bootstrap replications. Bootstrap values ≥ 70% are shown at the nodes. The sequence obtained from the maned wolf (Chrysocyon brachyurus) in this study (GenBank deposit PV 977395) clusters within the Leishmania infantum clade (highlighted in bold). Reference sequences from GenBank are labeled with their accession numbers followed by species names. Trypanosoma cruzi (GenBank accession JF746736.1) was used as the outgroup.
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Discussion
Zoological institutions play a critical role in biodiversity conservation, environmental education, and captive breeding programs, particularly for threatened and endangered species [27]. Among these, the maned wolf (Chrysocyon brachyurus) s listed as Vulnerable on The Brazilian Red List and may be considered Critically Endangered in specific biomes such as the Brazilian Pampa/Uruguayan Savanna [28]. The conservation significance of this species underscores the importance of understanding and managing infectious diseases within zoological settings.
Given their frequent location in urban or peri-urban areas and the high diversity of susceptible species they house, zoos may also function as sentinels for emerging infectious diseases (EIDs) at the human-animal-environment interface [15]. Zoonotic infections detected in zoos often overlap with recognized EID hotspots, reflecting complex ecological interactions that facilitate pathogen spillover [29]. It is estimated that approximately 60% of EIDs are zoonotic, and nearly three-quarters originate in wildlife [30]. Conversely, captive wildlife may also contribute to pathogen dissemination through animal translocations, reintroduction programs, and exposure to competent vectors [16,31,32]
Over the past 15 years, Leishmania infections have been documented in captive wild mammals across multiple continents, demonstrating that zoos and wildlife rescue centers are not exempt from transmission cycles. Visceral leishmaniasis (VL) has been reported in marsupials, canids, felids, and non-human primates (NHPs), involving both New and Old World’s species. While some infections progressed to overt and sometimes fatal [33–37], others were identified solely through molecular or serological screening in apparently healthy individuals [38–40].
The Belo Horizonte Zoo (BH Zoo) is situated in a visceral leishmaniases-endemic area, raising concerns about the potential role of captive wild animals as silent hosts within the local transmission cycle. This concern is reinforced by previous detection of Leishmania DNA in blood samples from 17 NHP species housed at the same institution, including Alouatta guariba, Aotus nigriceps, Callicebus nigrifrons, Cebus xanthosternos, Leontopithecus crysomelas, Pithecia irrorate, and Saguinus imperator [38]. Notably, one C. nigrifrons individual later developed fatal disease with clinical and pathological features compatible with VL. Moreover, xenodiagnosis experiments conducted in 52 NHPs at the BH Zoo demonstrated their capacity to infect Lutzomyia longipalpis, with Le. infantum promastigotes recovered from sand flies fed on eight different NHP species [41].
Captive canids may be particularly vulnerable to Leishmania infection due to stress-induced immunosuppression, increased exposure to vectors, and heightened susceptibility to clinical disease [31,38,42]. In Brazil, Leishmania infection has been reported in both captive and free-ranging wild canids, including hoary foxes (Lycalopex vetulus), crab-eating foxes (Cerdocyon thous), bush dogs (Speothos venaticus), and maned wolves (C. brachyurus) [31,42–45]. Although the reservoir role of these species remains unclear, maned wolves and bush dogs appear susceptible to clinical VL and can infect Lu. longipalpis when naturally infected—even in the absence of overt clinical signs [46]. Nevertheless, low parasite loads detected in engorged sand flies suggest their reservoir competence may be limited.
Entomological surveillance conducted at the BH Zoo between 2019 and 2021 identified Lu. longipalpis as the predominant sand fly species, accounting for more than 65% of captured specimens [47]. High vector densities were recorded in enclosures housing gorillas and maned wolves, including the enclosure of the Le. infantum–positive individual identified in this study. Although the detection of Le. infantum DNA in a clinically healthy adult female maned wolf, combined with elevated local vector density, supports the ecological plausibility of parasite circulation, this study did not assess infectivity or reservoir competence. Therefore, interpretations are presented cautiously, without extrapolation beyond the available data.
Future investigations should incorporate xenodiagnosis, parasite load quantification, and parasitological confirmation to clarify whether maned wolves can effectively contribute to transmission cycles in zoological settings. Notably, after the conclusion of this study, the Leishmania-positive maned wolf was euthanized due to severe clinical deterioration compatible with VL.
Although most evidence derives from canine visceral leishmaniasis, several studies demonstrate that skin tissue is a reliable substrate for the molecular detection of Leishmania infantum, supporting its translational application to wildlife species. In dogs, skin biopsies have shown consistent PCR positivity regardless of the presence of visible cutaneous lesions, with both lesioned and normal-looking skin harboring detectable parasite DNA [48–52]. Histopathological and parasitological analyses further indicate that macroscopically normal skin may contain substantial parasite loads across different clinical stages, highlighting the diagnostic value of this tissue even in the absence of dermatological manifestations. Comparative tissue analyses in naturally infected dogs have demonstrated that skin biopsies perform similarly to lymphoid tissues, such as lymph nodes, in PCR-based diagnosis and infection monitoring [53]. Moreover, quantitative studies in vertically infected dogs have shown that parasite burden in dog skin were significantly higher than those measured in the blood and approaching parasite burdens found in splenic tissue [54]. These findings collectively support the use of skin samples as a practical and informative matrix for molecular surveillance in wildlife, particularly in zoological settings where animals are sampled under anesthesia and minimally invasive procedures are prioritized.
The discordant results observed in this study—positive by LnPCR and K26-LAMP but negative by qPCR—are consistent with known differences in analytical sensitivity among molecular assays. qPCR performance may be affected by DNA degradation, PCR inhibitors, or an insufficient number of targeted minicircles, whereas LnPCR (with nested amplification) and LAMP (using multiple primer-binding sites) offer enhanced sensitivity under suboptimal conditions [55,56]. The successful amplification of the host IRBP gene confirmed adequate DNA integrity, suggesting that low parasite load —rather than technical failure—most likely explains the qPCR-negative result. These findings reinforce the value of employing complementary molecular approaches when screening wildlife populations.
This study has several limitations that should be acknowledged. First, the low number of positive animals precludes any inference regarding the role of maned wolves or other captive species as reservoirs of Le. infantum. Second, the absence of xenodiagnosis and parasite load quantification limits conclusions regarding infectivity to sand fly vectors. Third, reliance on blood and skin samples may have reduced sensitivity in animals with very low parasite burdens or localized infections. Nevertheless, these limitations reflect ethical and institutional constraints inherent to zoological surveillance and underscore the need for complementary diagnostic approaches and longitudinal monitoring to better elucidate transmission dynamics in captive wildlife populations.
In conclusion, the detection of Leishmania infantum DNA in a captive maned wolf within a visceral leishmaniasis–endemic urban zoo highlights the relevance of zoological institutions as critical interfaces for One Health surveillance. Elevated densities of competent vectors in close proximity to susceptible wildlife reinforce the need for integrated monitoring strategies that combine animal health surveillance, entomological monitoring, and environmental management.
Supporting information
S1 Fig. Graphical abstract.
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(TIFF)
Acknowledgments
We are grateful to the Programa de Pós-Graduação em Ciências da Saúde do Instituto René Rachou/FIOCRUZ. We acknowledge the technical support of the technicians and animal caretakers from the BH Zoo.
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