Spotted fever group rickettsiae (SFGR) detection in ticks following reported human case of Japanese spotted fever in Niigata Prefecture, Japan

Japanese spotted fever, a tick-borne disease caused by Rickettsia japonica, was firstly described in southwestern Japan. There was a suspicion of Rickettsia japonica infected ticks reaching the non-endemic Niigata Prefecture after a confirmed case of Japanese spotted fever in July 2014. Therefore, from 2015 to 2017, 38 sites were surveyed and rickettsial pathogens were investigated in ticks from north to south of Niigata Prefecture including Sado island. A total of 3336 ticks were collected and identified revealing ticks of three genera and ten species: Dermacentor taiwanensis, Haemaphysalis flava, Haemaphysalis hystricis, Haemaphysalis longicornis, Haemaphysalis megaspinosa, Ixodes columnae, Ixodes monospinosus, Ixodes nipponensis, Ixodes ovatus, and Ixodes persulcatus. Investigation of rickettsial DNA showed no ticks infected by R. japonica. However, three species of spotted fever group rickettsiae (SFGR) were found in ticks, R. asiatica, R. helvetica, and R. monacensis, confirming Niigata Prefecture as a new endemic area to SFGR. These results highlight the need for public awareness of the occurrence of this tick-borne disease, which necessitates the establishment of public health initiatives to mitigate its spread.


Methods
Area of the study and collection of ticks. The ticks were collected by flagging method in 38 sites from north to south of Niigata Prefecture including Sado island from June 2015 to November 2017, completing a total of 77 field surveys ( Table 1, Fig. 1). Collection sites where humans were likely to be exposed to ticks such as parks, forests with hiking courses, and camping areas were chosen for sampling.
Ticks identification. Collected ticks were identified morphologically under stereoscope based on the key by Yamaguti 7 and separated by the species, sex and growth stages, collection day and the collection sites. The ticks were separated in micro tubes and stored at − 80 °C until further processing. The identification of ticks with insufficient morphologic characteristics was confirmed by DNA sequencing of the mitochondrial 16S rDNA gene, as previously described 8 (data not shown).
DNA extraction. The DNA extraction and purification were done individually for ticks in the adult stage.
For ticks in larvae or nymph stages, the DNA was extracted individually or from a pool of 2 to 5 individuals. Ticks were thawed and homogenized using a cell crasher (FastPrep-24, M. P. Biomedicals) in tubes with six steel   www.nature.com/scientificreports/ I. ovatus was collected in 31 sites, though R. asiatica was detected from I. ovatus collected in 11 of these sites ( Table 5, Fig. 3A). I. ovatus harboring R. asiatica were collected in the central and western part of the prefecture and Sado Island, but not from northeast and mountainous area of the prefectural border with Gunma (collection sites Nos. [20][21][22][24][25][26][27]. Overall, R. asiatica was detected in 8.4% of the I. ovatus adult samples; however, the infection rate varied by collection site such as in Sado Island (site No. 37 and 38 in total) with an infection rate of 50%, Mt. Kakuda (Site No. 13) with 36%, and in Satogaike Sports Field (Site No. 28) with 26%.
I. monospinosus was present in eight sites. From three sites (site No. 6, 9 and 10) in the northeast region of the prefecture, 4 out of 5 I. monospinosus presented Rickettsia helvetica. In the other five sites with I. monospinosus, R. helvetica was not found (Table 5

Discussion
The last tick survey in Niigata Prefecture was done in the '50s 6 15 . SFGR was detected in ticks collected in 20 of 38 sites from all the collection sites in Niigata Prefecture. In 16 of 19 sites where SFGR positive ticks were not collected, there was a low number of collected ticks (lower than 20), and it might have influenced the SFGR detection rates, as seen in the low prevalence of the SFGR in ticks. To understand the SFGR prevalence in the prefecture, continuous tick collection is needed, especially in sites where the collection number is low. SFGR positivity in adult ticks in Niigata Prefecture was 5.6%, and it is similar to the positivity rate of the neighboring prefecture, Toyama, with 3.3% 15 . However, when the SFGR detection rate is compared to other prefectures, such as Fukui (22.0%) and six western prefectures including Shizuoka (21.6%) 16,17 , the SFGR positivity in Niigata Prefecture is still low. In the western part of Japan, SFGR positivity was reported to be as high as 40.5% in H. longicornis 17 ; in contrast, in hokuriku region of Honshu (incl. Niigata, Toyama, Ishikawa, and Fukui Prefectures), SFGR positivity rates are high in I. monospinosus, with 50% in Toyama 15 , 43.8% in Fukui 16 and 43.8% in Niigata (this study). The tick species prevalence depends on the area/region, therefore the prevalence of the SFGR, and Rickettsia spp. could also vary. Rickettsia spp. have strong host-specificity [15][16][17] and, SFGR detected in this study confirmed this feature. Ticks and Rickettsia sp. were: R. asiatica from I. ovatus, R.helvetica from I. monospinosus, and R. monacensis from I. nipponensis. The first report of R. asiatica was in Fukushima Prefecture in 1993, described as Rickettsia sp. IO-1 in I. ovatus with subsequent reports in other areas 18,19 . Moreover, R. asiatica was detected in other tick species, such as H. flava, H. japonica, and H. hystricis 9 , showing a diverse ticks host preference. Regarding mammalian hosts, R. asiatica was detected in blood samples of Japanese deer (Cervus nippon); however, the pathogenicity in these hosts is unknown 20 . SFGR detection in I. ovatus in the neighboring prefecture is varied, with rates of 0.0% in Toyama 15 , 7.9% in Fukui 16 , and 8.4% in Niigata (this study). Also, in this case, the positivity rates may vary according to Table 5. Prevalence of rickettsial genes detected from adult ticks by collection sites. The Northern (Kaetsu), Central (Chuetsu) and Southern (Joetsu) areas of Niigata Prefecture corresponds to the collection sites 1 to 13, 14 to 30, and 31 to 36, respectively. Sado Island area corresponds to the collection sites 37 and 38.   www.nature.com/scientificreports/ the number of sampling sites and sampling size. It is not clear if the R. asiatica positivity is influenced by the ecology of I. ovatus, environmental factors, or ticks' susceptibility for pathogens. Continuous research is needed including studies on environmental change and ticks endemicity. R. helvetica was reported as Japanese spotted fever pathogen in Fukui Prefecture 21 , and it is also detected in I. ovatus, I. persulcatus, and H. japonica 19 . In this study, R. helvetica was detected only from I. monospinosus, with a positivity rate of 33.3%. In Toyama Prefecture, R. helvetica was detected from 2 of 4 I. monospinosus 15 . In this study, R. helvetica positive I. monospinosus was present only in the northeast area of Niigata Prefecture (Site No 6, 9 and 10) (Fig. 3B); however, there was a limited number of I. monospinosus adults (N = 12), present in 8 out of 38 collection sites. To confirm these host specificity and region preferences, further tick collection and field surveys are necessary.

Species of tick
There is only one report of SFGR detected from I. nipponensis in the Toyama prefectural area, reported as Rickettsia sp. In56 9 . In this study, seven samples were positives to SFGR in I. nipponensis with 100% identity with Rickettsia sp. In56 (AB114819, AB114820) in gltA and rOmpA regions. Therefore Rickettsia sp. In56 might be R. monacensis. In Europe, R. monacensis is indicated as a spotted fever pathogen 22,23 and was also isolated from a spotted fever patient in Korea 24 . The tick species harboring R. monacensis is Ixodes ricinus in Europe, and in China, the same pathogen was described in I. persulcatus and Ixodes sinensis [25][26][27] . In Korea, similar to this study, R. monacensis was detected from I. nipponensis 28 . In this study, I. nipponensis presented the highest SFGR positivity in all the collected tick species; SFGR positive I. nipponensis were found from 7 out of 10 sites, indicating R. monacensis might be widely prevalent in Niigata Prefecture.
In Rickettsia sp. Hj126 (AB114803) and Candidatus Rickettsia principis Kh-79_Hj (MG544986), 2 SFGR detected in adult H. flava, the gltA region presented 100% identity and were classified as genotype III by Ishikura's categorization 9 (Fig. 2). The SFGR of the genotype III detected in Japan 9 , presented the same characteristics of the two SFGR detected in this study, indicating SFGR of the genotype III might be widely prevalent in Japan.
In this study, R. japonica was not detected in the ticks, despite having a case of Japanese spotted fever in 2014, and D. taiwanensis and H. hystricis which are known vectors for R. japonica 29 were collected. More wide sampling and/or larger sample size could be necessary to detect a low prevalent species in the arthropod hosts. Additionally, from a clinical point of view, the implementation of serology and DNA isolation might improve the diagnosis and management of patients with spotted fever like illnesses, as recommended in Europe in case of Mediterranean spotted fever like patients 30 .
Three causative agents of human spotted fever R. asiatica, R. helvetica, and R. monacensis, were detected in this study. The major SFGR positive ticks were Ixodes spp. followed by Haemaphysalis spp. High tick-pathogen specificity was also observed in Ixodes sp. and Rickettsia sp.
Continuous precaution is recommended in activities where there is a potential risk of contact with ticks, and the healthcare system should be aware of spotted fever, particularly since Niigata Prefecture can now be considered an SFGR endemic area and human cases may be occurring.