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Tick surveillance was conducted annually from February–November 2019–2021 at US Army Garrison (USAG) Humphreys, Pyeongtaek, Republic of Korea (ROK). Three general habitats, cut grasses, uncut grasses + herbaceous vegetation, and young mixed (deciduous and conifer) forest groves, were surveyed using tick drags. Ticks also were collected from Korean water deer, Hydropotes inermis argyropus, killed by vehicles at USAG Humphreys. A total of 53,428 ticks (18,004, 16,921, and 18,503 during 2019–2021, respectively) belonging to two genera and five species were collected by tick drag. Haemaphysalis longicornis (48,880; 91.49%) was the most frequently collected tick, followed by H. flava (2,501; 4.68%), Ixodes nipponensis (2,019; 3.78%), H. phasiana (27; 0.05%), and H. japonica (1

The extended distribution and potential introduction of exotic ticks and associated tick-borne pathogens along the northern and southern routes of migratory birds pose zoonotic tick-borne disease r...

As part of a tick-borne disease surveillance program, ticks were collected using tick drags and by making incidental collections from humans and animals in six provinces (Gyeonggi, Gangwon, Chungnam, Gyeongbuk, Gyeongnam, and Jeonbuk) of the Republic of Korea (ROK) during 2020–2021. A total of 57,751 Haemaphysalis longicornis, 2,020 H. flava, 1,861 Ixodes nipponensis, 14 H. phasiana, 9 H. japonica, and 9 Amblyomma testudinarium were collected. Cases of ectromely were observed in two H. longicornis with six legs (one female and one nymph), and six H. longicornis (two females and four nymphs), two female Haemaphysalis flava and one male Ixodes nipponensis with seven legs. Additionally, two female H. longicornis were found with extra legs (tarsus), and two female H. longicornis and one male H. japonica were observed with atrophied legs.

There are currently >300 malaria cases reported annually in the Republic of Korea (ROK), with most cases attributed to exposure in northern Gangwon and Gyeonggi provinces near the demilitarized zone (DMZ). The species diversity and malaria infection rate were determined for a sample of Anopheles mosquitoes collected from May to early November 2020 for six sites in a malaria high-risk area in/near the DMZ and two malaria low-risk areas in southern Gyeonggi province using Mosquito Magnet traps in the ROK. A total of 1864 Anopheles spp. were identified to species by PCR. Overall, An. kleini (31.4%, 510/1622) was the most frequently species assayed, followed by An. pullus (25.5%, 413/1622), An. sineroides (23.9%, 387/1622), and An. sinensis (10.2%, 165/1622), while the other four species only accunted for 9.1% (147/1622) collected in/near the DMZ. Only three species, An. pullus, An. sinensis, and An. sineroides were collected at Humphreys US Army Garrison (USAG) (235 individuals), while only An. sinensis was collected at Yongsan USAG (7 individuals). A total of 36 Anopheles specimens belonging to five species collected in/near the DMZ were positive for Plasmodium vivax by PCR. Anopheles kleini (9) was the most frequent species positive for P. vivax, followed by An. belenrae (8), An. pullus (8), An. sinensis (5), An. sineroides (5), and a member of the Anopheles Lindesayi Complex in the ROK (1). This is the first report of P. vivax in a member of the An. Lindesayi Complex in the ROK. These findings can assist in guiding future malaria vector management in the ROK.

Tick-borne disease surveillance was conducted by tick drag among uncut grasses/herbaceous vegetation and mixed forest habitats from 2018–2020 at US Army Garrison Humphreys, Pyeongtaek, the Republic of Korea (ROK). While identifying ticks collected in the spring of 2020, teratological anomalies were observed in nymphs of Haemaphysalis longicornis (in one specimen, partial twinning of the posterior idiosoma, resulting in 2 anuses; in a second, asymmetry of the idiosoma) and Ixodes nipponensis (7 legs). These teratological observations in H. longicornis and I. nipponensis are reported for the first time in the ROK.

Deer and horse flies, Family Tabanidae, are biological, mechanical, and potential vectors of approximately 27 viral, bacterial, and helminthic pathogens of veterinary and medical importance. Biting fly surveillance was conducted using New Jersey light traps and Mosquito Magnet® traps from May-October during 2015–2018 at eight sites (villages and US military installations and training areas) near/in the Demilitarized Zone and at a US military installation in southern Gyeonggi province, Republic of Korea. A total of 9985 deer and horse flies, comprising 4 genera and 17 species, were collected. The predominant species collected was Chrysops mlokosiewiczi (95.39%), followed by Chrysops suavis (1.48%) and Haematopota koryoensis (1.07%), while the remaining species accounted for only 2.06% of the specimens collected. A bimodal peak was observed for C. mlokosiewiczi in June and late-July, but not for the other two predominant species during the whole of the study period. This study provides and insight into ecological behavior and seasonal abundance of deer and horse flies in Gyeonggi province, Republic of Korea.

Two-point mutations (V419L and L925I) on the voltage-sensitive sodium channel of bed bugs (Cimex lectularius) are known to confer pyrethroid resistance. To determine the status of pyrethroid resistance in bed bugs in Korea, resistance allele frequencies of bed bug strains collected from several US military installations in Korea and Mokpo, Jeollanamdo, from 2009-2019 were monitored using a quantitative sequencing. Most bed bugs were determined to have both of the point mutations except a few specimens, collected in 2009, 2012 and 2014, having only a single point mutation (L925I). No susceptible allele was observed in any of the bed bugs examined, suggesting that pyrethroid resistance in bed bug populations in Korea has reached a serious level. Large scale monitoring is required to increase our knowledge on the distribution and prevalence of pyrethroid resistance in bed bug populations in Korea. Based on present study, it is urgent to restrict the use of pyrethroids and to introduce effective alternative insecticides. A nation-wide monitoring program to determine the pyrethroid resistance level in bed bugs and to select alternative insecticides should be implemented.

Tick-borne pathogens are contributing factors for the increased incidence of vector-borne diseases throughout the world, including Lyme borreliosis, one of the most prevalent spirochetes belonging to the Borrelia burgdorferi sensu lato group. The present study focused on the detection of Borrelia species from hard ticks collected at U.S. Army Garrison Humphreys, Republic of Korea (ROK), using molecular and genotypic analyses. Tick-borne disease surveillance was conducted from January to December, 2018–2019. A total of 24,281 ticks (2 genera and 5 species) were collected from road-killed Korean Water deer (KWD) and by tick drag. Haemaphysalis longicornis (92.0%) was the most commonly collected species, followed by Haemaphysalis flava (4.9%), Ixodes nipponensis (3.1%), Haemaphysalis phasiana (0.07%), and Haemaphysalis japonica (

Background Genus Anopheles mosquitoes are the primary vectors of human malaria, which is a serious threat to public health worldwide. To reduce the spread of malaria and identify the malaria infection rates in mosquitoes, accurate species identification is needed. Malaria re-emerged in 1993 in the Republic of Korea (ROK), with numbers peaking in 2004 before decreasing to current levels. Eight Anopheles species (Anopheles sinensis, Anopheles pullus, Anopheles belenrae, Anopheles lesteri, Anopheles kleini, Anopheles sineroides, Anopheles koreicus, Anopheles lindesayi) are distributed throughout Korea. Members of the Anopheles Hyrcanus group currently cannot be identified morphologically. The other species of Anopheles can be identified morphologically, except when specimens are damaged in traps. The purpose of this study was to develop a rapid and accurate method for simultaneous molecular identification of the eight Anopheles species present in the ROK. Methods Anopheles spp. used in this study were collected near/in the demilitarized zone in ROK, where most malaria cases are reported. DNA from 165 of the Anopheles specimens was used to develop a multiplex PCR assay. The internal transcribed spacer 2 (ITS2) region of each species was sequenced and analysed for molecular identification. Results DNA from a total of 165 Anopheles specimens was identified to species using a multiplex diagnostic system. These included: 20 An. sinensis, 21 An. koreicus, 17 An. lindesayi, 25 An. kleini, 11 An. lesteri, 22 An. sineroides, 23 An. belenrae, and 26 An. pullus. Each species was clearly distinguished by electrophoresis as follows: 1,112 bp for An. sinensis; 925 bp for An. koreicus; 650 bp for An. lindesayi; 527 bp for An. kleini; 436 bp for An. lesteri; 315 bp for An. sineroides; 260 bp for An. belenrae; and, 157 bp for An. pullus. Conclusion A multiplex PCR assay was developed to identify Anopheles spp. distributed in ROK. This method can be used to accurately identify Anopheles species that are difficult to identify morphologically to determine species distributions and malaria infection rates.

This study was done to characterize distribution of Rickettsia spp. in ticks in the northwestern and southwestern provinces in the Republic of Korea. A total of 2,814 ticks were collected between May and September 2009. After pooling, 284 tick DNA samples were screened for a gene of Rickettsia-specific 17-kDa protein using nested PCR (nPCR), and produced 88 nPCR positive samples. Of these positives, 75% contained 190-kDa outer membrane protein gene (ompA), 50% 120-kDa outer membrane protein gene (ompB), and 64.7% gene D (sca4). The nPCR products of ompA, ompB, and sca4 genes revealed close relatedness to Rickettsia japonica, R. heilongjiangensis, and R. monacensis. Most Rickettsia species were detected in Haemaphysalis longicornis. This tick was found a dominant vector of rickettsiae in the study regions in the Republic of Korea.

Biting midges (Culicoides: Ceratopogonidae) were collected using New Jersey light traps at Yongsan US Army Garrison (USAG;urban), Seoul Metropolitan city and Camp Humphreys USAG (rural), Pyeongtaek, Gyeonggi-do (province), Republic of Korea , from May-October 2010-2013 and 2015-2017, to determine species composition and seasonal distribution patterns in urban and rural habitats. A total of 9,958 female (53.85%) and 8,533 male (46.15%) Culicoides comprising 16 species were collected. Overall, the most commonly collected species was Culicoides arakawae (74.3%), followed by C. circumscriptus (16.2%), C. kibunensis (2.5%), C. nasuensis (2.2%), C. clavipalpis (1.4%), and C. pallidulus (1.3%), while the remaining 10 species accounted for

Background Genus Anopheles mosquitoes are the primary vectors of human malaria, which is a serious threat to public health worldwide. To reduce the spread of malaria and identify the malaria infection rates in mosquitoes, accurate species identification is needed. Malaria reemerged in 1993 in the Republic of Korea (ROK), with numbers peaking in 2004 before decreasing to current levels. Eight Anopheles species (Anopheles sinensis, An. pullus, An. belenrae, An. lesteri, An. kleini, An. sineroides, An. koreicus, and An. lindesayi) are distributed throughout Korea. Members of the Anopheles Hyrcanus Group currently cannot be identified morphologically. The other species of Anopheles can be identified morphologically, except when specimens are damaged in traps. The purpose of this study was to develop a rapid and accurate method for simultaneous molecular identification of the eight Anopheles species present in the ROK. Methods Anopheles spp. used in this study were collected near/in the demilitarized zone in the ROK, where most malaria cases are reported. DNA from 165 of the Anopheles specimens was used to develop a multiplex PCR assay. The internal transcribed spacer 2 (ITS2) region of each species was sequenced and analyzed for molecular identification. Results DNA from a total of 165 Anopheles specimens was identified to species using a multiplex diagnostic system. These included: 20 An. sinensis, 21 An. koreicus, 17 An. lindesayi, 25 An. kleini, 11 An. lesteri, 22 An. sineroides, 23 An. belenrae, and 26 An. pullus. Molecular weights determined by electrophoresis were 1112 bp for An. sinensis, 925 bp for An. koreicus, 650 bp for An. lindesayi, 527 bp for An. kleini, 436 bp for An. lesteri, 315 bp for An. sineroides, 260 bp for An. belenrae, and 157 bp for An. pullus. Conclusion A multiplex PCR assay was developed to identify Anopheles spp. distributed in the ROK. This method can be used to accurately identify Anopheles species that are difficult to identify morphologically to determine species distributions and malaria infection rates.

The extended distribution and potential introduction of exotic ticks and associated tick-borne pathogens along the northern and southern routes of migratory birds pose zoonotic tick-borne disease risks to wild and domestic animals and incidentally to humans. A knowledge of bird migratory patterns, species of attached ticks, and associated pathogens during their migrations to and from their feeding and nesting grounds is central to understanding associated tick-borne disease risks. Tick-borne disease surveillance was conducted from 2010 to 2011 and 2016 at Hong-do (do = island), Heuksan-do, and Nan-do, major stopovers for migratory birds in Republic of Korea (ROK), as part of the Migratory Birds Research Center bird-banding program for studying bird migration patterns in the ROK. A total of 877 ticks belonging to three genera and nine species were collected

In a follow-up to the investigations of soft ticks identified from seabird nest soil and litter collected from coastal islands of the Republic of Korea (ROK), Ornithodoros sawaii and Ornithodoros capensis were assessed for the presence and identification of rickettsiae. Ticks collected from samples of 50–100 g of nest litter and soil from seabird nests were identified individually by morphological techniques, and species confirmed by sequencing of the mt-rrs gene. Subsequently, tick DNA preparations were screened for the presence of rickettsiae using a genus-specific nested PCR (nPCR) assay targeting the 17 kDa antigen gene. The amplicons from the 17 kDa assay and two additional nPCR assays targeting the gltA and ompB gene fragments were sequenced and used to identify the rickettsiae. A total of 134 soft ticks belonging to two species, O. sawaii Kitaoka & Suzuki 1973 (n = 125) and O. capensis Neumann 1901 (n = 9), were collected. Rickettsia lusitaniae DNA was detected and identified among O. sawaii ticks (n = 11, 8.8%) collected from nest litter and soil of the Japanese murrelet (Synthliboramphus wumizusume Temminck 1836) at Gugul Island along the western coastal area of the ROK. This study confirmed for the first time the presence of R. lusitaniae associated with O. sawaii collected from migratory seabird nests in the ROK.

Haemaphysalis longicornis, the cattle tick or bush tick, has an extended distribution throughout Asia and the Pacific region, including China, Russia, the Republic of Korea (ROK), Japan, Australia, New Zealand, and the South Pacific islands. It is an obligate ectoparasite found commonly on medium to large sized wild and domestic animals, with humans as an accidental host. Haemaphysalis longicornis transmits a number of pathogens, including severe fever with thrombocytopenia syndrome and tick-borne encephalitis viruses, bacteria, helminths, and protozoans, that impact on veterinary (wild and domestic animals) and human health. Surveys of rickettsial pathogens associated with H. longicornis from China, the ROK, and Japan have resulted in the discovery of more than 35 incompletely characterized molecular isolates of Rickettsia. In response to the increased global threat of tick-borne rickettsial diseases, H. longicornis collected in the ROK and China were assessed in our laboratory and two additional Rickettsia spp. isolates (ROK-HL727 and XinXian HL9) were identified. These agents were fully characterized by multilocus sequence typing using partial gene fragment sequences of rrs, gltA, ompA, ompB, and sca4. Phylogenetic comparisons of these Rickettsia isolates with known Rickettsia species and other molecular isolates identified from H. longicornis were performed to better understand their interrelationships. Phylogenetic analysis of the sequences from these 5 gene fragments showed that ROK-HL727 was closely related to rickettsial isolates of H. longicornis previously reported from China, the ROK and Japan, but distinct from any currently recognized Rickettsia species. It therefore qualifies genetically as a new species, introduced herein as Candidatus Rickettsia longicornii. The XinXian-HL9 isolate detected from China was determined to be genetically similar to the human pathogen Rickettsia heilongjiangensis. People living and working in areas where H. longicornis is endemic should be aware of the potential for rickettsial diseases.

Ticks are vectors of a wide range of zoonotic viruses of medical and veterinary importance. Recently, metagenomics studies demonstrated that they are also the source of potentially pathogenic novel viruses. During the period from 2015 to 2017, questing ticks were collected by dragging the vegetation from geographically distant locations in the Republic of Korea (ROK) and a target-independent high-throughput sequencing method was utilized to study their virome. A total of seven viruses, including six putative novel viral entities, were identified. Genomic analysis showed that the novel viruses were most closely related to members in the orders Jingchuvirales and Bunyavirales. Phylogenetic reconstruction showed that the Bunyavirales-like viruses grouped in the same clade with other viruses within the Nairovirus and Phlebovirus genera, while the novel Jingchuvirales-like virus grouped together with other viruses within the family Chuviridae. Real-time RT-PCR was used to determine the geographic distribution and prevalence of these viruses in adult ticks. These novel viruses have a wide geographic distribution in the ROK with prevalences ranging from 2% to 18%. Our study expands the knowledge about the composition of the tick virome and highlights the wide diversity of viruses they harbor in the ROK. The discovery of novel viruses associated with ticks in the ROK highlights the need for an active tick-borne disease surveillance program to identify possible reservoirs of putative novel human pathogens.

Encounters with ticks harboring pathogenic agents have demonstrated increasing public health implications. Tick surveillance in the Republic of Korea (ROK) is essential for determining tick distributions and the potential regions where tick-borne pathogens may be found. Extensive tick collections (tick drags and tick flagging) were previously performed by Force Health Protection & Preventive Medicine (FHP&PM), Medical Activity-Korea (MEDDAC-K)/65th Medical Brigade (MED BDE) personnel, in collaboration with the Public Health Activity-Korea in the ROK. A total of 144,131 ticks were collected from 2,019 locations during 2004 to 2016. The associated location data (GPS coordinates) for each of the collection sites were incorporated into distribution maps using ArcGIS and combined with environmental data in the Maxent ecological niche modeling program (n = 733 geographical unique locations from 1,429 presence records/collection locations) to produce estimates of tick distributions for each species. The predominant tick species found and modeled were, in order of prevalence: Haemaphysalis longicornis, H. flava, Ixodes nipponensis, H. phasiana, I. turdus, Amblyomma testudinarium, H. japonica, and I. persulcatus. Haemaphysalis longicornis, H. flava, and I. nipponensis were the most widely distributed and most commonly collected species of ticks. The maps and models of suitable habitat regions produced in this study provide a better understanding of where there are potential risks of encountering a particular tick species, and which, as demonstrated herein with rickettsiae, can be used to study tick-pathogen dynamics of diseases. Knowledge of the distribution of ticks is important in the ROK because of the presence of tick-borne diseases, such as severe fever with thrombocytopenia syndrome, tick-borne encephalitis, rickettsioses, and borrelioses.

A total of 6,255 ticks belonging to three genera and six species (Haemaphysalis flava Neumann, Haemaphysalis longicornis Neumann, Haemaphysalis phasiana Saito, Ixodes nipponensis Kitaoka & Saito, Ixodes persulcatus Schulze, and Amblyomma testudinarium Koch) collected from May-August, 2013, at four southwestern provinces in the Republic of Korea (ROK) were submitted to the Armed Forces Research Institute of Medical Sciences and assayed for selected tick-borne pathogens. One pool each of H. flava and H. phasiana was positive by PCR and sequencing for a Francisella-like endosymbiont, while all pools were negative for Francisella tularensis, the causative agent of tularemia.

Flaviviruses comprise a large and diverse group of positive-stranded RNA viruses, including tick-, mosquito- and unknown-vector-borne flaviviruses. A novel flavivirus was detected in pools of Aedes vexans nipponii (n=1) and Aedes esoensis (n=3) collected in 2012 and 2013 near the demilitarized zone (DMZ), Republic of Korea (ROK). Phylogenetic analyses of the NS5, E gene and complete polyprotein coding sequence (CDS) showed that the novel virus fell within the Aedes-borne flaviviruses (ABFVs), with nucleotide identity ranging from 57.8-75.1 %, 46.1-74.2 % and 51.1-76.2 %, respectively. While the novel ABFV was distant from other flaviviruses within the group, it formed a clade with Ilomantsi virus (ILOV). Sequence alignments of the partial NS5 gene, full-length E gene and polyprotein CDS between the novel virus and ILOV showed approximately 76.2 % nucleotide identity and 90 % amino acid identity, respectively. The ABFV identified in Aedes mosquitoes from the ROK is a novel ABFV based on the sequence analyses and is designated as Panmunjeom flavivirus (PANFV).

Pyrethroid (PYR) and organophosphate (OP) insecticides have been extensively used for mosquito control for several decades in South Korea, and has resulted in the rapid development of resistance in the field. In this study, quantitative sequencing (QS) protocols were developed for the frequency prediction of insecticide resistance alleles [e.g., the L1014F/C mutation on the voltage sensitive sodium channel as a PYR resistance allele and the G119S mutation on the acetylcholinesterase 1 as OP resistance alleles] in four regional populations of Anopheles Hyrcanus Group and Culex pipiens complex. Both of the L1014F/C and G119S mutations were observed in all examined regional populations of An. Hyrcanus Group, suggesting a wide distribution of both PYR and OP resistance. In contrast, populations of the Cx. pipiens complex were determined to possess almost no G119S mutation, but relatively higher frequencies of the L1014F mutation, showing differential resistance patterns between different mosquito groups. The mutation frequencies were also monitored throughout a mosquito season (May-October) at one collection site to determine the seasonal changes of resistance mutation frequency in mosquito populations. Dramatic decreases of both L1014F/C and G119S mutation frequencies were observed in the An. Hyrcanus Group toward the fall, with no mutations observed in the early spring, suggesting a connection between the fitness costs of overwintering and insecticide resistance. However, no apparent trends were detectable in the Cx. pipiens complex populations due to low or zero mutation frequencies.

To identify spotted fever group (SFG) rickettsiae among ticks collected by dragging at eight sites in three provinces of the midwestern region of the Republic of Korea (ROK), genus- and species-specific quantitative real-time PCR (qPCR) assays and sequencing were performed. DNA was extracted from a total of 2,312 ticks that were assayed individually (n=140) or in pools (n=444), resulting in a total of 584 individual and pooled tick samples. The 584 tick samples were screened with the genus-specific qPCR assay (Rick17b) and produced 265 (45.38%) positive reactions [individual (n=64) and pooled (n=101) samples]. Of these genus-specific positive samples, 57 (21.51%) were identified as Candidatus Rickettsia longicornii and 48 (18.11%) were identified as R. monacensis by species-specific qPCR assays. Subsequently, nested PCR (nPCR) was performed with 120 samples, which tested positive samples for genus-specific, but not species-specific, qPCR assays. The sequences of ompA and ompB genes showed how many close relatedness to Ca. R. longicornii and Ca. R. jingxinensis isolate Xian Hl-79, uncultured Rickettsia sp. Y27-1, Ca. R. tasmanensis strain T152, R. endosymbiont of H. longicornis tick 47, and R. koreansis strain CNH17-7. In conclusion, we successfully detected specific rickettsial agents using qPCR and a sequence-based analysis approach that demonstrated the prevalence of various tick-borne Rickettsia spp. in midwestern ROK.

Severe fever with thrombocytopenia syndrome (SFTS) is caused by SFTS virus (SFTSV), a novel bunyavirus reported to be endemic to central-northeastern China, southern Japan, and the Republic of Korea (ROK). To investigate SFTSV infections, we collected serum samples and ticks from wild animals. Using serum samples and ticks, SFTSV-specific genes were amplified by one-step RT-PCR and nested PCR and sequenced. Indirect immunofluorescence assay (IFA) was performed to analyze virus-specific antibody levels in wild animals. Serum samples were collected from a total of 91 animals: 21 Korean water deer (KWD), 3 Siberian roe deer, 5 gorals, 7 raccoon dogs, 54 wild boars (WBs), and 1 carrion crow. The SFTSV infection rate in wild animals was 3.30% (3 of 91 animals: 1 KWD and 2 WBs). The seropositive rate was 6.59% (6 of 91 animals: 5 KWD and 1 WB). A total of 891 ticks (3 species) were collected from 65 wild animals (9 species). Of the attached tick species, Haemaphysalis longicornis (74.86%) was the most abundant, followed by Haemaphysalis flava (20.20%) and Ixodes nipponensis (4.94%). The average minimum infection rate (MIR) of SFTSV in ticks was 4.98%. The MIRs of H. longicornis, H. flava, and I. nipponensis were 4.51%, 2.22%, and 22.73%, respectively. The MIRs of larvae, nymphs, and adult ticks were 0.68%, 6.88%, and 5.53%, respectively. In addition, the MIRs of fed and unfed ticks were 4.67% and 4.96%, respectively. We detected a low SFTSV infection rate in wild animals, no differences in SFTSV infection rate with respect to bloodsucking in ticks, and SFTSV infection for all developmental stages of ticks. This is the first report describing the detection of SFTSV in wild animals in the ROK.

The 65th Medical Brigade and Public Health Command District-Korea, in collaboration with the Migratory Bird Research Center, National Park Research Institute, conducted migratory bird tick surveillance at Sogugul and Gaerin Islands (small rocky bird nesting sites), Jeollanam-do (Province), Republic of Korea (ROK), on 30 July and 1 August 2009. Breeding seabirds captured by hands in their nesting burrows were banded, identified to species, and carefully examined for ticks during the nesting season. A total of 9 Ornithodoros sawaii larvae were removed from 4 adult Hydrobates monorhis (Swinhoe’s storm petrel). The identification of the larvae of O. sawaii collected from migratory seabirds were molecularly confirmed using mitochondrial 16S rDNA primer sets.

Deer serve as reservoirs of tick-borne pathogens that impact on medical and veterinary health worldwide. In the Republic of Korea, the population of Korean water deer (KWD, Hydropotes inermis argyropus) has greatly increased from 1982 to 2011, in part, as a result of reforestation programs established following the Korean War when much of the land was barren of trees. Eighty seven Haemaphysalis flava, 228 Haemaphysalis longicornis, 8 Ixodes nipponensis, and 40 Ixodes persulcatus (21 larvae, 114 nymphs, and 228 adults) were collected from 27 out of 70 KWD. A total of 89/363 ticks (266 pools, 24.5% minimum infection rate) and 5 (1.4%) fed ticks were positive for Anaplasma phagocytophilum using nested PCR targeting the 16S rRNA and groEL genes, respectively. The 16S rRNA gene fragment sequences of 88/89 (98.9%) of positive samples for A. phagocytophilum corresponded to previously described gene sequences from KWD spleen tissues. The 16S rRNA gene fragment sequences of 20/363 (5.5%) of the ticks were positive for A. bovis and were identical to previously reported sequences. Using the ITS specific nested PCR, 11/363 (3.0%) of the ticks were positive for Bartonella spp. This is the first report of Anaplasma and Bartonella spp. detected in ticks collected from KWD, suggesting that ticks are vectors of Anaplasma and Bartonella spp. between reservoir hosts in natural surroundings.

Rodent-borne pathogens pose a critical public health threat in urban areas. An epidemiological survey of urban rodents was conducted from 2006 to 2010 at the U.S. Army Garrison (USAG), Seoul, Republic of Korea (ROK), to determine the prevalence of Seoul virus (SEOV), a rodent-borne hantavirus. A total of 1,950 rodents were captured at USAG, Yongsan, near/in 19.4% (234/1,206) of the numbered buildings. Annual mean rodent infestation rates were the highest for food service facilities, e.g., the Dragon Hill Lodge complex (38.0 rodents) and the Hartell House (18.8 rodents). The brown rat, Rattus norvegicus, accounted for 99.4% (1,939/1,950) of all the rodents captured in the urban area, whereas only 0.6% (11/1,950) of the rodents was house mice (Mus musculus). In November 2006, higher numbers of rats captured were likely associated with climatic factors, e.g., rainfall and temperatures as rats sought harborage in and around buildings. Only 4.7% (34/718) of the rodents assayed for hantaviruses was serologically positive for SEOV. A total of 8.8% (3/34) R. norvegicus were positive for SEOV RNA by reverse transcription polymerase chain reaction, of which two SEOV strains were completely sequenced and characterized. The 3' and 5' terminal sequences revealed incomplete complementary genomic configuration. Seoul virus strains Rn10-134 and Rn10-145 formed a monophyletic lineage with the prototype SEOV strain 80-39. Seoul virus Medium segment showed the highest evolutionary rates compared with the Large and Small segments. In conclusion, this report provides significant insights into continued rodent-borne disease surveillance programs that identify hantaviruses for analysis of disease risk assessments and development of mitigation strategies.

The seasonal abundance of horse and deer flies (family Tabanidae) was analyzed using Mosquito Magnet(®) traps at 5 sites located near/in the demilitarized zone, northern Gyeonggi-do, Republic of Korea from late April to early October for 4 consecutive years (2010-2013). A total of 2,999 horse and deer flies (tabanids) belonging to 5 genera and 20 species were collected. Chrysops mlokosiewiczi (90.9%) was the most frequently collected, followed by Haematopota koryoensis (4.8%) and C. suavis (1.0%). The remaining 17 species comprised only of 3.3% of all species collected. C. mlokosiewiczi demonstrated bimodal peak populations during mid-June and early August, while H. koryoensis demonstrated a unimodal peak during mid-July. Overall numbers of tabanids collected were influenced by the previous year's winter temperatures and precipitation. Population abundance was influenced by habitat with most of tabanids collected from habitats near forested areas, followed by rice paddies, and a beef farm.

A total of 4,576 Anopheles (Anopheles) Hyrcanus Group larvae belonging to 6 species (An. belenrae, An. kleini, An. sinensis, An. pullus, An. lesteri, and An. sineroides) were collected from 7 different habitat types in 3 provinces of the Republic of Korea. The occurrence and relative abundance of 6 Anopheles species were noted. The descriptions in the article of the waxy body ornamentations or patterns of An. (Ano.) Hyrcanus Group larvae and pupae may be useful for rapid field species identification when conducting larval mosquito surveillance.

Small mammal surveillance was conducted (2008-2010, 2012) at Camp (Cp) Humphreys, a US Army installation and new expansion site, Republic of Korea (ROK), to identify hemorrhagic fever with renal syndrome health threats to US military/civilian populations during its ongoing expansion phase. Small mammals were collected using Sherman live capture traps and transported to Korea University where they were euthanized, tissues removed, and assayed to determine hantavirus IgG antibody-positive and hantavirus-positive rates by RT-PCR. A total of 2,364 small mammals were captured over 11,300 trap nights (capture rate = 20.92%). Apodemus agrarius was the most commonly collected (76.65%), with capture rates of 9.62% and 21.70% for Cp Humphreys and the expansion site, respectively. Overall, Hantaan virus (HTNV) IgG antibody-positive (Ab+) rate for A. agrarius was 2.15% (39/1,812). A total of 5.43% (10/184) Crocidura lasiura, 0.79% (2/254) Microtus fortis and 2.44% (1/41) Micromys minutus were serologically IgG Ab+ for hantaviruses. HTNV-specific RT-PCR demonstrated that 28.2% (11/39) HTNV Ab+ A. agrarius harbored the 328-nt sequence of the GC glycoprotein-encoding M segment of HTNV. Among them, the whole genome sequences of 3 HTNV strains were obtained by conventional RT-PCR and Rapid Amplification cDNA Ends PCR. Phylogenetic analyses of the HTNV strains from Cp Humphreys and the expansion site, Pyeongtaek, show a greater diversity of rodent-borne hantaviruses compared to HTNV previously identified in Gyeonggi province of the ROK. Thus, this study provides significant insights for raising HFRS threat awareness, analysis, and risk reduction strategies in southern Gyeonggi province.

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.

In total, 183,602 female culicine mosquitoes were captured by Mosquito Magnet, black light, and New Jersey light traps, and manual aspiration of resting blood-fed mosquitoes, in the Republic of Korea from 2008 to 2011. Culicine mosquitoes were identified to species, placed in pools of up to 30 mosquitoes each, and screened for flavivirus RNA by using an SYBR green I-based reverse transcription-polymerase chain reaction assay. Thirty-two of the 8,199 pools assayed were positive by quantitative polymerase chain reaction for Chaoyang virus (CHAOV), an insect-specific virus [26 Aedes vexans nipponii Theobald, 3 Culex pipiens L., 1 Aedes albopictus (Skuse), 1 Aedes bekkui Mogi, and 1 Armigeres subalbatus (Coquillett)]. The maximum likelihood estimations (estimated number of virus-positive mosquitoes/1,000 mosquitoes) for Ae. bekkui, Ae. albopictus, Ar. subalbatus, Ae. vexans nipponii, and Cx. pipiens positive for CHAOV were 5.37, 3.29, 0.77, 0.27, and 0.26, respectively. CHAOV is an insect-specific virus, and there is currently no evidence to suggest a role in animal or human disease.

Rodents and soricomorphs are animal hosts of fleas and associated zoonotic microbial pathogens. A total of 4,889 small mammals were collected from Gyeonggi and Gangwon Provinces, Republic of Korea, from 2008 through 2010, including: Apodemus agrarius (4,122, 84.3%), followed by Crocidura lasiura (282, 5.8%), Microtus fortis (257, 5.3%), Myodes regulus (77, 1.6%), Micromys minutus (71, 1.5%), Mus musculus (63, 1.3%), and 4 other species (17, 0.3%). A total of 1,099 fleas belonging to 10 species and 7 genera were collected. Ctenophthalmus congeneroides (724, 65.9%) was the most commonly collected flea, followed by Stenoponia sidimi (301, 27.4%), Neopsylla bidentatiformis (29, 2.6%), and Rhadinopsylla insolita (25, 2.3%). The remaining species accounted for only 1.8% (20, range 1-6) of all fleas collected. The 2 dominant flea species, C. congeneroides and S. sidimi, showed an inverse seasonal pattern, with higher populations of C. congeneroides from January-September, whereas S. sidimi was more frequently collected during October-December. The overall flea infestation rates (FIR) and flea indices (FI) were 14.1% and 0.22, respectively, and were highest during April-June (19.7% and 0.30, respectively). A total of 735 of the 1,099 fleas were assayed for the detection of Bartonella spp. by PCR using Bartonella-specific primers, of which 515 were positive for Bartonella, with an overall maximum likelihood estimate (MLE) of 700.7/1,000. The highest MLE values were observed during April-June (899.2) and July-September (936.2) trapping periods and, although lower, were similar for January-March (566.7) and October-December (574.1). C. congeneroides demonstrated high MLEs for all seasons (range 752.5-934.8), while S. sidimi was positive for Bartonella only during January-March (MLE=342.1) and October-December (MLE=497.2) collection periods. Continued long-term surveillance of small mammals and associated ectoparasites is needed to improve our understanding of the prevalence of Bartonella spp. in fleas and the role of fleas in the zoonotic maintenance and transmission of Bartonella to humans.

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 1,305 ticks were collected from wild rodents captured monthly, except July and August, during 2008 at three US-ROK operated military training sites and three US military installations in Gyeonggi and Gangwon Provinces, the Republic of Korea (ROK). Ixodes nipponensis was the most frequently collected tick (n = 1,299, 99.5 %), followed by Ixodes pomerantzevi (n = 6, 0.5 %). The ticks were pooled (1-15/sample) and tested by nested polymerase chain reaction (nPCR) for spotted fever group (SFG) rickettsiae with primer sets targeting the outer membrane protein B (ompB), citrate synthase (gltA), and 17-kDa antigen gene loci. A total of 115/197 (58.4 %) pools were positive by nPCR for the outer membrane protein ompB. Nucleotide sequence analysis of 105/115 (91.3 %) ompB targeted nPCR positive products showed a high degree of similarity to Rickettsia monacensis (99.3-100 %, n = 87) and R. japonica (99.5-100 %, n = 18). From the 87 positive samples demonstrating a high degree of similarity to R. monacensis, 15 were selected and analyzed by nPCR for gltA and the 17-kDa genes. A total of 12/15 pooled samples were positive for by nPCR for gltA, with amplicons demonstrating a high degree of similarity to R. monacensis (99.3-99.7 %). A total of 13/15 pooled samples were positive by nPCR for the 17-kDa gene, with amplicons demonstrating a high degree of similarity to R. monacensis (99.4-100 %). These findings demonstrate that R. monacensis is distributed throughout Gyeonggi and Gangwon Provinces in the ROK. Furthermore, data suggest a relative high prevalence of R. monacensis in the tick, I. nipponensis.

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. longicornis and 32 H. flava, were collected from these areas. A total of 9,028 hard ticks were collected from wild boars of 8 habitats, including 4,347 H. longicornis, 4,172 H. flava, 508 I. nipponensis, and 1 I. turdus. A. testudinarium was collected only from wild boars, and I. nipponensis and I. turdus were collected only from the habitats of wild boars.

Background 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. Methods 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). Results 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 larvae), and Ixodes nipponensis (431; 25 adults, 5 nymphs and 401 larvae). The minimum field infection rate/100 ticks (assuming 1 positive tick/pool) was 0.93% for the 17 kDa antigen gene and 0.82% for the ompA nPCR assays. The partial 17 kDa antigen and ompA gene sequences from positive pools of H. longicornis were similar to: Rickettsia sp. HI550 (99.4–100%), Rickettsia sp. FUJ98 (99.3–100%), Rickettsia sp. HIR/D91 (99.3–100%), and R. japonica (99.7%). One sequence of the partial 17 kDa antigen gene for H. flava was similar to Rickettsia sp. 17kd-005 (99.7%), while seven sequences of the 17 kDa antigen gene obtained from I. nipponensis ticks were similar to R. monacensis IrR/Munich (98.7–100%) and Rickettsia sp. IRS3 (98.9%). Conclusions SFG rickettsiae were detected in three species of ixodid ticks collected in the southwestern provinces of the ROK during 2013. A number of rickettsiae have been recently reported from ticks in Korea, some of which were identified as medically important. Results from this study and previous reports demonstrate the need to conduct longitudinal investigations to identify tick-borne rickettsiae and better understand their geographical distributions and potential impact on medical and veterinary health, in addition to risk communication and development of rickettsial disease prevention strategies.

As part of the 65th Medical Brigade, U.S. Army, arthropod-borne disease surveillance program and in collaboration with the Korea National Institute of Biological Resources (NIBR), bats were captured from caves and abandoned mines in the Republic of Korea. A total of 39 adult bat flies including five species of Nycteribiidae [Penicillidia jenynsii, Nycteribia parvula, N. formosana, N. allotopa mikado, and an unidentified species of Nycteribia (N. cf. formosana)], and one species of Streblidae, Brachytarsina kanoi, were collected from bats belonging to two families, Rhinolophidae and Vespertilionidae. This is the first report of N. allotopa mikado and N. formosana from the Republic of Korea.

Black light traps were used to measure the seasonal and geographical distribution of Culicoides spp. (biting midges or no-see-ums) at 9 cowsheds in the southern half of the Republic of Korea (ROK) from June through October 2010. A total of 25,242 Culicoides females (24,852; 98.5%) and males (390; 1.5%) comprising of 9 species were collected. The most commonly collected species was Culicoides punctatus (73.0%) followed by C. arakawae (25.7%), while the remaining 7 species accounted for

One genus, Ornithoica Rondani, and two species, Ornithoica momiyamai Kishida, and O. unicolor Speiser, are reported for the first time from the Republic of Korea. A total of six species, including two new records, of louse flies were collected from 78 birds belonging to 35 species from Seoul and Incheon Metropolitan Areas and Gyeonggi, Gangwon, Gyeongsangbuk, Jeollabuk, and Jeollanam Provinces, 2005–2010. An updated checklist of Korean hippoboscids consisting of seven genera (Hippobosca L., Icosta Speiser, Lipoptena Nitzsch, Ornithoica Rondani, Ornithoctona Speiser, Ornithomya Lattreille, and Ornithophila Rondani) and 11 species is presented, including hosts, collection records, and repositories.

A survey of reptile-associated ticks and their infection status with severe fever with thrombocytopenia syndrome (SFTS) virus was conducted to determine the relative abundance and distribution among lizards, skinks, and snakes in the Republic of Korea (ROK). In total, 132 reptiles, including 49 lizards (two species), 15 skinks (one species), and 68 snakes (eight species) were collected. In total, 84 ixodid ticks belonging to two genera (Ixodes and Amblyomma) were collected from 28/132 (21.2%) lizards, skinks, and snakes. Ixodes nipponensis Kitaoka & Saito was only collected from lizards and skinks, while Amblyomma testudinarium Koch was only collected from snakes. Takydromus wolteri had the highest tick index (0.7; total number ticks/total number collected hosts) among lizards and skinks, while Rhabdophis tigrinus had the highest tick index (2.2) among the snakes. Ixodes nipponensis larvae and nymphs accounted for 11.1% and 88.9%, respectively, of all ticks collected from lizards and skinks, while only A. testudinarium nymphs were collected from snakes. Nymphs of both species of ticks were collected from lizards and skinks from April to October, while I. nipponensis larvae were collected only from September to October. Ixodes nipponensis larvae and nymphs were preferentially attached to the lateral trunk (83.3%) and the foreleg axillae (16.7%) of lizards and skinks. SFTS virus was detected in both I. nipponensis and A. testudinarium collected from lizards and snakes. Phylogenetic analysis of SFTS viruses of ticks collected from two lizards and one snake demonstrated close relationships with SFTS virus strains observed from humans and ticks in the ROK, China, and Japan. These results implicate lizards and snakes as potential hosts of SFTS virus.

In total, 22,846 (17,793 culicines and 5,053 Anopheles spp.) female mosquitoes were captured by a Mosquito Magnet trap at Daeseongdong, a small village adjacent to the military demarcation line (center of the demilitarized zone) in northern Gyeonggi Province, Republic of Korea (ROK). Culicine mosquitoes were identified to species, placed in pools of up to 30 mosquitoes each, and screened for flavivirus using a SYBR Green I-based real-time polymerase chain reaction. In total, 51/660 pools positive for flaviviruses and confirmed by DNA sequencing of the NS5 region, were positive for Japanese encephalitis virus (family Flaviviridae, genus Flavivirus, JEV) (50 Culex tritaeniorhynchus Giles and one Culex bitaeniorhynchus Giles). The JEV maximum likelihood estimations (MLEs) (estimated number of viral RNA-positive mosquitoes per 1,000) for Cx. tritaeniorhynchus and Cx. bitaeniorhynchus were 9.7 and 0.9, respectively. This is the first report of a Cx. bitaeniorhynchus positive for JEV in the ROK. JEV is a local civilian and military health threat and a significant concern for nonimmune (unvaccinated) U.S. soldiers, civilians, and family members deployed to the ROK.