Bats are a potential reservoir of pathogenic Leptospira species in Colombia

Introduction: Bats have become an epidemiologically significant source of pathogenic microorganisms, such as leptospires, the causative agents of leptospirosis. However, little information exists about bats and their potential role as a reservoir of pathogenic Leptospira spp. in Colombia. The aim of this study was to evaluate the presence of pathogenic Leptospira spp. in the kidneys of bats from the Caribbean region of Colombia deposited in the collection of mammals of the Museo Javeriano de Historia Natural (MPUJ-MAMM). Methodology: DNA was extracted from twenty-six kidney samples from a total of 13 species of bats captured in Colombia. First, 16S ribosomal RNA conventional PCR was performed to detect the presence of Leptospira spp. Then, in samples that tested positive, LipL32 PCR was performed to detect pathogenic Leptospira spp. by sequencing and phylogenetic analysis. Results: The presence of Leptospira spp. was observed in 7/26 (26.9%) bats from the following 6 species: Carollia perspicillata, Glossophaga soricina, Dermanura phaeotis, Uroderma bilobatum, Desmodus rotundus, and Lophostoma silvicolum, and pathogenic Leptospira spp. were detected in 4/26 samples (15.4%). Conclusions: This study suggests that bats present in the Caribbean region of Colombia could be potential reservoirs of pathogenic Leptospira spp.


Introduction
Leptospirosis, a bacterial zoonotic disease with a worldwide distribution, is caused by infection with pathogenic spirochetes of the genus Leptospira and affects humans and domestic and wild animals [1].This disease is a public health problem that affects 0.5 to 1 million people per year, with a mortality rate ranging from 5 to 10%.Leptospirosis is more prevalent in urban slum-dwellers and subsistence farmers in developing countries that are located in tropical and subtropical areas [2,3].Spirochetes reside in the kidneys of infected mammals and can cause infections that vary in severity from asymptomatic to acute infection in humans and animals.Leptospira spp.are mainly transmitted by contact with infected animal urine (direct) or urinecontaminated environments (mud or water; indirect) via mucosal membranes or open skin wounds [4].The disease may be underestimated because it is often misdiagnosed as other flu-like febrile illnesses [5].However, due to hematogenous spreading, the bacteria may cause other more severe complications, such as Weil's syndrome, meningoencephalitis or pulmonary hemorrhage [4].
Leptospira spp.are part of a diverse bacterial complex that includes pathogenic, intermediate, and saprophytic or nonpathogenic species.The bacteria can be classified into serovars, which are commonly defined as groups of Leptospira species with a specific serological variety; approximately 300 serovars have been identified, including 200 pathogenic serovars of epidemiological importance [1], and pathogenic and nonpathogenic serovars occur within several species [6].In Colombia, although few studies have evaluated the prevalence of Leptospira infection in animals and humans, it has been found that humans can acquire pathogenic serovars by contact with infected animals or contaminated water, suggesting that it is important to identify the major serovars of Leptospira spp.and their reservoirs to help prevent the spread of the disease by implementing public health intervention measures [7,8].
Different mammals have been identified in the transmission cycle of leptospirosis, such as wild, peridomestic and insectivorous rodents, as well as other wild and domestic animals.Rodents and dogs are often identified as potential sources of human infection, but other animals, such as cattle, birds, reptiles and bats, have also been suggested as reservoirs of pathogenic Leptospira spp.[9,10].Bats, which are mammals of the order Chiroptera, are involved in many biological functions within an ecosystem (seed dispersal, pollination activity and pest control).Due to some of their life history traits, such as high mobility, broad distribution and social behavior, the relevance of bats as reservoirs of zoonotic pathogens has been highlighted [11].Bats are one of the most diverse and abundant groups of mammal species in Neotropical ecosystems.These animals represent approximately 20% of the mammal species in the world, and approximately 205 species have been described in Colombia, corresponding to 36% of the bat species in the world [12].Bats are natural reservoirs for viral, protozoal, fungal, and bacterial human pathogens, such as the rabies virus, trypanosomes and leptospires [13].It was recently shown that tropical regions with a high richness of bat species are infected by pathogenic Leptospira spp.[14].For instance, a diverse group of leptospires, including pathogenic (L.interrogans, L. kirschneri, and L. borgpetersenii) and intermediate (L.fainei) species, was detected in the kidneys of bats from the Peruvian Amazon, suggesting that bats are a reservoir of Leptospira species [15].However, little information is available about bats and their potential role as a reservoir of pathogenic Leptospira spp. in Colombia.
Herein, we evaluated the presence of Leptospira spp. in the kidneys of bats from the Caribbean region in Colombia deposited in the collection of mammals of the Museo Javeriano de Historia Natural (MPUJ-MAMM).

Bat samples
The bat samples (kidneys of bats) were obtained from the collection of mammals at the MPUJ-MAMM.The requirements to use samples from the collection were completed according to the MPUJ-MAMM.The bats were captured in tropical dry forest areas of the Caribbean region of Colombia as described previously [16].Kidney samples from each bat were stored in 70% ethanol at 4 ºC prior to DNA extraction.The Research and Ethics Committees of the Pontificia Universidad Javeriana approved this study.

Detection of Leptospira spp.
Genomic DNA was extracted from bat kidney samples using a High Pure PCR Template Preparation kit according to the manufacturer's instructions (Roche, Mannheim, Germany).Afterwards, to evaluate the DNA integrity and to rule out the presence of inhibitors in the sample, conventional PCR was performed with the CytB Uni forward (5'-TCATCMTGATGAAAYTTYGG-3') and CytB Uni reverse (5'-ACTGGYTGDCCBCCRATTCA-3') primers, which amplify cytochrome B from small mammals, according to previously reported procedures [17].Then, to detect DNA from the genus Leptospira in samples that tested positive for CytB by PCR, PCR was performed with the Lep 1 (5'-GGCGGCGCGTCTTAAACATG-3') and Lep 2 (5'-TTCCCCCCATTGAGCAAGATT-3') primers, which amplify a 331 bp fragment of the 16S ribosomal RNA (rRNA) gene from L. interrogans serovar Canicola, using previously described PCR conditions [18].For both types of PCR, the following controls were included: reaction (water added in the room containing the reaction mixture), gray (water added in the room where the sample was added to the reaction) and positive (genomic DNA from Leptospira spp.).The reaction products were resolved using 1% agarose gel electrophoresis followed by staining with the SYBR™ Safe DNA Gel Stain (Invitrogen, Waltham, MA, USA).

Detection of pathogenic leptospires
To detect DNA from pathogenic Leptospira spp., PCR was performed with the LipL32-270F (5'-CGCTGAAATGGGAGTTCGTATGATT-3') and LipL32-692R5-CCAACAGATGCAACGAAAGATCCTTT-3') primers, which amplify the gene for the major outermembrane lipoprotein LipL32, which is an important virulence factor present in all pathogenic strains of Leptospira spp.[19,20].As mentioned above, the reaction, gray and positive controls were included, and each conventional PCR was repeated at least twice for reproducibility.PCR products were visualized using 1% agarose gel electrophoresis, followed by staining with the SYBR Safe DNA Gel Stain (Invitrogen).

Phylogenetic analysis
Samples that tested positive in 16S rRNA and LipL32 conventional PCR were further identified by sequencing analyses.The amplicons were purified using a Wizard® DNA Clean-Up System kit (Promega, Madison, WI, USA) and then sequenced in both directions using a 3500 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA).The forward and reverse sequences obtained in the present study were assembled, edited and compared among themselves and with reference sequences available in GenBank after alignment using the Clustal algorithm.A phylogenetic analysis was performed using the maximum likelihood (ML) method based on the Kimura 2-parameter model [21], and 1000 bootstrap replicates were performed using the complete deletion option and the Close-Neighbor-Interchange algorithm of the MEGA software, Version 6 [22].

Detection of Leptospira spp.
A total of 26 bat kidney samples from 11 genera and 12 species were analyzed.The analyzed samples of each species and their corresponding feeding habits are summarized in Table 1.Leptospira spp.were observed in 7 of 26 (26.9%) bats from the following 6 species: C. perspicillata, G. soricina, D. phaeotis, U. bilobatum, D. rotundus, and L. silvicolum.Interestingly, bats with Leptospira spp.had different feeding habits, which included frugivorous, insectivorous, nectarivorous, and hematophagous habits (Table 1).
To analyze the phylogenetic classification of the Leptospira spp.into pathogenic, intermediate, and nonpathogenic groups, a phylogenetic analysis of 16S rRNA genes was performed.Alignment of the Leptospira sequences from bats captured in this region  The tree with the highest log likelihood is shown and is drawn to scale, with branch lengths representing the number of substitutions per site.The sequences retrieved in this study are indicated by black triangles followed by the species name of the bat, and the GenBank numbers from the reference sequences are indicated in brackets.The Leptospira spp.and their groups are listed to the right of each branch.
of Colombia showed an overall identity of 92.0 -96.7% among the sequences.In addition, these sequences had identity values of 95.5%, 88.6%, and 72.4% with the pathogenic, intermediate, and nonpathogenic leptospire sequences reported in GenBank, respectively.ML analysis of the 16S rRNA genes generated a tree that showed a clear clustering of the Leptospira sequences into three distinct branches, as previously described [6]: (1) pathogenic species, (2) intermediate species and (3) nonpathogenic species.In particular, the bat Leptospira sequences clustered with the pathogenic species (Figure 1).

Detection of pathogenic leptospires
Amplification of the LipL32 gene was observed in 4 of 26 kidney samples (15.4%) (Table 1).The sequences isolated from D. rotundus and G. soricina were identical (GenBank accession number MF281056), and they showed identity values of 94.6% and 90.7% with the sequences isolated from U. bilobatum (GenBank accession number MF281054) and C. perspicillata (GenBank accession number MF281055), respectively.The identity between the sequences isolated from U. bilobatum and C. perspicillata was 93.6%.As shown in Figure 2, these sequences were close to those of some Leptospira spp. that are known zoonotic pathogens.The LipL32 sequence obtained from C. perspicillata (MF281055) was close to that obtained from the L. borgpetersenii clade, and it had an identity of 95.9% with the reference sequences.The sequence isolated from G. soricina and D. rotundus (MF281056) and the sequence from U. bilobatum (MF281054) were closest to the sequence of the L. interrogans clade; these sequences had genetic identity values of 94.9% and 99.6% with the L. interrogans reference sequences, respectively.In addition, the Leptospira sequence obtained in the present study from C. perspicillata was related to the variants KX420711 and KX420712 (from Myotis blythii and Miniopterus schreibersii, respectively), detected in bats from Georgia (USA), and was similar to that of L. borgpetersenii [23].Thus, these results suggest that the sequences obtained from LipL32 gene amplification in kidney samples from bats corresponded to the pathogenic group of Leptospira.

Discussion
Leptospirosis is a bacterial zoonotic disease caused by infection with spirochetes known as Leptospira spp.In humans, the disease has been associated with socioeconomically disadvantaged populations and it has been reported as an infection associated with adventure traveling [3,24].Leptospira spp.have been detected in domestic and wild animals, such as rodents, canines, cattle, pigs, sheep, sea lions, marsupials, and bats [4,10].Recently, several studies have highlighted the role of bats as potential reservoirs of different pathogenic microorganisms, including Leptospira spp.[13].A wider variety of bats has been described in Colombia than in the rest of Latin America [25].However, to our knowledge, there are currently no reports of the presence of Leptospira spp. in this group of small mammals in Colombia.Thus, to evaluate the presence of these bacteria in bats in Colombia, we detected pathogenic Leptospira spp. in the kidneys of different species of bats from the Caribbean region that were deposited in the MPUJ-MAMM.
In this study, Leptospira spp.were detected in 7 of 26 (26.92%) bats, including 6 species with different feeding habits.This spirochete has been detected in bats from Latin American countries, such as Brazil and Peru, with a prevalence of 2% and 3.4%, respectively [15,18].However, a study in the United States did not detect the presence of Leptospira spp. in bats [26], which suggests that the prevalence of these bacteria in bat populations may vary according to the region of study.Interestingly, in the present work, the presence of these bacteria was detected in D. phaeotis and L. silvicolum, in which the presence of Leptospira spp.had not been detected previously in bats from America [14,27].Moreover, it is estimated that the presence of Leptospira spp.has been detected in approximately 50 species of bats with different feeding habits [14,28].In this study, Leptospira spp.were detected in different species of bats independent of their feeding habits.Recently, it was shown that bats from Southern Brazil exhibit no associations among the frequency of positive results for pathogenic Leptospira and the age, sex, species, season of collection, location, or feeding habits of the bats [28]; this observation is similar to the results obtained in the present study.However, these results may be related to the number of bats analyzed in the sample used in the present work.
In Colombia, different studies have been carried out at the local level to determine the prevalence of leptospirosis in humans in different tropical areas.According to the National Health Institute of Colombia, in 2005, the prevalence of leptospirosis was 13.1% in a group of butchers and rice workers in the Caribbean region in Colombia [29].However, a recent study showed that in this department, the circulation and transmission of different serovars of L. interrogans occur between animals (pigs and dogs), humans and the environment, and it has been described that, in this same region, the prevalence of leptospirosis in humans was 67.9% in 2013, with a significant increase in human leptospirosis cases compared to the results reported in 2005 [7,8].In this study, the presence of Leptospira spp. was detected in bats from this same region, which suggests that bats could be involved in the transmission cycle of the disease and that it is important to study the role of bats in the epidemiology of leptospirosis in this region.This work is the first to detect pathogenic species of Leptospira spp. in populations of bats from the Caribbean region of Colombia, and we highlight the importance of evaluating the species of bats that are dominant at the local level [30] since, due to their abundance, they may have a greater probability of contacting humans, persisting in transformed environments in the Caribbean region and occupying the homes of people [31].
To date, approximately 22 species of Leptospira have been described and are classified according to their pathogenicity, as follows: pathogenic, intermediate (with low pathogenicity), and saprophytic or nonpathogenic species (free-living bacteria that are found in the microenvironment and are generally considered not to infect animals) [1].Although it has been commonly described that the pathogenic Leptospira spp.have similar degrees of pathogenicity, a recent phylogenomic analysis identified 4 subgroups of pathogenic leptospires, which could explain the degree of virulence of each species [32].Interestingly, in the present study, analysis of 16S rRNA conventional PCR amplicons by sequencing and phylogeny showed that these leptospires detected in bats from the tropical dry forests of the Caribbean region of Colombia are grouped in the pathogenic group of Leptospira spp.In addition, using the same methodological strategy mentioned above, the sequences identified by LipL32 conventional PCR amplification showed a high identity with those of the most pathogenic Leptospira spp., as described previously [20].
Thus, the findings of the present work suggest that bats present in tropical dry forests of the Caribbean region of Colombia could be potential reservoirs of pathogenic Leptospira spp.

Conclusions
Bats from the Caribbean region of Colombia harbor pathogenic Leptospira spp.These preliminary data show that bats could play an important role in maintaining these bacteria in nature.These data also reinforce the need for increased efforts to understand the roles of wild animals in the maintenance, spread, and transmission of zoonotic microorganisms and, therefore, to propose measures for their prevention and control.However, it remains necessary to also consider the irreplaceable ecological (seed dispersal and pollination), economic (insect pest control) and public health (control of insects that transmit diseases to people) benefits that bats offer.

Figure 1 .
Figure 1.Phylogenetic analysis via maximum likelihood (ML) analyses of the 16S rRNA genes of Leptospira spp.

Figure 2 .
Figure 2. Phylogenetic analysis via ML analyses of the lipL32 genes of Leptospira spp.

Table 1 .
Bat species from the Caribbean region in Colombia, their ecological data and Leptospira spp.Detection.
†Corresponds to the site where the bats were captured; § 16S rRNA and LipL32 PCR was performed as described in the Materials and Methods; ¶ The frequency was determined from the results obtained by 16S rRNA PCR; ND, not detectable by PCR.