Prevalence And Genetic Diversity of Bartonella Spp. In Northern Bats (Eptesicus Nilssonii) And Their Blood-Sucking Ectoparasites In Hokkaido, Japan

doi:10.21203/rs.3.rs-670515/v1

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Prevalence And Genetic Diversity of Bartonella Spp. In Northern Bats (Eptesicus Nilssonii) And Their Blood-Sucking Ectoparasites In Hokkaido, Japan

https://doi.org/10.21203/rs.3.rs-670515/v1

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published 03 Jan, 2022

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We investigated the prevalence of Bartonella in northern bats (Eptesicus nilssonii) and their ectoparasites from Hokkaido, Japan. Two batbugs (Cimex japonicus) and 174 bat fleas (Ischnopsyllus needhami) were collected from the bats. Bartonella bacteria were isolated from 32 (26.0%) of 123 bats examined. Though Bartonella DNA was detected in 79 (45.5%) of bat fleas, the bacterium was isolated from only one bat flea (0.6%). The gltA sequences of the isolates were categorized into genotypes I, II, and III, which were found in common between the bats and fleas. The gltA sequences of genotypes I and II showed 97.6% similarity with the gene from E. nilssonii in Finland and a bat flea from E. serotinus in the Netherlands. The rpoB sequences of the genotypes showed 98.9% similarity with strain 44722 from E. serotinus in Georgia. The gltA and rpoB sequences of genotype III showed 95.9% and 96.7% similarity with strains from two different species of shrews in Kenya and France, respectively. Phylogenetic analysis revealed that Bartonella isolates of genotypes I and II clustered with strains from Eptesicus bats in Georgia and Finland, Myotis bats in Romania and the UK, and a bat flea from an Eptesicus bat in Finland. In contrast, genotype III formed a clade with B. florencae, B. acomydis, and B. birtlesii. These data suggest that northern bats in Japan harbor two Bartonella species and I. needhami likely serves as a potential vector of Bartonella transmission in the bats.

Behavioral Ecology

Evolutionary Biology

Bartonella

bat flea

Japan

Northern bats

A total of 1,314 bat species are currently recognized in the world [1]. The largest bat family Vespertilionidae, known as Vesper bats, consists of 426 bat species and Pipistrellus, Myotis, Vespertilio, Plecotus, Murina, and Eptesicus are the common genera in Japan [2]. Recently, it has been reported that Vesper bats carry pathogenic bacteria, including Campylobacter, Clostridium, Listeria, Salmonella, Shigella, Vibrio, Yersinia, Borrelia, Grahamella, Neorickettsia, Leptospira, and Bartonella [3].

Bartonella are small, fastidious Gram-negative bacteria that parasitize erythrocytes and endothelial cells of various mammals. Bartonella infections have been found in various bat species belonging to Vesper bats, genera Myotis and Eptesicus in Europe, Asia, and the Americas [4]. Bartonella mayotimonensis was first detected from an aortic valve of a patient with endocarditis [5], and then a B. mayotimonensis-like organism was also found in Eptesicus bats in Finland and Myotis bats in Finland, France, and Spain [6, 7]. Additionally, it has been reported that the gltA sequences of Bartonella bacteria isolated from Eptesicus and Myotis bats in Georgia are closely related to those from forest workers in Poland [8]. These data suggest that Bartonella bacteria in Eptesicus and Myotis bat species are transmissible between the bats and humans and are pathogenic for humans.

Ectoparasites such as cat fleas [9], body lice [10], sand flies [11], and the deer ked [12] are known to be competent vectors of B. henselae, B. quintana, B. bacilliformis, and deer associated strains, respectively. Previous studies have shown that some ectoparasites, such as bat flies may be involved in Bartonella transmission among bats. Bartonella DNA has been frequently detected in nycteribiid and streblid bat flies collected in Japan [13] and other 19 countries [4, 14–20]. In particular, Bartonella bacteria were isolated from a Cyclopodia bat fly on Eidolon helvum in Ghana [19] and from three Nycteribia species that infested Miniopterus fuliginosus in Japan [13]. These findings suggest that bat flies may serve as the major vector for the transmission of Bartonella among bats regardless of country.

We previously reported that Bartonella bacteria are prevalent in 24% (12/50) of Miniopterus fuliginosus in Japan [21]. Furthermore, detection of Bartonella have been also reported in various Asian bat species at high prevalence, such as Taphozous melanopogon (100%; 1/1), Hipposideros sp. (37.2%; 16/43), and Chaerephon plicatus (39.5%; 17/43) in Thailand [22], Hipposideros sp. (45.5%; 5/11), Rhinolophus spp. (43.3%; 13/30), Megaderma spp. (33.3%; 1/3), and Megaerops niphanae (50%; 1/2) in Vietnam [23], Miniopterus schreibersii (42.9%; 6/14) in Taiwan [24], and Rhinolophus spp. (23%; 3/13) and Myotis spp. (30.4%; 24/79) in China [25]. Despite Myotis and Eptesicus bats are suspected of having pathogenic Bartonella species for humans [6], no epidemiological studies of the bacteria and the vectors have not been conducted in these bat species in Japan so far.

The aims of the present study were to investigate the prevalence and genetic properties of Bartonella bacteria in northern bats (Eptesicus nilssonii) and to identify a potential vector for the organism in the bat species in Japan.

2.1 Collection of blood and ectoparasites from bats

A total of 123 northern bats (Eptesicus nilssonii) were captured in Yakumo City (42°29`N, 140°18`E), Hokkaido Prefecture, located in the northern part of Japan. Bat samples were separately collected in August 2017 (N = 49), June 2018 (N = 38), and August 2018 (N = 36). The bats were living within the exterior walls of an abandoned building in dense colonies as shown in the supplementary video. Before performing the present study, we obtained individual permissions to capture bats from the Oshima general sub-prefectural bureau, Hokkaido government (license #: Oshima 27, 28, 29, 122, 123, and 124).

Blood samples were aseptically collected from the bats via heart puncture using the same procedures as previously reported [21]. The blood samples and the carcasses were immediately sent to the Laboratory of Veterinary Public Health at the Department of Veterinary Medicine, College of Bioresource Sciences, Nihon University under frozen conditions with dry ice. All of the samples were stored at − 70°C until they were examined and tested.

A total of 174 bat fleas and two batbugs were obtained from the surfaces of the carcasses in the laboratory. The ectoparasites were morphologically identified at the species level under a stereo microscope SZX16 (Olympus, Tokyo, Japan) with taxonomic keys [26, 27]. Rates of parasitism by species were compared using chi-square tests with P values < 0.01 and considered to be statistically significant.

2.2 Isolation of Bartonella bacteria from bats

Isolation of Bartonella bacteria from the bat blood samples exactly followed a previously described method [21]. Bartonella bacteria were tentatively identified by colony morphology (small, white, round shape) and three colonies per sample were sub-cultured on a fresh blood agar plate using the same conditions as the primary culture.

2.3 Detection of Bartonella DNA from ectoparasites

To avoid bacterial and fungal contamination on the surface of the ectoparasites, each sample was sterilized by immersing in 500 µl of 70% ethanol containing 0.35% povidone-iodine (Shionogi & Co., Ltd, Osaka, Japan) for 10 minutes. Then, the samples were once washed with 1 ml of 0.01 M PBS with 0.5% FBS and transferred into shatter-resistant 2.0 ml tubes (SSIbio, Lodi CA, USA). After adding 400 µl of sucrose phosphate glutamate (SPG; 10 mM sodium phosphate, 220 mM sucrose, and 0.5 mM L-glutamic acid), each sample was homogenized using a bead crusher µT-12 (TAITEC corp., Saitama, Japan) at 3,000 rpm for 1 min. Genomic DNA was extracted from the homogenate aliquot (100 µl) by InstaGene Matrix (Bio-Rad Laboratories, Inc. Hercules, CA, USA).

Real-time PCR targeting the transfer-mRNA (ssrA) gene of the genus Bartonella was used for screening Bartonella DNA [28]. Real-time PCR assays were performed in 20 µl reaction mixtures containing 10 µl of TB Green Premix Ex Taq II (Takara Bio Inc., Shiga, Japan), 2 µL of DNA sample (approximately 10 to 20 ng/µl), 1 µl of each primer (10 nM), and 6 µl of nuclease-free water. The PCR conditions were as follows: 95°C for 30 seconds, followed by 40 cycles at 95°C for 5 seconds and 60°C for 30 seconds. The genomic DNA from the northern bat isolate (EN2-1) was used as a positive control and nuclease-free water as a negative control in this study. The targeted DNA was amplified with a Thermal Cycler Dice Real Time System II (Takara Bio Inc). Melting curve analysis was applied to the results with a Ct value lower than 35 cycles. When a sample showed 80 ± 1°C melting temperature, the sample was defined as positive by the assay. Each positive sample was additionally analyzed by conventional PCR targeting the citrate synthase gene (gltA) [29]. The conventional PCR products were separated on 3% agarose gels by electrophoresis and were visualized by staining with ethidium bromide under UV light. When samples tested positive for both PCRs, the samples were defined as being infected with Bartonella bacteria. A band showing the expected size for gltA (approximately 380 bp) was purified by using the Wizard SV Gel and PCR Clean-Up System (Promega, Madison, WI, USA) following the manufacturer’s instructions.

The purified PCR products of the gltA gene were directly sequenced using a BigDye Terminator Cycle Sequencing Ready Reaction kit and a Genetic Analyzer model 3130 (Applied Biosystems). When double peaks were detected in the DNA sequencing chromatograms, the PCR products were cloned using the plasmid pGEM-T Easy vector system (Promega) and resequenced. The plasmids were purified from the transformed cell lysates with a plasmid purification kit (PureYield Plasmid Miniprep System; Promega) and then were sequenced using the primers SP6 (5’-CAAGCTATTTAGGTGACACTATAG-3’) and T7 (5’-TAATACGACTCACTATAGGG-3’).

2.4 Isolation of Bartonella bacteria from ectoparasites

The homogenates of Bartonella DNA-positive ectoparasites were subsequently used for the isolation of Bartonella bacteria following the previously described method [13].

2.5 PCR amplification of the gltA and rpoB genes from Bartonella isolates

Genomic DNA was extracted from each sub-cultured colony by using InstaGene Matrix (Bio-Rad Laboratories) and subjected to genus-specific PCR targeting the gltA [29] and RNA polymerase beta-subunit-encoding (rpoB) genes [30]. The PCR products were separated on 2% agarose gels by electrophoresis and visualized by staining with ethidium bromide under UV light. Any samples showing the expected band sizes for gltA and rpoB (approximately 850 bp) were considered as members of the genus Bartonella. The PCR products were then purified using the Wizard SV Gel and PCR Clean-Up System (Promega) and directly sequenced as previously described [21].

2.6 Genotyping.

Genotypes of the gltA genes were determined when unique sequence variants with ≥ 1 nucleotide difference were found in each isolate by comparing the gltA sequences using Genetyx software Ver 12 (Genetyx Corporation, Tokyo, Japan). Representative isolates from each genotype were further analyzed by sequencing their rpoB genes.

2.7 Sequence homology analysis

The gltA and rpoB sequences of representative strains were compared with genomic sequences of prokaryotes registered in the GenBank/EMBL/DDBJ database using the BLAST program.

2.8 Phylogenetic analysis

Based on evolutionary model selection using JModelTest2 [31] with Akaike’s information criterion corrected for finite sample sizes (AICc) [32], the generalized time-reversible substitution model with four gamma-distributed categories and a proportion of invariant sites (GTR + G + I) model was the best available model for the phylogenetic analyses based on the gltA sequences.

A phylogenetic tree of the gltA sequences was constructed using the maximum-likelihood method based on the GTR + G + I model in MEGA 7 [33]. Known Bartonella species (N = 40), bat-associated Bartonella strains (N = 420) derived from 36 countries, and Brucella melitensis 16M as an outgroup were included in this analysis. Strain names, host types, host scientific names, countries where the bats and ectoparasites were collected are summarized together with references and the gltA accession numbers in supplementary Table 1.

3.1 Ectoparasite fauna in the northern bats

All bat fleas (N = 174) were morphologically identified as Ischnopsyllus needhami and the two batbugs as Cimex japonicus. Parasite prevalence of I. needhami and C. japonicus in bats were 59.3% (71/123) and 2.4% (2/123), respectively and the prevalence of I. needhami was significantly higher than that of C. japonicus (p < 0.01). Parasite prevalence of bat fleas varied by sampling period:63.2% (31/49) in August 2017, 71.1% (27/38) in June 2018, and 36.1% (13/36) in August 2018 (Table 1).

Table 1 Prevalence of Bartonella and I. needhami in Japanese northern bats by sample collection date

Sample collection date	Number of bats examined	*Number of Bartonella* positive bats (%)**	*Number of I. needhami* positive bats (%)**
2017-Aug	49	7 (14.2)	31 (63.2)
2018-June	38	11 (28.9)	27 (71.1)
2018-Aug	36	14 (38.9)	13 (36.1)
Total	123	32 (26.0)	71 (59.3)

3.2 Isolation of Bartonella bacteria from northern bats and their ectoparasites, and gltA genotypes of the isolates

Bartonella bacteria were isolated from 26% (32/123) of the northern bats and 0.6% (1/174) of I. needhami, but not from C. japonicus. The prevalences of Bartonella in the bats by sampling period were 14.2% (7/49) in August 2017, 28.9% (11/38) in June 2018, and 38.9% (14/36) in August 2018 (Table 1). A total of 99 isolates were obtained from 32 bat and one flea samples (3 isolates per one positive sample). In 71 northern bats with I. needhami, 28.2% (20/71) were positive for Bartonella bacteria, whereas 23.1% (12/52) of the bats without the bat fleas were positive for Bartonella bacteria. There was no significant difference in the rates between I. needhami positive and negative bats.

The bat isolates were conveniently classified into three genotypes as genotypes I (33 isolates), II (33 isolates), and III (30 isolates) based on the gltA sequences. None of the bats was co-infected with multiple genotypes. Three isolates from one I. needhami were classified into the genotype I. The gltA and rpoB sequences of representative bat strains (EN2-1, EN19-2, and EN36-1) were registered in the GenBank/EMBL/DDBJ database (Table 2).

Table 2 Genotypes and accession numbers of gltA and rpoB genes of representative strains from Japanese northern bats and bat fleas

Genotype	No. of isolates	Representative strain	gltA accession #	rpoB accession #
I	36^*	EN2-1	LC596942	LC596945
II	33	EN19-2	LC596943	LC596946
III	30	EN36-1	LC596944	LC596947

* Three of the isolates were derived from bat fleas.

3.3 Detection of Bartonella DNA from bat ectoparasites and gltA genotypes of Bartonella DNA

Bartonella DNA was detected from 79 I. needhami (45.5%; 79/174), but not from C. japonicus. The gltA sequences from the bat fleas were also classified into genotypes I, II, and III, which are common with those of the bat isolates. The numbers of I. needhami harboring only one genotype were 43 for genotype I, 21 for genotype II, and 10 for genotype III, respectively (Table 3). Fleas harboring two genotypes were found in five individuals: genotypes I and II in two fleas, genotypes II and III in one flea, and genotypes I and III in two fleas.

Table 3 The gltA genotypes of Bartonella DNA detected in bat fleas (I. needhami)

gltA genotypes	No. of bat fleas
I	43
II	21
III	10
I+II	2
II+III	1
I+III	2
Total	79

3.4 Sequence similarities of the gltA and rpoB genes between the three genotypes and other Bartonella strains

The sequence similarities between the three genotypes and the closest Bartonella strains based on BLAST searches are shown in Table 4.

Table 4 Sequence similarities in the gltA and rpoB genes between the representative northern bat strains and closest strains.

Strain (genotype)		Closest strain (Host) in gltA sequences	% similarity	Closest strains (Host) in rpoB sequences	% similarity
EN2-1 (I)	Bartonella sp. 1157/3 (Eptesicus nilssonii) Uncultured Bartonella sp. clone 1F40 (Ischnopsyllus variabilis)		97.9%	Bartonella sp. 44722 (Eptesicus serotinus)	98.8%
EN19-2 (II)
EN36-1 (III)	Bartonella sp. B28303 (Crocidura olivieri)		95.9%	B. florencae R4^T(Crocidura russula)	96.7%

The gltA sequences of strains EN2-1 (genotype I) and EN19-2 (genotype II) showed 97.6% similarity with those of strain 1157/3 from a northern bat in Finland and strain 1F40 from a bat flea (Ischnopsyllus variabilis) in the Netherlands. Only one nucleotide base in the gltA was different between strains EN2-1 and EN19-2. The gltA sequence of strain EN36-1 (genotype III) showed 95.9% similarity with that of strain B28303 from an African giant shrew (Clocidura olivieri) in Kenya.

The rpoB sequences of strains EN2-1 and EN19-2 were identical each other and showed 98.9% similarity with that of strain 44722 from a serotine bat (E. serotinus) in Georgia. The rpoB sequence of EN36-1 showed 96.7% similarity with B. florencae R4^T from a greater white-toothed shrew (Clocidura russula) in France.

3.5 Phylogenetic analysis of bat isolates based on the gltA sequences

Three representative strains were divided into two different clades. Strain EN2-1 (genotype I) and strain EN19-2 (genotype II) formed clade A with strains from Myotis bats in the UK and Romania, Eptesicus bats in Georgia and Finland, and a flea (I. variabilis) infesting E. serotinus in the Netherlands. On the other hand, strain EN36-1 (genotype III) clustered in clade B with B. acomydis KS2-1^T from a golden spiny mouse (Acomys russatus), B. florencae R4^T, and B. birtlesii IBS325^T from a wood mouse (Apodemus speciosus) (Fig. 1).

In the present study, 26% (32/123) of Japanese northern bats, E. nilssonii were found to be infected with Bartonella bacteria. According to previous studies, Bartonella bacteria were also isolated from Eptesicus bats: E. nilssonii (33.3%; 1/3) in Finland [6] and E. serotinus (25%; 4/20) in Georgia [8]. The prevalence rate of Bartonella bacteria in Japanese northern bats seems to be quite similar to those of previous two countries. In contrast, no Bartonella was detected in E. serotinus in China [25] or France [7]. Since these results suggest that Bartonella prevalence in Eptesicus bats may vary depending on the locations of the bat populations and/or bat species, more investigations will be necessary to clarify the causes of the differences in Bartonella prevalence in different regions and bat species.

Our study showed that Japanese northern bats were infested with two species of ectoparasites: bat fleas (Ischnopsyllus needhami) and batbugs (Cimex japonicus). The parasitic rate of I. needhami was significantly higher than that of C. japonicus (p < 0.01), indicating that a dominant ectoparasite in Japanese northern bats is I. needhami. Previous studies have shown that various ectoparasites such as Ischnopsyllus spp. and Myodopsylla borealis (bat fleas), Cimex spp. (batbug), Carios kelleyi (soft tick), and Basilia forcipata (bat fly) parasitize Eptesicus bats in Russia and the USA [34, 35]. However, we did not find any other ectoparasites other than I. needhami and C. japonicus in this study. Hence, the fauna of ectoparasites on Eptesicus bats may depend on the season, habitat, or bat species.

Bartonella DNA was frequently detected from bat fleas (45.4%; 79/174) and Bartonella bacteria were isolated from one bat flea. In the present study, the isolation rate is much lower than the detection rate by PCR. The isolation of Bartonella from fleas was performed after finishing identification of all ectoparasites and detection of Bartonella DNA, and it took about 1 to 2 months. In the previous studies, the isolation rate of Bartonella from fresh deer ked samples was reported to be considerably high as 51.5% [12] and 73.3% [36]. Therefore it is necessary to use samples as fresh as possible for understanding the actual prevalence of the viable bacteria in ectoparasites. Since the northern bats that we sampled were living in dense conditions within the exterior walls of a building (Supplementary video), it seems that the fleas can easily move between bat bodies in the colony. When comparing the gltA genotypes of Bartonella between the bats and the fleas, multiple genotypes were detected in the fleas, but the bats were infected with only one genotype. The evidence supports the possibility that the bat fleas actively move and suck blood of northern bats in the colony, and the bat fleas are likely to transmit Bartonella bacteria to individual bats.

In the BLAST searches, the gltA and rpoB sequences of strains EN2-1 (genotype I) and EN19-2 (genotype II) showed the highest similarities (97.9% in gltA and 98.8% in rpoB) with strains 1157/3 from a northern bat in Finland, 1F40 from Ischnopsyllus variabilis in the Netherlands, and 44722 from Eptesicus serotinus in Georgia. On the other hand, EN36-1 (genotypes III) showed the highest similarities (95.9% in gltA and 96.7% in rpoB) with a shrew (Clocidura spp.) strain B28303 in Kenya and B. florencae R4^T in France.

In the phylogenetic analysis based on the gltA sequences, strains EN2-1 (genotype I) and EN19-2 (genotype II) formed clade A with strains from bat fleas collected from an Eptesicus bat in the Netherlands, Eptesicus bats in Georgia and Finland, and Myotis bats in the UK and Romania. Strain EN36-1 (genotypes III) formed clade B with B. florencae, B. acomydis, and B. birtlesii from shrews, wood mouse, and golden spiny mouse, respectively.

These results suggest that genotypes I and II are Vesper bat-specific Bartonella species and genotype III is closely related to shrew and rodent-derived Bartonella species. Interestingly, strains in clade B were distinct from other known bat-associated Bartonella strains. A previous study [37] suggested that host switching of Bartonella is a common event as has occured at least five times between bats and other small mammals. From the results of BLAST search and phylogenetic analysis based on the gltA sequences, such an event might have occurred in genotype III between Japanese northern bats and shrews. However, the identity of a “bridge” vector capable of transmitting Bartonella bacteria between bats and other small mammal species is currently unknown. Further investigation will be necessary to clarify the bridge vectors of northern bats.

Our study showed that northern bats in Japan harbor two species of Bartonella: Vesper bat-specific Bartonella species and a B. florencae-like bacterium. Additionally, bat fleas (Ischnopsyllus needhami) were found to be dominant ectoparasites in the bats and serve as a potential vector of Bartonella transmission among northern bats in Japan.

Acknowledgements

This work was supported by Grant-in Aid for Scientific Research from Japan Society for the Promotion of Science [Grant number 18K06003]. We would like to thank Dr. Yuki Kobayashi, Nihon University for her support of sample collections.

Funding

This work was supported by Grant-in Aid for Scientific Research (No. 18K06003) from the Japan Society for the Promotion of Science.

Conflicts of interest/Competing interests

The authors have no conflicts of interest directly relevant to the content of this article.

Availability of data and material, Code availability

Not applicable.

Authors' contributions

Kei Nabeshima: Writing - Original Draft, Methodology, Investigation, Shingo Sato: Methodology, Investigation, Writing - Review & Editing, Murasaki Amano: Investigation, Jory R. Brinkerhoff: Methodology, Investigation, Writing - Review & Editing, Hidenori Kabeya: Writing - Review & Editing, Itou Takuya: Investigation, Soichi Maruyama: Resources, Conceptualization, Supervision, Writing - Review & Editing

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Prevalence And Genetic Diversity of Bartonella Spp. In Northern Bats (Eptesicus Nilssonii) And Their Blood-Sucking Ectoparasites In Hokkaido, Japan

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