Date Published: March 31, 2017
Publisher: Public Library of Science
Author(s): Kiwa Furuno, Kyunglee Lee, Yukie Itoh, Kazuo Suzuki, Kenzo Yonemitsu, Ryusei Kuwata, Hiroshi Shimoda, Masahisa Watarai, Ken Maeda, Ai Takano, Brian Stevenson.
The genus Borrelia comprises arthropod-borne bacteria, which are infectious agents in vertebrates. They are mainly transmitted by ixodid or argasid ticks. In Hokkaido, Japan, Borrelia spp. were found in deer and Haemaphysalis ticks between 2011 and 2013; however, the study was limited to a particular area. Therefore, in the present study, we conducted large-scale surveillance of ticks and wild animals in the western part of the main island of Japan. We collected 6,407 host-seeking ticks from two regions and 1,598 larvae obtained from 32 engorged female ticks and examined them to elucidate transovarial transmission. In addition, we examined whole blood samples from 190 wild boars and 276 sika deer, as well as sera from 120 wild raccoons. We detected Borrelia spp. in Haemaphysalis flava, Haemaphysalis megaspinosa, Haemaphysalis kitaokai, Haemaphysalis longicornis, and Haemaphysalis formosensis. In addition, we isolated a strain from H. megaspinosa using Barbour-Stoenner-Kelly medium. The minimum infection rate of ticks was less than 5%. Transovarial transmission was observed in H. kitaokai. Phylogenetic analysis of the isolated strain and DNA fragments amplified from ticks identified at least four bacterial genotypes, which corresponded to the tick species detected. Bacteria were detected in 8.4%, 15%, and 0.8% of wild boars, sika deer, and raccoons, respectively. In this study, we found seasonal differences in the prevalence of bacterial genotypes in sika deer during the winter and summer. The tick activity season corresponds to the season with a high prevalence of animals. The present study suggests that a particular bacterial genotype detected in this study are defined by a particular tick species in which they are present.
Members of the genus Borrelia in the family Spirochaetaceae are arthropod-borne infectious agents in vertebrates , and they are classified into three major groups based on phylogenetic analyses: Lyme disease borreliae, relapsing fever borreliae, and reptile-associated borreliae [2, 3]. Relapsing fever borreliae are mostly found in ticks, and only Borrelia recurrentis is found in lice. Tick-borne relapsing fever caused by Borrelia crocidurae, Borrelia duttonii, Borrelia hermsii, and other related Borrelia spp. is a disease with worldwide distribution . Tick-borne relapsing fever is mostly transmitted by soft-bodied ticks belonging to the genera Ornithodoros and Argas. By contrast, several species are transmitted by hard-bodied ticks; Borrelia miyamotoi, B. theileri, Borrelia lonestari, Borrelia sp. AGRF, and Borrelia sp. BR were detected in Ixodes spp., Rhipicephalus spp., Amblyomma americanum, Amblyomma geoemydae, and Rhipicephalus microplus, respectively [5–13]. In addition, a Borrelia sp. similar to B. lonestari was recently found in sika deer (Cervus nippon yesoensis) and Haemaphysalis spp. in Hokkaido, Japan [14, 15]. Among the hard-bodied tick-borne relapsing fever (hTBRF) borreliae, B. miyamotoi has been recognized as a human pathogen in Russia , the USA , Europe [18, 19], and Japan , and B. theileri has been found as the causative agent of bovine spirochetosis . In the USA, B. lonestari was hypothesized to be the causative agent of southern tick-associated rash illness (STARI), which is a Lyme-like disease . However, a later study did not detect B. lonestari in STARI patients . Thus, the pathogenicity of B. lonestari remains unclear. Moreover, the isolation of hTBRF borreliae is difficult in vitro, except for B. miyamotoi from Japan and the USA and a strain of B. lonestari co-cultivated with a tick cell line [7, 23, 24]. Therefore, analyses of the genetic relationships and pathological mechanisms of hTBRF borreliae are limited.
We collected 6,407 host-seeking ticks from Yamaguchi and Wakayama prefectures (Fig 1 and S1 Table). From Shimonoseki, Yamaguchi Prefecture, we collected 387 Haemaphysalis ticks, which were individually prepared for DNA detection and cultivation (Table 1). Borrelial DNA fragments were detected in two H. megaspinosa ticks (a male and female), and the prevalence was 3.6% (2/55) in adult H. megaspinosa. In these PCR-positive ticks, a strain was successfully isolated from female ticks using BSK-M, and the strain was designated as tHM16w. This is the first Borrelia sp. isolate detected in Haemaphysalis ticks using BSK-M. In Shunan, Yamaguchi Prefecture, 1,678 ticks in 155 pools were examined and borrelial DNA was not detected. In Wakayama, 4,342 ticks were collected and processed in 530 pools (Table 2). Among the 530 tick pools, 21 were positive and the minimum prevalence was 0.48% (21/4,342): four, one, and four pools from H. flava males, females, and nymphs (4/96, 1/126, and 4/490, and minimum prevalence of 4.17%, 0.79%, and 0.82%), respectively; one pool from H. formosensis nymphs (1/339; 0.29%); one pool from H. kitaokai females (1/38; 2.63%); one and three pools from H. longicornis males and nymphs (1/145 and 3/1,828; 0.69% and 0.16%), respectively; and six pools from H. megaspinosa nymph (6/408; 1.47%). We also examined unfed larval ticks prepared from engorged females. From 32 engorged females, we examined 1,598 larval ticks in 32 pools (Table 3). Borrelia was not isolated using BSK-M, but a DNA fragment was detected in a pool from H. kitaokai.
We detected several bacterial genotypes of Borrelia spp. from Haemaphysalis spp. collected from two regions in the western part of the main island of Japan. The prevalence was 0%–0.5% in all ticks collected and 0%–4% in each tick species (Tables 1 and 2). In a previous study, Lee et al. found that the prevalence of Borrelia spp. in Haemaphysalis adult ticks was 0.7% . Several surveys of B. lonestari derived from unfed A. americanum adults have shown that the prevalence in the USA was less than 6% [32–37]. However, B. theileri has been detected in host-attached ticks. McCoy et al. reported that the prevalence of B. theileri in cattle infested with Rhipicephalus geigyi was 0.5% in Mari, while Cutler et al. detected B. theileri in 12.5% pools of animal-associated Amblyomma and Rhipicephalus spp. in Ethiopia [38, 39]. Moreover, the prevalence of human pathogenic hTBRF borreliae, B. miyamotoi, in unfed ixodid ticks was less than 5% in the USA, Eurasia, and Japan [26, 40, 41]. Our results and those of previous investigations suggest that the prevalence of hTBRF borreliae in unfed ticks is generally less than 5%. Transovarial transmission of Borrelia sp. HK in H. kitaokai was demonstrated in the present study (Table 3), which has also been examined in B. lonestari and B. miyamotoi [35, 42]. Thus, we suggest that hTBRF borreliae might be maintained in the environment via transovarial transmission.