Joon-Seok Chae

Tick-Borne Rickettsial Pathogens in Ticks and Small Mammals in Korea

ABSTRACT 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.

Identification of Ehrlichia chaffeensis, Anaplasma phagocytophilum, and A. bovis in Haemaphysalis longicornis and Ixodes persulcatus Ticks from Korea

A total of 1,467 tick (1,463 of Haemaphysalis longicornis, three of Ixodes persulcatus and one of I. turdus) collected from nine provinces of Korea were examined by TaqMan real-time PCR for the presence of Ehrlichia and Anaplasma species. One set of primers and a probe were designed for detection of all of the Ehrlichia and Anaplasma species. Template DNAs (total 803) were prepared either from pools of larvae, nymphs, adult males and females, or from the salivary gland and midgut of adult ticks. Only DNAs positive in TaqMan PCR were examined for A. phagocytophilum with nested PCR and for E. chaffeensis with PCR. Four A. phagocytophilum 16S rRNA gene PCR products were sequenced for comparison with sequences previously reported. Amplification of a 16S rRNA gene fragment of Ehrlichia and Anaplasma species was observed in 364 tick DNAs (45.3% of the total). Of these 364 positive ticks, species-specific PCRs confirmed that 35 H. longicornis and one I. persulcatus were positive for A. phagocytophilum and one I. persulcatus was positive in E. chaffeensis. Except for one (AB-GGHL, GenBank accession number [GAN] AF470698), three of the four 16S rRNA gene fragment sequences of the A. phagocytophilum-positive samples were similar or identical to the sequences of variants of A. phagocytophilum deposited in GenBank. The 16S rRNA gene fragment sequence of AB-GGHL was similar to that of Anaplasma (Ehrlichia) bovis 16S rRNA (GAN U03775). The identities of the Anaplasmataceae genus and species DNA in the 327 ticks that could not be confirmed infected with either E. chaffeensis, A. phagocytophilum, or A. bovis are not known. This study is the first to demonstrate the presence of E. chaffeensis, A. phagocytophilum and A. bovis in Korean ticks.

Serologic and molecular detection of Ehrlichia chaffeensis and Anaplasma phagocytophila (human granulocytic ehrlichiosis agent) in Korean patients.

ABSTRACT Sera from 491 Korean patients with acute febrile diseases were tested for Ehrlichia chaffeensis and Anaplasma phagocytophila antibodies by indirect immunofluorescence assay (IFA), Western blotting, and TaqMan real-time PCR. Overall, 0.4% of sera reacted with E. chaffeensis, and 1.8% reacted with A. phagocytophila in IFAs. This is the first report of detection of antibodies to A. phagocytophila and E. chaffeensis in Korea and suggests the presence of A. phagocytophila and E. chaffeensis or antigenically similar species.

A study of the systematics of Theileria spp. based upon small-subunit ribosomal RNA gene sequences

Abstract The systematics of benign and moderately pathogenic Theileria isolates from cattle and deer originating from different geographic regions was undertaken by small-subunit ribosomal RNA (SSU rRNA) gene nucleotide-sequence analysis. A maximum-likelihood phylogenetic tree constructed from these sequences resulted in two major divisions, each with a common ancestor. One major division branches into four relatively divergent groups, including (1) bovine Theileria sp. Type D (USA and Korea), (2) T. mutans Intona and Theileria sp. MSD (Africa), (3) T. cervi (USA), and (4) well-characterized pathogenic Theileria spp. (Africa). The other major division branches into two groups: (1) T. buffeli Warwick and T. buffeli Marula and (2) a second branch of closely related isolates with SSU rRNA gene Types B, B1, C, E, and H. Putative geographically associated diversity was noted only in the Korean bovine Theileria spp. with SSU rRNA gene types C and H and in African T. mutans Intona and Theileria sp. MSD. The current results show that the United States bovine Theileria isolates are not T. mutans because they have T. buffeli Marula (Type A) and/or Type D (species undesignated) SSU rRNA gene sequences. The taxonomic separation of T. buffeli Warwick from African T. mutans is confirmed in this study.

Microbial pathogens in ticks, rodents and a shrew in northern Gyeonggi-do near the DMZ, 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.

Digenetic trematodes, Acanthatrium sp. and Lecithodendrium sp., as vectors of Neorickettsia risticii, the agent of Potomac horse fever.

Neorickettsia (formerly Ehrlichia) risticii, the agent of Potomac horse fever (PHF), has been recently detected in trematode stages found in the secretions of freshwater snails and in aquatic insects. Insectivores, such as bats and birds, may serve as the definitive host of the trematode vector. To determine the definitive helminth vector, five bats (Myotis yumanensis) and three swallows (Hirundo rustica, Tachycineta bicolor) were collected from a PHF endemic location in northern California. Bats and swallows were dissected and their major organs examined for trematodes and for N. risticii DNA using a nested polymerase chain reaction (PCR) assay. Adult digenetic trematodes, Acanthatrium sp. and/or Lecithodendrium sp., were recovered from the gastrointestinal tract of all bats and from one swallow. The intestine of three bats, the spleen of two bats and one swallow as well as the liver of one swallow tested PCR positive for N. risticii. From a total of seven pools of identical digenetic trematodes collected from single hosts, two pools of Acanthatrium sp. and one pool of Lecithodendrium sp. tested PCR positive. The results of this investigation provide preliminary evidence that at least two trematodes in the family Lecithodendriidae are vectors of N. risticii. The data also suggest that bats and swallows not only act as a host for trematodes but also as a possible natural reservoir for N. risticii.

Evaluation of the Methicillin-Resistant Staphylococcus aureus (MRSA)-Screen Latex Agglutination Test for Detection of MRSA of Animal Origin

ABSTRACT Methicillin (oxacillin)-resistant staphylococci (MRS) have emerged as major clinical and epidemiological pathogens, and there have been frequent reports of MRS infections in the veterinary field. The MRSA-Screen latex agglutination test (Denka Seiken Co., Ltd., Tokyo, Japan) was compared with an oxacillin agar screen test, MIC determination, and mecA PCR assay, the “gold standard.” In an analysis of 15 mecA-positive and 48 mecA-negative S. aureus animal isolates, as well as 9 mecA-positive and 147 mecA-negative, coagulase-negative staphylococcal animal isolates, the latex agglutination test surpassed the widely used oxacillin agar screen method and MIC determination, with a sensitivity and a specificity of 100%. The MRSA-Screen test is a reliable and rapid method of detecting MRS in the veterinary field.

Molecular Epidemiological Study for Tick-Borne Disease (Ehrlichia and Anaplasma spp.) Surveillance at Selected U.S. Military Training Sites/Installations in Korea

Abstract: Vector‐borne diseases are a potential public health threat to U.S. Forces Korea (USFK). Ehrlichia and Anaplasma spp., transmitted by ticks, are only two of several diseases that may affect military readiness and operations. Rodents were collected at selected U.S. military installations and training sites in the Republic of Korea. DNA was extracted from spleen tissues and assayed by PCR methods for Ehrlichia and Anaplasma species. From rodents and mustelids collected during 1999 and 2000, a total of 196 Apodemus agrarius (striped field mouse), 2 Mustela sibirica (weasel), and 1 Cricetulus triton nestor (Korean greater long‐tailed hamster) were assayed for Ehrlichia and Anaplasma species‐specific DNA fragments. Rodent surveillance indicated a very high prevalence of Ehrlichia and Anaplasma spp. at selected training sites. Ehrlichia/Anaplasma DNA were identified from spleen tissue from 157 Apodemus agrarius, 1 Mustela sibirica, and 1 Cricetulus riton nestor. Species‐specific DNA fragments of E. canis (45), E. ewingii (16), A. phagocytophila (5), and A. platys (62) were amplified by PCR techniques. Seventy‐one striped field mice had single infections, while 24 had mixed infections of 2 (17 specimens), 3 (7 specimens), or 4 (1 specimen) pathogens. The striped field mouse plays a role as a reservoir for latent infections of various Ehrlichia or Anaplasma species.

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.

Transmission of Ehrlichia risticii, the agent of Potomac horse fever, using naturally infected aquatic insects and helminth vectors: preliminary report.

Ehrlichia risticii, the agent of Potomac horse fever (PHF), has been recently detected in trematode stages found in snail secretions and in aquatic insects. Based on these findings, horses could conceivably be exposed to E. risticii by skin penetration with infected cercariae, by ingestion of infected cercariae in water or via metacercariae in a second intermediate host, such as an aquatic insect. In order to test this hypothesis, horses were challenged with infectious snail secretions and aquatic insects collected from a PHF endemic region in northern California. Two horses stood with their front feet in water harbouring E. risticii-infected cercariae, 2 horses drank water harbouring E. risticii-infected cercariae, and 6 horses were fed pools of different aquatic insects harbouring E. risticii-infected metacercariae. In this preliminary study, only the one horse infected orally with mature caddisflies (Dicosmoecus gilvipes) developed the clinical and haematological disease syndrome of PHF. The agent was isolated from the blood of the infected horse in a continuous cell line and identified as E. risticii by characterisation of the 16S rRNA gene. Therefore, E. risticii is maintained in nature in a complex aquatic ecosystem and transmission to horses can occur through accidental ingestion of insects such as caddisflies containing infected metacercariae. At present, the small number of horses used in this study does not exclude other insects and free trematode stages as potential sources of infection.