The Culex pipiens complex in Asia includes a temperate subspecies, Culex pipiens pallens, of unce... more The Culex pipiens complex in Asia includes a temperate subspecies, Culex pipiens pallens, of uncertain taxonomic status. The shape of the male genitalia suggests it is a hybrid between Cx. pipiens and Cx. quinquefasciatus. We studied populations of Cx. p. pallens in Japan, Korea, and China and compared them to local populations of Cx. quinquefasciatus and Cx. p. pipiens. We examined variation in a nuclear intron in the acetylcholinesterase-2 gene [ACE] and eight microsatellite loci. We found a distinct microsatellite signature for Cx. p. pallens indicating restricted gene flow between Eastern and Western populations of Cx. pipiens, supporting the existence of two subspecies. Furthermore, a multilocus genotype analysis revealed current hybridization between Cx. p. pallens and Cx. quinquefasciatus in southern Japan, Republic of Korea, and China but not in Hokkaido, in northern Japan. Surprisingly, however, we found that the sex-linked ACE locus in chromosome I has introgressed asymmetrically through the males such that all male Cx. p. pallens have a copy of the Cx. quinquefasciatus ACE locus. This result highlights some of the potential consequences of hybridization between local and introduced species to disease transmission worldwide.
Tick is one of the most important arthropods in the transmission of vector-borne diseases. In thi... more Tick is one of the most important arthropods in the transmission of vector-borne diseases. In this study, we investigated the abundance and species of ticks associated with swine and their habitats to assess the risk of spread of tick-borne diseases in host species, such as wild boars. Ticks were collected from 24 grazing or traditionally reared domestic pig farms and 8 habitats of wild boars in 8 provinces and 1 city in the Republic of Korea, by using the dragging and flagging methods. Ticks were also collected directly from 49 wild boars by using fine forceps. A total of 9,846 hard ticks were collected, including 4,977 Haemaphysalis longicornis, 4,313 Haemaphysalis flava, 508 Ixodes nipponensis, 1 Ixodes turdus, and 47 Amblyomma testudinarium. A total of 240 hard ticks were collected from 49 wild boars, including 109 H. flava, 84 H. longicornis, and 47 A. testudinarium. A total of 578 hard ticks were collected from areas around domestic pig farms. Only 2 hard tick species, 546 H. ...
Hantaviruses (family Bunyaviridae) are enveloped negative-sense tripartite RNA viruses. The natur... more Hantaviruses (family Bunyaviridae) are enveloped negative-sense tripartite RNA viruses. The natural hosts of hantaviruses include rodents, shrews, moles, and bats. Imjin virus (MJNV) is a shrew-borne hantavirus identified from the Ussuri white-toothed shrews (Crocidura lasiura) in the Republic of Korea (ROK) and China. We have isolated MJNV and determined its prevalence and molecular diversity in Gyeonggi province, ROK. However, the distribution and phylogeography of MJNV in other regions of ROK remain unknown. A total of 96 C. lasiura were captured from Gangwon and Gyeonggi provinces, ROK, during 2011-2014. Among them, four (4.2%) shrews were positive for anti-MJNV IgG and MJNV RNA was detected from nine (9.4%), respectively. Based on the prevalence of MJNV RNA, the preponderance of infected shrews was male and adult, consistent with the gender-and weightspecific prevalence of hantaviruses in other species. We monitored the viral load of MJNV RNA in various tissues of shrews, which would reflect the dynamic infectious status and circulation of MJNV in nature. Our phylogeographic and genomic characterization of MJNV suggested natural occurrences of recombination and reassortment in the virus population. Thus, these findings provide significant insights into the epidemiology, phylogeographic diversity, and dynamic circulation and evolution of shrew-borne hantaviruses. Hantaviruses (genus Hantavirus, family Bunyaviridae) are zoonotic, negative-sense, single-stranded RNA viruses containing large (L), medium (M), and small (S) segments that encode RNA-dependent RNA polymerase (RdRp), glycoproteins (Gn and Gc), and nucleocapsid (N) protein, respectively 1. Hantaviruses cause hemorrhagic fever with renal syndrome (HFRS) with fatality rates of 1-15% in Eurasia and hantavirus pulmonary syndrome (HPS) with mortality rates of 30-50% in Americas 2,3. Natural hosts of hantaviruses include rodents (Rodentia), bats (Chiroptera), and insectivores (Soricomorpha) 4-6. To date, hantaviruses have been identified in shrews and moles (Eulipotyphla). These viruses include Thottapalayam virus (TPMV) from the Asian house shrew (Suncus murinus), Imjin virus (MJNV) from the Ussuri white-toothed shrew (Crocidura lasiura), Jeju virus (JJUV) from the Asian lesser white-toothed shrew (C. shantungensis), Camp Ripley virus (RPLV) from the northern short-tailed shrew (Blarina brevicauda), Cao Bang virus (CBNV) from the Chinese mole shrew (Anourosorex squamipes), Boginia virus (BOGV) from the Eurasian water shrew (Neomys fodiens), Seewis virus (SWSV) from the Eurasian common shrew (Sorex araneus), and Artybash virus (ARTV) from the Laxmann's shrew (S. caecutiens) 7-15. In our previous study, we first discovered and isolated a shrew-borne hantavirus, MJNV, from C. lasiura captured near Imjin River in Gyeonggi province, Republic of Korea (ROK) 8. MJNV was also found to circulate in Southeastern China 16. A phylogeographic study of MJNV revealed the molecular diversity of the virus in a limited area, Gyeonggi province 17. Infection with MJNV elicited a robust expression of pro-inflammatory cytokines in human macrophages and endothelial cells 18. In a Syrian hamster model, MJNV infection causes a lethal disease
Rickettsiae constitute a group of arthropod-borne, Gram-negative, obligate intracellular bacteria... more Rickettsiae constitute a group of arthropod-borne, Gram-negative, obligate intracellular bacteria that are the causative agents of diseases ranging from mild to life threatening that impact on medical and veterinary health worldwide. A total of 6,484 ticks were collected by tick drag from June-October 2013 in the southwestern provinces of the Republic of Korea (ROK) (Jeollanam, n = 3,995; Jeollabuk, n = 680; Chungcheongnam, n = 1,478; and Chungcheongbuk, n = 331). Ticks were sorted into 311 pools according to species, collection site, and stage of development. DNA preparations of tick pools were assayed for rickettsiae by 17 kDa antigen gene and ompA nested PCR (nPCR) assays and the resulting amplicons sequenced to determine the identity and prevalence of spotted fever group rickettsiae (SFGR). Haemaphysalis longicornis (4,471; 52 adults, 123 nymphs and 4,296 larvae) were the most commonly collected ticks, followed by Haemaphysalis flava (1,582; 28 adults, 263 nymphs and 1,291 larva...
Hantaan virus (HTNV), a negative sense tripartite RNA virus of the Family Bunyaviridae, is the mo... more Hantaan virus (HTNV), a negative sense tripartite RNA virus of the Family Bunyaviridae, is the most prevalent hantavirus in the Republic of Korea (ROK). It is the causative agent of Hemorrhagic Fever with Renal Syndrome (HFRS) in humans and maintained in the striped field mouse, Apodemus agrarius, the primary zoonotic host. Clinical HFRS cases have been reported commonly in HFRS-endemic areas of Gyeonggi province. Recently, the death of a member of the ROK military from Gangwon province due to HFRS prompted an investigation of the epidemiology and distribution of hantaviruses in Gangwon and Gyeonggi provinces that border the demilitarized zone separating North and South Korea. To elucidate the geographic distribution and molecular diversity of HTNV, whole genome sequences of HTNV Large (L), Medium (M), and Small (S) segments were acquired from lung tissues of A. agrarius captured from 2003-2014. Consistent with the clinical incidence of HFRS established by the Korea Centers for Dise...
Ticks play an important role in transmission of arboviruses responsible for emerging infectious d... more Ticks play an important role in transmission of arboviruses responsible for emerging infectious diseases, and have a significant impact on human, veterinary, and wildlife health. In the Republic of Korea (ROK), little is known about information regarding the presence of tick-borne viruses and their vectors. A total of 21,158 ticks belonging to 3 genera and 6 species collected at 6 provinces and 4 metropolitan areas in the ROK from March to October 2014 were assayed for selected tick-borne pathogens. Haemaphysalis longicornis (n=17,570) was the most numerously collected, followed by Haemaphysalis flava (n=3317), Ixodes nipponensis (n=249), Amblyomma testudinarium (n=11), Haemaphysalis phasiana (n=8), and Ixodes turdus (n=3). Ticks were pooled (adults 1-5, nymphs 1-30, and larvae 1-50) and tested by one-step reverse transcription polymerase chain reaction (RT-PCR) or nested RT-PCR for the detection of severe fever with thrombocytopenia virus (SFTSV), tick-borne encephalitis virus (TBE...
Emerging and re-emerging infectious diseases caused by RNA viruses pose a critical public health ... more Emerging and re-emerging infectious diseases caused by RNA viruses pose a critical public health threat. Next generation sequencing (NGS) is a powerful technology to define genomic sequences of the viruses. Of particular interest is the use of whole genome sequencing (WGS) to perform phylogeographic analysis, that allows the detection and tracking of the emergence of viral infections. Hantaviruses, Bunyaviridae, cause hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS) in humans. We propose to use WGS for the phylogeographic analysis of human hantavirus infections. A novel multiplex PCR-based NGS was developed to gather whole genome sequences of Hantaan virus (HTNV) from HFRS patients and rodent hosts in endemic areas. The obtained genomes were described for the spatial and temporal links between cases and their sources. Phylogenetic analyses demonstrated geographic clustering of HTNV strains from clinical specimens with the HTNV strains circulating ...
The Democratic People's Republic of Korea (DPRK) is located on the northern part of the Korea... more The Democratic People's Republic of Korea (DPRK) is located on the northern part of the Korean Peninsula in East Asia. While tick-borne pathogens of medical and veterinary importance have been reported from China and the Republic of Korea, they have not been reported from the DPRK. To screen for zoonotic tick-borne pathogens in the DPRK, ticks were collected from domestic goats. A total of 292 (27 nymph, 26 male, 239 female) Haemaphysalis longicornis were collected and assayed individually for selected tick-borne pathogens. Using the 16S ribosomal RNA and ITS species-specific nested PCR, a total of 77 (26.4%) were positive for Anaplasma bovis, followed by Bartonella grahamii (15, 5.1%), Anaplasma phagocytophilum (12, 4.1%), Bartonella henselae (10, 3.4%), and Borrelia spp. (3, 1.0%). Using the groEL-based nested PCR, a total of 6 and 1 H. longicornis were positive for B. grahamii and B. henselae. All products were sequenced and demonstrated 100% identity and homology with previo...
Established colonies of Anopheles campestris, An. cracens, An. dirus, An. kleini, An. minimus, An... more Established colonies of Anopheles campestris, An. cracens, An. dirus, An. kleini, An. minimus, An. sawadwongporni, and An. sinensis are maintained at the Armed Forces Research Institute of Medical Sciences (AFRIMS). Females were provided blood meals on human blood containing citrate as an anticoagulant using an artificial membrane feeder. The mean wing length, used as an estimate of body size, for each species was compared to blood-feeding duration (time), blood meal volume, and numbers of eggs oviposited. Except for An. campestris and An. cracens, there were significant interspecies differences in wing length. The mean blood meal volumes (mm 3) of An. kleini and An. sinensis were significantly higher than the other 5 species. For all species, the ratios of unfed females weights/blood meal volumes were similar (range: 0.76-0.88), except for An. kleini (1.08) and An. cracens (0.52), that were significantly higher and lower, respectively. Adult females were allowed to feed undisturbed for 1, 3, and 5 minute intervals before blood feeding was interrupted. Except for An. campestris and An. sawadwongporni, the number of eggs oviposited were significantly higher for females that fed for 3 minutes when compared to those that only fed for 1 min. This information is critical to better understand the biology of colonized Anopheles spp. and their role in the transmission of malaria parasites as they relate to the relative size of adult females, mean volumes of blood of engorged females for each of the anopheline species, and the effect of blood feeding duration on specific blood meal volumes and fecundity.
A seroepidemiological study of selected rodent-borne diseases (hantavirus [Seoul [SEO] virus], sc... more A seroepidemiological study of selected rodent-borne diseases (hantavirus [Seoul [SEO] virus], scrub typhus [Orientia tsutsugamushi], murine typhus [Rickettsia typhi], and leptospirosis [Leptospira interrogans]), as part of the U.S. military rodent surveillance and control program, was conducted from 2001 through 2005 at Yongsan Garrison, Seoul, Republic of Korea. Rodents were collected to determine the prevalence of rodent-borne diseases at a U.S. military installation in an urban environment. A total of 1,750 rodents representing three species was collected by using baited live traps (Tomahawk), glue boards, and poison baits (dead rodents observed but not assayed). The Norway rat, Rattus norvegicus (99.8%), accounted for nearly all of the rodents captured/observed. Only three roof rats, Rattus rattus (0.2%), and one house mouse, Mus musculus (Ͻ0.1%), were collected. R. norvegicus rats were the only rodents that were serologically positive for SEO virus (9.6%), scrub typhus (2.8%), murine typhus (3.8%), and leptospirosis (4.6%). One of six rodents that were positive for SEO virus by immunofluorescent antibody test was positive for SEO virus antigen by reverse transcriptase-polymerase chain reaction. Infection rates for SEO virus, scrub typhus, murine typhus, and leptospirosis varied annually. Rodents were captured from 228 (20.7%) of 1,104 total buildings in Yongsan Garrison. The Yongsan commissary had the highest annual infestation rate (22 rodents per year), followed by Commisky's Club (18 rodents per year). Annual infestation rates were high for food service facilities, which often store perishable food products outdoors for short periods of time, attracting rodent populations; refuse from these facilities provides harborage and food for rodents. The effect of rodent populations outside the boundary of Yongsan Garrison was not determined.
A survey to determine the geographical distribution and relative abundance of potential vectors o... more A survey to determine the geographical distribution and relative abundance of potential vectors of scrub typhus was conducted from October to November 2006 at 13 localities throughout the Republic of Korea. Apodemus agrarius accounted for 97.6% (80/82) of all rodents, while only 2 Myodes regulus (2/82) were collected. A total of 10,860 chiggers were collected from A. agrarius belonging to 4 genera and 8 species, while only Walchia fragilis (40) was collected from Myodes regulus. Leptotrombidium pallidum (8,137; 74.9%), a vector of scrub typhus, was the predominant species collected from A. agrarius followed by Leptotrombidium scutellare (2,057, 18.9%), Leptotrombidium palpale (279; 2.7%), Leptotrombidium orientale (232; 2.1%), and Leptotrombidium zetum (79; 0.7%), Neotrombicula tamiyai (58; 0.5%), Euschoengastica koreaensis (16; 0.1%), and Cheladonta ikaoensis (2; < 0.1%). L. pallidum was the predominant chigger collected at collection sites in Gangwon (100%), Gyeonggi (87.2%), C...
A total of 1,708 small mammals (1,617 rodents and 91 soricomorphs), including Apodemus agrarius (... more A total of 1,708 small mammals (1,617 rodents and 91 soricomorphs), including Apodemus agrarius (n = 1,400), Microtus fortis (167), Crocidura lasiura (91), Mus musculus (32), Myodes (= Eothenomys) regulus (9), Micromys minutus (6), and Tscherskia (= Cricetulus) triton (3), were live-trapped at US/Republic of Korea (ROK) military training sites near the demilitarized zone (DMZ) of Paju, Pocheon, and Yeoncheon, Gyeonggi Province from December 2004 to December 2009. Small mammals were examined for their intestinal nematodes by necropsy. A total of 1,617 rodents (100%) and 91 (100%) soricomorphs were infected with at least 1 nematode species, including Nippostrongylus brasiliensis, Heligmosomoides polygyrus, Syphacia obvelata, Heterakis spumosa, Protospirura muris, Capillaria spp., Trichuris muris, Rictularia affinis, and an unidentified species. N. brasiliensis was the most common species infecting small mammals (1,060; 62.1%) followed by H. polygyrus (617; 36.1%), S. obvelata (370; 21.7%), H. spumosa (314; 18.4%), P. muris (123; 7.2%), and Capillaria spp. (59; 3.5%). Low infection rates (0.1-0.8%) were observed for T. muris, R. affinis, and an unidentified species. The number of recovered worms was highest for N. brasiliensis (21,623 worms; mean 20.4 worms/infected specimen) followed by S. obvelata (9,235; 25.0 worms), H. polygyrus (4,122; 6.7 worms), and H. spumosa (1,160; 3.7 worms). A. agrarius demonstrated the highest prevalence for N. brasiliensis (70.9%), followed by M. minutus (50.0%), T. triton (33.3%), M. fortis (28.1%), M. musculus (15.6%), C. lasiura (13.2%), and M. regulus (0%). This is the first report of nematode infections in small mammals captured near the DMZ in ROK.
Comprehensive quarterly serosurveillance on scrub typhus in small mammals collected from military... more Comprehensive quarterly serosurveillance on scrub typhus in small mammals collected from military training sites located near the Demilitarized Zone (DMZ), northern Gyeonggi-do (Province), ROK was conducted to determine the potential rodent-borne and associated ectoparasite disease risks to military personnel. A total of 1,196 rodents and insectivores representing 8 species, Apodemus agrarius (87.3%, n = 1,044), Mus musculus (5.4%, n = 65), Crocidura lasiura (3.3%, n = 40), Microtus fortis (2.6%, n = 31), Micromys minutus (0.3%, n = 4), Tscherskia triton (0.3%, n = 4), Rattus norvegicus (0.3%, n = 4), and Myodes regulus (0.3%, n = 4) were assayed for the presence of antibodies to Orientia tsutsugamushi. O. tsutsugamushi antibodies were detected in 6 of 8 species and seroprevalence determined; A. agrarius (45.6%), M. musculus (23.1%), M. fortis (48.4%), M. minutus (50.0%), T. triton (50.0%), and R. norvegicus (25.0%). A total of 31,184 chigger mites collected from 508 rodents and ins...
Adult mosquito collections using New Jersey light traps and Black-hole light traps were conducted... more Adult mosquito collections using New Jersey light traps and Black-hole light traps were conducted to determine the potential vectors and the relative mosquito infection rates of Dirofilaria immitis and Dirofilaria repens in Gyeonggi and Gangwon Provinces, Republic of Korea, 2005. Dirofilaria spp. were confirmed by polymerase chain reaction (PCR) using species-specific primers for D. immitis and D. repens. Minimum field infection rates (MFIR) [MFIR = (number infected pools/total number of mosquitoes) Â 1000] of 12 pools/2059 total number mosquitoes (5.8) and 21 pools (10.1) of the wild caught mosquitoes were positive by PCR for D. immitis and D. repens, respectively. Dual infections of both D. immitis and D. repens were detected by PCR in eight pools (3.9). Anopheles sinensis sensu lato (includes An. sinensis s.s., An. pullus, An. kleini, An. belenrae, and An. lesteri), An. sineroides, Aedes vexans nipponi, Culex pipiens and Armigeres subalbatus were found to be infected and are potential vectors of D. immitis and D. repens in Korea. Infections observed in mosquitoes represent potential health risks for domestic animal and human populations.
Recently, Imjin virus (MJNV), a genetically distinct hantavirus, was isolated from lung tissues o... more Recently, Imjin virus (MJNV), a genetically distinct hantavirus, was isolated from lung tissues of the Ussuri white-toothed shrew (Crocidura lasiura) captured near the demilitarized zone in the Republic of Korea. To clarify the genetic diversity of MJNV, partial M- and L-segment sequences were amplified from lung tissues of 12 of 37 (32.4%) anti-MJNV IgG antibody-positive Ussuri white-toothed shrews captured between 2004 and 2010. A 531-nucleotide region of the M segment (coordinates 2,255 to 2,785) revealed that the 12 MJNV strains differed by 0-12.2% and 0-2.3% at the nucleotide and amino acid levels, respectively. A similar degree of nucleotide (0.2-11.9%) and amino acid (0-3.8%) difference was found in a 632-nucleotide length of the L segment (coordinates 962 to 1,593) of nine MJNV strains. Phylogenetic analyses, based on the partial M and L segments of MJNV strains generated by the neighbor-joining and maximum likelihood methods, showed geographic-specific clustering, akin to t...
This study describes the seasonal distribution of larvae, nymph, and adult life stages for 3 spec... more This study describes the seasonal distribution of larvae, nymph, and adult life stages for 3 species of ixodid ticks collected by tick drag and sweep methods from various habitats in the Republic of Korea (ROK). Grasses less than 0.5 m in height, including herbaceous and crawling vegetation, and deciduous, conifer, and mixed forests with abundant leaf/needle litter were surveyed at United States (US) and ROK operated military training sites and privately owned lands near the demilitarized zone from April-October, 2004 and 2005. Haemaphysalis longicornis Neumann adults and nymphs were more frequently collected from April-August, while those of Haemaphysalis flava Neumann and Ixodes nipponensis Kitaoka and Saito were collected more frequently from April-July and again during October. H. longicornis was the most frequently collected tick in grass habitats (98.9%), while H. flava was more frequently collected in deciduous (60.2%) and conifer (57.4%) forest habitats. While more H. flava (54.1%) were collected in mixed forest habitats than H. longicornis (35.2%), the differences were not significant. I. nipponensis was more frequently collected from conifer (mean 8.8) compared to deciduous (3.2) and mixed (2.4) forests.
A total of 9,281 larval chigger mites were collected from small mammals captured at Hwaseong-gun,... more A total of 9,281 larval chigger mites were collected from small mammals captured at Hwaseong-gun, Gyeonggi-do (Province) (2,754 mites from 30 small mammals), Asan city, Chungcheongnam-do (3,358 mites from 48 mammals), and Jangseong-gun, Jeollanam-do (3,169 for 62 mammals) from April-November 2009 in the Republic of Korea (= Korea) and were identified to species. Leptotrombidium pallidum was the predominant species in Hwaseong (95.8%) and Asan (61.2%), while Leptotrombidium scutellare was the predominant species collected from Jangseong (80.1%). Overall, larval chigger mite indices decreased from April (27.3) to June (4.9), then increased in September (95.2) and to a high level in November (169.3). These data suggest that L. pallidum and L. scutellare are the primary vectors of scrub typhus throughout their range in Korea. While other species of larval chigger mites were also collected with some implications in the transmission of Orientia tsutsugamushi, they only accounted for 11.2% of all larval chigger mites collected from small mammals.
A tick survey was conducted to determine the relative abundance and distribution of ticks associa... more A tick survey was conducted to determine the relative abundance and distribution of ticks associated with selected mammals in the Republic of Korea (ROK) during 2008-2009. A total of 918 ticks were collected from 76 mammals (6 families, 9 species) captured at 6 provinces and 3 Metropolitan Cities in ROK. Haemaphysalis longicornis (54.4%) was the most frequently collected tick, followed by Haemaphysalis flava (28.5%), Ixodes nipponensis (7.6%), Ixodes pomerantzevi (4.8%), Ixodes persulcatus (4.6%), and Haemaphysalis japonica (0.1%). Adults (57.0%) and nymphs (28.7%) of Ixodes and Haemaphysalis spp. were collected most frequently from medium or large mammals in this survey, while few larvae (14.3%) were collected. Hydropotes inermis was the most frequently captured mammal (52.6%), with a 16.4 tick index and 5 of 6 species of ticks collected during this survey. H. longicornis (69.7%) was the predominant tick collected from H. inermis, followed by H. flava (22.2%), I. persulcatus (6.1%)...
Ticks were collected from 35 animals from 5 provinces and 3 metropolitan cities during 2012. Tick... more Ticks were collected from 35 animals from 5 provinces and 3 metropolitan cities during 2012. Ticks also were collected by tick drag from 4 sites in Gyeonggi-do (2) and Jeollabuk-do (2) Provinces. A total of 612 ticks belonging to 6 species and 3 genera were collected from mammals and a bird (n= 573) and by tick drag (n= 39). Haemaphyalis longicornis (n= 434) was the most commonly collected tick, followed by H. flava (158), Ixodes nipponensis (11), Amblyomma testudinarium (7), H. japonica (1), and H. formosensis (1). H. longicornis and H. flava were collected from all animal hosts examined. For animal hosts (n> 1), the highest Tick Index (TI) was observed for domestic dogs (29.6), followed by Siberian roe deer (17.4), water deer (14.4), and raccoon dogs (1.3). A total of 402 H. longicornis (adults 86, 21.4%; nymphs 160, 39.8%; larvae 156, 38.9%) were collected from wild and domestic animals. A total of 158 H. flava (n= 158) were collected from wild and domestic animals and 1 ring-necked pheasant, with a higher proportion of adults (103, 65.2%), while nymphs and larvae only accounted for 12.7% (20) and 22.2% (35), respectively. Only 7 A. testudinarium were collected from the wild boar (6 adults) and Eurasian badger (1 nymph), while only 5 I. nipponensis were collected from the water deer (4 adults) and a raccoon dog (1 adult). One adult female H. formosensis was first collected from vegetation by tick drag from Mara Island, Seogwipo-si, Jeju-do Province.
The Culex pipiens complex in Asia includes a temperate subspecies, Culex pipiens pallens, of unce... more The Culex pipiens complex in Asia includes a temperate subspecies, Culex pipiens pallens, of uncertain taxonomic status. The shape of the male genitalia suggests it is a hybrid between Cx. pipiens and Cx. quinquefasciatus. We studied populations of Cx. p. pallens in Japan, Korea, and China and compared them to local populations of Cx. quinquefasciatus and Cx. p. pipiens. We examined variation in a nuclear intron in the acetylcholinesterase-2 gene [ACE] and eight microsatellite loci. We found a distinct microsatellite signature for Cx. p. pallens indicating restricted gene flow between Eastern and Western populations of Cx. pipiens, supporting the existence of two subspecies. Furthermore, a multilocus genotype analysis revealed current hybridization between Cx. p. pallens and Cx. quinquefasciatus in southern Japan, Republic of Korea, and China but not in Hokkaido, in northern Japan. Surprisingly, however, we found that the sex-linked ACE locus in chromosome I has introgressed asymmetrically through the males such that all male Cx. p. pallens have a copy of the Cx. quinquefasciatus ACE locus. This result highlights some of the potential consequences of hybridization between local and introduced species to disease transmission worldwide.
Tick is one of the most important arthropods in the transmission of vector-borne diseases. In thi... more Tick is one of the most important arthropods in the transmission of vector-borne diseases. In this study, we investigated the abundance and species of ticks associated with swine and their habitats to assess the risk of spread of tick-borne diseases in host species, such as wild boars. Ticks were collected from 24 grazing or traditionally reared domestic pig farms and 8 habitats of wild boars in 8 provinces and 1 city in the Republic of Korea, by using the dragging and flagging methods. Ticks were also collected directly from 49 wild boars by using fine forceps. A total of 9,846 hard ticks were collected, including 4,977 Haemaphysalis longicornis, 4,313 Haemaphysalis flava, 508 Ixodes nipponensis, 1 Ixodes turdus, and 47 Amblyomma testudinarium. A total of 240 hard ticks were collected from 49 wild boars, including 109 H. flava, 84 H. longicornis, and 47 A. testudinarium. A total of 578 hard ticks were collected from areas around domestic pig farms. Only 2 hard tick species, 546 H. ...
Hantaviruses (family Bunyaviridae) are enveloped negative-sense tripartite RNA viruses. The natur... more Hantaviruses (family Bunyaviridae) are enveloped negative-sense tripartite RNA viruses. The natural hosts of hantaviruses include rodents, shrews, moles, and bats. Imjin virus (MJNV) is a shrew-borne hantavirus identified from the Ussuri white-toothed shrews (Crocidura lasiura) in the Republic of Korea (ROK) and China. We have isolated MJNV and determined its prevalence and molecular diversity in Gyeonggi province, ROK. However, the distribution and phylogeography of MJNV in other regions of ROK remain unknown. A total of 96 C. lasiura were captured from Gangwon and Gyeonggi provinces, ROK, during 2011-2014. Among them, four (4.2%) shrews were positive for anti-MJNV IgG and MJNV RNA was detected from nine (9.4%), respectively. Based on the prevalence of MJNV RNA, the preponderance of infected shrews was male and adult, consistent with the gender-and weightspecific prevalence of hantaviruses in other species. We monitored the viral load of MJNV RNA in various tissues of shrews, which would reflect the dynamic infectious status and circulation of MJNV in nature. Our phylogeographic and genomic characterization of MJNV suggested natural occurrences of recombination and reassortment in the virus population. Thus, these findings provide significant insights into the epidemiology, phylogeographic diversity, and dynamic circulation and evolution of shrew-borne hantaviruses. Hantaviruses (genus Hantavirus, family Bunyaviridae) are zoonotic, negative-sense, single-stranded RNA viruses containing large (L), medium (M), and small (S) segments that encode RNA-dependent RNA polymerase (RdRp), glycoproteins (Gn and Gc), and nucleocapsid (N) protein, respectively 1. Hantaviruses cause hemorrhagic fever with renal syndrome (HFRS) with fatality rates of 1-15% in Eurasia and hantavirus pulmonary syndrome (HPS) with mortality rates of 30-50% in Americas 2,3. Natural hosts of hantaviruses include rodents (Rodentia), bats (Chiroptera), and insectivores (Soricomorpha) 4-6. To date, hantaviruses have been identified in shrews and moles (Eulipotyphla). These viruses include Thottapalayam virus (TPMV) from the Asian house shrew (Suncus murinus), Imjin virus (MJNV) from the Ussuri white-toothed shrew (Crocidura lasiura), Jeju virus (JJUV) from the Asian lesser white-toothed shrew (C. shantungensis), Camp Ripley virus (RPLV) from the northern short-tailed shrew (Blarina brevicauda), Cao Bang virus (CBNV) from the Chinese mole shrew (Anourosorex squamipes), Boginia virus (BOGV) from the Eurasian water shrew (Neomys fodiens), Seewis virus (SWSV) from the Eurasian common shrew (Sorex araneus), and Artybash virus (ARTV) from the Laxmann's shrew (S. caecutiens) 7-15. In our previous study, we first discovered and isolated a shrew-borne hantavirus, MJNV, from C. lasiura captured near Imjin River in Gyeonggi province, Republic of Korea (ROK) 8. MJNV was also found to circulate in Southeastern China 16. A phylogeographic study of MJNV revealed the molecular diversity of the virus in a limited area, Gyeonggi province 17. Infection with MJNV elicited a robust expression of pro-inflammatory cytokines in human macrophages and endothelial cells 18. In a Syrian hamster model, MJNV infection causes a lethal disease
Rickettsiae constitute a group of arthropod-borne, Gram-negative, obligate intracellular bacteria... more Rickettsiae constitute a group of arthropod-borne, Gram-negative, obligate intracellular bacteria that are the causative agents of diseases ranging from mild to life threatening that impact on medical and veterinary health worldwide. A total of 6,484 ticks were collected by tick drag from June-October 2013 in the southwestern provinces of the Republic of Korea (ROK) (Jeollanam, n = 3,995; Jeollabuk, n = 680; Chungcheongnam, n = 1,478; and Chungcheongbuk, n = 331). Ticks were sorted into 311 pools according to species, collection site, and stage of development. DNA preparations of tick pools were assayed for rickettsiae by 17 kDa antigen gene and ompA nested PCR (nPCR) assays and the resulting amplicons sequenced to determine the identity and prevalence of spotted fever group rickettsiae (SFGR). Haemaphysalis longicornis (4,471; 52 adults, 123 nymphs and 4,296 larvae) were the most commonly collected ticks, followed by Haemaphysalis flava (1,582; 28 adults, 263 nymphs and 1,291 larva...
Hantaan virus (HTNV), a negative sense tripartite RNA virus of the Family Bunyaviridae, is the mo... more Hantaan virus (HTNV), a negative sense tripartite RNA virus of the Family Bunyaviridae, is the most prevalent hantavirus in the Republic of Korea (ROK). It is the causative agent of Hemorrhagic Fever with Renal Syndrome (HFRS) in humans and maintained in the striped field mouse, Apodemus agrarius, the primary zoonotic host. Clinical HFRS cases have been reported commonly in HFRS-endemic areas of Gyeonggi province. Recently, the death of a member of the ROK military from Gangwon province due to HFRS prompted an investigation of the epidemiology and distribution of hantaviruses in Gangwon and Gyeonggi provinces that border the demilitarized zone separating North and South Korea. To elucidate the geographic distribution and molecular diversity of HTNV, whole genome sequences of HTNV Large (L), Medium (M), and Small (S) segments were acquired from lung tissues of A. agrarius captured from 2003-2014. Consistent with the clinical incidence of HFRS established by the Korea Centers for Dise...
Ticks play an important role in transmission of arboviruses responsible for emerging infectious d... more Ticks play an important role in transmission of arboviruses responsible for emerging infectious diseases, and have a significant impact on human, veterinary, and wildlife health. In the Republic of Korea (ROK), little is known about information regarding the presence of tick-borne viruses and their vectors. A total of 21,158 ticks belonging to 3 genera and 6 species collected at 6 provinces and 4 metropolitan areas in the ROK from March to October 2014 were assayed for selected tick-borne pathogens. Haemaphysalis longicornis (n=17,570) was the most numerously collected, followed by Haemaphysalis flava (n=3317), Ixodes nipponensis (n=249), Amblyomma testudinarium (n=11), Haemaphysalis phasiana (n=8), and Ixodes turdus (n=3). Ticks were pooled (adults 1-5, nymphs 1-30, and larvae 1-50) and tested by one-step reverse transcription polymerase chain reaction (RT-PCR) or nested RT-PCR for the detection of severe fever with thrombocytopenia virus (SFTSV), tick-borne encephalitis virus (TBE...
Emerging and re-emerging infectious diseases caused by RNA viruses pose a critical public health ... more Emerging and re-emerging infectious diseases caused by RNA viruses pose a critical public health threat. Next generation sequencing (NGS) is a powerful technology to define genomic sequences of the viruses. Of particular interest is the use of whole genome sequencing (WGS) to perform phylogeographic analysis, that allows the detection and tracking of the emergence of viral infections. Hantaviruses, Bunyaviridae, cause hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS) in humans. We propose to use WGS for the phylogeographic analysis of human hantavirus infections. A novel multiplex PCR-based NGS was developed to gather whole genome sequences of Hantaan virus (HTNV) from HFRS patients and rodent hosts in endemic areas. The obtained genomes were described for the spatial and temporal links between cases and their sources. Phylogenetic analyses demonstrated geographic clustering of HTNV strains from clinical specimens with the HTNV strains circulating ...
The Democratic People's Republic of Korea (DPRK) is located on the northern part of the Korea... more The Democratic People's Republic of Korea (DPRK) is located on the northern part of the Korean Peninsula in East Asia. While tick-borne pathogens of medical and veterinary importance have been reported from China and the Republic of Korea, they have not been reported from the DPRK. To screen for zoonotic tick-borne pathogens in the DPRK, ticks were collected from domestic goats. A total of 292 (27 nymph, 26 male, 239 female) Haemaphysalis longicornis were collected and assayed individually for selected tick-borne pathogens. Using the 16S ribosomal RNA and ITS species-specific nested PCR, a total of 77 (26.4%) were positive for Anaplasma bovis, followed by Bartonella grahamii (15, 5.1%), Anaplasma phagocytophilum (12, 4.1%), Bartonella henselae (10, 3.4%), and Borrelia spp. (3, 1.0%). Using the groEL-based nested PCR, a total of 6 and 1 H. longicornis were positive for B. grahamii and B. henselae. All products were sequenced and demonstrated 100% identity and homology with previo...
Established colonies of Anopheles campestris, An. cracens, An. dirus, An. kleini, An. minimus, An... more Established colonies of Anopheles campestris, An. cracens, An. dirus, An. kleini, An. minimus, An. sawadwongporni, and An. sinensis are maintained at the Armed Forces Research Institute of Medical Sciences (AFRIMS). Females were provided blood meals on human blood containing citrate as an anticoagulant using an artificial membrane feeder. The mean wing length, used as an estimate of body size, for each species was compared to blood-feeding duration (time), blood meal volume, and numbers of eggs oviposited. Except for An. campestris and An. cracens, there were significant interspecies differences in wing length. The mean blood meal volumes (mm 3) of An. kleini and An. sinensis were significantly higher than the other 5 species. For all species, the ratios of unfed females weights/blood meal volumes were similar (range: 0.76-0.88), except for An. kleini (1.08) and An. cracens (0.52), that were significantly higher and lower, respectively. Adult females were allowed to feed undisturbed for 1, 3, and 5 minute intervals before blood feeding was interrupted. Except for An. campestris and An. sawadwongporni, the number of eggs oviposited were significantly higher for females that fed for 3 minutes when compared to those that only fed for 1 min. This information is critical to better understand the biology of colonized Anopheles spp. and their role in the transmission of malaria parasites as they relate to the relative size of adult females, mean volumes of blood of engorged females for each of the anopheline species, and the effect of blood feeding duration on specific blood meal volumes and fecundity.
A seroepidemiological study of selected rodent-borne diseases (hantavirus [Seoul [SEO] virus], sc... more A seroepidemiological study of selected rodent-borne diseases (hantavirus [Seoul [SEO] virus], scrub typhus [Orientia tsutsugamushi], murine typhus [Rickettsia typhi], and leptospirosis [Leptospira interrogans]), as part of the U.S. military rodent surveillance and control program, was conducted from 2001 through 2005 at Yongsan Garrison, Seoul, Republic of Korea. Rodents were collected to determine the prevalence of rodent-borne diseases at a U.S. military installation in an urban environment. A total of 1,750 rodents representing three species was collected by using baited live traps (Tomahawk), glue boards, and poison baits (dead rodents observed but not assayed). The Norway rat, Rattus norvegicus (99.8%), accounted for nearly all of the rodents captured/observed. Only three roof rats, Rattus rattus (0.2%), and one house mouse, Mus musculus (Ͻ0.1%), were collected. R. norvegicus rats were the only rodents that were serologically positive for SEO virus (9.6%), scrub typhus (2.8%), murine typhus (3.8%), and leptospirosis (4.6%). One of six rodents that were positive for SEO virus by immunofluorescent antibody test was positive for SEO virus antigen by reverse transcriptase-polymerase chain reaction. Infection rates for SEO virus, scrub typhus, murine typhus, and leptospirosis varied annually. Rodents were captured from 228 (20.7%) of 1,104 total buildings in Yongsan Garrison. The Yongsan commissary had the highest annual infestation rate (22 rodents per year), followed by Commisky's Club (18 rodents per year). Annual infestation rates were high for food service facilities, which often store perishable food products outdoors for short periods of time, attracting rodent populations; refuse from these facilities provides harborage and food for rodents. The effect of rodent populations outside the boundary of Yongsan Garrison was not determined.
A survey to determine the geographical distribution and relative abundance of potential vectors o... more A survey to determine the geographical distribution and relative abundance of potential vectors of scrub typhus was conducted from October to November 2006 at 13 localities throughout the Republic of Korea. Apodemus agrarius accounted for 97.6% (80/82) of all rodents, while only 2 Myodes regulus (2/82) were collected. A total of 10,860 chiggers were collected from A. agrarius belonging to 4 genera and 8 species, while only Walchia fragilis (40) was collected from Myodes regulus. Leptotrombidium pallidum (8,137; 74.9%), a vector of scrub typhus, was the predominant species collected from A. agrarius followed by Leptotrombidium scutellare (2,057, 18.9%), Leptotrombidium palpale (279; 2.7%), Leptotrombidium orientale (232; 2.1%), and Leptotrombidium zetum (79; 0.7%), Neotrombicula tamiyai (58; 0.5%), Euschoengastica koreaensis (16; 0.1%), and Cheladonta ikaoensis (2; < 0.1%). L. pallidum was the predominant chigger collected at collection sites in Gangwon (100%), Gyeonggi (87.2%), C...
A total of 1,708 small mammals (1,617 rodents and 91 soricomorphs), including Apodemus agrarius (... more A total of 1,708 small mammals (1,617 rodents and 91 soricomorphs), including Apodemus agrarius (n = 1,400), Microtus fortis (167), Crocidura lasiura (91), Mus musculus (32), Myodes (= Eothenomys) regulus (9), Micromys minutus (6), and Tscherskia (= Cricetulus) triton (3), were live-trapped at US/Republic of Korea (ROK) military training sites near the demilitarized zone (DMZ) of Paju, Pocheon, and Yeoncheon, Gyeonggi Province from December 2004 to December 2009. Small mammals were examined for their intestinal nematodes by necropsy. A total of 1,617 rodents (100%) and 91 (100%) soricomorphs were infected with at least 1 nematode species, including Nippostrongylus brasiliensis, Heligmosomoides polygyrus, Syphacia obvelata, Heterakis spumosa, Protospirura muris, Capillaria spp., Trichuris muris, Rictularia affinis, and an unidentified species. N. brasiliensis was the most common species infecting small mammals (1,060; 62.1%) followed by H. polygyrus (617; 36.1%), S. obvelata (370; 21.7%), H. spumosa (314; 18.4%), P. muris (123; 7.2%), and Capillaria spp. (59; 3.5%). Low infection rates (0.1-0.8%) were observed for T. muris, R. affinis, and an unidentified species. The number of recovered worms was highest for N. brasiliensis (21,623 worms; mean 20.4 worms/infected specimen) followed by S. obvelata (9,235; 25.0 worms), H. polygyrus (4,122; 6.7 worms), and H. spumosa (1,160; 3.7 worms). A. agrarius demonstrated the highest prevalence for N. brasiliensis (70.9%), followed by M. minutus (50.0%), T. triton (33.3%), M. fortis (28.1%), M. musculus (15.6%), C. lasiura (13.2%), and M. regulus (0%). This is the first report of nematode infections in small mammals captured near the DMZ in ROK.
Comprehensive quarterly serosurveillance on scrub typhus in small mammals collected from military... more Comprehensive quarterly serosurveillance on scrub typhus in small mammals collected from military training sites located near the Demilitarized Zone (DMZ), northern Gyeonggi-do (Province), ROK was conducted to determine the potential rodent-borne and associated ectoparasite disease risks to military personnel. A total of 1,196 rodents and insectivores representing 8 species, Apodemus agrarius (87.3%, n = 1,044), Mus musculus (5.4%, n = 65), Crocidura lasiura (3.3%, n = 40), Microtus fortis (2.6%, n = 31), Micromys minutus (0.3%, n = 4), Tscherskia triton (0.3%, n = 4), Rattus norvegicus (0.3%, n = 4), and Myodes regulus (0.3%, n = 4) were assayed for the presence of antibodies to Orientia tsutsugamushi. O. tsutsugamushi antibodies were detected in 6 of 8 species and seroprevalence determined; A. agrarius (45.6%), M. musculus (23.1%), M. fortis (48.4%), M. minutus (50.0%), T. triton (50.0%), and R. norvegicus (25.0%). A total of 31,184 chigger mites collected from 508 rodents and ins...
Adult mosquito collections using New Jersey light traps and Black-hole light traps were conducted... more Adult mosquito collections using New Jersey light traps and Black-hole light traps were conducted to determine the potential vectors and the relative mosquito infection rates of Dirofilaria immitis and Dirofilaria repens in Gyeonggi and Gangwon Provinces, Republic of Korea, 2005. Dirofilaria spp. were confirmed by polymerase chain reaction (PCR) using species-specific primers for D. immitis and D. repens. Minimum field infection rates (MFIR) [MFIR = (number infected pools/total number of mosquitoes) Â 1000] of 12 pools/2059 total number mosquitoes (5.8) and 21 pools (10.1) of the wild caught mosquitoes were positive by PCR for D. immitis and D. repens, respectively. Dual infections of both D. immitis and D. repens were detected by PCR in eight pools (3.9). Anopheles sinensis sensu lato (includes An. sinensis s.s., An. pullus, An. kleini, An. belenrae, and An. lesteri), An. sineroides, Aedes vexans nipponi, Culex pipiens and Armigeres subalbatus were found to be infected and are potential vectors of D. immitis and D. repens in Korea. Infections observed in mosquitoes represent potential health risks for domestic animal and human populations.
Recently, Imjin virus (MJNV), a genetically distinct hantavirus, was isolated from lung tissues o... more Recently, Imjin virus (MJNV), a genetically distinct hantavirus, was isolated from lung tissues of the Ussuri white-toothed shrew (Crocidura lasiura) captured near the demilitarized zone in the Republic of Korea. To clarify the genetic diversity of MJNV, partial M- and L-segment sequences were amplified from lung tissues of 12 of 37 (32.4%) anti-MJNV IgG antibody-positive Ussuri white-toothed shrews captured between 2004 and 2010. A 531-nucleotide region of the M segment (coordinates 2,255 to 2,785) revealed that the 12 MJNV strains differed by 0-12.2% and 0-2.3% at the nucleotide and amino acid levels, respectively. A similar degree of nucleotide (0.2-11.9%) and amino acid (0-3.8%) difference was found in a 632-nucleotide length of the L segment (coordinates 962 to 1,593) of nine MJNV strains. Phylogenetic analyses, based on the partial M and L segments of MJNV strains generated by the neighbor-joining and maximum likelihood methods, showed geographic-specific clustering, akin to t...
This study describes the seasonal distribution of larvae, nymph, and adult life stages for 3 spec... more This study describes the seasonal distribution of larvae, nymph, and adult life stages for 3 species of ixodid ticks collected by tick drag and sweep methods from various habitats in the Republic of Korea (ROK). Grasses less than 0.5 m in height, including herbaceous and crawling vegetation, and deciduous, conifer, and mixed forests with abundant leaf/needle litter were surveyed at United States (US) and ROK operated military training sites and privately owned lands near the demilitarized zone from April-October, 2004 and 2005. Haemaphysalis longicornis Neumann adults and nymphs were more frequently collected from April-August, while those of Haemaphysalis flava Neumann and Ixodes nipponensis Kitaoka and Saito were collected more frequently from April-July and again during October. H. longicornis was the most frequently collected tick in grass habitats (98.9%), while H. flava was more frequently collected in deciduous (60.2%) and conifer (57.4%) forest habitats. While more H. flava (54.1%) were collected in mixed forest habitats than H. longicornis (35.2%), the differences were not significant. I. nipponensis was more frequently collected from conifer (mean 8.8) compared to deciduous (3.2) and mixed (2.4) forests.
A total of 9,281 larval chigger mites were collected from small mammals captured at Hwaseong-gun,... more A total of 9,281 larval chigger mites were collected from small mammals captured at Hwaseong-gun, Gyeonggi-do (Province) (2,754 mites from 30 small mammals), Asan city, Chungcheongnam-do (3,358 mites from 48 mammals), and Jangseong-gun, Jeollanam-do (3,169 for 62 mammals) from April-November 2009 in the Republic of Korea (= Korea) and were identified to species. Leptotrombidium pallidum was the predominant species in Hwaseong (95.8%) and Asan (61.2%), while Leptotrombidium scutellare was the predominant species collected from Jangseong (80.1%). Overall, larval chigger mite indices decreased from April (27.3) to June (4.9), then increased in September (95.2) and to a high level in November (169.3). These data suggest that L. pallidum and L. scutellare are the primary vectors of scrub typhus throughout their range in Korea. While other species of larval chigger mites were also collected with some implications in the transmission of Orientia tsutsugamushi, they only accounted for 11.2% of all larval chigger mites collected from small mammals.
A tick survey was conducted to determine the relative abundance and distribution of ticks associa... more A tick survey was conducted to determine the relative abundance and distribution of ticks associated with selected mammals in the Republic of Korea (ROK) during 2008-2009. A total of 918 ticks were collected from 76 mammals (6 families, 9 species) captured at 6 provinces and 3 Metropolitan Cities in ROK. Haemaphysalis longicornis (54.4%) was the most frequently collected tick, followed by Haemaphysalis flava (28.5%), Ixodes nipponensis (7.6%), Ixodes pomerantzevi (4.8%), Ixodes persulcatus (4.6%), and Haemaphysalis japonica (0.1%). Adults (57.0%) and nymphs (28.7%) of Ixodes and Haemaphysalis spp. were collected most frequently from medium or large mammals in this survey, while few larvae (14.3%) were collected. Hydropotes inermis was the most frequently captured mammal (52.6%), with a 16.4 tick index and 5 of 6 species of ticks collected during this survey. H. longicornis (69.7%) was the predominant tick collected from H. inermis, followed by H. flava (22.2%), I. persulcatus (6.1%)...
Ticks were collected from 35 animals from 5 provinces and 3 metropolitan cities during 2012. Tick... more Ticks were collected from 35 animals from 5 provinces and 3 metropolitan cities during 2012. Ticks also were collected by tick drag from 4 sites in Gyeonggi-do (2) and Jeollabuk-do (2) Provinces. A total of 612 ticks belonging to 6 species and 3 genera were collected from mammals and a bird (n= 573) and by tick drag (n= 39). Haemaphyalis longicornis (n= 434) was the most commonly collected tick, followed by H. flava (158), Ixodes nipponensis (11), Amblyomma testudinarium (7), H. japonica (1), and H. formosensis (1). H. longicornis and H. flava were collected from all animal hosts examined. For animal hosts (n> 1), the highest Tick Index (TI) was observed for domestic dogs (29.6), followed by Siberian roe deer (17.4), water deer (14.4), and raccoon dogs (1.3). A total of 402 H. longicornis (adults 86, 21.4%; nymphs 160, 39.8%; larvae 156, 38.9%) were collected from wild and domestic animals. A total of 158 H. flava (n= 158) were collected from wild and domestic animals and 1 ring-necked pheasant, with a higher proportion of adults (103, 65.2%), while nymphs and larvae only accounted for 12.7% (20) and 22.2% (35), respectively. Only 7 A. testudinarium were collected from the wild boar (6 adults) and Eurasian badger (1 nymph), while only 5 I. nipponensis were collected from the water deer (4 adults) and a raccoon dog (1 adult). One adult female H. formosensis was first collected from vegetation by tick drag from Mara Island, Seogwipo-si, Jeju-do Province.
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