Infestation of introduced raccoons (Procyon lotor) with indigenous ixodid ticks on the Miura Peninsula, Kanagawa Prefecture, Japan

Since the raccoon (Procyon lotor) was introduced to Japan, studies have established that they are infested with native Japanese tick species. However, the quantity of ticks infesting raccoons is unknown. We conducted a survey of ticks on invasive raccoons captured on the Miura Peninsula, Kanagawa Prefecture, Japan, from April 2015 through June 2016 to determine the species of ticks and to quantify the intensity of tick infestation in order to obtain basal information related to the ecology of host–parasite relationships among indigenous tick species and an alien mammalian species. We collected and identified 15,931 ticks of two genera and six species, namely, Haemaphysalis flava, H. megaspinosa, H. longicornis, H. japonica, Ixodes ovatus, and I. tanuki, from 100 out of 115 raccoons. The dominant tick species was H. flava (96.8%) and individuals were mainly adults. Seasonal patterns of infestation intensity of adults and nymphs peaked in the autumn and winter and decreasing in the late spring and summer, May to August, while larvae peaked in August. Our results indicated that host–parasite relationships between invasive raccoons and Japanese tick species, especially H. flava, were established in Kanagawa Prefecture. The ticks infest invasive raccoons for their blood-meal and also for overwintering. The results of this study extend our understanding of the ecology of tick-borne diseases.


Introduction
The first report of feral raccoons (Procyon lotor) in Japan was reported in 1962 in Aichi Prefecture and the raccoon was imported to Japan as a pet and increased number in the late 1970s (Ikeda, 2006). A survey performed by the Japanese government in 2006 confirmed that raccoons have established populations throughout Japan (Ikeda, 2006). They compete for resources with native mammals, such as the raccoon dog (Nyctereutes procyonoides) and the red fox (Vulpes vulpes japonica; Ikeda, 2006;Ikeda et al., 2004), prey on endangered reptiles (Kaneda and Kato, 2011), damage crops (Ikeda, 2006), break into houses (Ikeda, 1999(Ikeda, , 2006 and are recognized as an invasive species (Ikeda, 1999). Furthermore, they are hosts to ticks and associated pathogens that impact the health of human, livestock, and other indigenous wildlife. Multiple reports indicate that raccoons have a role as a reservoir of tickborne diseases (TBD), (e.g., Japanese spotted fever; JSF) (Inoue et al., 2011), tularemia (Berrada et al., 2006;Fujita, 2009a;Inoue et al., 2011), babesiosis (Kawabuchi et al., 2005), and severe fever and thrombocytopenia syndrome (SFTS) (Takahashi et al., 2014). Since both human infestation and TBD case reports, such as JSF, concentrate in seasons, ticks are mostly active in the environment (Mahara, 1997). Also, TBD pathogens circulate between wildlife and ticks and the distribution of host wildlife is overlapped with vector ticks (Tsukada et al., 2014). Thus, the seasonal patterns of ticks and distributional patterns of ticks and host wildlife are strongly interacting each other. Moreover, on-host ticks observation indicates how vector ticks widen their distribution. However, the seasonal patterns of on-host ticks are not fully understood especially among introduced wildlife. and Haemaphysalis flava, H. megaspinosa, H. longicornis, Amblyomma testudinarium, Ixodes nipponenis, I. tanuki, and I. ovatus infest invasive raccoons in Japan (Fujita, 2009b;Kobayashi et al., 2012). These ticks contribute to the risk of transmitting TBD pathogens to raccoons (Furuno et al., 2017). The possibility that indigenous tick species and invasive raccoon developed a new host-parasite relationship in recent decades implies that a new route of TBD transmission has been developed by vector ticks transmitting TBD pathogens from native reservoir animals to a new host species (Fujita, 2009b). The feral raccoons in Kanagawa Prefecture, Japan may have resulted in changes of host availability for local ticks. These ticks may have experienced a rapid expansion of newly available hosts, while original hosts in the sympatric niche, such as raccoon dogs and red foxes, were driven away from the habitat (Ikeda, 1999;Ikeda et al., 2004). However, the details of the host-parasite relationships of raccoons and ticks in Kanagawa Prefecture are not well known, despite the recommendation that monitoring wildlife hosts and vector ticks are important to define the ecoepidemiology of TBD (Alexander et al., 2012).
The purpose of our research was to identify and quantify the species of ticks infesting raccoons. This survey is a first step to obtaining a full understanding the host-parasite relationship between invasive raccoons and Japanese ticks.
Fauna in the study area included raccoon dog, Japanese badger (Meles anakuma), and invasive raccoons. The Japanese Sika deer (Cervus nippon), Japanese macaque (Macaca fuscata), and Japanese wild boar (Sus scrofa leucomystax) were not present based on an animal survey conducted by the Biodiversity Center of Ministry of the Environment of Japan (2004).

Raccoon samples
Since the Invasive Alien Species Act in 2005 listed the raccoon as an invasive alien species, local governments of Japan have implemented countermeasures against the biological and economical damage caused by raccoons (Ama, 2007

Tick collection and identification
Ticks were collected from 115 raccoon carcasses using forceps and combing all skin surfaces using a cat flea comb. Ticks were preserved in 70% ethanol and then identified to species, stages of development, and sex of adults based on morphological examination under a stereo-microscope and a biological microscope using standard keys (Yamaguti et al., 1971).

Data analysis
The mean burden and proportion of each tick species were

Seasonal patterns
Temporal analyses of H. flava on the invasive raccoons demonstrated a relatively intense infestation in the autumn and winter which decreased in the late spring and summer, May to August ( Fig. 2; Table 1). Adult ticks peaked in the winter, while larvae peaked in August. In addition, despite seasonal changes, H. flava used raccoons as a host throughout the year for every developmental stage (Table 1).

Discussion
Our results and previous studies which reported tick infestation on raccoons in Japan (Fujita, 2009b;Kobayashi et al., 2012) indicated that invasive raccoons and the Japanese native tick, H. flava, have a well established host-parasite relationship.
The previous study in Chiba Prefecture, Japan, about 80 km east of our study area, also reported that larval H. flava were the most abundant in the summer and adult H. flava were the most abundant in the autumn and winter (Asanuma, 1956;Saito, 1959). In addition, two previous tick surveys in the western region of the Republic of Korea during April and October by Cobrun et al. (2016), and in the southeastern region of the Republic of Korea during March and October by Johnson et al. (2017), reported that adult and nymphal H. flava were frequently collected in April, May and October, and decreased during August, while larvae were collected starting July and peaked in August. Moreover, Johnson et al. (2017) reported that the habitat preference of H. flava was the forest environment.
Our results indicated a relatively high intensity of adult and nymphal H. flava infestation that started to increase in November and peaked in February while larval H. flava peaked in August (Fig. 2). Asanuma and Sakurai (1958), Yoshida (1980), Fujimoto et al. (1987), and Kakuda et al. (1990) reported larval H. flava and H. longicornis overwintered on the host. Thus, our results of intense infestation on raccoons during winter indicated on-host overwinter habitat of adult and nymphal H. flava. H. flava is known that they frequently infest domestic dogs (Canis lupus familiaris) going into forest environment (Choe et al., 2011). Also, surveys in previous studies were conducted before invasive raccoons spread across Japan, infestations of H. flava (Saito, 1959;Saito et al., 1965), H. longicornis (Saito et al., 1965), I. tanuki (Saito, 1964), and I. ovatus (Saito, 1959) were found from medium-sized carnivores, including raccoon dogs, red foxes, Japanese weasels (Mustela sibirica itatsi), and Japanese badgers. These ticks established host-parasite relationships with indigenous medium-sized carnivores in the Miura Peninsula before or at the time of the introduction of raccoons. In addition, our study area, Miura Peninsula was the area that Japanese Sika deer and Japanese wild boar have not been observed in recent decades, which were known to be hosts of various tick species, such as H. longicornis, H. megaspinosa, A. testudinarium, and I. ovatus, (Biodiversity Center of Ministry of the Environment of Japan, 2005; Yamaguti et al., 1971;Tsukada et al., 2014). This implies that H. flava, H. longicornis, I. ovatus, and I. tanuki may have selected raccoons or at least used raccoons as hosts in addition to indigenous medium-sized carnivores because of its availability as the host. Although, Yamaguti et al. (1971) reported that larval H. flava prefer small mammals and small birds, we observed larvae H. flava infestation in August (Fig. 2). This indicated larval H. flava may be able to infest larger mammals (e.g., raccoon) if they were more abundant in the area or used the environment of the area frequently.

Conclusion
The relationships between native ticks and invasive raccoons in Table 1 Life stage* and sex of ticks infesting the raccoons captured in Kanagawa (Ishikura et al., 2000;Katayama et al., 1996Katayama et al., , 2001 and cases of human infestation (Seishima et al., 2000;Yamauchi et al., 2010) were reported in the past. Raccoon, which was infested by ticks in our study area, is known as an urban wildlife breaking into houses and using various environment (e.g. forest, grassland, urban) (Ikeda, 2009). Thus, it is highly possible that ticks spread its distribution toward human living environment by infesting a raccoon and those ticks possibly harbor TBDs. It is necessary to perform tick surveys of medium-sized carnivores living in our study area to have a better understanding of the host preferences of various tick species and their associated pathogens they harbor in Japan.

Conflicts of interest
None.