Genetic Evidence for a Potentially New Pathogenic <i>Leptospira</i> sp. Circulating in Bats from Brazilian Amazon

Di Azevedo, Maria Isabel Nogueira; Verde, Rair de Souza; Ezepha, Camila; Carvalho-Costa, Filipe Anibal; D’Andrea, Paulo Sérgio; Medeiros, Luciana dos Santos; Lilenbaum, Walter

doi:https://doi.org/10.1155/2023/9677047

Transboundary and Emerging Diseases

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Abstract Introduction Methods Results Discussion Data Availability Conflicts of Interest Acknowledgments Supplementary Materials References Copyright Related Articles

Research Article | Open Access

Volume 2023 | Article ID 9677047 | https://doi.org/10.1155/2023/9677047

Genetic Evidence for a Potentially New Pathogenic Leptospira sp. Circulating in Bats from Brazilian Amazon

Maria Isabel Nogueira Di Azevedo,¹Rair de Souza Verde,²Camila Ezepha,¹Filipe Anibal Carvalho-Costa,³Paulo Sérgio D’Andrea,⁴Luciana dos Santos Medeiros,⁵and Walter Lilenbaum¹

Academic Editor: Zongfu Wu

Received04 May 2023

Revised19 Aug 2023

Accepted26 Aug 2023

Published19 Sept 2023

Abstract

Leptospirosis is a neglected zoonosis frequently reported worldwide, caused by a spirochete of the genus Leptospira. It is capable of infecting domestic animals, free-living animals, and humans. Among wild animals, the role of bats in the epidemiology of leptospirosis has been investigated but is far from being elucidated. The Amazonian biome has the ideal conditions for maintaining and disseminating leptospires and, despite efforts that have been made to better understand leptospires’ occurrence in wild animals in the region, few studies aimed to explore and genetically characterize leptospires in bats. Based on this, the aim of the present study is to deeper investigate and genetically characterize leptospires detected in bats from the southwest Amazon. Animals were trapped by mist netting at five sites in the state of Acre, Brazil. Kidney samples were obtained and stored for molecular analysis. Polymerase chain reaction (PCR) was conducted first based on the LipL32 gene, and positive samples were submitted to rrs and secY-PCR and sequencing. Sequences were then submitted to phylogenetic analyses through multiple bioinformatic tools. The rrs sequences from the present study formed one single haplotype, different from any other previously deposited, grouped in a highly supported cluster with sequences from bats from Madagascar and China. The initial secY screening revealed no identity with previously deposited sequences. The phylogenetic trees revealed the sequences from the present study in an isolated branch, clearly separated from all previously known pathogenic Leptospira spp., suggesting the existence of a potentially undescribed species. The haplotype network including only leptospires from the Amazon region confirmed two new haplotypes from the same taxon unity, isolated from the others, with a probable origin of the species from L. noguchii. The characterization of this potentially new species in bats reinforces the complexity of the transmission dynamics of leptospires, including wild, periurban, and urban environments, emphasizing the need for an integrative look at leptospirosis vigilance within the context of One Health.

1. Introduction

Leptospirosis is a neglected zoonosis frequently reported worldwide, caused by a spirochete of the genus Leptospira sp. [1]. Several species are recognized as pathogenic agents, including over 250 serovars [2]. It is endemic in tropical and subtropical regions of the world and capable of infecting domestic animals, free-living animals, and humans [3]. The bacterium is transmitted through water, soil, or mud contaminated by the urine of infected animals and circulates in urban, rural, and wild environments, with a wide variety of animal species acting as carriers/hosts [4].

Among wild mammals, although rodents are the most important and studied reservoirs [5], bats have been proven to carry Leptospira sp. [6], and their role in the epidemiology of leptospirosis has been increasingly investigated over the last two decades [7]. These flying mammals are distributed worldwide and present essential and complex ecological roles, with different feeding habits as, insectivorous, nectarivores, frugivorous, carnivorous, and hematophagous [8].

The Amazonian biome has the ideal conditions for maintaining and disseminating leptospires, such as a warm and humid climate, the presence of great mammal biodiversity, and a variety of potential host species [9]. Many efforts have been made to better explore leptospires’ occurrence in wild animals in the region [10–14]. Regarding bats, few studies aimed to explore and genetically characterize leptospires in this region, although a high prevalence has been reported [15, 16].

Recently, our group performed a field study involving the investigation of pathogens in bats from the Brazilian Amazon biome (data not published). Among the many results found, one was particularly intriguing, regarding the possible identification of a new species of Leptospira (Leptospira n. sp.). Based on this, the aim of the present study is to deeper investigate and genetically characterize these leptospires detected in bats from the southwest Amazon.

2. Material and Methods

This study is part of a long-term ecological and parasitological wild mammal project in the Amazon Biome, and has captured 129 bats specimens. All capture, handling, and euthanasia procedures were approved by the Ethics Committee on Animal Use of the Federal University of Acre (CEUA/UFAC under no 28/2019) and under the capture license granted by the competent environmental agency (SISBio under no 71451). The animals were euthanized for other parasitological and taxonomic studies and not only for the purposes of the present work.

2.1. Study Area

This study was conducted during the spring of 2019 and 2021 in four Amazon Forest conservation units, in the state of Acre, Brazil: (1) Chandless State Park (9° 91′ 84″S/70° 15′ 96″W), at municipality of Manoel Urbano, that is characterized as a continuous forest with an area of 695.303 hectares, surrounded by a mosaic of preserved areas; (2) Zoobotanical Park (9° 95′ 70″S/67° 87′ 45″W) at UFAC, of 150 hectares; (3) Chico Mendes Park (10°03′ 71″S/67° 79′ 68″W), of 50 hectares; and (4) the Roberval Cardoso Forest School (10° 05′ 05″S/67°59′15.14″W), and Piracema Forest, of 153 hectares (10° 01′ 42″S/67°55′23.80″W) being the last two at the municipality of Rio Branco, and both characterized as very altered urban forest fragments (Figure 1). The east of the Acre state is dominated by induced pastures for cattle production, where forest cover (primary forest and secondary vegetation) is limited to numerous small patches. In contrast, Chandless State Park, which served as the control area for this study, is characterized by dense shoulder length forests dominated by palm trees.

2.2. Capture, Taxonomic Identification and Bat Sample Collection

For the sampling, eight mist nets (12 × 3 m, mesh 19 mm, Ecotone®) were installed for two consecutive nights in two plots in each area at ground level. The captures began at sunset and ended six hours after the installation of the nets, with inspections every 20 min. The captured bats were placed in containment cotton bags for weighing, body measuring, and preliminary taxonomic identification at the genus level. Bats were identified in the field according to the following keys: Gardner [17], Díaz et al. [18], and López-Baucells [19].

The animals were taken to a field laboratory for euthanasia (9 : 1, 10% ketamine hydrochloride and 2% acepromazine) and biological sample collection. The specimens were identified at the species level by external morphological characters and confirmed by molecular analysis (CytB gene) and DNA sequence comparisons, and further deposited as voucher specimens in the Laboratory of Biology and Parasitology of Reservoirs of Wild Mammals Collection (Oswaldo Cruz Foundation/Rio de Janeiro, Brazil).

2.3. DNA Extraction and Molecular Detection of Pathogenic Leptospira

All molecular and genetic analyses were based on the previous protocols of our group, as described in [20]. Kidney samples were obtained during the necropsy and stored in sterile 2.0 mL microtubes at −20°C, which were destined for molecular analysis. DNA extraction (kidney samples) was performed using the DNeasy® Blood & Tissue Kit (Qiagen, California, USA), according to the manufacturer’s instructions. Specific primers of the LipL32 gene, reported to be present in pathogenic leptospires were used for the reactions [21] (Supplementary 1), performed as previously described by Hamond et al. [22]. For each test, ultrapure water was used as a negative control in all reactions, while 10 fg of DNA extracted from Leptospira interrogans serovar Copenhageni (Fiocruz L1-130) was used as a positive control. The polymerase chain reaction (PCR) products were analyzed by electrophoresis in 1.5%–2% agarose gel after gel red staining and then visualized under ultraviolet (UV) light.

2.4. DNA Sequencing and Phylogenetic Analysis

Samples were submitted to a nested PCR targeting the 16 S rRNA gene (rrs) [23] and secY gene [24]. Detailed information about primers used for Leptospira sp. identification in the present study is shown in Supplementary 1. Amplicons were purified with the Wizard® SV Gel Kit and PCR Clean-Up System (Promega, USA), according to the manufacturer’s instructions and intended for sequencing. Sequencing reactions were performed using the Big Dye Terminator v. 3.1 Cycle Sequencing Kit (Applied Biosystems, USA) on a 3100 automatic DNA sequencer according to the manufacturer’s instructions. Regarding sequencing analysis, Pairwise/Blast/NCBI software, SeqMan v. 7.0, ClustalW v. 1.35, and BioEdit v. 7.0.1 were used to edit and analyze the sequences.

Reference sequences of pathogenic Leptospira sp. from bats identified worldwide and from different hosts from the Amazon region were obtained from GenBank for rrs and secY analyses, respectively. Unpublished and too short sequences (<400 bp) were excluded to avoid biases in the analyzes. Neighbor-joining (NJ) and maximum-likelihood (ML) trees were constructed using the Tamura–Nei model (TN92) in MEGA X software [25], as it was determined to be the best-fitting model of DNA substitution using the Bayesian information criterion. Genetic distances were calculated using the TN92 model on MEGA X. Nucleotide sequences were translated to amino acid sequences on BioEdit v. 7.0.1 software.

A haplotype network based on secY sequences from Leptospira spp. from the Amazon region was constructed through the population genetics software PopART [26], using the media-joining inference method [27], in order to better visualize the number of single nucleotide polymorphisms (SNPs) and to evaluate haplotypes distribution and evolutionary origins.

3. Results

3.1. Detection of Pathogenic Leptospira sp. in Bats

Pathogenic Leptospira sp. was detected through positive LipL32-PCR in bats’ kidneys from the species Carollia perspicillata (n = 1), Myotis riparius (n = 1), Choeroniscus minor (n = 1), Artibeus planirostris (n = 2), Uroderma bilobatum (n = 1), Gardnerycteris crenulatum (n = 1), and Desmodus rotundus (n = 1). Details about positive hosts are summarized in Table 1.

3.2. Phylogenetic Analysis

LipL32-PCR positive samples were submitted to rrs and secY-nested PCR. Amplicons with the expected size were produced (600 and 409 bp, respectively), purified, and submitted to DNA sequencing. In all samples, it was possible to obtain high-quality secY gene sequences, while for the rrs gene, it was possible to obtain only three sequences. Sequences were deposited on GenBank under accession numbers OQ793707 and OQ793714. Initially, Pairwise/Blast/NCBI comparisons of the rrs sequences from the present study with the GenBank dataset revealed 98% of identity with L. noguchii, L. kirshnerii, and L. interrogans, while no high identity (>95%) was observed with any previously deposited sequences based on secY sequences.

The phylogenetic tree based on bat Leptospira sp. rrs sequences from different geographical origins revealed two main clades, the first including L. alexanderi, L. weilli, L. mayottensis, L. borgpetersenii, and L. santarosai and the second including L. noguchii, L. kirshnerii and L. interrogans (Figure 2). Sequences from the present study formed a single haplotype, different from any other previously deposited (red circle with glow in Figure 2), grouped in a high supported cluster with sequences from bats from Madagascar and China identified only as Leptospira sp. (gray circle in Figure 2). Details about Leptospira sp. rrs sequences from bats used in this analysis are shown in Supplementary 2.

Figure 2

Neighbor-joining (NJ) phylogenetic tree inferred from rrs gene sequences (n = 249, 440 bp), including pathogenic Leptospira sp. previously identified in bats from different geographical localizations in the world (countries are represented by colors, as shown in the legend). The circles represent haplotypes and the numbers inside represent the “n” of sequences. The smaller circles without numbers indicate haplotypes with only one sequence. Sequences from the present study are represented by a red circle with a glow. The gray circle indicates the clade formed by sequences from the present study and those previously deposited on GenBank. Numbers at nodes are bootstrap values greater than 50%. Leptospira biflexa is the outgroup taxa. Detailed information about sequences included in the analysis is shown in Supplementary 2.

Regarding secY genetic distances, sequences from the present study presented the lowest distance with L. noguchii (TN92 = 0.12 ± 0.02), reflecting only 88% of identity. For the remaining pathogenic species, genetic distances vary from 0.13 to 0.25 (87%–75% of identity) (Table 2). Concerning only the sequences from the present study, intraspecific genetic distance was 0.01, reflecting a high identity between them (99%) (Table 2). The intraspecific genetic distance of L. noguchii, the genetically closest species, increased from 0.02 to 0.05 when sequences from the present study were included, a value higher than all intraspecific distances of all species (see values in italics in Table 2).

The phylogenetic trees revealed the sequences from the present study in an isolated, highly supported branch (NJ = 99%, ML = 99%), basal to L. noguchii species cluster, clearly separated from all previously known pathogenic Leptospira sp., suggesting a potential existence of an undescribed species (Figure 3). Moreover, it is possible to observe the cluster from the present study included in a major clade that includes, beyond L. noguchii, L. interrogans, and L. kirshnerii (Figure 3). The secY amino acid alignment using L. interrogans strain Fiocruz L1-130 (AE016823) sequence as a reference and including all L. noguchii sequences, showed three genetic signatures, that is, polymorphisms shared only by strains from Leptospira n. sp.: T3M, I4V, and V47I. Additionally, the haplotype including the strains R21960, R22023, and R21980 showed an additional signature: L96F. Importantly, the amino acid composition was distinct between L. noguchii and Leptospira n. sp. from the present study (Supplementary 3).

The haplotype network including only Leptospira sp. from the Amazon region confirmed two new haplotypes including sequences from bats of the present study, isolated from the others, with a probable origin from L. noguchii (Figure 4). It is important to note that the number of SNPs separating the present Leptospira n. sp. from L. noguchii (n = 31) is much bigger than any intraspecific SNP and in accordance with SNPs between species (Figure 4). Moreover, the number of SNPs between the two haplotypes from the present Leptospira n. sp. (n = 11) is in agreement with SNPs observed intraspecifically in other species, like L. santarosai and L. borgpetersenii (Figure 4). Importantly, haplotypes from the present study are clearly separated from others belonging to an undescribed Leptospira species isolated from bovine in the same region (Figure 4).

4. Discussion

The present study reinforces the role of bats as reservoirs and dispersers in the dynamics of the transmission cycle of Leptospira sp., and sheds light on the knowledge about a still underexplored genetic diversity of leptospires in the Amazon region. We provided, for the first time, genetic sequences from the secY gene of a potential new pathogenic Leptospira n. sp. infecting bats in the region, and, importantly, include new bat hosts species, Choeroniscus minor (lesser long-tailed bat) and Gardnerycteris crenulatum (striped hairy-nosed bat), for this bacterium. Regarding the remaining hosts identified in the present study, leptospires were previously identified in Carollia perspicillata from Colombia, in a cave in the Andes Mountains [28] and from a Caribbean region [29]; in Artibeus planirostris and Myotis riparius from Peruvian Amazon [16]; in Artibeus planirostris from Uraba region, Colombia [30]; in Uroderma bilobatum from Caribbean Colombia [29] and Peruvian Amazon [16]; and in Desmodus rotundus from Caribbean Colombia [29].

We herein provided genetic evidence based on rrs and secY gene sequences, using different phylogenetic methods and models, for the existence of a new pathogenic species. Although the 16S rRNA gene, the first genetic marker applied for Leptospira sp. identification, has been used for a long time as a target for many diagnostic PCR assays [31], it presents a low-taxonomic resolution to differentiate between Leptospira species within a clade. This was confirmed in the present study, where no species-specific clusters were observed (Figure 2). Anyway, it was possible to clearly observe the distribution of Leptospira sp. haplotypes from bats according to geographic location, bringing important epidemiological inferences (Figure 2). Our sequences from the Amazon region identified in Uroderma bilobatum, Gardnerycteris crenulatum, and Desmodus rotundus clustered together with sequences from Triaenops menamena from Madagascar [32] and from Chinese Myotis spp. [33]. Further analyses based on markers with better taxonomic resolution are needed to know whether these species in fact constitute a single genetic entity or are distinct. Unfortunately, reference leptospires from bats included in the present study identified by the rrs gene do not have associated secY gene information.

Currently, the secY gene has been prioritized as a genetic marker for Leptospira sp. identification, since it presents a good discriminatory power, and sequence analysis of the gene allows the identification of species, strains, and occasionally genotypes [34]. Interspecific secY genetic distances between sequences from the present study and the remaining pathogenic species included are equal to or greater than the others previously known (Table 2). Moreover, when including the present sequences within the L. noguchii group, the closest species, the intraspecific distance of the clade increases by 150%, reaching a value greater than any Leptospira intraspecies distance. Phylogenetic trees and haplotype network corroborated and illustrated clearly the presence of a distinct, highly supported clade, close but separated from L. noguchii (Figures 3 and 4). Together, the results suggest genetic evidence for a potentially new species of Leptospira circulating in bats from the Brazilian Amazon.

Undoubtedly, a formal naming of the new species involves an integrative approach, including culture and isolation, morphology, serology, and genetic characterization based on multilocus sequence typing (MLST) and whole-genome sequencing (WGS). Unfortunately, leptospires are fastidious and slow-growing organisms, and cultures must be kept and checked for up to 14 weeks [35]. Moreover, we worked in closed forest areas of the Amazon rainforest, and it is not possible to culture the material collected on-site in a sterile environment to optimize culturing. The kidneys were sent frozen to the laboratory, which, added to the fastidious growth of the bacteria, made it unfeasible to cultivate and obtain isolates. Therefore, a complete characterization became a challenge, but efforts will be directed to obtain this formal description.

A high genetic diversity of Leptospira sp. circulating in the Amazon region has already been demonstrated based on secY gene, and the species L. interrogans, L. noguchii, L. kirshnerii. L. santarosai, and L. borgpetersenii have been identified in bovine, pigs, rodent, and marsupials [13, 36–39]. Moreover, similarly to the results from the present study, an independent clade, close but separated from L. kirshnerii was identified in cattle from the region after phylogenetic analyses based on secY gene [36].

Leptospirosis is an infectious disease that requires studies on human–animal–environment interface, based on the One Health approach [40]. Evidence for another pathogenic leptospiral species circulating in periurban areas deserves attention, since Brazilian Amazon has been going through intense processes of deforestation, and this generates environmental changes and ecological disturbances that can facilitate human contact with wild species of vectors and reservoirs, favoring the incidence of zoonotic diseases [9]. This becomes even more worrying when the host/carrier is a flying mammal, which can reach long distances, including urban areas, and reinforces the importance of bats as important reservoir hosts and disseminators of multiple pathogenic Leptospira sp. [30].

Through a robust genetic analysis, we present evidence for the occurrence of a potentially new species of pathogenic Leptospira circulating in the Amazonian biome. Our findings support the high diversity of this genus and contribute to new Leptospira taxonomic data about the bat reservoir role that deserves and should be further explored. The characterization of this Leptospira n. sp. in bats reinforces the complexity of the transmission dynamics of leptospires, including wild, periurban, and urban environments, emphasizing the need for an integrative look at leptospirosis vigilance within the context of One Health.

Data Availability

The data that support the findings of this study are openly available in GenBank (https://www.ncbi.nlm.nih.gov/genbank/), under acession numbers OQ793707 and OQ793714.

Conflicts of Interest

The authors declare that they have no conflicts of interest.

Acknowledgments

We would like to thank the Program for Technological Development in Tools for Health- PDTIS/FIOCRUZ for the use of its facilities. We are grateful to the field team of Dr. LM (UFAC/UFF) and Dr. PSD’A (FIOCRUZ), in particular for their help in the taxonomic identification of bats. MINDA is a FAPERJ fellow. W. L. and P. S. D. are CNPq and FAPERJ fellows. R. S. V. is a CAPES fellow. Field and lab work was supported by grants from Institutional Agreement IOC-Fiocruz/UFAC and from CNPq to Dr. LM (CNPq 428213/2016-2) and to Dr. PSD’A (CNPq 439208/2018-1).

Supplementary Materials

Supplementary 1. Detailed information about primers used for Leptospira sp. identification in the present study.

Supplementary 2. Detailed information of Leptospira sp. GenBank sequences from bats included in the present study. Rows in green indicate sequences that clustered together with sequences from the present study. Accession numbers in bold are reference sequences from other hosts.

Supplementary 3. Amino acid alignment of secY gene from Leptospira n. sp. from this study and GenBank sequences from L. noguchii. Points indicate identical position with L. interrogans strain Fiocruz L1-130 reference sequence. Genetic signatures of Leptospira n. sp. are in gray.

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Copyright © 2023 Maria Isabel Nogueira Di Azevedo et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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