key: cord-0939707-r0pdg96m
authors: Lin, Jen-Wei; Hsu, Yuan-Man; Chomel, Bruno B.; Lin, Liang-Kong; Pei, Jai-Chyi; Wu, Sheng-Hai; Chang, Chao-Chin
title: Identification of novel Bartonella spp. in bats and evidence of Asian gray shrew as a new potential reservoir of Bartonella
date: 2012-04-23
journal: Vet Microbiol
DOI: 10.1016/j.vetmic.2011.09.031
sha: dd99f94a203d0769003a64c678ae81a5945e2c62
doc_id: 939707
cord_uid: r0pdg96m

Many studies indicated that small mammals are important reservoirs for Bartonella species. Using molecular methods, several studies have documented that bats could harbor Bartonella. This study was conducted to investigate the relationship of Bartonella spp. identified in bats and small mammals living in the same ecological environment. During May 2009 and March 2010, a total of 102 blood specimens were collected. By whole blood culture and molecular identification, a total of 6 bats, 1 rodent and 9 shrews were shown to be infected by Bartonella species. After sequencing and phylogenetic analyses of the sequences of gltA, ftsZ, rpoB and ribC genes, these specific isolates from bats were not similar to the known Bartonella species (the similarity values were less than 91.2%, 90.5%, 88.8%, and 82.2%, respectively); these isolates formed an independent clade away from other known Bartonella type strains. The Bartonella spp. isolated from small mammals, which were closely related to Bartonella tribocorum, Bartonella elizabethae, Bartonella grahamii, Bartonella rattimassiliensis and Bartonella queenslandensis, were similar to the findings in previous studies worldwide. Therefore, the results implied that the species of Bartonella strains isolated from small mammals were different from those identified in bats. Our results strongly suggested that the bat isolate could be a new Bartonella species. This study is also the first one to isolate Bartonella organisms from Asian gray shrews, Crocidura attenuata tanakae.

, and Bartonella silvatica (Inoue et al., 2010) . The order Rodentia has been reported as the reservoir for several zoonotic Bartonella species, such as B. elizabethae (Daly et al., 1993) , and B. washoensis (Kosoy et al., 2003) and B. grahamii (Kerkhoff et al., 1999) .

Up to date, more than 4600 species of mammals have been identified in Class Mammalia. The top two orders within mammalian consist of Rodentia and Chiroptera, which are with over 2000 and 930 species, respectively (Wong et al., 2007) . Since the Middle Ages (A.D. 500-1400), many historical records and recent studies have shown that rodents play major roles for transmission of important zoonotic diseases, such as plague and hemorrhagic fever (Meerburg et al., 2009) . Since the 1990s, some studies have also shown that bats were associated with several emerging zoonotic agents, including Hendra, Nipah, rabies, Ebola, and coronavirus-like viruses (Calisher et al., 2006; Maeda et al., 2008; Wong et al., 2007) . Concannon et al. (2005) first demonstrated that bats could be infected with Bartonella in the United Kingdom. In the study using molecular identification of the gltA gene, 5 (two Pipistrellus spp., one Nyctalus noctula, one Myopterus daubentonii, and one Myotis mystacinus) of 60 blood samples collected from bats were detected to harbor Bartonella DNAs (Concannon et al., 2005) . Reeves et al. (2005 Reeves et al. ( , 2007 further detected Bartonella DNA in ectoparasites of bats, such as Trichobius major, Cimex adjunctus, and Sternopsylla texanus, using PCR of the 16S-23S rDNA intergenic spacer (ITS) region. Recently, using whole blood culture, Bartonella organisms were successfully isolated from bats in Kenya, and 106 of the 331 bats tested by whole blood culture were shown Bartonella bacteremic .

Therefore, this study was conducted to investigate if bats could be reservoirs of Bartonella spp. in Taiwan, and to further understand if novel Bartonella spp. could be identified in bats and small mammals trapped from the same ecological environment. Moreover, this study was the first time to investigate if Bartonella organisms could be isolated from Asian gray shrews, Crocidura attenuata tanakae.

During the period of May 2009 and March 2010, a total of 54 bats, 23 rodents and 25 shrews were collected in Taiwan. The animals were trapped from several sites, including Taipei, Taichung, Nantou, Pingtung, Hualian counties, and Dongsha island. The trapping sites for rodents and bats included caves, air-aid shelters, abandoned farm houses, chicken/pig/cattle farms and deep trenches. The specimens were initially collected for the purposes of understanding the ecology of these small mammals, and investigating zoonotic pathogens (i.e., rabies virus, influenza virus, Japanese encephalitis virus and hantavirus) in these animals. The captured animals were humanely anesthetized with zoletil 50 (Virbac Laboratories, 06516 Carros, France) and blood was collected via cardiac puncture using 3 ml syringes fitted with 22 gauge, 1.5 in. needles. The blood specimen was collected in an EDTA tube (10 ml, 1.5 mg/ml) and stored at À80 8C for further isolation of Bartonella species.

A total of 102 whole blood samples collected from bats, rodents, and shrews lived in the same ecological environment were used for Bartonella isolation. For bacterial isolation, 50 ml of thawed whole blood sample was plated onto chocolate agar. The plates were kept at 35 8C in 5% CO 2 incubator for at least 1 month (Boulouis et al., 2005) . The plates were examined weekly for the growth of putative colonies of Bartonella. At least two colonies were randomly selected from the suspected plates and each colony was separately sub-cultured onto two different chocolate agar plates. After harvesting pure bacteria culture, the DNA was extracted and used for further molecular confirmation of Bartonella species by polymerase chain reaction (PCR) and sequence analysis.

The DNA of each isolate was extracted using Viogene DNA/RNA Extraction Kit (Viogene Biotek Corp., Taipei, Taiwan) following the manufacturer's instructions. The primers BhCS.781p and BhCS.1137n were used for amplifying the partial fragment (approximately 390 bp) of the gltA gene of Bartonella species (Norman et al., 1995) . The PCR-positive samples for the gltA gene were further analyzed by PCR of the 16S/23S rRNA intergenic spacer region (ITS) (Jensen et al., 2000) . For further phylogenetic analysis, confirmed Bartonella isolates were analyzed by partial sequences of the ftsZ, rpoB and ribC genes. For PCR amplification of the ftsZ gene, a set of primers Bfp1 and Bfp2 was used to amplify an expected 900-bp product (Zeaiter et al., 2002) . Primers 1400F and 2300R were used to amplify a 900-bp fragment of the rpoB gene (Renesto et al., 2001) . For PCR amplification of the ribC gene, a set of primers ribC-F and ribC-R was used to amplify an expected 800-bp DNA fragment (Inoue et al., 2009) . As the 16S rRNA gene has been well-accepted for species identification of bacteria, the primers P8 and Pc1544 were used for amplifying approximately 1300-1400 bp of the 16S rRNA gene (Heller et al., 1997) .

The confirmed PCR products were sent for automated sequencing (Mission Biotech, Taipei, Taiwan). By phylogenetic analysis, the sequences obtained from the isolates were compared to the sequences of Bartonella type/ reference strains. The accession numbers of the Bartonella type/reference strains used for comparison were listed in Table 2 . The sequences of the 16S ribosomal DNA, ftsZ, gltA, ribC and rpoB genes, and the 16S/23S rRNA ITS of the Bartonella strain isolated in this study have been submitted to the GenBank. The accession numbers of the partial sequences of the gltA, ribC, rpoB, ftsZ and 16S rRNA genes for the isolates in this study were serial numbers of JF500507-JF500522, JF500523-JF500538, JF500539-JF500554, JF500491-JF500506 and JF500555-JF500560, respectively. The sequences of the 16S/23S rRNA ITS region and gltA gene of the isolates in this study were analyzed first by using the blastn program of the NCBI website to search for the closest Bartonella species in the GenBank database. Then, the sequences of 16S ribosomal DNA, ftsZ, gltA, ribC and rpoB genes were aligned by Clustal W method of BioEdit program version 7.0.8 (Tom Hall, Ibis Biosciences, Isis Pharmaceuticals, Carlsbad, CA, USA).

Using sequences of the gltA gene and the merged set of sequences of five genes (16S ribosomal DNA, ftsZ, gltA, ribC and rpoB), further phylogenetic analysis was performed to show the relationship of the Bartonella type/reference strains and the bat isolates in this study. Phylogenetic analysis was performed on the aligned DNA sequences using maximum-parsimony as implemented in PHYLIP version 3.6 (Joseph Felsenstein, Department of Genome Sciences and Department of Biology, University of Washington, Seattle, WA, USA). Bootstrap support was calculated by using 1000 bootstrap data replicates as implemented by SEQBOOT of the PHYLIP program.

As reported by Fournier et al. (2006) , the prokaryote genomic DNA G+C content can be inferred from the sequences of ftsY genes. Therefore, the primers BartftsyF and BartftsyR were used to amplify the fragment of the ftsY gene of Bartonella species by the procedures developed by Fournier et al. (2007) .

Among the 102 small mammals tested, Bartonella organisms were isolated from 16 animals (15.7%) using whole blood culture (Table 1 ). The isolates were from six bats (Miniopterus schreibersii), one rodent (Rattus coxinga niviventer) and nine shrews (including seven Crocidura attenuata tanakae and two without species information). All isolates were confirmed to be Bartonella after sequence analysis of the 16S/23S rRNA ITS region and gltA gene. Bartonella isolates Nos. 5, 6, 7, 8, 15, and 16 were from M. schreibersii and No. 9 was from Rattus coxinga niviventer. The isolates from Crocidura attenuata tanakae were numbered as Nos. 1, 2, 3, 4, 12, 13, and 14. The two isolates from the unknown species of shrews were numbered as Nos. 10 and 11 (Table 1) .

The gltA sequences obtained from the bat isolates were very similar to each other ranging from 99.0% to 100.0%. Further compared to the sequences of the Bartonella type/ reference strains listed in Table 2 , the results indicated that the isolates from bats were not close to any known Bartonella species, with DNA similarity values less than 91.2%. However, the isolate from Crocidura attenuata tanakae and the isolate from Rattus coxinga niviventer were closely related to Bartonella queenslandensis with DNA similarity values of 99.0% and 99.7%, respectively. In comparison with the sequences of the gltA gene of the Bartonella type/reference strains, except for the isolate No. 1 from Crocidura attenuata tanakae, other isolates from shrews were shown DNA similarity values of 95.1-95.5%, closest to B. tribocorum, followed by B. grahamii (DNA similarity value: 94.2-94.5%), and B. elizabethae (DNA similarity value: 93.5-93.8%).

With regard to the sequences of the ftsZ gene, the bat isolates were shown DNA similarity values of 97.1-100% between each other and were distant from all known Bartonella type/reference strains (DNA similarity values <90.5%). One isolate from Crocidura attenuata tanakae and one isolate from Rattus coxinga niviventer were close to B. queenslandensis with DNA similarity values from 99.5% to 99.9%. Except for one isolate (No. 1) from Crocidura attenuata tanakae, other isolates from shrews were shown closest to B. tribocorum (96.7%), followed by B. elizabethae (96.4%), and B. queenslandensis (96.2%).

More divergence of the sequences of the rpoB and ribC genes were observed for the bat isolates, as the DNA similarity values were shown to be 95.1-99.9% and 94.9-100%, respectively. The sequences of these bat isolates were still distinct from the sequences of all known Bartonella type/reference strains obtained in this study; the DNA similarity value for the rpoB gene was less than 88.8% and that for the ribC gene was less than 82.2%). The rpoB gene sequences obtained from one isolate from Crocidura attenuata tanakae and one isolate from Rattus The phylogenetic tree constructed by the gltA gene ( Fig. 1) showed that the bat isolates were grouped into an independent clade, separated from all Bartonella type/ reference strains. The rodent and shrew isolates belonged to the same clade of B. tribocorum, B. elizabethae, B. grahamii, B. rattimassiliensis and B. queenslandensis (Fig. 1) . In this specific clade, one isolate from Crocidura attenuata tanakae and one isolate from Rattus coxinga niviventer were formed a specific group with B. queenslandensis (bootstrap value >90%). Using the merged set of concatenated sequences of five genes (16S ribosomal DNA, ftsZ, gltA, ribC, and rpoB) to construct the phylogenetic relationship, the results also shown that the isolates from bats formed a significantly distinct clade comparing to the other Bartonella type/reference strains (Fig. 2) .

After sequence analysis of the ftsY gene, the genomic DNA G+C content of the Bartonella isolates in bats in Taiwan were estimated to be from 30.29% to 31.15%.

In this study, we investigated the epidemiology of Bartonella infections in bats and other small mammals from the same ecological environment. This is the first report that Bartonella organisms were isolated from bats, M. schreibersii, in Taiwan. Through sequence and phylogenetic analyses of various genes, the results indicated that the isolates from M. schreibersii were not similar to the known Bartonella species as previously described. However, Bartonella spp. isolated from small mammals, were closely related to B. tribocorum, B. elizabethae, B. grahamii, B. rattimassiliensis, and B. queenslandensis, similar to the 

findings in previous studies worldwide Gundi et al., 2009; Hsieh et al., 2010; Liu et al., 2010) . In this study, it was also found that the Bartonella infections could occur among Asian gray shrew (Crocidura attenuata tanakae). To the best of our knowledge, this is the first study to demonstrate the Bartonella infection in Crocidura attenuata tanakae. This shrew is a subspecies belonged to the species of Crocidura attenuate. The species is generally found in the tropical and subtropical zone of Asia, such as India and China.

According to the study by Concannon et al. (2005) , two blood specimens of bats from Pipistrellus spp. have been detected by molecular identification. In our study, a total of 35 blood specimens from the bats, Pipistrellus abramus, belonging to the same genus, were collected. However, no Bartonella isolates have been obtained from these specimens by whole blood culture. It might be related to the limited samples tested in our study, or related to the sensitivity of different detection methods. Using the sequences of the partial gltA gene, when the sequences found in Pipistrellus spp. by Concannon et al. (2005) were compared to the sequences of the bat isolates in our study, the DNA similarity values of 86.7-90.9% indicated that Bartonella species in bats in the United Kingdom were not closely related to the isolates in Taiwan. Our phylogenetic analysis further supported that bat isolates in Taiwan formed a unique clade, which was separated from the sequences of Bartonella in bats in the United Kingdom (Fig. 1) . Recently, Kosoy et al. (2010) reported Bartonella isolates from bats in Kenya. Among the 331 bats tested, Bartonella were isolated from 106 animals using whole blood culture. The Bartonella-bacteremic bat species were Eidolon helvum (23/88), Rousettus aegyptiacus (22/105), Coleura afra (4/9), Triaenops persicus (7/8), Hipposideros commersoni (1/4), and Miniopterus spp. (49/87) . In our study, we also tested the bats from the same genus of Miniopterus (M. schreibersii) and Hipposideros (Hipposideros terasensis). Due to only one sample of H.

[ ( F i g . _ 1 ) T D $ F I G ] terasensis tested, no Bartonella isolate was obtained in our study. However, we cultured Bartonella isolates from the blood of 42.9% (6/14) of the M. schreibersii. After sequence analysis, the results indicated that the DNA identity of these isolates from M. schreibersii in our study was not closely related to the strains isolated in Miniopterus spp. in to the strain HM545139, and 92.5-92.9% to the strain HM545140).

Of major interests, after comparing the sequences of the partial gltA gene of the bat isolates in Taiwan to the available sequence data in the GenBank, it was found that the bat isolates were most similar to the uncultured Bartonella spp. isolates KK49 and KK61, with DNA similarity value of 95.5-96.8%. The KK49 and KK61 sequences were amplified from stray dogs in Thailand in 2010 . Therefore, further investigation should be conducted to determine whether these Bartonella organisms could be possibly transmitted between bats and dogs.

Arthropods play an essential role in the transmission cycle of Bartonella organisms between hosts. The studies by Reeves et al. (2005 Reeves et al. ( , 2007 collected ectoparasites from bats and successfully detected Bartonella DNA from the T. major, C. adjunctus, and S. texanus using PCR of the 16S-23S rRNA intergenic spacer (ITS) region. Further research should be conducted to investigate whether the Bartonella isolates found in bats in Taiwan could be transmitted in bats or between bats and dogs through common vectors.

In this study, it was further identified at least two genotypes of the bat isolates in Taiwan. Based on the [ ( F i g . _ 2 ) T D $ F I G ] Fig. 2 . Phylogenetic analysis of different Bartonella species based on the sequences of a merged set of concatenated sequences of five genes (16S rRNA, ftsZ, gltA, ribC and rpoB genes). The phylogenetic relationship was constructed by using the maximum-parsimony method of the PHYLIP version 3.6 program, and bootstrap analysis was performed with 1000 trials of bootstrap data (bootstrap values not shown if lower than 70%). sequence comparison, the sequences of the gltA genes and 16S ribosomal DNA were highly similar among the Bartonella isolates in bats in Taiwan (DNA similarity values: 99-100%,  and 99 .9-100%, respectively). However, the sequences of the ftsZ, rpoB and ribC genes were slightly divergent (DNA similarity values: 97.1-100%, 95.1-99.9% and 94.9-100%, respectively). On the basis of sequencing and phylogenetic analyses of gltA, ftsZ, rpoB, ribC genes and 16S ribosomal DNA, the bat isolates No. 7 and No. 16 could belong to one genotype, as their rpoB and ribC sequences were not similar to the other Bartonella isolates in bats in Taiwan (divergence values: 4.3-4.8% and 4.7-5.1%, respectively).

It is of major interests to understand if the bat isolates in Taiwan could be a new Bartonella species. In our study, to study the bat isolates, except for the gltA gene, sequences of the 16S rRNA, ftsZ, ribC, and rpoB gene were also analyzed. As reported by the ad hoc committee for the re-evaluation of the species definition in bacteriology, descriptions of a new species could be based on housekeeping gene (encoded metabolic functions) sequence analysis with a comparison of at least five genes (Stackebrandt et al., 2002) . The study by La Scola et al. (2003) also documented that newly encountered Bartonella isolates should be considered as a new species if the 327-bp gltA fragment and 825-bp rpoB fragment shared <96.0% and <95.4% sequence similarity with those of validated species, respectively. In comparison with the sequences of the gltA, ftsZ, rpoB and ribC genes of the Bartonella type/reference strains, the DNA similarity values of the bat isolates in Taiwan were less than 91.2%, 90.5%, 88.8%, and 82.2% to all known Bartonella species, respectively. After phylogenetic analyses using the merged set of concatenated sequences of the 16S ribosomal DNA, gltA, ftsZ, rpoB, ribC genes, all the results strongly suggested that the isolates from M. schreibersii in Taiwan could be considered as a new Bartonella species.

The results of the estimated genomic DNA G+C content of the Bartonella isolates in bats in Taiwan using the sequences of the ftsY gene were from 30.29% to 31.15%, which were much less than the Bartonella type/reference strains (ranging from 37% to 41%) (Bermond et al., 2000 (Bermond et al., , 2002 . It was also found that the segment of the ftsY genes in the bat isolates in Taiwan was shorter than Bartonella species tested, including Bartonella australis, Bartonella henselae, Bartonella quintana, Bartonella grahamii, Bartonella bacilliformis, and B. tribocorum (Fournier et al., 2006 (Fournier et al., , 2007 . Although it is still unclear why the ftsY gene of the bat isolates in Taiwan differs from the Bartonella type/ reference strains, further studies will be conducted to examine the possible relationship between the divergence of the ftsY gene and host specificity.

At present, no bat-associated Bartonella infection was documented in humans. In Taiwan, bats and small mammals live very closely to the human living environment. As bats play important roles for the transmission of certain emerging zoonoses, physicians and veterinarians should be aware of accidental Bartonella infections from bats.

Our results implied that the species of Bartonella strains isolated from small mammals were different from those identified in bats. Furthermore, the bat isolate could be a new Bartonella species. This study is also the first one to isolate Bartonella organisms from Asian gray shrews, Crocidura attenuata tanakae.

Prevalence and genetic heterogeneity of Bartonella strains cultured from rodents from 17 provinces in Thailand

Enrichment culture and molecular identification of diverse Bartonella species in stray dogs

Bartonella birtlesii sp. nov., isolated from small mammals (Apodemus spp.)

Bartonella bovis sp. nov. and Bartonella capreoli sp. nov., isolated from European ruminants

Proposals to unify the genera Grahamella and Bartonella, with descriptions of Bartonella talpae comb. nov., Bartonella peromysci comb. nov., and three new species, Bartonella grahamii sp. nov., Bartonella taylorii sp. nov., and Bartonella doshiae sp. nov

Factors associated with the rapid emergence of zoonotic Bartonella infections

Bats: important reservoir hosts of emerging viruses

Molecular characterization of haemoparasites infecting bats (Microchiroptera) in Cornwall

Rochalimaea elizabethae sp. nov. isolated from a patient with endocarditis

Rats of the genus Rattus are reservoir hosts for pathogenic Bartonella species: an Old World origin for a New World disease?

Estimation of prokaryote genomic DNA G+C content by sequencing universally conserved genes

Bartonella australis sp. nov. from kangaroos

Isolation of Bartonella rattimassiliensis sp. nov. and Bartonella phoceensis sp. nov. from European Rattus norvegicus

Bartonella rattaustraliani sp. nov., Bartonella queenslandensis sp. nov. and Bartonella coopersplainsensis sp. nov., identified in Australian rats

Prevalence of Bartonella henselae and Bartonella clarridgeiae in stray cats

Bartonella tribocorum sp. nov., a new Bartonella species isolated from the blood of wild rats

Epidemiology of Bartonella infection in rodents and shrews in Taiwan

Evolutional and geographical relationships of Bartonella grahamii isolates from wild rodents by multi-locus sequencing analysis

Bartonella japonica sp. nov. and Bartonella silvatica sp. nov., isolated from Apodemus mice

Rapid identification and differentiation of Bartonella species using a single-step PCR assay

Demonstration of Bartonella grahamii DNA in ocular fluids of a patient with neuroretinitis

Bartonella strains from ground squirrels are identical to Bartonella washoensis isolated from a human patient

Bartonella spp. in bats

Gene-sequence-based criteria for species definition in bacteriology: the Bartonella paradigm

Isolation of Bartonella species from rodents in Taiwan including a strain closely related to 'Bartonella rochalimae' from Rattus norvegicus

Detection of Bartonella species in small mammals from Zhejiang Province

Isolation of novel adenovirus from fruit bat (Pteropus dasymallus yayeyamae)

Rodent-borne diseases and their risks for public health

Differentiation of Bartonella-like isolates at the species level by PCRrestriction fragment length polymorphism in the citrate synthase gene

Molecular evidence for novel Bartonella species in Trichobius major (Diptera: Streblidae) and Cimex adjunctus (Hemiptera: Cimicidae) from two southeastern bat caves, USA

Association of Bartonella with the fleas (Siphonaptera) of rodents and bats using molecular techniques

Use of rpoB gene analysis for detection and identification of Bartonella species

Report of the ad hoc committee for the re-evaluation of the species definition in bacteriology

Isolation of a new subspecies, Bartonella vinsonii subsp. arupensis, from a cattle rancher: identity with isolates found in conjunction with Borrelia burgdorferi and Babesia microti among naturally infected mice

Bats as a continuing source of emerging infections in humans

Genetic and ecologic characteristics of Bartonella communities in rodents in southern China

Genetic classification and differentiation of Bartonella species based on comparison of partial ftsZ gene sequences

This project was supported by the grant of NSC 95-2313-B-005-028-MY2 from the National Science Council, Taiwan.