Introduction

Ixodid ticks are ectoparasites of a broad range of hosts, including reptiles, e.g., lizards, skinks, tortoises, and snakes (Yoneda 1981, Bauwens et al. 1983, Krinsky 1983, Hammond & Dorsett 1988, Fujita & Takada 1997, Durden et al. 2002, Eisen et al. 2004, Fajfer 2012). Noh (1965) first reported Ixodes granulatus Supino blood feeding on a Tsushima Ground Skink, Scincella vandenburghi(Schmidt), in the Republic of Korea (ROK), and later Ra et al. (2011) reported Ixodes nipponensis Kitaoka and Saito nymphs on four lizard species collected at 22 sites in the ROK. More recently, I. nipponensis nymphs and larvae and Amblyomma testudinarium Koch nymphs were reported blood feeding on three species of lizards and three species of snakes, respectively, collected in 6 provinces and one metropolitan city of the ROK (Suh et al. 2016).

The role of reptiles as hosts of ticks and as reservoir hosts of bacterial pathogens, e.g., Borreliaspp. and spotted fever group rickettsiae, has been investigated in some detail (Wright et al. 1998, Dsouli et al. 2006, Reeves et al. 2006), while studies of their role as reservoir hosts of arboviruses, e.g., severe fever with thrombocytopenia syndrome virus (SFTSV), have been limited (Reisen et al. 2007, Suh et al. 2016). SFTSV, a tick-borne Bunyavirus, was first identified in China in 2009 and retrospectively in a patient that died in Korea in 2012. With better recognition, the number of SFTS cases increased from 36 (17 deaths; 47.2%) in 2013 (including 1 retrospective case in a patient that died in 2012), 55 (16 deaths; 29.1%) in 2014, 79 (21 deaths; 26.6%) in 2015, 165 (19 deaths; 11.5%) in 2016, and >250 (54 deaths; 20.0%) in 2017 (KCDC 2017). To better understand the relationship between reptilian hosts and associated ticks and pathogens affecting human and veterinary health, a program was developed to determine tick relative abundance and infestation rates, as well as the stage(s) of development associated with particular host species, and the geographical distributions of both reptiles and associated ticks.

Materials and methods

Detection of SFTSV

Following identification, all ticks (40 nymphs and 26 larvae) were placed in a secure Styrofoam container of dry ice and transported to the KNIH, where they were stored at -80°C until assayed for the detection of the partial medium (M) gene segment of SFTSV by reverse transcription-polymerase chain reaction (RT-PCR). Tick samples were homogenized individually in 600 μl of phosphate-buffered saline (pH 7.0) using a Precellys® 24 high-throughput tissue homogenizer (Bertin Technologies, Bretonneux, France) and 2.8-mm stainless steel beads. A viral RNA extraction kit (iNtRON Biotechnology, Seongnam, ROK) was used to extract RNA from the supernatant of the tick homogenates. To detect the partial M segment of SFTSV, a 1-step RT-PCR was performed using a DiaStar™ 2X OneStep RT-PCR Pre-Mix Kit (SolGent, Daejeon, ROK) via a previously described method (Yun et al. 2014).

FIGURE 1.

Map showing collection sites of lizards and skinks infested with Ixodes nipponensis nymphs and larvae (A): Takydromus wolteri, Takydromus amurensis, Eremias argus (Squamata: Lacertidae), and Scincella vandenburghi (Squamata: Scincidae); and snakes infested with Amblyomma testudinarium nymphs (B): Elaphe dione (Squamata: Colubridae), Gloydius brevicaudus, and Gloydius ussuriensis (Squamata: Viperidae). (GG = Gyeonggi Province; GW = Gangwon Province; CB = Chungbuk Province; CN = Chungnam Province; JB = Jeonbuk Province; JN = Jeonnam Province; GB = Gyeongbuk Province; GN = Gyeongnam Province).

Results

Tick collections

A total of 98 reptiles, including lizards (30, 3 species belonging to two genera), skinks (5, 1 species), and snakes (63, 10 species belonging to 7 genera) were collected (Table 1). The Mountain Grass Lizard (Takydromus wolteri Fischer) (14, 14.3% of all hosts) and the Steppe Rat Snake (Elaphe dione (Pallas)) (14, 14.3%) were the most frequently collected species, followed by the Ussuri Mamushi (Gloydius ussuriensis (Emelianov)) (13, 13.3%), the Mongolia Racerunner (Eremias argusPeters) (11, 11.2%), the Amur Rat Snake (Elaphe schrenckii (Strauch)) (8, 8.2%), the Short-tailed Mamushi (Gloydius brevicaudus (Stejneger)), the Tiger Keelback (Rhabdophis tigrinus (Boie)) (7, 7.1%), the Tsushima Ground Skink (S. vandenburghi), the Amur Grass Lizard (Takydromus amurensis Peters) (5, 5.1%), the Rock Mamushi (Gloydius saxatilis (Emelianov)) (4, 4.1%), the Red-banded Snake (Lycodon rufozonatus Cantor) (3, 3.1%), the Frog-eating Rat Snake (also called the Red-backed Rat Snake) (Oocatochus rufodorsatus (Cantor)) (3, 3.1%), the Japanese Keelback (Hebius vibakari (Boie)) (2, 2.0%), and the Slender Racer (Orientocoluber spinalis (Peters)) (2, 2.0%).

A total of 66 ixodid ticks belonging to two genera and two species, I. nipponensis and A. testudinarium, were collected from 11/30 (36.7%) lizards, 2/5 (40.0%) skinks, and 6/63 (9.5%) snakes. Takydromus amurensis (4/5, 80.0%) was the most frequently infested reptile, followed by S. vandenburghi (2/5, 40.0%), T. wolteri (5/14, 35.7%), G. ussuriensis (4/13, 30.8%), E. argus (2/11, 18.2%), G. brevicaudus (1/7, 14.3%), and E. dione (1/14, 7.1%), while the remaining less frequently collected species of snakes were negative for ticks (Table 1).

Ixodes nipponensis larvae and nymphs were collected only from lizards (21 nymphs, 26 larvae) and skinks (9 nymphs), while A. testudinarium nymphs (10) were collected only from snakes (Table 1). Among the lizards and skinks, T. amurensis had the highest tick index (total number ticks/total number hosts) (3.00), followed by E. argus (1.91), S. vandenburghi (1.80), T. wolteri (0.79) (Table 1). Ixodes nipponensis larvae and nymphs were preferentially attached to the foreleg axillae (66.1%), followed by the lateral trunk (23.2%), and head and eye (10.7%) body region of lizards and skinks (Figure 2).

FIGURE 2.

Takydromus amurensis (A, B), Takydromus wolteri (C), Scincella vandenburghi (D), and Eremias argus (E) with Ixodes nipponensis nymphs and larvae.

Amblyomma testudinarium was the only species of tick collected from snakes — none were collected from lizards and skinks. A total of 10 A. testudinarium nymphs were collected from three of ten snake species (E. dione, G. brevicaudus, and G. ussuriensis). Gloydius ussuriensis was the snake most frequently infested with ticks (4/13 snakes; 30.8%), followed by G. brevicaudus (1/7; 14.3%), and E. dione (1/14; 7.1%). Gloydius brevicaudus had the highest tick index (0.57) among the snakes, followed by G. ussuriensis (0.38), and E. dione (0.07). Amblyomma testudinariumnymphs were preferentially attached to the head and eye (40.0%), followed by the lateral trunk (60.0%) of snakes (Table 1, Figure 3). In two other recent studies of amblyommine ticks collected from East Asian snakes, three females of Amblyomma helvolum Koch were removed from the head of a Taiwan Stink Snake, Elaphe carinata (Günther), in southern Taiwan (Chao et al. 2013), while two and three Amblyomma nymphs were collected, respectively, from the lateral sides of the body of a Taiwanese Rat Snake (also called the Beauty Rat Snake), Orthriophis taeniurus friesi (Werner) and of a Chinese Cobra, Naja atra Cantor, in west-central Taiwan (Norval et al. 2009).

FIGURE 3.

Gloydius ussuriensis (A), Gloydius brevicaudus (B), and Elaphe dione (C) with Amblyomma testudinarium nymphs.

TABLE 1.

Numbers of lizards, skinks, and snakes captured, numbers infested with ticks, and numbers of ticks (Ixodes nipponensis and Amblyomma testudinarium) collected, by stage of development and species from March to October 2016, Republic of Korea.

Seasonal distribution

Ixodes nipponensis nymphs were collected from lizards and skinks during March (5/30; 16.7%), April (20/30; 66.7%), May (1/30; 3.3%) and June (4/30; 13.3%), while larvae were collected only during June (22/26; 84.6%), August (3/26; 11.5%) and September (1/26; 3.8%) (Table 2). Amblyomma testudinarium nymphs were collected from snakes during April (2/10; 10.0%), May (4/10; 40.0%), June (3/10; 30.0%) and August (1/10; 10.0%) (Table 2).

TABLE 2.

Reptile species, month of collection, province of collection, and number of larvae and nymphs of Ixodes nipponensis and Amblyomma testudinarium collected from reptiles from March to October 2016, Republic of Korea.

Discussion

Habitat restoration and alteration, including a major reforestation program initiated in the 1960s and rapid urbanization following the end of the Korean War in 1953, have led to changes in landscape ecology conducive to higher populations of wild animals, such as small mammals (e.g., rodents, soricomorphs, rabbits, and weasels), larger mammals (e.g., deer, wild pigs, raccoon dogs, badgers, and feral dogs and cats), local and migratory birds, and reptiles (e.g., lizards, skinks, and snakes) that are hosts to various species of ticks (Kim et al. 2010b, 2011, 2013, Chong et al. 2013a, 2013b, Kang et al. 2013, Shin et al. 2013, Park et al. 2014). Ticks harbor numerous zoonotic pathogens, e.g., viruses, bacteria and protozoa, that are of veterinary and medical importance, and as humans encroach upon the habitats of wild animals and birds, they and their pets/domestic animals may be exposed to ticks and associated tick-borne pathogens (Kang et al. 1982, Park et al. 2011, Yun et al. 2012). Based on recent case increases for a number of tick-borne diseases, e.g., SFTS, which increased from 36 cases in 2013 to >250 cases in 2017, tick bites and illnesses due to tick-borne pathogens appear to be underreported in the ROK (Jang et al. 2004, Choi et al. 2005, Shin et al. 2013, Yi et al. 2016). Because human tick bites from I. nipponensis and A. testudinarium are reported more frequently for patients seen at medical clinics in the ROK, these two species may play an important role in the transmission of tick-borne pathogens to humans and domestic animals (Kang et al. 1982, Lee et al. 1989, Cho et al. 1994, 1995, Yamada et al. 1996, Ryu et al. 1998, Chae et al. 2000, Yun et al. 2001, Ko et al. 2002, Chang et al. 2006, Kim et al. 2010a, 2014b, Suh et al. 2013).

In order to provide a descriptive analysis of disease risks to human and domestic animal populations, it is important to identify the relative abundance of ticks, their host associations, stages of development found on various hosts, geographic distributions, and potential for the maintenance and transmission of zoonotic pathogens. Disease threat assessments and risk analyses are central to the development of disease mitigation strategies (e.g., use of insecticide-impregnated uniforms) for US and ROK civilian and military populations, and to efforts to increase awareness of disease risks, thus reducing the impact of zoonotic tick-borne diseases.

Small mammals, birds and reptiles are hosts of I. nipponensis larvae, which are commonly collected by tick drag in tall grasses and herbaceous vegetation bordering forested hillsides and mountains, wetland and dryland farms, and military training areas. Nymphs and adults are found on larger mammals, e.g., deer, and are much less frequently collected from vegetation by tick drag (Kim et al. 2010b, 2013, 2014a, Ra et al. 2011, Kang et al. 2013, HC Kim personal communication). Ixodes nipponensis nymphs and adults are more frequently reported to bite humans, possibly because of their larger mouthparts (i.e., longer hypostomes), which are probably more irritating to human hosts than the relatively smaller mouthparts of Haemaphysalis spp. (Cho et al. 1995, Ryu et al. 1998, Yun et al. 2001, Ko et al. 2002, Jeon et al. 2014). During a 2015 reptile tick survey, I. nipponensisnymphs accounted for 88.9% of ticks collected from lizards and skinks, while larvae accounted for only 11.1% (Suh et al. 2016), whereas during this survey, I. nipponensis larvae accounted for 46.4% of the ticks collected from lizards and skinks. This difference was due, in part, to the high numbers of I. nipponensis larvae (20 larvae) collected from Eremias argus in mid-June. However, in previous studies the number of I. nipponensis larvae increased in July, peaked in August, and then declined in September (Kim et al. 2013, Coburn et al. 2016, Suh et al. 2016) (Table 1).

Amblyomma testudinarium adults are relatively large ticks that are frequently reported to bite humans, which is not unexpected, given this species' biting behavior (blood feeding on hosts for up to 30 days) as well as its large mouthparts that are capable of producing painful bites (Kim et al. 2010a, 2014b, Suh et al. 2013). In Korea, A. testudinarium was infrequently collected by tick drag or from small mammals (>2,500 rodents and soricomorphs) during a comprehensive survey in various habitats, perhaps in part because of its biting behavior and preference for larger mammals (Yamaguti et al. 1971, Kim et al. 2011, 2013, 2014a, Coburn et al. 2016, Yun et al. 2016, Johnson et al. 2017).

In this survey, a total of 98 reptiles, including lizards, skinks, and snakes, were collected from March-October. As in the 2015 reptile survey, I. nipponensis nymphs were collected only from lizards and skinks, while A. testudinarium nymphs were collected only from snakes (Suh et al. 2016). Suh et al. (2016) collected two genera of lizards (2 species) and skinks (1 species) and five genera of snakes (8 species). Similarly, this survey resulted in the collection of two genera of lizards (3 species) and skinks (1 species), and seven genera of snakes (10 species). The lower number of A. testudinarium collected from snakes in 2016 (10) compared to 2015 (48) and the relatively low infectivity rate of ticks (1/48; 2.1%) may have resulted in none of the A. testudinarium collected during 2016 being positive for SFTS virus. While similar numbers of I. nipponensis nymphs were collected (2015, 32; 2016, 30), only two (6.3%) were positive for SFTS virus in 2015. Moreover, both positive I. nipponensis nymphs were from the same locality, indicating focal distribution of SFTSV. Although larval I. nipponensis were collected, and there is evidence of transovarial transmission of SFTSV, none were positive during either 2015 or 2016.

While Haemaphysalis longicornis Neumann is considered the primary vector of SFTSV, this virus also has been detected in I. nipponensis biting humans and in specimens collected by tick drag, as well as in A. testudinarium biting humans (Park et al. 2014, Yun et al. 2014). On average, there are approximately 40 tick bites reported by the Korea Centers for Disease Control and Prevention annually, with many unreported based on the number of SFTSV infections identified annually from 2013–2017 (36, 55, 79, 165 and >250 cases, respectively). Although I. nipponensis and A. testudinarium collected from lizards (T. wolteri; Hapcheon County, Gyeongsangnam Province) and one snake (R. tigrinus; Wanju County, Jeollabuk Province) were positive for SFTSV in a 2015 survey (Suh et al. 2016), none of the ticks collected from lizards and snakes during the 2016 reptile tick survey were positive for SFTSV.

Further collections in other areas (e.g., Gyeonggi, Gangwon, and Gyeongbuk provinces and Jeju Island) are necessary to better understand the geographical and host distributions of ticks associated with reptiles and the potential impact of tick-associated pathogens on human and animal health.