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Bats are important reservoirs for many zoonotic pathogens. However, no surveys of bacterial pathogens in bats have been performed in the Caucasus region. To understand the occurrence and distribution of bacterial infections in these mammals, 218 bats belonging to eight species collected from four regions of Georgia were examined for Bartonella, Brucella, Leptospira, and Yersinia using molecular approaches. Bartonella DNA was detected in 77 (35%) bats from all eight species and was distributed in all four regions. The prevalence ranged 6–50% per bat species. The Bartonella DNA represented 25 unique genetic variants that clustered into 21 lineages. Brucella DNA was detected in two Miniopterus schreibersii bats and in two Myotis blythii bats, all of which were from Imereti (west-central region). Leptospira DNA was detected in 25 (13%) bats that included four M. schreibersii bats and 21 M. blythii bats collected from two regions. The Leptospira sequences represented five genetic variants with one of them being closely related to the zoonotic pathogen L. interrogans (98.6% genetic identity). No Yersinia DNA was detected in the bats. Mixed infections were observed in several cases. One M. blythii bat and one M. schreibersii bat were co-infected with Bartonella, Brucella, and Leptospira; one M. blythii bat and one M. schreibersii bat were co-infected with Bartonella and Brucella; 15 M. blythii bats and three M. schreibersii bats were co-infected with Bartonella and Leptospira. Our results suggest that bats in Georgia are exposed to multiple bacterial infections. Further studies are needed to evaluate pathogenicity of these agents to bats and their zoonotic potential.
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RESEARCH ARTICLE
Molecular Survey of Bacterial Zoonotic Agents
in Bats from the Country of Georgia
(Caucasus)
Ying Bai
1
*, Lela Urushadze
2,3
, Lynn Osikowicz
1
, Clifton McKee
1,4
, Ivan Kuzmin
5
,
Andrei Kandaurov
6
, Giorgi Babuadze
2,3
, Ioseb Natradze
6
, Paata Imnadze
2
, Michael Kosoy
1
1Division of Vector-Borne Disease, Centers for Disease Control and Prevention, Fort Collins, Colorado,
United States of America, 2National Center for Disease Control and Public Health, Tbilisi, Republic of
Georgia, 3Institute of Chemical Biology, Ilia State University, Tbilisi, Republic of Georgia, 4Department of
Biology, Colorado State University, Fort Collins, Colorado, United States of America, 5Department of
Pathology, University of Texas Medical Branch, Galveston, Texas, United States of America, 6Institute of
Zoology, Ilia State University, Tbilisi, Republic of Georgia
These authors contributed equally to this work.
*bby5@cdc.gov
Abstract
Bats are important reservoirs for many zoonotic pathogens. However, no surveys of bacte-
rial pathogens in bats have been performed in the Caucasus region. To understand the
occurrence and distribution of bacterial infections in these mammals, 218 bats belonging to
eight species collected from four regions of Georgia were examined for Bartonella,Brucella,
Leptospira, and Yersinia using molecular approaches. Bartonella DNA was detected in 77
(35%) bats from all eight species and was distributed in all four regions. The prevalence ran-
ged 6–50% per bat species. The Bartonella DNA represented 25 unique genetic variants
that clustered into 21 lineages. Brucella DNA was detected in two Miniopterus schreibersii
bats and in two Myotis blythii bats, all of which were from Imereti (west-central region). Lep-
tospira DNA was detected in 25 (13%) bats that included four M.schreibersii bats and 21 M.
blythii bats collected from two regions. The Leptospira sequences represented five genetic
variants with one of them being closely related to the zoonotic pathogen L.interrogans
(98.6% genetic identity). No Yersinia DNA was detected in the bats. Mixed infections were
observed in several cases. One M.blythii bat and one M.schreibersii bat were co-infected
with Bartonella,Brucella, and Leptospira; one M.blythii bat and one M.schreibersii bat were
co-infected with Bartonella and Brucella; 15 M.blythii bats and three M.schreibersii bats
were co-infected with Bartonella and Leptospira. Our results suggest that bats in Georgia
are exposed to multiple bacterial infections. Further studies are needed to evaluate patho-
genicity of these agents to bats and their zoonotic potential.
PLOS ONE | DOI:10.1371/journal.pone.0171175 January 27, 2017 1 / 12
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OPEN ACCESS
Citation: Bai Y, Urushadze L, Osikowicz L, McKee
C, Kuzmin I, Kandaurov A, et al. (2017) Molecular
Survey of Bacterial Zoonotic Agents in Bats from
the Country of Georgia (Caucasus). PLoS ONE 12
(1): e0171175. doi:10.1371/journal.pone.0171175
Editor: Wanda Markotter, University of Pretoria,
SOUTH AFRICA
Received: November 3, 2016
Accepted: January 16, 2017
Published: January 27, 2017
Copyright: This is an open access article, free of all
copyright, and may be freely reproduced,
distributed, transmitted, modified, built upon, or
otherwise used by anyone for any lawful purpose.
The work is made available under the Creative
Commons CC0 public domain dedication.
Data Availability Statement: All relevant data are
within the paper.
Funding: The authors received no specific funding
for this work.
Competing Interests: The authors have declared
that no competing interests exist.
Introduction
Bats (Chiroptera) represent one of the most successfully evolved mammalian groups on Earth
for their unique characteristics, such as a long lifespan, the capability to fly long distances dur-
ing foraging and particularly during seasonal migrations, the ability to inhabit a multitude of
diverse ecological niches, and the colonial habitation. The role of bats in epidemiology of zoo-
notic diseases is very important as they frequently live in close proximity to humans and serve
as reservoirs to different pathogens that include viruses, bacteria, fungi and parasites [1,2].
Many previous and ongoing research activities predominantly focused on viral agents in bats
[3,4], but little is known about a presence of bacterial pathogens [5].
The bacterial genera Bartonella,Brucella,Leptospira, and Yersinia each consist of multiple
species, some of which are zoonotic pathogens causing diseases in domestic or companion ani-
mals and in humans. Bartonella infections have been reported from a variety of animals occur-
ring over a broad geographic distribution. Around 30 species have been described within the
genus and the number is still increasing [6]. Controversy has been raised in several studies
regarding host specificity of Bartonella [79]. Importantly, bats in the Northern Hemisphere
have been implicated as a reservoir of B.mayotimonensis that was described from a human
case of endocarditis in the USA [10,11], although the mechanism of transmission between bats
and humans remains unresolved.
Brucellosis is an important zoonotic disease caused by bacteria of the genus Brucella.
Domestic animals such as cattle, goats, sheep, pigs, camel, buffalo and dogs serve as reservoir
hosts. Humans can be infected after contacting infectious animals or drinking raw milk [12].
Knowledge of Brucella ecology in wildlife is limited although several species were described in
rodents, foxes, and marine mammals [1317]. Except for an old report of anti-Brucella aggluti-
nins in vampire bats (Desmodus rotundus) in Brazil [18], no other studies have reported Bru-
cella infection in bats.
Leptospirosis is a bacterial zoonosis caused by L.interrogans and other pathogenic spiro-
chetes of the genus Leptospira. Animals and humans acquire the infection through contact
with water or soil contaminated with the urine of infected animals or by direct contact with
these animals [19,20]. Leptospira spp. are distributed worldwide in rats and many other mam-
malian species [21]. Recently, leptospiral infections have been identified in bats from several
countries [2224].
Among infections caused by Yersinia species, plague (Y.pestis) causes the most notorious
disease, infecting many mammalian species along with humans. Yersiniosis, occurring as an
enteric disease in humans, is caused by Y.pseudotuberculosis and Y.enterocolitica, both of
which have a broad distribution [25]. These bacteria have been frequently isolated from a vari-
ety of wild and domestic animals [26,27], but compared to Y.pestis their association with wild-
life is not as well studied. Recently, bats have been reported to be infected with Yersinia [28,29].
The country of Georgia is located between the Greater Caucasus and Lesser Caucasus
mountain ridges at the intersection of Europe and Asia. There are 109 mammalian species and
many associated zoonotic agents in this region [30,31]. Recent studies conducted in this coun-
try have demonstrated the presence of diverse Bartonella species in wild rodents [32] and sug-
gested the role of rat-associated Bartonella as a causative agent for a human illness [33].
Brucellosis is endemic in the area, with B.abortus and B.melitensis actively circulating in live-
stock and affecting local residents [34]. Leptospirosis is also broadly distributed in the country
with increasing morbidity in recent years [35].
Bats are abundant in Georgia with at least 29 species identified [36]. However, information
on bacterial infectious agents in bats from this region was absent. Understanding the preva-
lence and distribution of zoonotic pathogens in local bats would be significant from the
Bai et al., Bacterial Zoonotic Agents in Bats, Georgia
PLOS ONE | DOI:10.1371/journal.pone.0171175 January 27, 2017 2 / 12
veterinary and public health perspectives. In this study, we evaluated the presence and distri-
bution of Bartonella,Brucella,Leptospira, and Yersinia in bats collected from eight colonies
located in four regions of Georgia.
Material and Methods
Ethics statement
The work was performed in compliance with the protocol approved by the CDC Institutional
Animal Care and Use Committee (protocol #2096FRAMULX-A3). Permits for the list of bat
species sought and the number of animals per colony available for sampling were obtained
from the Ministry of Environmental and Natural Resources Protection of Georgia.
Study sites and bat tissues collection
In June 2012, bats were captured manually or by using nets from eight colonies (found in
caves, building attics, or monasteries) within four sites located in four regions of Georgia: one
colony in Martvili (42˚N, 42˚E, Samegrelo-Zemo Svaneti region in western Georgia); three
colonies in Tskaltubo (42˚N, 42˚E, Imereti region in west-central Georgia); one colony in Gar-
dabani district (41˚N, 45˚E, Kvemo Kartli region in southern Georgia); and three colonies in
David Gareja (41˚N, 45˚E, Kakheti region in eastern Georgia). Martvili and Tskaltubo, Garda-
bani and David Gareja, are neighboring sites, respectively (Fig 1).
Fig 1. Bat sampling sites, Georgia, June 2012.
doi:10.1371/journal.pone.0171175.g001
Bai et al., Bacterial Zoonotic Agents in Bats, Georgia
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Captured bats were delivered to the processing site in individual cotton bags. The bats were
sexed, weighed, and identified to species based on external morphological characteristics. Bats
were anesthetized using ketamine (0.05–0.1 mg/g body mass) and exsanguinated by cardiac
puncture. Tissue samples (including spleen, kidney, intestine, and others) were collected from
the bats. Samples were stored on dry ice in the field, then transferred to a -80˚C freezer in the
laboratory of Georgian NCDC before shipping to the US CDC’s laboratory in Fort Collins,
Colorado for bacterial testing.
DNA extraction and PCR detection
A small piece (~10 mg) of spleen, kidney, and intestine of each bat were homogenized sepa-
rately using a Bullet Blender1Gold homogenizer (Next Advance, Averill Park, NY) following
the protocols provided by the manufacturer. The homogenates were then transferred to a
QIAxtractor (Qiagen, Valencia, CA) platform for DNA extraction using the tissue protocol,
and the DNA was used as the template for downstream analyses. The kidney DNA was tested
for Bartonella and Leptospira; the spleen DNA was tested for Brucella and Yersinia; and the
intestine DNA was tested for Yersinia only. Molecular detection was performed using a con-
ventional PCR assay carried out in a C1000 Touch Thermal Cycler (Bio-Rad, Hercules, CA)
and/or real-time PCR assay carried out in a CFX96 Real-Time System (Bio-Rad, Hercules,
CA). The relevant genes targeted were 16S 23S internal transcribed spacer (ITS), insertion
sequence (IS711), 32-kDa lipoprotein (lipL32) gene, and peptidoglycan-associated lipoprotein
(pal) gene for Bartonella,Brucella,Leptospira, and Yersinia, respectively. The reactions for ITS
were run under conventional PCR settings; the reactions for IS711, pal and lipL32were run
under the real-time multiplex PCR settings, following protocols published elsewhere [3739].
Ct value <36 with an amplification curve is recorded as positive. Positive samples for Brucella
DNA (IS711) and Leptospira DNA (lipL32) identified by real-time PCR were further tested by
conventional PCR targeting a 223 base pair-fragment in 31 kDa gene (bcsp31) for Brucella [40]
and a 423 base pair-fragment in lipL32 for Leptospira using primers [41] different from the
real-time PCR. All primers and probes used in this study are listed in Table 1. The pal primers
and probes for detection of Yersinia species were developed for this study based on a whole-
genome scan (M. Diaz, unpublished data). For any conventional PCR, the PCR products were
Table 1. Molecular detection of bacterial agents in bats from Georgia, June 2012.
Agents Gene target PCR assay Primer/probe sequences Reference
Bartonella ITS conventional Forward: CTT CAG ATG ATG ATC CCA AGC CTT CTG GCG [39]
Reverse: GAA CCG ACG ACC CCC TGC TTG CAA AGC A
Forward: GCT TGA AGC TTG CGG ACA GT
Brucella IS711 real-time Reverse: GGC CTA CCG CTG CGA AT [37]
Probe: AAG CCA ACA CCC GGC CAT TAT GGT
Brucella bcsp31 conventional Forward: TGG CTC GGT TGC CAA TAT CAA [40]
Reverse: CGC GCT TGC CTT TCA GGT CTG
Forward: AAG CAT TAC CGC TTG TGG TG
Leptospira lipL32 real-time Reverse: GAA CTC CCA TTT CAG CGA TT [38]
Probe: AA AGC CAG GAC AAG CGC CG
Leptospira lipL32 conventional Forward: CGC TGA AAT GGG AGT TCG TAT GAT T [41]
Reverse: CCA ACA GAT GCA ACG AAA GAT CCT TT
Forward: CGC AAA TAA TGA CCA ATC TGG
Yersinia Pal real-time Reverse: CGT GGC CTT CAA CAA CAA C This study
Probe: CGG TTC TGA CTT CGC TCA AAT GCT GG
doi:10.1371/journal.pone.0171175.t001
Bai et al., Bacterial Zoonotic Agents in Bats, Georgia
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analyzed for the presence of amplicons of the expected size by electrophoresis on 1.5% agarose
gels containing GelGreen stain (Biotium, Hayward, CA). Positive and negative controls were
included in each PCR assay to evaluate the presence of appropriately sized amplicons and to
rule out potential contamination, respectively.
Leptospira infection rate was compared between study sites and between bat species using
chi-square tests.
Sequencing and phylogenetic analysis for Bartonella species and
Leptospira species
Samples positive for Bartonella (ITS) and Leptospira (lipL32) were further identified by
sequencing analyses. The PCR amplicons were purified using a QIAquick PCR Purification
Kit (Qiagen, Valencia, CA) according to manufacturer’s instructions, and then sequenced in
both directions using ABI 3130 Genetic Analyzer (Applied Biosystems, Foster City, CA). For-
ward and reverse sequences were assembled using the SeqMan Pro program in Lasergene v.12
(DNASTAR, Madison, WI). Assembled sequences obtained from all samples in the present
study were compared between themselves and with reference sequences available in GenBank
after alignment using the Clustal algorithm in the MegAlign program in Lasergene. Using the
neighbor-joining method, cladogram (showing the cladistics relationship rather than phyloge-
netic relationship) was generated for Bartonella among the ITS sequences. Sequences were
assigned to clades visually based on monophyletic clusters. Phylogenetic tree was constructed
for Leptospira, Branch support was estimated using 1000 bootstrap replicates. Newly identified
sequence variants were submitted to GenBank.
Results
Bat sampling
A total of 236 bats were captured from the trapping sites. Samples with incomplete or missing
information were excluded, which resulted in 218 bats available for analysis. The animals
belonged to eight species of five genera, including Eptesicus serotinus (n = 17), Miniopterus
schreibersii (n = 27), Myotis blythii (n = 68), Myotis emarginatus (n = 42), Myotis mystacinus
(n = 1), Pipistrellus pygmaeus (n = 11), Rhinolophus euryale (n = 28), and R.ferrimequinum
(n = 24) (Table 2). The Myotis spp. bats accounted for more than half of the tested bats. Other
bat species accounted for a smaller portion, ranging from 5% to 13%.
The number of bat species varied by site, with three, four, five, and three in Martvili, Tskal-
tubo, Gardabani, and David Gareja, respectively. All M.schreibersii bats (n = 27) were collected
in Tskaltubo. M.blythii bats were mainly captured in Tskaltubo and David Gareja (Table 3).
Table 2. Detection of Bartonella,Brucella, and Leoptospira in bats from Georiga, 2012.
Bat species # Tested Bartonella Brucella Leptospira Yersinia
# Pos Prevalence (%) # Pos Prevalence (%) # Pos Prevalence (%) # Pos Prevalence (%)
Eptesicus serotinus 17 1 6 0 0 0 0 0 0
Miniopterus schreibersii 27 13 48 2 7 4 15 0 0
Myotis blythii 68 26 38 2 3 21 31 0 0
Myotis emarginatus 42 12 29 0 0 0 0 0 0
Myotis mystacinus 1 0 0 0 0 0 0 0 0
Pipistrellus pygmaeus 11 1 9 0 0 0 0 0 0
Rhinolophus euryale 28 12 43 0 0 0 0 0 0
Rhinolophus ferrimequinum 24 12 50 0 0 0 0 0 0
Total 218 77 35 4 2 25 11 0 0
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Bai et al., Bacterial Zoonotic Agents in Bats, Georgia
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Molecular detection
Bartonella DNA was detected in 77 of the 218 tested bat kidneys, giving the overall prevalence
of 35%. Bartonella DNA was detected in all bat species but M.mystacinus, for which there was
only one sample available for testing. The prevalence of Bartonella infection ranged from 6%
in E.serotinus to 50% in R.ferrimequinum (Table 2). The Bartonella-positive bats were present
in all colonies, with variable numbers of 4–23 per colony (the proportion of infected bats per
colony cannot be estimated as the size of the colonies was not evaluated).
Brucella DNA was detected in spleen samples of four bats by real-time PCR (IS711) and
confirmed by conventional PCR (bcsp31). The positive batsinclude two M.schreibersii and two
M.blythii. All of the four Brucella-positive bats were obtained from one site (Tskaltubo) with
three of them from one colony and the last one (M.schreibersii) from a neighboring colony.
Leptospira DNA was amplified from kidneys of 25 bats by real-time PCR (lipL32) and con-
firmed by conventional PCR (lipL32). The positive bats were M.schreibersii (n = 4) or M.
blythii (n = 21). The prevalence in M.blythii (31%; 21/68) was significantly higher (χ
2
= 1.89,
p<0.05) than that in M.schreibersii (15%; 4/27). The Leptospira-positive bats were found in
four colonies within two sites–one colony in David Gareja and the other three colonies in
Tskaltubo. All four positive M.schreibersii were collected in Tskaltubo; the M.blythii were dis-
tributed in David Gareja (n = 8) and Tskaltubo (n = 13). The infection rate of Leptospira in M.
blythii was 36% (8/22) in David Gareja and 30% (13/44) in Tskaltubo, with no statistical differ-
ence observed between the two sites (χ
2
= 0.20, p >0.05).
Yersinia DNA was detected in none of the bats, neither in spleen nor intestine.
Mixed infection
Two or three pathogens were detected in M.blythii and M.schreibersii. Two bats (one M.
blythii and one M.schreibersii) captured in different colonies within site Tskaltubo were co-
infected with all three pathogens–Bartonella,Brucella, and Leptospira. Two bats (one M.blythii
and one M.schreibersii) captured in the same colony within site Tskaltubo were co-infected
with Bartonella and Brucella; and eighteen bats (15 M.blythii and three M.schreibersii) cap-
tured in one colony in site David Gareja and in two colonies in site Tskaltubo were co-infected
with Bartonella and Leptospira. No mixed infections were observed in the remaining bat
species.
Sequencing analysis
Sequencing analyses were performed on ITS sequences of Bartonella and lipL32 sequences of
Leptospira, both of which were amplified from bat kidney DNA.
Table 3. Geographic distribution of the captured bats, Georgia, June 2012.
Bat species Martvili Tskaltubo Gardabani David Gareja Total
Eptesicus serotinus 17 17
Miniopterus schreibersii 27 27
Myotis blythii 2 44 22 68
Myotis emarginatus 14 15 13 42
Myotis mystacinus 1 1
Pipistrellus pygmaeus 11 11
Rhinolophus euryale 13 15 28
Rhinolophus ferrimequinum 5 1 18 24
Total 20 100 45 53 218
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Bai et al., Bacterial Zoonotic Agents in Bats, Georgia
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The ITS sequences of Bartonella exhibited considerable heterogeneity. The 77 Bartonella-
positive DNA represented 25 genetic variants (a variant is defined when at least one nucleotide
difference is observed between compared sequences). These variants were all unique and sub-
mitted to GenBank (accession numbers KX420713—KX420737). Only a few variants were rel-
atively close (96.3% similarity) between themselves, while the majority were largely distant
from each other and clustered into 21 clades (a clade is a cluster of sequences following neigh-
bor-joining analysis) (Fig 2). Except for E.serotinus and P.pygmaeus in which Bartonella was
detected in one individual for each species, each of the other bat species was associated with 4
to 9 Bartonella clades. For example, M.blythii was associated with nine lineages (I—IV, XV,
Fig 2. Cladistics relationship of the Bartonella variants detected in bats from Georgia based on ITS sequences. A total of 77 Bartonella ITS
sequences were obtained. The sequences belonged to 25 variants (each indicated by a unique GenBank accession number), and the variants clustered into
21 clades (marked by a unique Roman number). After each variant, it is host species and number of Bartonella sequences obtained from the host species.
The cladogram was generated by neighboring-joining method. ES: Eptesicus serotinus; MB: Myotis blythii; ME: Myotis emarginatus; MS: Miniopterus
schreibersii; PP: Pipistrellus pygmaeus; RE: Rhinolophus euryale; RF: Rhinolophus ferrimequinum.
doi:10.1371/journal.pone.0171175.g002
Bai et al., Bacterial Zoonotic Agents in Bats, Georgia
PLOS ONE | DOI:10.1371/journal.pone.0171175 January 27, 2017 7 / 12
and XVII—XXI); M.schreibersii was associated with six lineages (III—VI, XVI, and XVIII); R.
euryale was associated with eight lineages (VII—XIII and XVII). Most Bartonella lineages were
specific to certain bat genus/species. In particular, lineages I—VI and lineages VII—XIII were
associated with Myotis spp. and Rhinolophus spp., respectively; while lineages XV—XXI could
be associated with multiple bat genera (Fig 2).
The 25 LipL32 sequences of Leptospira detected in the bats represented five variants with
sequence distances of 3.7% - 7.3% (Fig 3). All variants were novel and assigned GenBank acces-
sion numbers KX420708 –KX420712. Of the 25 sequences, the four sequences recovered from
M.schreibersii were identical (variant KX420712); while the other 21 sequences from M.blythii
were of four variants, each of which was detected in 11, 4, 2, and 4 individuals, respectively.
These variants were close to some Leptospira species that are known zoonotic pathogens. Spe-
cifically, variant KX420710 that was detected in 11 M.blythii was closest to L.interrogans with
genetic identity of 98.6%; while a few other variants were relatively close to L.borgpetersenii
with genetic identity of 96% - 97%.
Discussion
Using molecular approaches, we report the detection of multiple potential zoonotic pathogens,
including Bartonella,Brucella, and Leptospira in bats from the country of Georgia.
Fig 3. Phylogenetic relationships of the five Leptospira variants detected in bats from Georgia based on lipL32 sequences. Each
variant is indicated by a unique GenBank accession number, and followed by bat species name and sequences obtained fromthe bat species.
Phylogenetic tree was constructed by neighboring-joining method, and bootstrap values were calculated with 1,000 replicates.
doi:10.1371/journal.pone.0171175.g003
Bai et al., Bacterial Zoonotic Agents in Bats, Georgia
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Similar to early reports from other parts of the world [8,42], Bartonella species are widely
distributed in bats in Georgia with very high diversity. All genetic variants discovered in the
study were novel and do not belong to any previously described Bartonella species. Further
characterization is necessary to verify whether the identified DNA sequences represent novel
Bartonella species. Most Bartonella variants described here show specific relationships to their
bat hosts at a genus level, particularly Myotis spp. and Rhinolophus spp. Conversely, some Bar-
tonella variants were shared among bats of different genera, which suggests frequent host-shift-
ing or cross-species transmission potentially related to exchange of ectoparasites between bats
[43,44]. We do not know whether the Bartonella variants identified in the bats are responsible
for any human disease in Georgia, but a recent report that bats in northern Europe harbor a
human pathogen B.mayotimonensis [11] indicates such a possibility. New information about
Bartonella infection in bats in Georgia can provide additional insights towards understanding
the interactions between humans, animals, and parasites.
Leptospira infection was detected in the tested bats. Interestingly, all Leptospira infected-
bats were either M.blythii or M.schreibersii. No Leptospira infection was detected in bats of
other species, including the M.emarginatus which is a quite common local species. Species-
specific variations in bacterial infection rates may indicate that certain bat species are more
exposed habitually (e.g. drinking the same contaminated water) or even more susceptible to
Leptospira than other bat species [23,45,46]. Our observation of similar Leptospira infection
rates in M.blythii in Tskaltubo and David Gareja suggests that these bats were equally exposed
to the infection at different sites; while higher prevalence in M.blythii than in M.schreibersii
may suggest that M.blythii is more susceptible to Leptospira. Field observations showed that
M.blythii and M.schreibersii share the same roosts and M.schreibersii usually incorporate into
the dense groups of M.blythii, which presumably results in close body to body contact between
animals of these two species. M.blythii may transmit the infection to M.schreibersii through
urinary shedding and other similar routes. The high prevalence observed suggests that these
bats might play a possible role in the maintenance of Leptospira spp. in the environment [47].
On the other hand, one Leptospira variant identified in M.blythii was closely related to L.inter-
rogans (98.6% identity), a well-known zoonotic pathogen frequently found in rats [48]. Con-
sidering the high frequency of this variant (detected in 11 of 25 or 44% infected bats), it warns
that M.blythii may serve as a natural reservoir to L.interrogans and can potentially transmit
the infection to humans, particularly when they roost synantropically, e.g. in monasteries. Fur-
thermore, some of the variants were relatively close to L.borgpetersenii that also is a zoonotic
pathogen.
The most intriguing finding of this work is probably the discovery of Brucella in bats.
Although there was a single report of Brucella agglutinins in Desmodus rotundus bats from Bra-
zil [18], our study represents the first detection of Brucella DNA in bats. Similar to the finding
of Leptospira spp., the Brucella infections were found only in M.schreibersii and M.blythii.
Interestingly, all Brucella positive bats were from the site Tskaltubo. The habitat preference
and geographic origin could influence the infection prevalence in these bats. It was evident for
M.schreibersii since this species was only captured in Tskaltubo; whereas absence of Brucella-
positive M.blythii bats in David Gareja (where no M.schreibersii were present) may suggest
that M.blythii contracted Brucella infection from M.schreibersii in Tskaltubo. Alternatively, as
was the case with Leptospira, bats of both species in Tskaltubo might be exposed to Brucella
independently by consuming the same contaminated source, such as water. The most studied
Brucella species (B.melitensis,B.abortus, and B.suis) are so genetically similar that some
researchers have argued for their combination into one species [49]. However, the epidemio-
logical and diagnostic benefits for separating the genus based on phenotypic and ecological
characteristics are more compelling. Therefore, it is important to distinguish genotypic and
Bai et al., Bacterial Zoonotic Agents in Bats, Georgia
PLOS ONE | DOI:10.1371/journal.pone.0171175 January 27, 2017 9 / 12
phenotypic differences between this newly discovered bat-associated Brucella and other Bru-
cella species. In the current presentation, we only report the detection of Brucella DNA in the
bats. Further characterization of the bacterium is in progress. Because most Brucella species
are highly pathogenic, it is important to determine whether this strain has a zoonotic potential.
We did not find any Yersinia infections in this study using recently designed primers. Since
the sensitivity of the utilized primers for detection of all Yersinia species in the field samples
has not been sufficiently evaluated in other field studies, the negative results for this bacterial
genus in Georgian bats might represent a lack of sensitivity of our assay or a need for more
broadly reactive primers.
In conclusion, bats from Georgia harbor several potential bacterial pathogens. These pre-
liminary data highlight that bats may play an important role in maintaining those agents in
nature. Further studies need to be carried out to understand the importance of these agents for
bats, other wildlife, veterinary and public health.
Acknowledgments
We wish to thank Nikoloz Tsertsvadze, Ketevan Sidamonidze and many others from the
National Center for Disease Control and Public Health, Tbilisi, Georgia for their assistance
with the bat trapping, sample collecting and processing. Thanks to Maureen Diaz from Divi-
sion of Bacterial Diseases, US CDC for helping with designing the primers/probes for Yersinia
detection. Special thanks to Ayimgul Kakenova and James Jubilee for supporting the travel of
Lela Urushadze to Fort Collins, Colorado to conduct the laboratory analysis in the Bartonella
Laboratory at US CDC.
Author Contributions
Conceptualization: YB MK PI.
Formal analysis: YB.
Investigation: YB LU LO CM.
Methodology: YB.
Supervision: MK YB PI.
Writing original draft: YB.
Writing review & editing: MK CM IK AK IN GB LO LU.
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PLOS ONE | DOI:10.1371/journal.pone.0171175 January 27, 2017 12 / 12
... Bacterial pathogen screening detected the presence of Bartonella sp. in a single I. vespertilionis (n = 1, EW83, Hungary) in the nymph stage that had fed on R. ferrumequinum (Figs 2 and 3). Sequence data showed the highest similarity with a Bartonella sequence found in R. ferrumequinum in Georgia (KX420722, percentage identity: 100%, query coverage: 97%) [44] (Fig 4). ...
... Bartonella species have been widely described from bats and associated ectoparasites, representing a wide range of genotypes [8,54]. We identified a single I. vespertilionis tick infected with these bacteria, with high similarity to a Bartonella infection from a R. ferrumequinum tested in Georgia (KX420722) [44]. The infected I. vespertilionis tick contained R. ferrumequinum DNA, and it was collected in Hungary, indicating that this Bartonella genotype may show a wide geographical range in this bat host species. ...
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... Indeed, several bacterial genera with zoonotic potential have been described in bats around the world, such as Bartonella, Brucella, Campylobacter, Clostridium, Escherichia, Leptospira, Listeria, Pasteurella, Salmonella, Staphylococcus, and Yersinia, from different samples (Mühldorfer, 2013;Bai et al., 2017;Stuckey et al., 2017a;Bai et al., 2018;Ferreira et al., 2018;André et al., 2019;Ferreira et al., 2021;Szentivanyi et al., 2023). Two reviews on the subject have already been published, but one is not a systematic review (Mühldorfer, 2013), and the other, more recently published (Szentivanyi et al., 2023), despite being a systematic review with meta-analysis, differs in the scope of the present one. ...
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Lymphadenopathy and fever that developed in a woman in Tbilisi, Georgia, most likely were caused by a ratborne Bartonella strain related B. tribocorum and B. elizabethae. The finding suggests that this Bartonella strain could be spread y infected rats and represents a potential human risk. © 2016, Centers for Disease Control and Prevention (CDC). All rights reserved.
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A growing number of recent studies have highlighted bats as a reservoir for Leptospira bacteria, pointing out the potential role of bats in the epidemiology of the most widespread zoonotic disease in the world [1]. Because leptospirosis is a largely neglected disease, a number of unan-swered questions remain about the ecology and evolution of Leptospira, especially those associated with bats. Here we summarize what has been recently learned about this emerging but enigmatic host–pathogen association. We show how this system can provide exciting new opportunities to obtain insights into the evolutionary ecology of bat-borne pathogens and propose future directions to disentangle the role of bats in human leptospirosis. What Do We Know, Briefly, about Leptospirosis and Leptospira?
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