Terry A. Klein

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.

Random amplified polymorphic DNA (RAPD) markers readily distinguish cryptic mosquito species (Diptera: Culicidae: Anopheles)

The usefulness of random amplified polymorphic DNA (RAPD) was examined as a potential tool to differentiate cryptic mosquito species. It proved to be a quick, effective means of finding genetic markers to separate two laboratory populations of morphologically indistinguishable African malaria vectors, Anopheles gambiae and An. arabiensis. In an initial screening of fiftyseven RAPD primers, 377 bands were produced, 295 of which differed between the two species. Based on criteria of interpretability, simplicity and reproducibility, thirteen primers were chosen for further screening using DNA from thirty individuals of each species. Seven primers produced diagnostic bands, five of which are described here. Some problematic characteristics of RAPD banding patterns are discussed and approaches to overcome these are suggested.

The AFHSC-Division of GEIS Operations Predictive Surveillance Program: a multidisciplinary approach for the early detection and response to disease outbreaks

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.

Anopheles kleini, Anopheles pullus, and Anopheles sinensis: Potential Vectors of Plasmodium vivax in the Republic of Korea

Abstract Anopheles sinensis Wiedemann (63.3%) was the most abundant Anopheles mosquito captured at cowshed resting collections in malaria high-risk areas (northern Gyeonggi Province) near the demilitarized zone (DMZ) in Korea during 2005, followed by Anopheles kleini Rueda (24.7%) and Anopheles pullus M. Yamada (8.7%). At cowshed resting collections in malaria low-risk areas (Jeonnam and Gyeongnam provinces), An. sinensis accounted for 96.8% of all Anopheles spp. collected, followed by An. kleini Rueda (2.7%), whereas no An. pullus were collected. Three species, An. kleini (50.9%), An. pullus (29.0%), and An. sinensis (13.8%), accounted for nearly all of the 224 Anopheles spp. captured by New Jersey light trap near the DMZ. In addition, An. pullus and An. kleini captured by New Jersey light trap near the DMZ and assayed by enzyme linked immunosorbent assay for Plasmodium vivax circumsporozoite antigen concentrations were higher than An. sinensis sensu stricto (s.s.), indicating higher levels of sporozoites. In laboratory studies of four concurrent artificial membrane feedings on malaria-infected blood from patients, F1 progeny of An. kleini and An. pullus had higher infection rates (8.8 and 7.5%, respectively) than An. sinensis s.s. (4.2%). These data suggest that An. kleini and An. pullus and An. sinensis are vectors of malaria in Korea. Further studies are required to determine the role of these species in the transmission of P. vivax in the Republic of Korea.

Vector Competence of Peruvian Mosquitoes (Diptera: Culicidae) for Epizootic and Enzootic Strains of Venezuelan Equine Encephalomyelitis Virus

Abstract Mosquitoes collected in the Amazon Basin, near Iquitos, Peru, were evaluated for their susceptibility to epizootic (IAB and IC) and enzootic (ID and IE) strains of Venezuelan equine encephalomyelitis (VEE) virus. After feeding on hamsters with a viremia of ≈108 plaque-forming units of virus per milliliter, Culex (Melanoconion) gnomatus Sallum, Huchings, & Ferreira, Culex (Melanoconion) vomerifer Komp, and Aedes fulvus (Wiedemann) were highly susceptible to infection with all four subtypes of VEE virus (infection rates ≥87%). Likewise, Psorophora albigenu (Peryassu) and a combination of Mansonia indubitans Dyar & Shannon and Mansonia titillans (Walker) were moderately susceptible to all four strains of VEE virus (infection rates ≥50%). Although Psorophora cingulata (Fabricius) and Coquillettidia venezuelensis (Theobald) were susceptible to infection with each of the VEE strains, these two species were not efficient transmitters of any of the VEE strains, even after intrathoracic inoculation, indicating the presence of a salivary gland barrier in these species. In contrast to the other species tested, both Culex (Melanoconion) pedroi Sirivanakarn & Belkin and Culex (Culex) coronator Dyar & Knab were nearly refractory to each of the strains of VEE virus tested. Although many of the mosquito species found in this region were competent laboratory vectors of VEE virus, additional studies on biting behavior, mosquito population densities, and vertebrate reservoir hosts of VEE virus are needed to incriminate the principal vector species.

Endemic Venezuelan Equine Encephalitis in Northern Peru

Since Venezuelan equine encephalitis virus (VEEV) was isolated in Peru in 1942, >70 isolates have been obtained from mosquitoes, humans, and sylvatic mammals primarily in the Amazon region. To investigate genetic relationships among the Peru VEEV isolates and between the Peru isolates and other VEEV strains, a fragment of the PE2 gene was amplified and analyzed by single-stranded conformation polymorphism. Representatives of seven genotypes underwent sequencing and phylogenetic analysis. The results identified four VEE complex lineages that cocirculate in the Amazon region: subtypes ID (Panama and Colombia/Venezuela genotypes), IIIC, and a new, proposed subtype IIID, which was isolated from a febrile human, mosquitoes, and spiny rats. Both ID lineages and the IIID subtype are associated with febrile human illness. Most of the subtype ID isolates belonged to the Panama genotype, but the Colombia/Venezuela genotype, which is phylogenetically related to epizootic strains, also continues to circulate in the Amazon basin.

Genetic evidence for the existence of cryptic species in the Anopheles albitarsis complex in Brazil: allozymes and mitochondrial DNA restriction fragment length polymorphisms.

Allozyme and mitochondrial DNA (mtDNA) restriction studies were undertaken to determine the extent of genetic divergence among field populations ofAnopheles albitarsis in Brazil. Two sympatric species,An. deaneorum andAn. marajoara, were identified in collections from Costa Marques (CM), Rondonia. Genetic evidence includes (1) the presence of two types of individuals, each with diagnostic allelic clusters (forHad-1, Pgi-1, Pep-1, Mpi-1, andIdh-1), (2) a deficiency of heterozygotes, and (3) characteristic mtDNA haplotypes. In addition, two allopatric cryptic species ofAn. marajoara were identified, one from Iguape (An. marajoara form IG), Sao Paulo state, and the other from the Island of Marajo (An. marajoara form MA). Though form IG and form-MA resemble form CM in wing spot morphology, they differ from it in diagnostic allozymes and mtDNA haplotypes.An. marajoara form CM had a higher variability (mean heterozygosity,H=0.22, and percentage of polymorphic loci,P=66.7) than did form IG and form MA (H=0.08 in both, andP=25.0 and 33.3, respectively). Form MA and form IG are genetically more similar to each other than both are to form CM. Based on wing morphology, estimates ofF statistics, and genetic similarities, we propose thatAn. albitarsis in Brazil is a species complex. It comprises at least two morphologically distinguishable species: (1)An. deaneorum (currently one taxon) and (2) theAn. marajoara species complex, which further consists of at least three cryptic forms,marajoara form MA,marajoara form IG, andmarajoara form CM.

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.

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.

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.