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Molecular Survey of Bacterial Zoonotic Agents in Bats from the Country of Georgia (Caucasus)

  • Ying Bai ,

    Contributed equally to this work with: Ying Bai, Lela Urushadze

    bby5@cdc.gov

    Affiliation Division of Vector-Borne Disease, Centers for Disease Control and Prevention, Fort Collins, Colorado, United States of America

    ORCID http://orcid.org/0000-0001-7634-4051

  • Lela Urushadze ,

    Contributed equally to this work with: Ying Bai, Lela Urushadze

    Affiliations National Center for Disease Control and Public Health, Tbilisi, Republic of Georgia, Institute of Chemical Biology, Ilia State University, Tbilisi, Republic of Georgia

  • Lynn Osikowicz,

    Affiliation Division of Vector-Borne Disease, Centers for Disease Control and Prevention, Fort Collins, Colorado, United States of America

  • Clifton McKee,

    Affiliations Division of Vector-Borne Disease, Centers for Disease Control and Prevention, Fort Collins, Colorado, United States of America, Department of Biology, Colorado State University, Fort Collins, Colorado, United States of America

  • Ivan Kuzmin,

    Affiliation Department of Pathology, University of Texas Medical Branch, Galveston, Texas, United States of America

  • Andrei Kandaurov,

    Affiliation Institute of Zoology, Ilia State University, Tbilisi, Republic of Georgia

  • Giorgi Babuadze,

    Affiliations National Center for Disease Control and Public Health, Tbilisi, Republic of Georgia, Institute of Chemical Biology, Ilia State University, Tbilisi, Republic of Georgia

  • Ioseb Natradze,

    Affiliation Institute of Zoology, Ilia State University, Tbilisi, Republic of Georgia

  • Paata Imnadze,

    Affiliation National Center for Disease Control and Public Health, Tbilisi, Republic of Georgia

  • Michael Kosoy

    Affiliation Division of Vector-Borne Disease, Centers for Disease Control and Prevention, Fort Collins, Colorado, United States of America

Molecular Survey of Bacterial Zoonotic Agents in Bats from the Country of Georgia (Caucasus)

  • Ying Bai, 
  • Lela Urushadze, 
  • Lynn Osikowicz, 
  • Clifton McKee, 
  • Ivan Kuzmin, 
  • Andrei Kandaurov, 
  • Giorgi Babuadze, 
  • Ioseb Natradze, 
  • Paata Imnadze, 
  • Michael Kosoy
PLOS
x

Abstract

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.

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 during 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 zoonotic 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 animals and in humans. Bartonella infections have been reported from a variety of animals occurring 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 agglutinins in vampire bats (Desmodus rotundus) in Brazil [18], no other studies have reported Brucella infection in bats.

Leptospirosis is a bacterial zoonosis caused by L. interrogans and other pathogenic spirochetes 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 mammalian 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 variety of wild and domestic animals [26,27], but compared to Y. pestis their association with wildlife 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 country have demonstrated the presence of diverse Bartonella species in wild rodents [32] and suggested 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 livestock 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 prevalence and distribution of zoonotic pathogens in local bats would be significant from the veterinary and public health perspectives. In this study, we evaluated the presence and distribution 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 Gardabani 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, Gardabani and David Gareja, are neighboring sites, respectively (Fig 1).

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 separately using a Bullet Blender® Gold 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 conventional 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 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.

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Table 1. Molecular detection of bacterial agents in bats from Georgia, June 2012.

https://doi.org/10.1371/journal.pone.0171175.t001

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). Forward 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 phylogenetic 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%.

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Table 2. Detection of Bartonella, Brucella, and Leoptospira in bats from Georiga, 2012.

https://doi.org/10.1371/journal.pone.0171175.t002

The number of bat species varied by site, with three, four, five, and three in Martvili, Tskaltubo, 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).

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Table 3. Geographic distribution of the captured bats, Georgia, June 2012.

https://doi.org/10.1371/journal.pone.0171175.t003

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 confirmed 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 (χ2 = 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 distributed 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 difference observed between the two sites (χ2 = 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) captured 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.

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 submitted to GenBank (accession numbers KX420713—KX420737). Only a few variants were relatively 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 neighbor-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, 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).

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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.

https://doi.org/10.1371/journal.pone.0171175.g002

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 accession 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. Specifically, 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%.

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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 from the bat species. Phylogenetic tree was constructed by neighboring-joining method, and bootstrap values were calculated with 1,000 replicates.

https://doi.org/10.1371/journal.pone.0171175.g003

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.

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 Bartonella variants were shared among bats of different genera, which suggests frequent host-shifting 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. interrogans (98.6% identity), a well-known zoonotic pathogen frequently found in rats [48]. Considering 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. Furthermore, 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 Brazil [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 epidemiological and diagnostic benefits for separating the genus based on phenotypic and ecological characteristics are more compelling. Therefore, it is important to distinguish genotypic and phenotypic differences between this newly discovered bat-associated Brucella and other Brucella 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 preliminary 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 Division 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

  1. Conceptualization: YB MK PI.
  2. Formal analysis: YB.
  3. Investigation: YB LU LO CM.
  4. Methodology: YB.
  5. Supervision: MK YB PI.
  6. Writing – original draft: YB.
  7. Writing – review & editing: MK CM IK AK IN GB LO LU.

References

  1. 1. Whitaker JO Jr, Ritzi CM, Dick CW. Collecting and preserving ectoparasites for ecological study. In: Kunz TH, Parsons S (eds) Ecological and Behavioral Methods for the Study of Bats. 2009; 2nd edn: 806–827. The Johns Hopkins University Press, Baltimore, MD.
  2. 2. Wibbelt G, Speck S, Field H. Methods for assessing diseases in bats. In: Kunz TH, Parsons S (eds) Ecological and Behavioral Methods for the Study of Bats. 2009; 2nd edn: 775–794. The Johns Hopkins University Press, Baltimore, MD.
  3. 3. Messenger SL, Smith JS, Orciari LA, Yager PA, Rupprecht CE. Emerging pattern of rabies deaths and increased viral infectivity. Emerg Infect Dis. 2003;9(2):151–4. pmid:12603983
  4. 4. Calisher CH, Childs JE, Field HE, Holmes KV, Schountz T. Bats: important reservoir hosts of emerging viruses. Clin Microbiol Rev. 2006;19(3):531–45. pmid:16847084
  5. 5. Mühldorfer K. Bats and bacterial pathogens: a review. Zoonoses Public Health. 2013;60(1):93–103. pmid:22862791
  6. 6. Carrasco SE, Chomel BB, Gill VA, Kasten RW, Maggi RG, Breitschwerdt EB, et al. Novel Bartonella infection in northern and southern sea otters (Enhydra lutris kenyoni and Enhydra lutris nereis). Vet Microbiol. 2014;170(3–4):325–34. pmid:24629902
  7. 7. Bai Y, Calisher C, Kosoy M, Root J, Doty J. Persistent infection or successive reinfection of deer mice with Bartonella vinsonii subsp. arupensis. Appl Environ Microbiol. 2011;77(5):1728–31. pmid:21239553
  8. 8. Bai Y, Kosoy M, Recuenco S, Alvarez D, Moran D, Turmelle A, et al. Bartonella spp. in bats, Guatemala. Emerg Infect Dis. 2011;17(7):1269–72. pmid:21762584
  9. 9. Bai Y, Malania L, Castillo DA, Moran D, Boonmar S, Chanlun A, et al. Global distribution of Bartonella infections in domestic bovine and characterization of Bartonella bovis strains using multi-locus sequence typing. PLoS One. 2013;8(11):e80894. pmid:24278342
  10. 10. Lin EY, Tsigrelis C, Baddour LM, Lepidi H, Rolain JM et al. Candidatus Bartonella mayotimonensis and endocarditis. Emerg Infect Dis. 2010;16(3):500–3. pmid:20202430
  11. 11. Veikkolainen V, Vesterinen EJ, Lilley TM, Pulliainen AT. Bats as reservoir hosts of human bacterial pathogen, Bartonella mayotimonensis. Emerg Infect Dis. 2014;20(6):960–7. pmid:24856523
  12. 12. Haque N, Bari MS, Hossain MA, Muhammad N, Ahmed S, Rahman A, et al. An overview of Brucellosis. Mymensingh Med J. 2011;20(4):742–7. pmid:22081201
  13. 13. Stoenner HG, Lackman DB. A new species of Brucella isolated from the desert wood rat, Neotoma lepida Thomas. Am J Vet Res. 1957;18(69):947–51. pmid:13470254
  14. 14. Ewalt DR, Payeur JB, Martin BM, Cummins DR, Miller WG. Characteristics of a Brucella species from a bottlenose dolphin (Tursiops truncates). J Vet Diagn Invest. 1994;6(4):448–52. pmid:7858024
  15. 15. Hubálek Z, Scholz HC, Sedlácek I, Melzer F, Sanogo YO, Nesvadbová J. Brucellosis of the common vole (Microtus arvalis). Vector Borne Zoonotic Dis. 2007;7(4):679–87. pmid:18021023
  16. 16. Scholz HC, Hofer E, Vergnaud G, Le Fleche P, Whatmore AM, Al Dahouk S. Isolation of Brucella microti from mandibular lymph nodes of red foxes, Vulpes vulpes, in lower Austria. Vector Borne Zoonotic Dis. 2009;9(2):153–6. pmid:18973444
  17. 17. Tiller RV, Gee JE, Frace MA, Taylor TK, Setubal JC, Hoffmaster AR, et al. Characterization of novel Brucella strains originating from wild native rodent species in North Queensland, Australia. Appl Environ Microbiol. 2010;76(17):5837–45. pmid:20639360
  18. 18. Ricciardi ID, Nunes MP, Andrade CM, Da Silva AG. Anti-brucella agglutinins in bats and "Callithrix" monkeys. J Wildl Dis. 1976;12(1):52–4. pmid:815569
  19. 19. Levett PN. Leptospirosis. Clin Microbiol Rev. 2001;14(2):296–326. pmid:11292640
  20. 20. Adler B, Moctezuma AP. Leptospira and leptospirosis. Vet Microbiol. 2010;140(3–4):287–96. pmid:19345023
  21. 21. Acha PN, Szyfres B. Zoonosis y enfermedades transmisibles comunes al hombre y a los animales (3. ed.), 1, Organización Panamericana de la Salud, Washington. 2001;pp. 175–186 (Publicación Científica y Técnica, 580).
  22. 22. Cox TE, Smythe LD, Leung LK. Flying foxes as carriers of pathogenic Leptospira species. J Wildl Dis. 2005;41(4):753–7. pmid:16456164
  23. 23. Matthias MA, Díaz MM, Campos KJ, Calderon M, Willig MR, Pacheco V, et al. Diversity of bat-associated Leptospira in the Peruvian Amazon inferred by bayesian phylogenetic analysis of 16S ribosomal DNA sequences. Am J Trop Med Hyg. 2005;73(5):964–74. pmid:16282313
  24. 24. Dietrich M, Mühldorfer K, Tortosa P, Markotter W (2015) Leptospira and Bats: Story of an Emerging Friendship. PLoS Pathog. 2015;11(11):e1005176. pmid:26562435
  25. 25. Dollinger P, Baumgartner R, Hatt JM, Isenbugel E, Pagan O. Zoonoses surveillance and safeguard measures in Swiss zoos. Proc EAZWV and BVSII Scientific Meeting, Chester, United Kingdom. 1998;1–12.
  26. 26. Mair NS. Pseudotuberculosis in free-living wild animals. Symp Zool Soc Lond. 1968;24: 107–117.
  27. 27. Mair NS. Yersiniosis in wildlife and its public health implications. Wildl Dis. 1973;9(1):64–71.
  28. 28. Childs-Sanford SE, Kollias GV, Abou-Madi N, McDonough PL, Garner MM, Mohammed HO. Yersinia pseudotuberculosis in a closed colony of Egyptian fruit bats (Rousettus aegyptiacus). J Zoo Wildl Med. 2009;40(1):8–14. pmid:19368235
  29. 29. Mühldorfer K, Wibbelt G, Haensel J, Riehm J, Speck S. Yersinia species isolated from bats, Germany. Emerg Infect Dis. 2010;16(3):578–80. pmid:20202457
  30. 30. Bondyrev IV, Davitashvili ZV, Singh VP. The geography of Georgia—problems and perspectives. 2015;228pp. Springer.
  31. 31. Han BA, Kramer AM, Drake JM. Global Patterns of zoonotic disease in mammals. Trends Parasitol. 2016 Jul;32(7):565–77. pmid:27316904
  32. 32. Malania L, Bai Y, Osikowicz LM, Tsertsvadze N, Katsitadze G, Imnadze P, et al. Prevalence and diversity of Bartonella species in rodents from Georgia (Caucasus). Am J Trop Med Hyg. 2016;95(2):466–71. pmid:27162268
  33. 33. Kandelaki G, Malania L, Bai Y, Chakvetadze N, Katsitadze G, Imnadze P, et al. (2016) Human lymphadenopathy caused by ratborne Bartonella, Tbilisi, Georgia. Emerg Infect Dis. 2016;22(3):544–6. pmid:26889959
  34. 34. Sanodze L, Bautista CT, Garuchava N, Chubinidze S, Tsertsvadze E, Broladze M, et al. Expansion of brucellosis detection in the country of Georgia by screening household members of cases and neighboring community members. BMC Pubic Health. 2015;15:459.
  35. 35. Mamuchishvili N, Kuchuloria T, Mchedlishvili I, Imnadze P. Leptospirosis in Georgia. Georgian Med News. 2014;(228):63–6. pmid:24743125
  36. 36. Yavruyan E, Rakhmatulina I, Bukhnikashvili A, Kandaurov A, Natradze I, Gazaryan S. Bats conservation action plan for the Caucasus. 2008;83pp. Universal, Tbilisi, Georgia.
  37. 37. Halling SM, Tatum FM, Bricker BJ. Sequence and characterization of an insertion sequence, IS711, from Brucella ovis. Gene. 1993;133(1):123–7. pmid:8224885
  38. 38. Stoddard RA. Detection of pathogenic Leptospira spp. through real-time PCR (qPCR) targeting the LipL32 gene. Methods Mol Biol. 2013;943:257–66. pmid:23104295
  39. 39. Diniz PP, Maggi RG, Schwartz DS, Cadenas MB, Bradley JM, Hegarty B, et al. Canine bartonellosis: serological and molecular prevalence in Brazil and evidence of co-infection with Bartonella henselae and Bartonella vinsonii subsp. berkhoffii. Vet Res. 2007;38(5):697–710. pmid:17583666
  40. 40. Baily GG, Krahn JB, Drasar BS, Stoker NG. Detection of Brucella melitensis and Brucella abortus by DNA amplification. J Trop Med Hyg. 1992;95(4):271–5. pmid:1495123
  41. 41. Levett PN, Morey RE, Galloway RL, Turner DE, Steigerwalt AG, Mayer LW. Detection of pathogenic leptospires by real-time quantitative PCR. J Med Microbiol. 2005;54(Pt 1):45–9. pmid:15591254
  42. 42. Kosoy M, Bai Y, Lynch T, Kuzmin IV, Niezgoda M, Franka R, et al. Bartonella spp. in bats, Kenya. Emerg Infect Dis. 2010;16(12):1875–81. pmid:21122216
  43. 43. Morse SF, Olival KJ, Kosoy M, Billeter S, Patterson BD, Dick CW, et al. Global distribution and genetic diversity of Bartonella in bat flies (Hippoboscoidea, Streblidae, Nycteribiidae). Infect Genet Evol. 2012;12(8):1717–23. pmid:22771358
  44. 44. McKee CD, Hayman DT, Kosoy MY, Webb CT (2016) Phylogenetic and geographic patterns of bartonella host shifts among bat species. Infect Genet Evol. 2016;44:382–94. pmid:27473781
  45. 45. Fennestad KL, Borg-Petersen C (1972) Leptospirosis in Danish wild mammals. J Wildl Dis. 1972;8(4):343–51. pmid:4564183
  46. 46. Bessa TA, Spichler A, Chapola EG, Husch AC, de Almeida MF, Sodré MM, et al. The contribution of bats to leptospirosis transmission in Sa˜o Paulo City, Brazil. Am J Trop Med Hyg. 2010;82(2):315–7. pmid:20134010
  47. 47. Everard CO, Fraser-Chanpong GM, Bhagwandin LJ, Race MW, James AC. Leptospires in wildlife from Trinidad and Grenada. J Wildl Dis. 1983;19(3):192–9. pmid:6644917
  48. 48. Cosson JF, Picardeau M, Mielcarek M, Tatard C, Chaval Y, Suputtamongkol Y, et al. Epidemiology of Leptospira transmitted by rodents in Southeast Asia. PLoS Negl Trop Dis. 2014;8(6):e2902. pmid:24901706
  49. 49. Olsen SC, Palmer MV (2014) Advancement of knowledge of Brucella over the past 50 years. Vet Pathol. 2014;51(6):1076–89. pmid:24981716