Sungjin Ko

New genetic variants of Anaplasma phagocytophilum and Anaplasma bovis from Korean water deer (Hydropotes inermis argyropus).

Wild deer are one of the important natural reservoir hosts of Anaplasma species, which cause granulocytic anaplasmosis in equines, canines, and humans. The objective of the present study was to determine whether and what species of Anaplasma naturally infect Korean water deer (KWD) in the Republic of Korea. A total of 66 spleens from KWD carcasses were collected by the Conservation Genome Resource Bank for Korean Wildlife in Korea between March 2008 and May 2009. Polymerase chain reaction (PCR) was performed using 16S ribosomal (r)RNA, with ankA, groEL, and msp2 gene primers to amplify the genes of Anaplasma and Ehrlichia. Using 16S rRNA-based nested PCR, Anaplasma phagocytophilum and Anaplasma bovis were detected in 42 (63.6%) and 23 (34.8%) of 66 KWD spleens, respectively. The 42 A. phagocytophilum were classified into five genotypes and the 23 A. bovis were classified into two genotypes by sequence analysis. By ankA-, groEL-, and msp2-based nested PCR, A. phagocytophilum was detected in 1 (1.5%), 7 (10.6%), and 3 (4.6%) of 66 samples, respectively. These gene sequences had only one genotype. Five of seven obtained 16S rRNA gene sequences have never been identified. The ankA, groEL, and msp2 obtained gene sequences represented new genotypes. This is the first report of A. phagocytophilum and A. bovis in KWD, suggesting that they may act as reservoirs for anaplasmosis zoonotic pathogens.

Molecular Detection of Anaplasma, Bartonella, and Borrelia Species in Ticks Collected from Migratory Birds from Hong-do Island, Republic of Korea

Bird migration is a recurring annual and seasonal event undertaken by more than 100 species of birds in the southeast Asian and northeast Palearctic regions that pass through or remain for short periods from April to May and September to November at Hong-do Island, Republic of Korea (ROK). A total of 212 ticks (40 Haemaphysalis flava, 12 H. longicornis, 146 Ixodes turdus, 13 I. nipponensis, and 1 I. ornithophila) were collected from 65/2,161 (3.0%) migratory birds consisting of 21 species that were captured from January, 2008, through December, 2009, as part of the Migratory Birds Center, Hong-do bird banding program for studying bird migration patterns. Adult ticks were assayed individually while larvae and nymphs were pooled (1-22 and 1-6 ticks per pool, respectively) into 31 and 65 pools, respectively. Ticks were assayed for zoonotic pathogens by PCR using 16S rRNA, heat shock protein (groEL), and internal transcribed spacer (ITS) gene primers to amplify genera specific for Anapalsma, Bartonella, and Borrelia PCR amplicons. Using the 16S rRNA-based nested PCR, A. phagocytophilum (n=1) was detected in I. nipponensis collected from Zoothera sibirica and A. bovis (n=1) was detected in I. turdus collected from Emberiza chrysophrys. Borrelia turdi 16S rRNA genes (n=3) were detected in I. turdus and I. nipponensis collected from Turdus pallidus and Zoothera aurea. Borrelia spp. 16S rRNA genes (n=4) were detected in Ixodes ticks collected from Emberiza tristrami, T. pallidus, and Z. aurea. The Bartonella grahamii ITS gene (n=1) was detected by nested PCR assay in I. turdus collected from Z. aurea. These results provide insight into the potential role of migratory birds in the dispersal of ticks and associated tick-borne pathogens throughout their ranges in Asia.

Prevalence of tick-borne encephalitis virus in ticks from southern Korea

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.

Ticks Collected from Selected Mammalian Hosts Surveyed in the Republic of Korea During 2008-2009

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%).

Predicting the Emergence of Tick-Borne Infections Based on Climatic Changes in Korea

Granulocytic anaplasmosis (GA) and monocytic ehrlichiosis (ME) are maintained in wild rodent reservoirs and tick vectors in the Republic of Korea. This study investigated the prevalence of 2 tick-borne pathogens, Anaplasma phagocytophilum and Ehrlichia chaffeensis, in wild rodents and ticks in central Korea to identify any significant associations with existing or changing climatic conditions. Specifically, the goal of this study was to develop simple models for the probability of occurrence of an epidemic of GA or ME as a function of climate in an area in a given year. Climatic data from 2 regions, Munsan and Dongducheon, Gyeonggi, in central Korea (between the Demilitarized Zone and Seoul, latitude between 37 degrees N-38 degrees N and longitude between 127 degrees E-128 degrees E), were analyzed with respect to the prevalence of GA and ME in Paju, Yoncheon, Pocheon, and Dongducheon for the period from 2001 to 2005. Rates of A. phagocytophilum and E. chaffeensis decreased as the total yearly precipitation levels and daily humidity increased, and as the daily mean sunshine hours decreased. Rates of A. phagocytophilum and E. chaffeensis from rodent ticks and rodents increased in the fall season. Linear regression analyses evaluating the numbers of positive samples by sample type found that rodent ticks were 6.64 times more likely to be actively infected with A. phagocytophilum than grass ticks or rodents, though the likelihood of any samples testing positive for this pathogen decreased by 0.17 as the annual mean level of precipitation increased by 1 mm. For E. chaffeensis, rodents were 15.67 times more likely to be infected than ticks. Logistic regression analyses evaluating each sample separately found that the odds of infection with A. phagocytophilum were nearly 5 times greater for rodents than ticks. In these analyses, precipitation was one potential factor to account for the prevalence of tickborne diseases.

Detection of Rickettsia typhi and seasonal prevalence of fleas collected from small mammals in the Republic of Korea.

Fleas were collected from live-captured small mammals to identify potential flea-borne pathogens, seasonal prevalence of flea species, and host preference as part of the US military rodent-borne diseases surveillance program conducted at one US military installation and 10 military training sites, northern Gyeonggi Province, Republic of Korea. During 2003–04, 948 fleas (563 females and 385 males) were recovered from 2,742 small mammals (seven rodent and one insectivore species). Apodemus agrarius (striped field mouse) accounted for 88.9% (2,439/2,742) of the small mammals, followed by Crocidura lasiura (4.2%), Mus musculus (2.9%), Microtus fortis (2.2%), Myodes regulus (0.6%), Micromys minutus (0.5%), Tscherskia triton (0.5%), and Rattus norvegicus (0.3%). Small mammal infestation rates (number with fleas/number captured) ranged from 7.7% (M. minutus and T. triton) to 31.3% (M. regulus). Flea indices were highest for M. regulus (0.69/captured rodent), followed by C. lasiura (0.54), M. fortis (0.41), A. agrarius (0.34), and R. norvegicus (0.33). Overall, Ctenophthalmus congeneroides (51.3%) was more frequently collected, followed by Stenoponia sidimi (42.6%), Rhadinopsylla insolita (5.5%), Neopsylla bidentatiformis (0.4%), Rhadinopsylla concava (0.1%), and Doratopsylla coreana (0.1%). Ctenophthalmus congeneroides was more frequently collected from small mammals during the spring and summer, while S. sidimi was more frequently collected during the winter season. Rickettsia typhi, the causative agent of murine typhus, was detected in 3.2% of specimens (7/220 pools from 654 fleas; minimum field infection rate [number of positive pools/total number of fleas] was 1.1%).

Molecular Detection of Bartonella grahamii and B. schoenbuchensis-Related Species in Korean Water Deer (Hydropotes inermis argyropus)

We determined the prevalence of Bartonella spp. and investigated which species of Bartonella naturally infects Korean water deer (KWD, Hydropotes inermis argyropus) in the Republic of Korea (ROK). A total of 70 spleens from KWD carcasses were collected by the Conservation Genome Resource Bank for Korean Wildlife (CGRB) in the ROK between 2008 and 2009. Nested PCRs were performed using the rpoB gene and internal transcribed spacer (ITS) region primers to amplify the DNA fragment of Bartonella. Using ITS-based nested PCR, Bartonella grahamii and Bartonella schoenbuchensis-related species were detected in 11 (15.8%) and 9 (12.9%) of 70 KWD spleens, respectively. The 11 B. grahamii amplicons were classified into 2 genotypes by sequence analysis. Using rpoB-based nested PCR, B. grahamii was detected in 5 (7.1%) of 70 KWD spleen samples. This is the first report of B. grahamii and B. schoenbuchensis in KWD, suggesting that KWD may act as reservoirs for the spreading of Bartonella spp. in the ROK.

Detection of Rickettsia felis and Rickettsia typhi and Seasonal Prevalence of Fleas Collected from Small Mammals at Gyeonggi Province in the Republic of Korea

Fleas were collected from live-captured small mammals to identify flea-borne pathogens, host associations, and seasonal prevalence of flea species, as part of the 65th Medical Brigade rodent-borne disease surveillance program at 20 military installations and training sites, Gyeonggi Province, Republic of Korea, 2005-2007. A total of 1251 fleas were recovered from 2833 small mammals. Apodemus agrarius, the striped field mouse, accounted for 93.1% (2,637/2,833) of all small mammals captured, followed by Crocidura lasiura (3.1%), Mus musculus (1.3%), Microtus fortis (0.7%), Myodes regulus (0.7%), Micromys minutus (0.5%), Rattus norvegicus (0.4%), Tscherskia triton (0.1%), Apodemus peninsulae (< 0.1%), Rattus rattus (< 0.1%), and Mogera robusta (< 0.1%). A total of 6/11 species of mammals captured were infested with fleas with infestation rates ranging from a high of 26.3% (A. agrarius and M. regulus) to a low of 5.3% (M. fortis). Flea indices among infested mammals were highest for R. norvegicus (2.50), followed by C. lasiura (2.20), A. agrarius (1.71), M. regulus (1.20), M. musculus (1.0), and M. fortis (1.0). The predominant flea species collected were Stenoponia sidimi (56.5%), followed by Ctenophthalmus congeneroides (38.3%) and Rhadinopsylla insolita (3.9%). The minimum field infection rates [number of positive pools/total number of fleas (600)] for Rickettsia typhi and for Rickettsia felis were 1.7% and 1.0%, respectively.

Prevalence of Anaplasma and Bartonella spp. in Ticks Collected from Korean Water Deer (Hydropotes inermis argyropus)

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.

Prevalence of Anaplasma, Bartonella and Borrelia Species in Haemaphysalis longicornis collected from goats in North Korea

North Korea 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 South Korea, they have not been reported from North Korea. To screen for zoonotic tick-borne pathogens in North Korea, ticks were collected from domestic goats. A total of 292 (27 nymph, 26 male, 239 female) Haemaphysalis (H.) longicornis were collected and assayed individually for selected tick-borne pathogens. A total of 77 (26.4%) were positive for Anaplasma bovis, followed by Bartonella (B.) grahamii (15, 5.1%), Anaplasma phagocytophilum (12, 4.1%), Bartonella henselae (10, 3.4%), and Borrelia spp. (3, 1.0%) based on 16S ribosomal RNA and ITS species-specific nested polymerase chain reaction. Using the groEL-based nested PCR, a total of 6 and 1 H. longicornis were positive for B. grahamii and B. henselae, respectively. All products were sequenced and demonstrated 100% identity and homology with previously reported sequences from other countries in GenBank. This is the first report of the detection of tick-borne pathogens in the North Korea and suggests that farm animals may act as reservoirs for zoonotic tick-borne pathogens.