1. Introduction
Leptospirosis is an emerging zoonotic disease caused by pathogenic bacteria of the genus Leptospira [1]. Previous studies have estimated that 1.03 million cases and 58,900 deaths occur due to leptospirosis worldwide annually [2]. Leptospirosis is considered a neglected disease, found mainly in the tropical regions of developing countries [3] and is now recognized as an emerging infectious disease due to large outbreaks in different regions of the world, which are associated with environmental disasters, and extreme climate change. In many endemic regions, severe forms of the disease, such as Weil’s disease and pulmonary hemorrhage syndrome have emerged as the leading cause of death [4]. Currently, about 65 genomic Leptospira species have been identified (NCBI database: https://www.ncbi.nlm.nih.gov/genome [accessed on 30 April 2021]), which are subdivided into four main clades according to the phylogenetic analysis of 1371 conserved genes: pathogens (P1), pathogens (P2), saprophytes (S1), and saprophytes (S2) [4][5]. Through serological classification about 300 Leptospira serovars have been described, which are grouped into approximately 30 serogroups and about 200 of these serovars have been considered pathogenic [6]. Colombia is an endemic country for leptospirosis with at least 500 cases every year [7]. Antioquia is the second department in Colombia with the highest number of confirmed cases of leptospirosis [7], with a seroprevalence close to 12.5% [8]. Leptospira interrogans and L. santarosai have been identified as the causative agents of this disease [9]. Therefore, this department in an important region in Colombia for the study of leptospirosis.
Rodents and dogs are often identified as potential sources of human infection, but other mammals have also been identified in the transmission cycle of leptospirosis
[1]. Globally, various studies have explored the biological role of bats as reservoirs of zoonotic pathogens due to their ability to fly long distances and disperse pathogens (viruses
[10], bacteria
[11], parasites
[12] and fungi
[13]) through urine, saliva, and feces. Bats are the only true flying mammals, belonging to the order Chiroptera
[14]. This order includes over 1400 different species in 21 families
[15], which are scattered throughout the world, except Antarctica
[16]. These mammals are oriented and hunt by echolocation
[17]. Depending on the species they can feed on arthropods, fruits, pollen, fish, blood, and other vertebrates (carnivores)
[10]. Some species can hibernate
[18], form large colonies
[19], migrate long distances
[20], and have long lifespans (approximately 35 years)
[21].
Bats have been identified worldwide as an important reservoir of different Leptospira species (L. interrogans, L. borgpetersenii, L. kirschneri, L. fainei) and their role in disease transmission, and spillover in the life cycle of this bacterium has yet to be defined [22]. Currently, more than 50 species of infected bats with Leptospira have been reported in different countries, including Peru [23], Brazil [24], Argentina [25], Australia [26], Comoros island and Madagascar [27], Reunion Island [28], Mayotte Island [22], Indonesia [23], Malaysia [24], Tanzania [25], Trinidad [26], Sudan [27], Democratic Republic of Congo [28], Africa [29], and Azerbaijan [30]. In Colombia, two studies have reported the presence of bats naturally infected with Leptospira [31][32]. Due to the above characteristics, bats could act as excellent spillover of Leptospira species to the environment, favoring contamination of water and soil, serving as a direct or indirect source of infection for other animals, which are the main reservoirs and disseminators of the bacteria. The objective of the present investigation was to detect Leptospira species infecting different bat species in the Urabá region (Antioquia-Colombia) and to evaluate the genetic diversity of the circulating Leptospira species. This information will illustrate the role of bats in the transmission cycle of human leptospirosis.
2. Places of Bat’s Capture
The investigation was carried out in the Urabá region (Antioquia-Colombia). The sampling was undertaken in five different municipalities (Chigorodó—43 captured bats), (Carepa—43 captured bats), (Apartadó—39 captured bats), (Turbo—40 captured bats), (Necoclí—41 captured bats). In total, 206 bats were captured. The map of the Urabá region and the exact location of the three sampling sites are shown in Figure 1.
Figure 1. The geographical location of capture sites. The map shows the geographical location of the five municipalities that were used to capture the 206 bats (blue-Necoclí, purple-Turbo, orange-Apartadó, dark green-Carepa and light green-Chigorodó). These capture sites are located in the Urabá region (Antioquia-Colombia). The map was generated using the environment and programming language R and packages (ggplot2, MappingGIS, sfMaps, spData, ggrepel, ggspatial, cowplot).
3. Families, Genera and Species of Captured Bats
Researchers captured 206 bats in the five municipalities of the Urabá region (Antioquia, Colombia). These bats were classified into three different families (Phyllostomidae, Molossidae, and Vespertilionidae), 10 different genera (Artibeus, Carollia, Dermanura, Glossophaga, Sturnira, Molossus, Myotis, Uroderma, Rhogeessa, Phyllostomus), and fifteen different species (Artibeus jamaicensis, A. lituratus, A. planirostris, Carollia brevicauda, C. castanea, C. perspicillata, Dermanura rava, Glossophaga soricina, Sturnira bakeri, Molossus molossus, Myotis caucensis, Uroderma convexum, Phyllostomus hastatus, P. discolor), and one unidentified species belonging to the genus Rhogeessa. The genera, families and species are shown in Figure 2. These species have different feeding habits, such as frugivorous (60.19%), insectivores (17.47%), omnivore (1.45%), nectarivores (20.87%) (Table 1).
Figure 2. Diversity and abundance of bats captured in the five municipalities of the Urabá region. Figure 2 shows the 3 families, 10 genera, and 15 species of bats that were captured in the five sampling areas. The number of individuals for each taxonomic group classification are also indicated.
Table 1. Diversity of bats captured in the study. This table shows information about the species, number, percentage, frequency and feeding habits of the 206 bats captured.
Species |
Number |
Percentage (%) |
Frequency |
Feeding Habits |
Artibeus jamaicensis |
1 |
0.49% |
0.005 |
frugivore |
Carollia brevicauda |
13 |
6.31% |
0.063 |
frugivore |
Carollia castanea |
1 |
0.49% |
0.005 |
frugivore |
Carollia perspicillata |
13 |
6.31% |
0.063 |
frugivore |
Dermanura rava |
4 |
1.94% |
0.019 |
frugivore |
Glossophaga soricina |
43 |
20.87% |
0.209 |
nectarivore |
Sturnira bakeri |
17 |
8.25% |
0.083 |
frugivore |
Molossus molossus |
26 |
12.62% |
0.126 |
insectivore |
Artibeus lituratus |
5 |
2.43% |
0.024 |
frugivore |
Myotis caucensis |
9 |
4.37% |
0.044 |
insectivore |
Artibeus planirostris |
55 |
26.70% |
0.267 |
frugivore |
Uroderma convexum |
15 |
7.28% |
0.073 |
frugivore |
Rhogeessa sp. |
1 |
0.49% |
0.005 |
insectivore |
Phyllostomus hastatus |
2 |
0.97% |
0.010 |
omnivore |
Phyllostomus discolor |
1 |
0.49% |
0.005 |
omnivore |
TOTAL |
206 |
100% |
1 |
|
4. Detection of Leptospira spp. in Bats by Conventional PCR
Researchers analyzed 206 bat kidneys by PCR by amplifying the 16S ribosomal gene for detection of Leptospira spp. Twenty individual bats were positive for Leptospira (20/206), obtaining a 9.7% of infected bats (Figure 3). Positive bats for Leptospira infection were found in the 5 municipalities studied (Chigorodó: 3 bats, Carepa: 2 bats, Apartadó: 3 bats, Turbo: 10 bats, and Necoclí: 2 bats). Additionally, 6 different species of bats were found to be infected: Carollia perspicillata, Dermanura rava, Glossophaga soricina, Molossus molossus, Artibeus planirostris, and Uroderma convexum. According to sex, 11 males (55%) and 9 females (45%) were found infected. Regarding feeding habits, 12 frugivores (60%), 6 nectarivores (30%), and 2 insectivores (10%) bats were found infected (Table 2).
Figure 3. Molecular detection of bats naturally infected with Leptospira. The figure shows a 1% agarose gel with the amplification products of 20 bats infected with Leptospira spp. The band (331 base pair) corresponding to a fragment of the 16S ribosomal gene. A 100 base pair molecular weight markers were used. Additionally, a positive control (C+: Leptospira interrogans) and a negative control (C-: PCR reagents without DNA) were used in all reactions.
Table 2. Natural infection of bats with different Leptospira species. This table shows the code of the positive samples, Leptospira species identified by amplification of the 16S ribosomal gene, bat species infected, and the municipality from which the sampling area originated.
Code |
Phylogenetic Identification (16S Ribosomal Gene) |
Infected Species |
Feeding Habits |
Gender |
Municipality |
ZM-022 |
Leptospira kirschneri |
Carollia perspicillata |
Frugivore |
Female |
Carepa |
ZM-025 |
Leptospira kirschneri |
Dermanura rava |
Frugivore |
Male |
Carepa |
ZM-047 |
Leptospira interrogans |
Glossophaga soricina |
Nectarivore |
Female |
Apartadó |
ZM-056 |
Leptospira kirschneri |
Glossophaga soricina |
Nectarivore |
Male |
Apartadó |
ZM-060 |
Leptospira noguchii |
Glossophaga soricina |
Nectarivore |
Female |
Apartadó |
ZN-083 |
Leptospira noguchii |
Uroderma convexum |
Frugivore |
Male |
Chigorodó |
ZN-087 |
Leptospira noguchii |
Uroderma convexum |
Frugivore |
Male |
Chigorodó |
ZN-107 |
Leptospira noguchii |
Uroderma convexum |
Frugivore |
Female |
Chigorodó |
ZN-125 |
Leptospira noguchii |
Molossus molossus |
Insectivore |
Female |
Turbo |
ZN-126 |
Leptospira kirschneri |
Molossus molossus |
Insectivore |
Male |
Turbo |
ZN-129 |
Leptospira kirschneri |
Artibeus planirostris |
Frugivore |
Male |
Turbo |
ZN-136 |
Leptospira borgpetersenii |
Glossophaga soricina |
Nectarivore |
Female |
Turbo |
ZN-138 |
Leptospira borgpetersenii |
Glossophaga soricina |
Nectarivore |
Female |
Turbo |
ZN-139 |
Leptospira interrogans |
Artibeus planirostris |
Frugivore |
Male |
Turbo |
ZN-141 |
Leptospira alexanderi |
Uroderma convexum |
Frugivore |
Female |
Turbo |
ZN-149 |
Leptospira alexanderi |
Glossophaga soricina |
Nectarivore |
Male |
Turbo |
ZN-150 |
Leptospira borgpetersenii |
Artibeus planirostris |
Frugivore |
Male |
Turbo |
ZN-163 |
Leptospira kirschneri |
Artibeus planirostris |
Frugivore |
Male |
Turbo |
ZN-168 |
Leptospira noguchii |
Uroderma convexum |
Frugivore |
Male |
Necoclí |
ZN-169 |
Leptospira interrogans |
Uroderma convexum |
Frugivore |
Female |
Necoclí |
5. Identification of Leptospira Species by Phylogenetic Analysis
Through the amplification, sequencing, and phylogenetic analysis of the 20 positive bat samples, the following Leptospira species were identified: Leptospira borgpetersenii (3/20–15%), Leptospira alexanderi (2/20–10%), Leptospira noguchii (6/20–30%), Leptospira interrogans (3/20–15%), and Leptospira kirschneri (6/20–30%). Results of the phylogenetic identification are shown in Figure 4.
Figure 4. Identification of Leptospira species infecting bats by phylogenetic analysis of the 16S ribosomal gene. Phylogenetic reconstruction of the 16S ribosomal gene of the genus Leptospira is shown. Red diamonds represent the bats infected with Leptospira spp. Leptospira borgpetersenii, Leptospira alexanderi, Leptospira noguchii, Leptospira interrogans, and Leptospira kirschneri were the Leptospira species found infecting this bat population.
6. Host-Pathogen Relationship between Bats and Leptospira
The host-pathogen association is as follows: Leptospira borgpetersenii infected 2 bats species (Glossophaga soricina and Artibeus planirostris), Leptospira alexanderi infected 2 bats species (Uroderma convexum and Glossophaga soricina), Leptospira noguchii infected 3 bats species (Glossophaga soricina, Uroderma convexum, and Molossus molossus), Leptospira interrogans infected 3 bats species (Glossophaga soricina, Artibeus planirostris, and Uroderma convexum) and Leptospira kirschneri infected 5 bats species (Carollia perspicillata, Dermanura rava, Glossophaga soricina, Molossus molossus, and Artibeus planirostris). The number of infected bats for each Leptospira species is shown in Table 3. Additionally, no renal infection was detected in 9 bat species (A. jamaicensis, C. brevicauda, C. castanea, S. bakeri, A. lituratus, M. caucensis, P. hastatus, P. discolor, and Rhogeessa sp.).
Table 3. Natural infection of bats with different Leptospira species. The table shows the host-pathogen relationship between 6 Leptospira species and 6 bats species susceptible to infection. The number of bats infected by each Leptospira species is shown in parentheses.
Leptospira Species |
Infected Bat Species |
Infected Bats |
Leptospira borgpetersenii |
Glossophaga soricina (2)
Artibeus planirostris (1)
|
3
|
Leptospira alexanderi |
Uroderma convexum (1)
Glossophaga soricina (1)
|
2
|
Leptospira noguchii |
Glossophaga soricina (1)
Uroderma convexum (4)
Molossus molossus (1)
|
6
|
Leptospira interrogans |
Glossophaga soricina (1)
Artibeus planirostris (1)
Uroderma convexum (1)
|
3
|
Leptospira kirschneri |
Carollia perspicillata (1)
Dermanura rava (1)
Glossophaga soricina (1)
Molossus molossus (1)
Artibeus planirostris (2)
|
6
|
|
|
Total: 20
|
7. Conclusion
Bats in the Urabá region (Antioquia-Colombia) are important reservoirs and disseminators of pathogenic Leptospira species. With changing habitats due to man-made interventions, bats are becoming a significant reservoir of many zoonotic pathogens.
This entry is adapted from the peer-reviewed paper 10.3390/microorganisms9091897