Introduction

Bacteria of the genus Leptospira serve as the etiological agents of leptospirosis, a disease that significantly impacts the economy, public health, and animal [1, 2]. Classified as a zoonosis, this disease has a global distribution and is particularly prevalent in Brazil [3, 4]. In endemic regions, the persistence of leptospirosis outbreaks is often linked to reservoir hosts capable of harboring Leptospira spp. for extended periods. These hosts may or may not exhibit clinical signs but contribute to the spread of the infectious agent in both rural and urban areas [1, 5, 6].

Environments contaminated with urine from infected animals—such as soil, mud, or water—act as transmission sources for the microorganism to animals and humans, primarily through mucous membranes or skin [7, 8]. While rodents are the main reservoirs of the etiological agent [1, 9], various animal species, including wild animals, can action as hosts and reservoirs for Leptospira spp. in specific regions [5, 6, 10,11,12]. Therefore, investigations into Leptospira spp. in wild animals are important to provide information about the epidemiology of this relevant zoonotic infection, since these animals often coexist with humans and domestic animals [11, 13,14,15].

Given that wild animals often share habitats with humans and domestic animals, studying Leptospira spp. in these species is crucial for understanding the epidemiology of this significant zoonotic [11, 13,14,15]. Understanding the animal host range and geographic distribution of Leptospira species is essential for identifying strains in local animal hosts that can infect people and other animals [16,17,18,19,20]. Tropical countries such as Brazil, which boast extensive biodiversity, provide numerous animal species that warrant investigation as potential Leptospira spp. reservoirs [21], as demonstrated in several Brazilian studies that directly or indirectly detected Leptospira spp. in wild mammals [3, 22, 23]. Therefore, this study aims to assess the presence of Leptospira spp. DNA in various species of free-living and captive wild animals in Rio Grande do Sul, Brazil and to analyze the phylogenetic relationships among Leptospira spp. identified.

Materials and methods

This study examined kidney tissue samples from 114 wild animals, comprising 75 free-living and 39 captive-bred specimens. All animals belonged to Mammalia class and died in Rio Grande do Sul State, in South of Brazil, between 2021 and 2023. They were sent for necropsy without suspicion of leptospirosis to the Laboratório de Patologia Veterinária (Veterinary Pathology Laboratory) at Universidade Federal de Santa Maria (Federal University of Santa Maria, UFSM) (Table 1). During necropsy, a single kidney from each animal was individually harvested and stored at -20°C until molecular analysis. Taxonomic identification was conducted according to the family, genus, and species, as described by Cubas et al. [24] and Hickman et al. [25].

Table 1 Detection of DNA from pathogenic Leptospira spp. in kidney tissue samples from wild animals of Mammalia class, collected from 2021 to 2023 in Rio Grande do Sul, Brazil
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Kidney tissue samples were sent to the Laboratório de Diagnóstico e Pesquisa em Leptospirose (Leptospirosis Diagnostic and Research Laboratory) at UFSM, where were homogenized, and an aliquot (~20 mg) was placed in polypropylene microtubes for total DNA extraction, following a protocol adapted for tissue samples [26]. Tissue fragments were lysed in a buffer containing 2-β-mercaptoethanol, 2% sodium dodecyl sulfate, 10% cetyltrimethylammonium bromide, and 5N sodium chloride. DNA was extracted using a phenol-chloroform method and reconstituted in 40 µL of sterile Tris-EDTA buffer. DNA concentrations were quantified via spectrophotometry.

A polymerase chain reaction (PCR) was performed to amplify a 242-base pair fragment of the lipL32 gene, which encodes for outer membrane proteins exclusively found in pathogenic Leptospira spp. [12]. The sensitivity of the PCR reaction was verified through the detection threshold of the positive control, which detected up to 1.5 × 103 bacteria/mL. The PCR sample was prepared to a final volume of 12.5 µL containing 1 x buffer (Ludwig Biotec, Brazil), 1.5 mM MgCl2 (Ludwig Biotec, Brazil), 0.2 mM dNTPs (Ludwig Biotec, Brazil), 2.5 U of Taq DNA polymerase (Ludwig Biotec, Brazil), 50 nM of each primer (Invitrogen, Brazil) lipL32-45F (5′-AAG CAT TACCGC TTG TGG TG-3′) and lipL32-286R (5′-GAA CTC CCA TTT CAG CGA TT-3′), and 2.5 µL (330 ng/µL) of the extracted DNA sample. The amplification was carried out in a PCR thermal cycler (K960, TION96, Brazil) using a specific set of cycling conditions, consisting of an initial denaturation of 94 °C for 2 min, 35 cycles of 94 °C for 30 s, 53 °C for 30 s, 72 °C for 1 min, followed by a final extension at 72 °C for 5 min. The PCR products were subsequently analyzed through horizontal electrophoresis on a 1% agarose gel, which was stained with non-mutagenic Safer dye (Kasvi, Brazil), observed under ultraviolet light, and photodocumented.

The samples amplified in the PCR were purified using a PCR purification kit (Ludwig Biotec, Brazil) according to the manufacturer’s instructions and sent for DNA sequencing (ACTGene Análises Moleculares, Brazil). The resulting sequences were aligned using the MEGA X software [27], compared among themselves, and with reference sequences available in the GenBank (MN906895, MK328874, MK568983, MK568984). A phylogenetic tree was constructed using Bayesian analysis [28], and the bootstrap resampling method was employed as a phylogeny test with 500 replications [29].

Results

LipL32 was detected in 9.2% (11/114) of the samples examined. Among these amplified samples, ten were identified in at least one distinct species of wild animal evaluated, as listed in Table 1.

Of the positive samples, nine species of wild animals were identified, including the white-eared opossum (D. albiventris) at 18.2% (2/11), skunk (C. chinga) at 18.2% (2/11), geoffroy’s cat (L. geoffroyi) at 9.1% (1/11), margay (L. wiedii) at 9.1% (1/11), pampas fox (L. gymnocercus) at 9.1% (1/11), capybara (H. hydrochaeris) at 9.1% (1/11), common marmoset (C. jacchus) at 9.1% (1/11), neotropical river otter (L. longicaudis) at 9.1% (1/11), and european hare (L. europaeus) at 9.1% (1/11), all from Rio Grande do Sul, Brazil. Regarding the sex of the animals that tested positive, 81.8% (9/11) were males and 18.2% (2/11) were females. In terms of age distribution, 90.9% (10/11) of the animals were adults.

The evaluated samples from free-living animals came from Santa Maria (67/114), Palmeira das Missões (5/114), Lagoa Vermelha (1/114), and Cruz Alta (1/114) municipalities. Captive-bred animal samples were collected from Cachoeira do Sul (28/114) and Santa Maria (12/114) cities. Among the animals that tested positive for pathogenic Leptospira spp. (11/114), 63.6% (7/11) were free-living— 71.4% (5/7) of which were from Santa Maria and 28.6% (2/7) from Palmeira das Missões. The remaining 36.4% (4/11) were captive-bred, with 50% (2/4) from Cachoeira do Sul and 50% (2/4) from Santa Maria.

In the phylogenetic analysis (Figure 1), eleven fragments of the gene lipL32 of Leptospira spp. were sequenced from nine different species of wild animals in Rio Grande do Sul State showed a grouping with sequences belonging to the pathogenic species L. interrogans (OR578518, OR578519, OR578521, OR578522, OR795078, OR795076, OR795077, OR795075) and L. borgptersenii (OR513921, OR513922, OR513923).

Fig. 1
figure 1

Phylogenetic analysis of gene lipL32 sequences from Leptospira interrogans and Leptospira borgptersenii obtained from wild animals in Rio Grande do Sul State, in South of Brazil. The analysis was carried out using the Bayesian method, with 500 bootstraps, in MEGA X software. L. interrogans and L. borgptersenii sequences from the samples analyzed are highlighted in bold

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Discussion

The presence of pathogenic Leptospira spp. DNA was predominantly detected in mammals of Carnivora order. Phylogenetic analysis revealed that species L. interrogans and L. borgptersenii are present in wild mammals in Rio Grande do Sul State, Brazil, a critical international transit region for both humans and animals moving between Brazil, Uruguay, and Argentina [30]. In Brazil, various studies have reported the presence of Leptospira spp. DNA in different biomes [3, 30].

Our study revealed the presence of pathogenic Leptospira spp. DNA in diverse wild animals that live in the southernmost state of Brazil, in areas of the Pampa biome, the Mata Atlantica biome and transition zones between these two biomes. This suggests the involvement of wild animals in the epidemiological chain of leptospirosis, highlighting a variety of wild hosts that can act as reservoirs for this pathogen [6]. Among studies employing molecular detection techniques for leptospirosis diagnosis, Carnivora order has been the most extensively studied [3, 11]. In our study, pathogenic Leptospira spp. DNA was detected in 54.55% (6/11) of carnivore samples (Table 1). This higher occurrence in carnivores could be attributed to their extensive terrestrial movements, including through flooded areas, primarily in search of food and preying on other potentially infected animal species [31].

In our study, the presence of L. interrogans (1/23) and L. borgpetersenii (1/23) DNA was detected in kidney tissue of white-eared opossum. These findings might be linked to the omnivorous diet of these animals [22], as well as their extensive habitat range, which includes forests, shrublands, grasslands, and both rural and urban areas [32,33,34], thereby increasing their exposure to Leptospira spp.

Rodents, particularly of the Rodentia order, have been extensively studied in various regions [29, 35,36,37]. However, in this study, L. borgpetersenii DNA was found in one kidney tissue sample from a capybara (1/11). This is, likely, because these large rodents inhabit flood-prone pastures [38], a significant environmental factor for Leptospira spp. transmission [39]. Thus, capybaras are considered important reservoirs for this pathogen, and given their proximity to farm animals and semi-urban areas, they represent a risk to both animal and public health [40].

Epidemiological studies in leptospirosis involving Lagomorpha and Primate orders are relatively scarce. Notably, this study detected L. borgpetersenii DNA in a captive European hare (L. europaeus) (1/3) and L. interrogans DNA in a captive common marmoset (C. jacchus) (1/2). For Artiodactyla order, no positive samples were found in this study, contrasting with findings from other regions. For example, in New Caledonia, deer species tested positive for L. interrogans and L. borgpetersenii DNA [41, 42]. Similarly, pampas deer (O. bezoarticus) from Brazil’s Pantanal biome showed a 3% positivity rate in blood PCR tests [43].

In this study, both L. interrogans and L. borgptersenii were detected. L. interrogans is considered the most widely distributed species globally and has been described in various hosts, including wild animals [30, 43, 44], synanthropic animals [45], domestic animals [46], humans [47, 48], and even environmental samples [49]. L. borgptersenii considered a bacterium that has already been found in rodents [50, 51] and in cattle [52], but is not expected its detection in different wild animal species. However, in this study it was possible to observe that this bacterial species is found circulating in species of wild mammals, such as white-eared opossums, capybara and neotropical river otters, probably due to the proximity of these animals to herds of cattle, as well as rodents possibly infected with Leptospira borgptersenii [51].

Due to the limited number of studies that address the epidemiological aspects of leptospirosis in different regions in Brazil, the importance of this investigation is owing to the detection of important pathogenic Leptospira species in wild animals from Rio Grande do Sul. Factors such as rainfall, water availability, and elevated temperatures significantly influence the survival of Leptospira spp. in the environment [53, 54]. Therefore, the high proportion of molecular detection of Leptospira spp. in free-living wild animals (7/11) from the cities of Santa Maria (5/7) and Palmeira das Missões (2/7) can be attributed to favorable ecological conditions. These include climatic elements that present four distinct seasons, with summer characterized by abundant solar radiation and higher temperatures and winter marked by lower average temperatures [55, 56]. The year and the resulting intense vegetation growth create favorable conditions for the survival of several species of wild animals and the maintenance of Leptospira spp. [56].

Beyond the ecological considerations, free-living animals present a health risk to other animals and humans in the evaluated areas. They also pose occupational risks to environmental police officers, veterinarians, biologists, and other professionals who may come into contact with these animals [57]. Likewise, captive animals constitute an occupational risk for those who work directly with them in settings such as breeding facilities and zoos [58]. In this study, pathogenic Leptospira spp. DNA was detected in samples from captive animals, including common marmosets, capybaras, margays, and European hares. This may be attributable to the stress and behavioral changes experienced by animals in captivity, leading to compromised health [59]. Moreover, these captive settings may be located in urban areas where synanthropic animals serve as important reservoirs for Leptospira spp. [60,61,62,63].

This study is the first to report the molecular detection of pathogenic Leptospira spp., including L. interrogans and L. borgpetersenii, in kidney tissue samples from free-living and captive wild animals in Rio Grande do Sul, Brazil.

Conclusion

This study demonstrates the presence of L. interrogans and L. borgpetersenii DNA in kidney tissue samples from free-living and captive wild animals, predominantly from Mammalia class, in Rio Grande do Sul, Brazil. Therefore, it can be inferred that these animals can act as reservoirs in the epidemiology of leptospirosis. Thus, this research also highlights the need for continuous epidemiological surveillance of leptospirosis in wild mammal populations to mitigate the risks of transmission of the etiological agent to humans and other species of domestic and wild animals. In addition, it is suggested that wild animals be included in the monitoring of the epidemiology of this important zoonotic disease with the aim of guiding leptospirosis control and prevention measures, especially in endemic regions.