key: cord-0689895-bwsmncpg authors: Nabeshima, Kei; Sato, Shingo; Kabeya, Hidenori; Kato, Chihiro; Suzuki, Kazuo; Maruyama, Soichi title: Isolation and genetic properties of Bartonella in eastern bent-wing bats (Miniopterus fuliginosus) in Japan date: 2020-05-05 journal: Infect Genet Evol DOI: 10.1016/j.meegid.2020.104354 sha: 52d8c3e92123ff335392e51837e5118a508973e3 doc_id: 689895 cord_uid: bwsmncpg Abstract The prevalence and genetic characteristics of Bartonella species in eastern bent-wing bats (Miniopterus fuliginosus) from Japan were investigated. Bartonella bacteria were isolated from 12/50 (24%) of bats examined. Analyses of sequence similarities of the citrate synthase gene (gltA) and RNA polymerase beta-subunit-encoding (rpoB) genes indicated that the isolates from M. fuliginosus were distinct from those present in known Bartonella species as the levels of similarity for both of the genes were lower than the cut-off values for species identification in Bartonella. A phylogenetic analysis of the gltA sequences revealed that the Miniopterus bat-associated strains fell into five genotypes (I to V). Though genotypes I to IV formed a clade with Bartonella from Miniopterus bats from Taiwan, genotype V made a monophyletic clade separate from other bat isolates. In a phylogenetic analysis with the concatenated sequences of the 16S rRNA, gltA, rpoB, cell division protein (ftsZ) gene, and riboflavin synthase gene (ribC) gene, isolates belonging to genotypes I to IV clustered with Bartonella strains from Taiwanese Miniopterus bats, similar to the outcome of the phylogenetic analysis with gltA, whereas genotype V also made a monophyletic clade separate from other bat-associated Bartonella strains. The present study showed that M. fuliginosus in Japan harbor both genus Miniopterus-specific Bartonella suggesting to be specific to the bats in Japan. Bats (Order Chiroptera) are widely distributed and are found on all continents except the polar regions. Thirty-five species of bats are now recognized to be present in Japan (Matsue et al., 2006) ; the genera Pipistrellus, Myotis, Rhinolophus, Eptesicus, and Miniopterus inhabit throughout the country. Bats can serve as reservoirs for various viral zoonotic pathogens such as rabies virus in Desmodus bats in South America, Nipah virus and Hendra virus in Pteropus bats in India and Australia, and SARS coronavirus in Rhinolophus bats in China (Calisher et al., 2006) . Other zoonotic bacteria, such as Campylobacter jejuni and Leptospira spp., have also been detected in Myotis bats in the Netherlands and in Artibeus, Phyllostomus, and Sturnira bats in Peru (Muhldorfer, 2013) . Bartonella bacteria are small, fastidious, and Gram-negative bacilli that parasitize erythrocytes and endothelial cells in many species of mammals. Several Bartonella spp. are known to cause human diseases including cat-scratch disease (B. henselae), trench fever (B. quintana), and Carrión's disease (B. bacilliformis). Blood-sucking ectoparasites have been shown to play an important role in the transmission of Bartonella species among their hosts: cat fleas transmit B. henselae between cats (Chomel et al., 1996) and lice transmit B. quintana between humans (Bonilla et al., 2009 ). There are evidences that bats also serve as a reservoir for (Lin et al., 2012) . To date, however, no information on the prevalence and genetic properties of Bartonella species is available in any bat species in Japan. The present study was conducted to determine the prevalence and genetic characteristics of Bartonella species in Miniopterus fuliginosus bats in Japan. In March 2013, fifty M. fuliginosus were captured at a headrace tunnel in Wakayama Prefecture located in the western part of Japan (33°40`N, 135°23`E) after gaining the permission (license # Nishi 4 and 5) of sample collection from the local government. Blood samples were aseptically collected via heart puncture from bats euthanized following the guidelines for euthanasia of the Japan Veterinary Medical Association. The blood samples were transferred to a blood collection tube containing EDTA, the samples immediately frozen in dry ice, and transported to the Laboratory of Veterinary Public Health, the Department of Veterinary Medicine, College of Bioresource Sciences, Nihon University. The blood samples were stored at −70°C until examined. Ectoparasites were also collected from the bat carcasses during autopsy in the laboratory. Frozen blood samples were thawed at room temperature and 100 μl of each blood sample was separately transferred to sterile 1.5 ml conical tubes. The blood was mixed with 100 μl of medium 199 supplemented with 1 mM sodium pyruvate solution and 20% fetal bovine serum (Life Technologies, Carlsbad, CA, USA). Aliquots (100 μl) of the mixture were placed on two heart infusion agar plates (Difco, Sparks Glencoe, MI, USA) containing 5% rabbit blood. The inoculated plates were incubated at 35°C in a moist atmosphere under 5% CO2 for up to 4 weeks. Bartonella bacteria were tentatively identified by colony morphology (small, gray or cream-yellow, round shape); three colonies were picked from each sample and sub-cultured on a fresh blood agar plate using the same conditions as the primary culture. The identification of ectoparasites on the bats was performed by morphological characteristics under a stereomicroscope SZX16 (Olympus, Tokyo, Japan) by the reference of bat flies in Japan (Sato and Mogi, 2008). Genomic DNA was extracted from each sub-culture colony using InstaGene Matrix (Bio-Rad approximately 390 bp for the gltA and approximately 900 bp for the rpoB were considered to be positive for the genus Bartonella. Nucleotide sequences of the gltA, rpoB and 16S rRNA were compared between the representative bat isolates and the strains of prokaryotes registered in the GenBank/EMBL/DDBJ database using the BLAST program. Sequence similarities in the gltA and rpoB genes were additionally compared with the type strains of Bartonella species. Table 1 . (Myotis blithii) and 89.1% similarity for the rpoB with Bartonella quintana strain Fuller T (Table 3 ). In addition, 16S rRNA sequences of genotypes I to IV were identical to that of Bartonella sp. No.5 from Miniopterus bats in Taiwan. On the other hand, genotype V showed 97.9% similarity with Bartonella sp. F2 isolated from Pteronotus parnellii in Mexico (data not shown). Sequence similarities for the gltA and rpoB between representative isolates and type strains of existing Bartonella species ranged from 87.2 to 91.3% and 88.2 to 89.1%, respectively (Table 4 ). In the phylogenetic analysis based on the gltA sequences, four representative isolates derived from Taiwan. However, genotype V formed a monophyletic clade clearly separate from any bat-associated Bartonella strains. Thus, Japanese M. fuliginosus may harbor a unique Bartonella lineage which has not been found in any other bat species. As reported by the ad hoc committee for the re-evaluation of species definition in bacteriology, the description of a new species should be based on the sequence analysis of housekeeping genes using at least five genes (Stackebrandt et al., 2002) . The phylogenetic tree based on the concatenated sequences of five genes showed that genotypes I to IV were closely related to the isolates from Miniopterus in Taiwan, as in the phylogenetic tree for the gltA. These data suggest that Miniopterus bats in both Japan and Taiwan harbor Bartonella bacteria with high genetic similarities. However, strain bat 24-1 of genotype V formed a clearly independent clade that was separate from other bat isolates and other known Bartonella species. These results suggest that M. fuliginosus in Japan harbor two novel Bartonella species: one is similar to species found in Taiwanese Miniopterus and another is specific to Japanese bats. Previous studies have shown that some of the blood-sucking arthropods that infest bats may be involved in Bartonella transmission among bats (Brook et al., 2015; Muhldorfer, 2013) . Bat flies have been suggested to be a potential vector for Bartonella transmission in bat population in Africa, South America, and Asia as Bartonella DNAs are often detected in these ectoparasites (Stuckey et al., 2017a) . In the present study, we identified bat flies belonging to Nycteribia sp. and P. jenynsii on Miniopterus bats; these bat fly In the present study, we showed that 24% (12/50) of eastern bent-wing bats (Miniopterus fuliginosus) harbored Bartonella bacteria in their blood for the first time in Japan. Phylogenetic analyses based on the gltA and the concatenated sequences with the gltA, rpoB, ribC, ftsZ, and 16S rRNA of the isolates clarified that two novel Bartonella species are present in the bats; one is close to the isolates from Taiwanese Miniopterus bats, and another is distinct from other known bat-associated Bartonella suggesting to be specific to the bats in Japan. J o u r n a l P r e -p r o o f Diversity of Bartonella spp. in bats, southern Vietnam Bartonella spp. in bats, Guatemala. 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