No detection of severe Fever with thrombocytopenia syndrome virus from ixodid ticks collected in seoul.

Larvae, nymphs, and adult stages of 3 species of ixodid ticks were collected by tick drag methods in Seoul during June-October 2013, and their infection status with severe fever with thrombocytopenia syndrome (SFTS) virus was examined using RT-PCR. During the period, 732 Haemaphysalis longicornis, 62 Haemaphysalis flava, and 2 Ixodes nipponensis specimens were collected. Among the specimens of H. longicornis, the number of female adults, male adults, nymphs, and larvae were 53, 11, 240, and 446, respectively. Ticks were grouped into 63 pools according to the collection site, species, and developmental stage, and assayed for SFTS virus. None of the pools of ticks were found to be positive for SFTS virus gene.

Haemaphysalis longicornis is an important vector of zoonotic tick-borne pathogens that impact on medical and veterinary health worldwide [1]. Severe fever with thrombocytopenia syndrome (SFTS) virus is a tick-borne virus transmitted by H. longicornis and included in the family Bunyaviridae (genus Phlebovirus) [2,3,4,5].

Tick-borne disease surveillance is becoming increasingly important as zoonotic tick-borne pathogens are recognized to affect man and wild and domestic animals worldwide [6]. Dragging vegetation for questing ticks are often employed where ticks are present and provides information on habitats and seasonal life stage distributions. The purpose of the present study was to provide estimates of the distribution of life cycle stages during periods when ixodid ticks are active and their infection status with SFTS virus. The data would serve to provide information that is necessary for the development of tick-borne disease threat assessments.

Tick surveillance was conducted in Seoul during June-October 2013. Tick drags consisted of a 1.0 m long and 1.0 m wide flannel cloth attached to a stainless dowel (1.2 m long, 2.0 cm diameter). Collections were made by slowly walking and dragging the flannel cloth on the ground for approximately 5-10 m, turning the drag over and then removing the attached ticks using a fine forceps from both sides of the cloth. Nymphs and adults were placed in 2 ml cryovials, while larvae were placed separately in 2 ml cryovials. This was repeated twice until each collector surveyed an area 5-10 m length. Nymphs, adults, and larvae were identified to species and developmental stages under a dissecting microscope according to Yamaguti et al. [7].

A total of 796 ticks in 63 pools were assayed for SFTS virus. Ticks were collected at 51 sites from 7 parks (8 sites of Jamshil, 5 sites of Gwangnaru, 8 sites of Nanji, 5 sites of Gangseo, 5 sites of Worldcup, 5 sites of Seoul Forest, and 15 sites of Seoul Grand parks). Pools of larvae (n=40), nymphs (n=20), and adults (n=5) were pretreated by precellys 24 homogenizer (Bertin Technology, Orsay, France) at 6,000 rpm, for 25 sec, twice at -20℃. Pools were then homogenized by MK28R (Bertin Technology) with 2.8 mm stainless-steel beads in 600 µl buffer solution containing autoclaved 10% (v/v) FBS and 5% (v/v) penicillin/streptomycin.

RNA was extracted from the tick suspensions using viral RNA mini kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions [8]. Detection of the M gene was conducted using Diastar™ 2X onestep RT-PCR pre-mix (Solgent, Seoul, Korea) and conventional RT-PCR arrayed for SFTS virus.

The foward sequence primer was 5'-GATGAGATGGTCCATGCTGATTCTAA-3', and the reverse primer was 5'-CTCATG GG GTGGAATGTCCTCAC-3'. RT-PCR was conducted in a 2720 thermal cycler (Applied Biosystems, Foster City, California, USA). The PCR products were identified by electrophoresis in 2.0% agarose gel, and the M gene was confirmed at 560 bp.

A total of 796 ticks (larvae, nymphs, and adults) belonging to 2 genera and 3 species, H. longicornis (732), H. flava (62), and I. nipponensis (2), were collected from 7 parks (Jamshil, Gwangnaru, Nanji, Gangseo, Worldcup, Seoul Forest, and Seoul Grand parks) in Seoul, Korea, June-October 2013 (Table 1). Overall, H. longicornis (adults 64, nymphs 240, and larvae 446) accounted for 91.9% of the 3 species collected, followed by H. flava 7.8% (adults 1, nymphs 43, and larvae 18) and I. nipponensis 0.3% (nymphs 2). Significantly more H. longicornis adults, nymphs, and larvae were collected than H. flava and I. nipponensis (Table 1).

Table 1

Morphologic identification of ticks collected from natural/city parks from June to October 2013 and assayed for tick-borne severe fever with thrombocytopenia syndrome (SFTS) virus, by species and developmental stage

aThe areas which had hard ticks in 51 sites of 7 parks: Jamshil (8 sites), Gwangnaru (5 sites), Nanji (8 sites), Gangseo (5 sites), Worldcup (5 sites), Seoul Forest (5 sites), and Seoul Grand (15 sites) parks.

And, the areas which had no hard ticks in 59 sites of 15 parks: Yeouido (3 sites), Ttukseom (9 sites), Ichon (4 sites), Mangwon (4 sites), Yeouido (3 sites), Independence (2 sites), Seoseoul Lake (2 sites), North Seoul Iris (3 sites), Boramae (3 sites), Namsan (8 sites), Yongsan Family (2 sites), Gildong Ecological (2 sites), Jungnang Camping Forest (3 sites), Yangjae Citizen (3 sites), and Children's Grand (8 sites) parks.

bIncluding 5 pools (83 ticks) from Goduk Ecological park.

cConventional RT-PCR.

★ Eight parks along Han river: Yeouido, Ttukseom, Ichon, Mangwon, Jamshil, Gwangnaru (included with Goduk Ecological), Nanji, and Gangseo parks.

★ Fourteen large scale Seoul citizen parks: Yeouido, Independence, Seoseoul Lake, Worldcup, North Seoul Iris, Boramae, Namsan, Yongsan Family, Seoul Forest, Gildong Ecological, Jungnang Camping Forest, Yangjae Citizen, Seoul Grand, and Children's Grand parks.

Similarly, H. longicornis was the most frequently collected ticks in northern Gyeonggi-do (Province) (75.8%), while H. flava and I. nipponensis accounted for 19.6% and 4.6% from April to October 2004 and 2005 [6]. In the southern province and Jeju-do (Island), H. longicornis was the most frequently collected ticks (73.4%), whereas H. flava and I. nipponensis accounted for 22.4% and 0.4% in 2007 [9]. However, in Hong-do, H. longicornis accounted only for 5.7% of all ticks collected, while H. flava and I. nipponensis accounted for 18.9% and 6.1%, respectively, from 2008 to 2009 [10]. The areas surveyed were grasses and not forested areas. Higher numbers of H. flava may have been collected if forested habitats were surveyed.

Also, Yamaguti [7] reported that H. longicornis and H. flava were the most commonly collected ticks in Korea and Japan. Lee [11] reported that H. longicornis were ectoparasites of mammals, whereas H. flava were ectoparasites of birds and mammals, None of these ticks were collected among grasses where people walk or sit. At the Seoul Forest and Seoul Grand parks, 116 and 490 ticks were collected, respectively. At the Seoul Forest Park, ticks were collected in grasses near the places where deers were kept, while at Seoul Grand Park, they were collected in grasses surrounding a wolf enclosure. No ticks were collected from 15 parks that were surveyed: Yeouido, Ttukseom, Ichon, Mangwon, Yeouido, Independence, Seoseoul Lake, North Seoul Iris, Boramae, Namsan, Yongsan Family, Gildong Ecological, Jungnang Camping Forest, Yangjae Citizen, and Children's Grand parks (Table 1; Fig. 1)

Fig. 1

The survey sites on ticks in citizen parks from April through October 2013 in Seoul, Korea by periodic surveillance.

All of the 63 pools of 796 ticks (larvae, nymphs, and adults) belonged to 3 species, and none were positive for SFTS virus by conventional RT-PCR. The reason that all the tick pools were negative, may be due, in part, to a small sample-size and lack of zoonotic hosts.

In Shandong Province, China, from April to November 2011, SFTS virus-specific antibodies were detected in 69.5% of sheep, 60.5% of cattle, 37.9% of dogs, 3.1% of pigs, and 47.4% of chickens [2]. In addition, ELISA showed that 3.6% and 47.7% of human and animal serum samples were positive for SFTS virus antibodies, indicating that SFTS virus has circulated widely among domestic animals and birds in China [12]. By the end of 2011, SFTS had been reported in 11 provinces including Henan [3,4]. In China, the initial fatality rate for SFTS was 30%. However, with improved diagnosis and supportive care, the fatality rate decreased to 10-15% with an annual incidence of approximately 5 cases among 100,000 rural populations according to the 2011 surveillance data [5].