As part of a field ecology study of arbovirus and malaria activity in the Amazon Basin, Loreto Department, Peru, we collected mosquitoes landing on humans at a forest site and inside and outside of residences and military barracks at periurban, rural, and village sites. We collected 11 Anopheles spp. from these four sites. An. darlingi, the principal malaria vector in the region, accounted for 98.7% of all Anopheles spp. collected at Puerto Almendra. Peaks in landing activity occurred during the December and April collection periods. However, the percent of sporozoite-positive Anopheles spp. was highest 1–2 months later, when landing activity decreased to approximately 10% of the peak activity periods. At all sites, peak landing activity occurred about 2 hours after sunset. These data provide a better understanding of the taxonomy, population density, and seasonal and habitat distribution of potential malaria vectors within the Amazon Basin region.

Japanese encephalitis virus (JEV), a mosquito-borne zoonotic pathogen, is one of the major causes of viral encephalitis. To reduce the impact of Japanese encephalitis among children in the Republic of Korea (ROK), the government established a mandatory vaccination program in 1967. Through the efforts of this program only 0–7 (mean 2.1) cases of Japanese encephalitis were reported annually in the ROK during the period of 1984–2009. However, in 2010 there was an outbreak of 26 confirmed cases of Japanese encephalitis, including 7 deaths. This represented a >12-fold increase in the number of confirmed cases of Japanese encephalitis in the ROK as compared to the mean number reported over the last 26 years and a 3.7-fold increase over the highest annual number of cases during this same period (7 cases). Surveillance of adult mosquitoes was conducted during the 2010 outbreak of Japanese encephalitis in the ROK. A total of 6,328 culicine mosquitoes belonging to 12 species from 5 genera were collected at 6 survey sites from June through October 2010 and assayed by reverse-transcription polymerase chain reaction (RT-PCR) for the presence of JEV. A total of 34/371 pooled samples tested positive for JEV (29/121 Culex tritaeniorhynchus, 4/64 Cx. pipiens, and 1/26 Cx. bitaeniorhynchus) as confirmed by sequencing of the pre-membrane and envelope protein coding genes. The maximum likelihood estimates of JEV positive individuals per 1,000 culicine vectors for Cx. tritaeniorhynchus, Cx. pipiens, and Cx. bitaeniorhynchus were 11.8, 5.6, and 2.8, respectively. Sequences of the JEV pre-membrane and envelope protein coding genes amplified from the culicine mosquitoes by RT-PCR were compared with those of JEV genotypes I-V. Phylogenetic analyses support the detection of a single genotype (I) among samples collected from the ROK in 2010.

Background

Culex tritaeniorhynchus is the primary vector of Japanese encephalitis virus (JEV), a leading cause of encephalitis in Asia. JEV is transmitted in an enzootic cycle involving large wading birds as the reservoirs and swine as amplifying hosts. The development of a JEV vaccine reduced the number of JE cases in regions with comprehensive childhood vaccination programs, such as in Japan and the Republic of Korea. However, the lack of vaccine programs or insufficient coverage of populations in other endemic countries leaves many people susceptible to JEV. The aim of this study was to predict the distribution of Culex tritaeniorhynchus using ecological niche modeling.

Methods/Principal Findings

An ecological niche model was constructed using the Maxent program to map the areas with suitable environmental conditions for the Cx. tritaeniorhynchus vector. Program input consisted of environmental data (temperature, elevation, rainfall) and known locations of vector presence resulting from an extensive literature search and records from MosquitoMap. The statistically significant Maxent model of the estimated probability of Cx. tritaeniorhynchus presence showed that the mean temperatures of the wettest quarter had the greatest impact on the model. Further, the majority of human Japanese encephalitis (JE) cases were located in regions with higher estimated probability of Cx. tritaeniorhynchus presence.

Conclusions/Significance

Our ecological niche model of the estimated probability of Cx. tritaeniorhynchus presence provides a framework for better allocation of vector control resources, particularly in locations where JEV vaccinations are unavailable. Furthermore, this model provides estimates of vector probability that could improve vector surveillance programs and JE control efforts.

Author Summary

Japanese encephalitis virus (JEV) is transmitted predominately by the mosquito, Culex tritaeniorhynchus. The primary reservoirs of the virus are wading birds, with swine serving as amplifying hosts. Despite the development of a JEV vaccine, people remain unvaccinated in endemic countries and are susceptible to JEV infection. The distribution of the JEV vector(s) provides essential information for preventive measures. This study used an ecological niche modeling program to predict the distribution of Cx. tritaeniorhynchus based on collection records and environmental maps (climate, land cover, and elevation). The model showed that the mean temperatures of the wettest quarter had the greatest impact on the model. Of the 25 countries endemic for Japanese encephalitis (JE) endemic countries, seven possessed greater than 50% land area with an estimated high probability of Cx. tritaeniorhynchus presence. Our model provides a useful tool for JEV surveillance programs that focus on vector control strategies.

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 <1.0% of all Culicoides spp. collected. The mean number of Culicoides spp. collected per trap night (Trap Index [TI]) was highest for C. punctatus (409.3), followed by C. arakawae (144.2), C. tainanus (4.1), C. oxystoma (1.2), C. circumscriptus (0.7), C. homotomus (0.6), C. erairai (0.4), C. kibunensis (0.3), and C. nipponensis (0.04). Peak TIs were observed for C. punctatus (1,188.7) and C. arakawae (539.0) during July and August, respectively. C. punctatus and C. arakawae have been implicated in the transmission of arboviruses and other pathogens of veterinary importance that adversely impact on animal and bird husbandry.

Background

Japanese encephalitis virus (JEV) genotype V reemerged in Asia (China) in 2009 after a 57-year hiatus from the continent, thereby emphasizing a need to increase regional surveillance efforts. Genotypic characterization was performed on 19 JEV-positive mosquito pools (18 pools of Culex tritaeniorhynchus and 1 pool of Cx. bitaeniorhynchus) from a total of 64 positive pools collected from geographically different locations throughout the Republic of Korea (ROK) during 2008 and 2010.

Findings

Two regions of the JEV genome were sequenced from 19 pools; the envelope gene and the nonstructural protein 5 (NS5)/3'-untranslated region (UTR). Eighteen pools of Culex tritaeniorhynchus and one pool of Cx. bitaeniorhynchus were positive for genotype I and genotype V, respectively. Sequence alignment of the complete E gene from Cx. bitaeniorhynchus showed high amino acid similarity (98.8%) to the Muar strain, characterized as the first report of genotype V, isolated from an encephalitis patient in Malaysia in 1952.

Conclusion

This study represents the first report of JEV genotype V in the ROK. The reemergence of genotype V in Asia (China and ROK) after more than a half-century and its discovery in Cx. bitaeniorhynchus, a mosquito species previously unknown to carry JEV in the ROK, emphasizes the need for enhanced JE surveillance to monitor the dynamics of JEV strains within the region. Future findings may have implications with regard to JEV vaccination/prevention strategies.

Vivax malaria is a significant military and civilian health threat in the north of the Republic of Korea (ROK). The island of Baengnyeong-do is the westernmost point of the ROK and is located close to the southwestern coast of the Democratic People's Republic of Korea (DPRK). Mosquitoes were collected using a black light trap on Baengnyeong-do, and Anopheles spp. were assayed by PCR, to identify the species, and screened for sporozoites of Plasmodium vivax. Of a subsample of 257 mosquitoes, Anopheles lesteri was the most frequently collected (49.8%), followed by Anopheles sinensis (22.6%), Anopheles pullus (18.7%), Anopheles kleini (7.8%), and Anopheles belenrae (1.2%). The overall sporozoite rate was 3.1%, with the highest rates observed in An. kleini (15.0%), An. sinensis (5.2%), and An. lesteri (1.6%). No sporozoite positive An. pullus or An. belenrae were observed. The results extend our knowledge of the distribution and potential role in malaria transmission of An. kleini, An. lesteri, and An. sinensis, for an area previously considered to be at a low risk for contracting vivax malaria.

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%), I. nipponensis (1.8%), and H. japonica (0.2%).

Nucleotide sequence analysis of the Plasmodium vivax PvMSP-3α gene was conducted on blood from 143 malaria patients admitted to Korea University Medical Center from 1996 to 2007 in the Republic of Korea (ROK). From 1996 to 2002, the PvMSP-3α alleles were of two types, SKOR-67 (2.53 kb) and SKOR-69 (1.78 kb), which differed in length and amino acid sequence. Two new variants with similar size to SKOR-67 were first observed in 2002 and in 2006–2007 accounted for nearly 50% (25/51) of the sampled isolates. The new variants had the same amino acid sequence as SKOR-69 in the N-terminal region, but in Blocks I and II and in the C-terminal region, they were similar to previously reported isolates from Thailand, Papua New Guinea, India, Brazil, and Ecuador strains.

The Armed Forces Health Surveillance Center, Division of Global Emerging Infections Surveillance and Response System Operations (AFHSC-GEIS) initiated a coordinated, multidisciplinary program to link data sets and information derived from eco-climatic remote sensing activities, ecologic niche modeling, arthropod vector, animal disease-host/reservoir, and human disease surveillance for febrile illnesses, into a predictive surveillance program that generates advisories and alerts on emerging infectious disease outbreaks. The program’s ultimate goal is pro-active public health practice through pre-event preparedness, prevention and control, and response decision-making and prioritization. This multidisciplinary program is rooted in over 10 years experience in predictive surveillance for Rift Valley fever outbreaks in Eastern Africa. The AFHSC-GEIS Rift Valley fever project is based on the identification and use of disease-emergence critical detection points as reliable signals for increased outbreak risk. The AFHSC-GEIS predictive surveillance program has formalized the Rift Valley fever project into a structured template for extending predictive surveillance capability to other Department of Defense (DoD)-priority vector- and water-borne, and zoonotic diseases and geographic areas. These include leishmaniasis, malaria, and Crimea-Congo and other viral hemorrhagic fevers in Central Asia and Africa, dengue fever in Asia and the Americas, Japanese encephalitis (JE) and chikungunya fever in Asia, and rickettsial and other tick-borne infections in the U.S., Africa and Asia.

Vector-borne infections (VBI) are defined as infectious diseases transmitted by the bite or mechanical transfer of arthropod vectors. They constitute a significant proportion of the global infectious disease burden. United States (U.S.) Department of Defense (DoD) personnel are especially vulnerable to VBIs due to occupational contact with arthropod vectors, immunological naiveté to previously unencountered pathogens, and limited diagnostic and treatment options available in the austere and unstable environments sometimes associated with military operations. In addition to the risk uniquely encountered by military populations, other factors have driven the worldwide emergence of VBIs. Unprecedented levels of global travel, tourism and trade, and blurred lines of demarcation between zoonotic VBI reservoirs and human populations increase vector exposure. Urban growth in previously undeveloped regions and perturbations in global weather patterns also contribute to the rise of VBIs. The Armed Forces Health Surveillance Center-Global Emerging Infections Surveillance and Response System (AFHSC-GEIS) and its partners at DoD overseas laboratories form a network to better characterize the nature, emergence and growth of VBIs globally. In 2009 the network tested 19,730 specimens from 25 sites for Plasmodium species and malaria drug resistance phenotypes and nearly another 10,000 samples to determine the etiologies of non-Plasmodium species VBIs from regions spanning from Oceania to Africa, South America, and northeast, south and Southeast Asia. This review describes recent VBI-related epidemiological studies conducted by AFHSC-GEIS partner laboratories within the OCONUS DoD laboratory network emphasizing their impact on human populations.

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 the phylogeography of rodent-borne hantaviruses.

The prevalence of tick-borne encephalitis virus (TBEV) in southern Korea was determined by collecting ticks using tick drags. A total of 4,077 of 6,788 ticks collected were pooled (649 pools) according to collection site, species, and developmental stage and assayed for TBEV. The TBEV protein E and NS5 gene fragments were detected using RT-nested PCR in six pools of nymphs collected from Jeju Island (2,491 ticks). The minimum field detection rates for TBEV were 0.17% and 0.14% for Haemaphysalis longicornis and Haemayphysalis flava nymphs, respectively. The 252 bp NS5 and 477 bp protein E gene amplicons were sequenced. Phylogenetic analysis showed that the NS5 and protein E genes of the Jeju strain were clustered with Western subtype (98.0% and 99.4% identity, respectively). The Western subtype of TBEV is endemic in Korea, including Jeju Island. The study of vector and zoonotic host susceptibility to TBEV is required to better understand its potential impact on public health.

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 insectivores were slide-mounted and 10 species belonging to 4 genera were identified. Leptotrombidium pallidum (53.4%) was the most frequently collected, followed by L. palpale (15.7%), Neotrombicula tamiyai (14.3%), L. orientale (10.7%), L. zetum (3.1%), Walchia fragilis (2.1%), and L. gemiticulum (0.8%), while the remaining 3 species, L. subintermedium, N. gardellai, and Euschoengastia koreaensis were rarely observed (prevalence < 10%). In contrast to previous surveys, higher chigger indices of the primary scrub typhus vectors, L. pallidum (165.4), L. orientale (45.0), and L. palpale (21.4), were observed during the spring season.

Background

Japanese encephalitis virus (JEV), the causative agent of Japanese encephalitis (JE), is endemic to the Republic of Korea (ROK) where unvaccinated United States (U.S.) military Service members, civilians and family members are stationed. The primary vector of the JEV in the ROK is Culex tritaeniorhynchus. The ecological relationship between Culex spp. and rice fields has been studied extensively; rice fields have been shown to increase the prevalence of Cx. tritaeniorhynchus. This research was conducted to determine if the quantification of rice field land cover surrounding U.S. military installations in the ROK should be used as a parameter in a larger risk model that predicts the abundance of Cx. tritaeniorhynchus populations.

Mosquito data from the U.S. Forces Korea (USFK) mosquito surveillance program were used in this project. The average number of female Cx. tritaeniorhynchus collected per trap night for the months of August and September, 2002-2008, was calculated. Rice fields were manually digitized inside 1.5 km buffer zones surrounding U.S. military installations on high-resolution satellite images, and the proportion of rice fields was calculated for each buffer zone.

Results

Mosquito data collected from seventeen sample sites were analyzed for an association with the proportion of rice field land cover. Results demonstrated that the linear relationship between the proportion of rice fields and mosquito abundance was statistically significant (R2 = 0.62, r = .79, F = 22.72, p < 0.001).

Conclusions

The analysis presented shows a statistically significant linear relationship between the two parameters, proportion of rice field land cover and log10 of the average number of Cx. tritaeniorhynchus collected per trap night. The findings confirm that agricultural land cover should be included in future studies to develop JE risk prediction models for non-indigenous personnel living at military installations in the ROK.

Background

Larval mosquito habitats of potential malaria vectors and related species of Anopheles from three provinces (Gyeonggi, Gyeongsangbuk, Chungcheongbuk Provinces) of the Republic of Korea were surveyed in 2007. This study aimed to determine the species composition, seasonal occurrence and distributions of Anopheles mosquitoes. Satellite derived normalized difference vegetation index data (NDVI) was also used to study the seasonal abundance patterns of Anopheles mosquitoes.

Methods

Mosquito larvae from various habitats were collected using a standard larval dipper or a white plastic larval tray, placed in plastic bags, and were preserved in 100% ethyl alcohol for species identification by PCR and DNA sequencing. The habitats in the monthly larval surveys included artificial containers, ground depressions, irrigation ditches, drainage ditches, ground pools, ponds, rice paddies, stream margins, inlets and pools, swamps, and uncultivated fields. All field-collected specimens were identified to species, and relationships among habitats and locations based on species composition were determined using cluster statistical analysis.

Results

In about 10,000 specimens collected, eight species of Anopheles belonging to three groups were identified: Hyrcanus Group - Anopheles sinensis, Anopheles kleini, Anopheles belenrae, Anopheles pullus, Anopheles lesteri, Anopheles sineroides; Barbirostris Group - Anopheles koreicus; and Lindesayi Group - Anopheles lindesayi japonicus. Only An. sinensis was collected from all habitats groups, while An. kleini, An. pullus and An. sineroides were sampled from all, except artificial containers. The highest number of Anopheles larvae was found in the rice paddies (34.8%), followed by irrigation ditches (23.4%), ponds (17.0%), and stream margins, inlets and pools (12.0%). Anopheles sinensis was the dominant species, followed by An. kleini, An. pullus and An. sineroides. The monthly abundance data of the Anopheles species from three locations (Munsan, Jinbo and Hayang) were compared against NDVI and NDVI anomalies.

Conclusion

The species composition of Anopheles larvae varied in different habitats at various locations. Anopheles populations fluctuated with the seasonal dynamics of vegetation for 2007. Multi-year data of mosquito collections are required to provide a better characterization of the abundance of these insects from year to year, which can potentially provide predictive capability of their population density based on remotely sensed ecological measurements.

Until recently, the single known exception to the rodent-hantavirus association was Thottapalayam virus (TPMV), a long-unclassified virus isolated from the Asian house shrew (Suncus murinus). Robust gene amplification techniques have now uncovered several genetically distinct hantaviruses from shrews in widely separated geographic regions. Here, we report the characterization of a newly identified hantavirus, designated Imjin virus (MJNV), isolated from the lung tissues of Ussuri white-toothed shrews of the species Crocidura lasiura (order Soricomorpha, family Soricidae, subfamily Crocidurinae) captured near the demilitarized zone in the Republic of Korea during 2004 and 2005. Seasonal trapping revealed the highest prevalence of MJNV infection during the autumn, with evidence of infected shrews' clustering in distinct foci. Also, marked male predominance among anti-MJNV immunoglobulin G antibody-positive Ussuri shrews was found, whereas the male-to-female ratio among seronegative Ussuri shrews was near 1. Plaque reduction neutralization tests showed no cross neutralization for MJNV and rodent-borne hantaviruses but one-way cross neutralization for MJNV and TPMV. The nucleotide and deduced amino acid sequences for the different MJNV genomic segments revealed nearly the same calculated distances from hantaviruses harbored by rodents in the subfamilies Murinae, Arvicolinae, Neotominae, and Sigmodontinae. Phylogenetic analyses of full-length S, M, and L segment sequences demonstrated that MJNV shared a common ancestry with TPMV and remained in a distinct out-group, suggesting early evolutionary divergence. Studies are in progress to determine if MJNV is pathogenic for humans.

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%), Chungnam (100%), Chungbuk (100%), Jeonbuk (73.9%), Jeonnam (77.0%), and Gyeongbuk (66.1%) provinces, whereas L. scutellare was the predominant chigger collected in Gyeongnam province (77.9%) and Jeju Island (62.3%). Data suggest a correlation between chigger population abundance and human cases of scrub typhus in Korea.

Four US soldiers acquired hemorrhagic fever with renal syndrome while training near the Demilitarized Zone, South Korea, in 2005. Hantaan virus sequences were amplified by reverse transcription–PCR from patient serum samples and from lung tissues of striped field mice (Apodemus agrarius) captured at training sites. Epidemiologic investigations specified the ecology of possible sites of patient infection.

Hydroxychloroquine (HCQ) is an antimalarial drug used as chemoprophylaxis against malaria caused by Plasmodium vivax in the Republic of Korea Army (ROKA). In this study, we evaluated the pharmacokinetics (PK) of HCQ and its metabolites and the relationship between the PK of HCQ and the effect of treatment of HCQ on vivax malaria in South Koreans. Three PK studies of HCQ were conducted with 91 healthy subjects and patients with vivax malaria. Plasma concentrations were analyzed by noncompartmental and mixed-effect modeling approaches. A two-compartment model with first-order absorption best described the data. The clearance and the central and peripheral volumes of distribution were 15.5 liters/h, 733 liters, and 1,630 liters, respectively. We measured the plasma concentrations of HCQ in patients with prophylactic failure of HCQ and compared them with the prediction intervals of the simulated concentrations for HCQ from the final PK model built in this study. In 71% of the patients with prophylactic failure, the plasma concentrations of HCQ were below the lower bounds of the 95% prediction interval, while only 8% of them showed higher levels than the upper bounds of the 95% prediction interval. We report that a significant cause of prophylactic failure among the individuals in ROKA was ascribed to plasma concentrations of HCQ lower than those predicted by the PK model. However, prophylactic failure despite sufficient plasma concentrations of HCQ was confirmed in several individuals, warranting continued surveillance to monitor changes in the HCQ susceptibility of Plasmodium vivax in the Republic of Korea.

A total of 1,498 small mammals (rodents and insectivores), including Apodemus agrarius (n = 1,366), Crocidura lasiura (54), Mus musculus (32), Micronytus fortis (28), Eothenomys regulus (9), Micronys minutes (6), and Cricetulus triton (3), were live-trapped in Gyeonggi-do (Province) (Paju-si, Pocheon-gun, and Yeoncheon-gun) near the demilitarized zone (DMZ) from December 2004 to September 2005. A. agrarius was found to be infected with 3 species of echinostomes (Echinostoma hortense, Echinostoma cinetorchis, and Euparyphium murinum), while C. lasiura was infected with 1 species (Echinochasmus japonicas) of echinostome. Other mammals were free from echinostome infections. Total 16 E. hortense were detected in 7 (0.5%) mice, 9 E. cinetorchis from 5 (0.4%), and 3 E. murinum from 2 (0.1%) out of 1.366 A. agrarius examined. E. japonicus was found only in 1 (1.9%; total 3 specimens) C. lasiura. These results demonstrate that A. agrarius and C. lasiura, inhabiting near the DMZ of Gyeonggi-do serve as the natural definitive hosts for several species of echinostomes, although their infection rates are low. This is the first record of natural infections of A. agrarius with E. cinetorchis and C. lasiura with E. japonicus in the Republic of Korea.

A total of 1,618 ticks [420 individual (adults) and pooled (larvae and nymphs) samples], 369 rodents (Apodemus agrarius, Rattus norvegicus, Tscherskia triton, Mus musculus, and Myodes regulus), and 34 shrews (Crocidura lasiura) that were collected in northern Gyeonggi-do near the Demilitarized Zone (DMZ) of Korea during 2004-2005, were assayed by PCR for selected zoonotic pathogens. From a total of 420 individual and pooled tick DNA samples, Anaplasma (A.) phagocytophilum (16), A. platys (16), Ehrlichia (E.) chaffeensis (63), Borrelia burgdorferi (16), and Rickettsia spp. (198) were detected using species-specific PCR assays. Out of 403 spleens from rodents and shrews, A. phagocytophilum (20), A. platys (34), E. chaffeensis (127), and Bartonella spp. (24) were detected with species-specific PCR assays. These results suggest that fevers of unknown causes in humans and animals in Korea should be evaluated for infections by these vector-borne microbial pathogens.

Acute-phase sera from >5 % of cases of haemorrhagic fever with renal syndrome occurring annually in Korea have been found to exhibit a fourfold or higher antibody titre to Puumala virus (PUUV) than to Hantaan virus (HTNV) by double-sandwich IgM ELISA, suggesting the existence of a PUUV-related hantavirus. Based on the phylogenetic relationships among arvicolid rodents, the royal vole (Myodes regulus) was targeted as a likely reservoir host of hantavirus. Using RT-PCR, a genetically distinct hantavirus, designated Muju virus (MUJV), was detected in lung tissue of royal voles, captured in widely separated geographical regions in Korea during 1996–2007. Pairwise analysis of the full-length S (1857 nt) and M (3634 nt) segments of MUJV indicated approximately 77 % sequence similarity with PUUV. At the amino acid level, MUJV differed from PUUV by 5.5–6.9 % (nucleocapsid) and 10.0–11.6 % (Gn and Gc envelope glycoproteins). Interstrain variation of MUJV sequences from royal voles captured in different regions suggested geographic-specific clustering. Neutralizing antibody titres against PUUV were two- to sixfold higher than to HTNV in sera of MUJV-infected Myodes regulus. Although virus isolation attempts were unsuccessful, the collective data indicate that MUJV is a distinct hantavirus species.

The small intestines of 6 species of rodents and 1 species of insectivore were examined seasonally for Plagiorchis muris infection in 3 different localities in northern Gyeonggi-do (Province), near the demilitarized zone (DMZ). A total of 1,496 animals, including 1,366 Apodemus agrarius, 54 Crocidura lasiura (insectivore), 32 Mus musculus, 28 Micronytus fortis, 9 Eothenomys regulus, 6 Micronys minutus, and 3 Cricetulus triton, were live-trapped at Yeoncheon-gun (n = 351), Paju-shi (804) and Pocheon-gun (343) at 3-mo intervals from December 2004 to September 2005. A total of 1,647 P. muris were collected from 72 (5.3%) A. agrarius. The infection rate was the highest in Pocheon-gun (8.2%), followed by Yeoncheon-gun (5.0%) and Paju-shi (4.2%). A higher infection rate was observed in A. agrarius captured during September (19.4%) than those captured during December (3.0%), June (2.6%), or April (0%). However, the worm burden was the highest in June (av. 32.1/animal), followed by September (24.7), December (4.0), and April (0). None of the other animal species were found infected with P. muris. The results reveal that A. agrarius is a natural definitive host for P. muris, and infection rates and worm burdens vary seasonally and geographically.

A total of 1,496 rodents and insectivores were live-trapped at Yeoncheon-gun (n = 351), Paju-shi (804), and Pocheon-gun (343), Gyeonggi-do (Province), and examined for intestinal helminths, including Neodiplostomum seoulense, seasonally from December 2004 to September 2005. Six species of rodents, including Apodemus agrarius (1,366), Mus musculus (32), Micronytus fortis (28), Eothenomys regulus (9), Micronys minutus (6), and Cricetulus triton (3), and 1 species of insectivores Crocidura lasiura (54) were collected. A total of 321 adult N. seoulense were collected from 19 (1.4%) A. agrarius. The worm burden ranged from 1 to 101 per A. agrarius (mean; 16.9). No N. seoulense was observed in other rodent or insectivore species examined. The infection rate during autumn (4.5%) was higher than those during spring (0.8%), summer (0.8%), and winter (0.5%). The average number of N. seoulense in infected A. agrarius was the highest in spring (66.0 specimens), followed by autumn (15.2), winter (4.5), and summer (3.3). This study first confirms that A. agrarius is a natural definitive host for N. seoulense, and demonstrates that the infection rates and intensities vary seasonally and geographically.

In order to investigate the prevalence of tick-borne infectious agents among ticks, ticks comprising five species from two genera (Hemaphysalis spp. and Ixodes spp.) were screened using molecular techniques. Ticks (3,135) were collected from small wild-caught mammals or by dragging/flagging in the Republic of Korea (ROK) and were pooled into a total of 1,638 samples (1 to 27 ticks per pool). From the 1,638 tick samples, species-specific fragments of Anaplasma phagocytophilum (1 sample), Anaplasma platys (52 samples), Ehrlichia chaffeensis (29 samples), Ehrlichia ewingii (2 samples), Ehrlichia canis (18 samples), and Rickettsia rickettsii (28 samples) were amplified by PCR assay. Twenty-one pooled and individual tick samples had mixed infections of two (15 samples) or three (6 samples) pathogens. In addition, 424 spleen samples from small captured mammals (389 rodents, 33 insectivores, and 2 weasels) were screened for selected zoonotic pathogens. Species-specific DNA fragments of A. phagocytophilum (110 samples), A. platys (68 samples), E. chaffeensis (8 samples), E. ewingii (26 samples), E. canis (51 samples), and Rickettsia sp. (22 samples) were amplified by PCR assay. One hundred thirty small mammals had single infections, while 4, 14, and 21 striped field mice (Apodemus agrarius) had mixed infections of four, three, and two pathogens, respectively. Phylogenetic analysis based on nucleotide sequence comparison also revealed that Korean strains of E. chaffeensis clustered closely with those from China and the United States, while the Rickettsia (rOmpA) sequences clustered within a clade together with a Chinese strain. These results suggest that these agents should be considered in differential diagnosis while examining cases of acute febrile illnesses in humans as well as animals in the ROK.