Sato et al. Parasites Vectors (2021) 14:73 https://doi.org/10.1186/s13071-021-04585-w Parasites & Vectors RESEARCH Open Access Lipoptena fortisetosa as a vector of Bartonella bacteria in Japanese sika deer (Cervus nippon) Shingo Sato1* , Hidenori Kabeya2, Sayuri Ishiguro1, Yasuhiro Shibasaki3,4 and Soichi Maruyama1 Abstract Background: Two species of deer ked (Lipoptena cervi and L. mazamae) have been identifed as vectors of Bartonella bacteria in cervids in Europe and the USA. In an earlier study we showed that Japanese sika deer (Cervus nippon) harbor three Bartonella species, namely B. capreoli (lineage A) and two novel Bartonella species (lineages B and C); however, there is currently no information on the vector of Bartonella bacteria in sika deer. The aim of this study was to clarify potential vectors of Bartonella in Japanese sika deer. Methods: Thirty-eight wingless deer keds (L. fortisetosa) and 36 ticks (Haemaphysalis and Ixodes species) were col- lected from sika deer. The prevalence of Bartonella in the arthropods was evaluated by real-time PCR targeting the 16S 23S internal transcribed spacer (ITS) and by culture of the organisms. The total number of Bartonella bacteria were− quantifed using real-time PCR. The distribution of Bartonella bacteria in deer ked organs was examined by immunofuorescence analysis. The relationship of Bartonella strains isolated from sika deer and arthropods were examined by a phylogenetic analysis based on concatenated sequences of the gltA, rpoB, ftsZ, and ribC genes, fol- lowed by a BLAST search for gltA and rpoB. Results: Bartonella prevalence in deer keds was 87.9% by real-time PCR and 51.5% in culture and that in the ticks was 8.3% by real-time PCR and 2.8% in culture. The mean number of Bartonella bacteria per ked was calculated to be 9.2 105 cells. Bartonella aggregates were localized in the midgut of the keds. The phylogenetic analysis and BLAST search× showed that both the host deer and the keds harbored two Bartonella species (lineages B and C), while B. capreoli (lineage A) was not detected in the keds. Two novel Bartonella species (lineages D and E) were isolated from one ked. Conclusions: Lipoptena fortisetosa likely serves as a vector of at least two Bartonella species (lineages B and C), whereas ticks do not seem to play a signifcant role in the transmission of Bartonella between sika deer based on the lower detection rates of Bartonella in ticks compared to keds. Bartonella species in lineages D and E appear to be L. fortisetosa-specifc strains. Keywords: Bartonella, Deer keds, Ticks, Sika deer, Japan Background hematophagous arthropods are known to serve as vec- Bartonella species are Gram-negative, facultative intra- tors for the transmission of Bartonella bacteria to mam- cellular bacteria. To date, more than 30 species and three malian hosts, including sand fy (Lutzomyia verrucarum) subspecies have been described in the genus [1]. Several for B. bacilliformis [2], human body louse (Pediculus humanus humanus) for B. quintana [3, 4] and cat fea *Correspondence: [email protected] (Ctenocephalides felis) for B. henselae [5]. Within the last 1 Laboratory of Veterinary Public Health, Department of Veterinary two decades, it has been shown that Lipoptena, Hippo- Medicine, College of Bioresource Sciences, Nihon University, 1866 bosca and Melophagus keds in the family Hippoboscidae Kameino, Fujisawa, Kanagawa 252-0880, Japan Full list of author information is available at the end of the article © The Author(s) 2021. 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The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/publi cdoma in/ zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Sato et al. Parasites Vectors (2021) 14:73 Page 2 of 10 are involved in the transmission of Bartonella bacteria to on sika deer that tested positive for Bartonella bacte- ruminants [6]. ria. Tese observations suggest that these ectoparasites Deer keds (Lipoptena spp.) are recognized as obli- are involved in the transmission of Bartonella bacteria gate ectoparasites of ruminants but they can acciden- between sika deer. To date, there have been no stud- tally infest humans and other mammals [7, 8]. Te keds ies investigating the role of hematophagous arthropods have an atypical life-cycle compared with that of other in the transmission of Bartonella bacteria between sika hematophagous arthropods. After a winged ked attaches deer. Te aim of the study reported here was to deter- to its host’s body, it sheds its wings and then loses the mine whether deer keds and/or ticks serve as vectors of ability to fy. Wingless keds are thought to remain on Bartonella bacteria in sika deer. To achieve this aim, we their initial hosts. One possible exception to this gen- used a variety of bacteriological, molecular biological and eral rule is the movement of the Neotropical deer ked immunohistochemical techniques. (L. mazamae) from a mother deer to its ofspring due to close contact [9]. After blood-feeding on the host, a Methods female ked lays a larva on the host body, and the larva Sample collection pupates immediately. Although the pupa is thought to Between 2009 and 2012, 17 Honshu deer were captured hatch in the summer or early autumn, precise informa- in Nara (N = 8) and Shizuoka (N = 9) Prefectures, Japan. tion on the hatch-timing is lacking. Blood samples were collected from the deer and immedi- In Europe, L. cervi is the commonest deer ked found on ately transferred into EDTA-containing collection tubes. red deer (Cervus elaphus), roe deer (Capreolus capreolus) Te blood samples collected from Nara Prefecture were and moose (Alces alces) [10]. Bartonella-derived DNA utilized for the isolation of Bartonella bacteria in our pre- was frst detected in wingless L. cervi collected from roe vious study [16]; the blood samples from Shizuoka Pre- deer in France [11], with the high detection rate (93.8%) fecture were used in the present study. Te frozen blood indicating that L. cervi may be epidemiologically asso- samples were sent to the Laboratory of Veterinary Pub- ciated with the transmission of Bartonella in this deer lic Health, Department of Veterinary Medicine, College species. Bartonella schoenbuchensis was subsequently of Bioresource Sciences, Nihon University and stored at isolated from 73.3% of wingless L. cervi collected from − 70 °C until examined. roe deer and red deer in Germany [12]. Immunohisto- A total of 38 wingless deer keds were collected from the chemical analysis has also revealed the presence of large deer captured in Nara (N = 10) and Shizuoka Prefectures bacterial masses consisting of B. schoenbuchensis in the (N = 28). All of the keds were morphologically identifed midguts of deer keds [12]. In addition, B. schoenbuchensis as Lipoptena fortisetosa. Five deer keds from Shizuoka DNA was detected in wingless L. cervi (83.3%) collected Prefecture were used for immunofuorescence analysis to from white-tailed deer in Massachusetts, USA [13], and determine the distribution of Bartonella bacteria within Bartonella DNA was detected not only in developing lar- the bodies of the insects. A total of 36 ticks were col- vae (71%) but also in winged L. cervi (6.4%) in Hungary lected from the deer in Nara (N = 33) and Shizuoka Pre- [14]. In Finland, B. schoenbuchensis-like DNA was also fectures (N = 3). Based on morphological analysis under detected from 13 pupae and one winged L. cervi collected stereomicroscopic observations these ticks were identi- in the feld [10]. Tis epidemiological evidence raises the fed as Haemaphysalis fava (N = 16), H. megaspinosa possibility that B. schoenbuchensis may be transstadially (N = 15), H. longicornis (N = 4) and Ixodes monospinosus transmitted from the pupal to the winged stages of L. (N = 1). Te live arthropod samples were immediately cervi. sent to the same laboratory under room temperature or Te Japanese sika deer (Cervus nippon) is native to refrigeration conditions (approx. 4 °C). Japan and genetically divided into six subspecies, among which Honshu deer (Cervus nippon centralis) is widely Isolation of Bartonella bacteria from deer blood samples distributed throughout the country, with the exception of and arthropods Hokkaido, Shikoku and Kyushu islands. Yezo deer is also Isolation of Bartonella bacteria from deer blood samples a subspecies (Cervus nippon yesoensis) of sika deer and was performed according to the procedure reported by inhabits only Hokkaido island [15]. In a previous study Sato et al. [16]. To isolate Bartonella bacteria from the [16], we isolated Bartonella bacteria from 67.6% of Hon- keds and ticks, frst the surface of each arthropod was shu deer and 50% of Yezo deer examined. A genetic anal- sterilized for 10 min with 500 μl of 70% ethanol contain- ysis showed that Honshu and Yezo deer harbor at least ing 0.1% povidone-iodine, following which the arthro- three Bartonella species, including B. capreoli and two pod was washed twice (1 min each wash) with 0.01 M novel Bartonella species.