Detection of Rickettsia felis and Rickettsia typhi and SeasonalPrevalence of Fleas Collected from Small Mammals

at Gyeonggi Province in the Republic of Korea

Sungjin Ko,1,* Heung-Chul Kim,2,* Young-Cheol Yang,3 Sung-Tae Chong,2 Allen L. Richards,4

William J. Sames,5 Terry A. Klein,6 Jun-Gu Kang,1 and Joon-Seok Chae1

Abstract

Fleas were collected from live-captured small mammals to identify flea-borne pathogens, host associations, andseasonal prevalence of flea species, as part of the 65th Medical Brigade rodent-borne disease surveillance pro-gram at 20 military installations and training sites, Gyeonggi Province, Republic of Korea, 2005–2007. A total of1251 fleas were recovered from 2833 small mammals. Apodemus agrarius, the striped field mouse, accounted for93.1% (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 speciesof mammals captured were infested with fleas with infestation rates ranging from a high of 26.3% (A. agrariusand 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). Thepredominant 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/totalnumber of fleas (600)] for Rickettsia typhi and for Rickettsia felis were 1.7% and 1.0%, respectively.

Key Words: Ctenophthalmus congeneroides, Stenoponia sidimi—Flea—Korea—Rickettsia felis—Rickettsia typhi.

Introduction

Fleas are vectors of zoonotic pathogens of publichealth importance, including plague, murine typhus, and

other flea-borne rickettsial agents (Mohr 1951, Parola et al.2005). The accidental or intentional introduction of flea-bornediseases poses serious public and military health risks, espe-cially during military training exercises, natural disasters, ormilitary operations. This is particularly true in locationswhere reservoir hosts (e.g., rodents and insectivores) and theirassociated ectoparasites occur in high densities, or when thehosts are displaced and leave their associated ectoparasites inabandoned nests and burrows or nearby surroundings

(Richards et al. 1997, Eisen et al. 2007). In the Republic ofKorea (ROK), small mammals are reservoir hosts to a numberof zoonotic pathogens transmitted by ticks (e.g., Rickettsia,Anaplasma, Ehrlichia, and Bartonella spp.), mites (Orientiatsutsugamushi), and fleas (Rickettsia typhi) (Chu and Hong1958, Ahn and Soh 1973, Hong et al. 1975, Walton and Hong1976, Lee et al. 1983, Hong and Shin 1990, Kim et al. 2003,2005, 2006, Chae et al. 2008, Kim et al. 2010a, 2010b, O’Guinnet al. 2010, Sames et al. 2010).

The first recorded survey of rat fleas in Korea was con-ducted by Kobayashi in 1931, with subsequent surveys re-porting a total of 37 flea species and associated hostrelationships (Nagahana 1954, Tipton et al. 1972, Hong 1994).

1Department of Veterinary Internal Medicine, Research Institute and BK21 Program for Veterinary Science, College of Veterinary Medicine,Seoul National University, Seoul, Korea.

25th Medical Detachment, 168th Multifunctional Medical Battalion, 65th Medical Brigade, APO AP96205.3Department of Environmental Health, Eulji University, Seongnam, Korea.4Viral and Rickettsial Diseases Department, Naval Medical Research Center, Silver Spring, Maryland.5OD/AFPMB, Walter Reed Army Medical Center, Washington, DC.6Force Health Protection and Preventive Medicine, 65th Medical Brigade/U.S. Army MEDDAC-Korea, AP APO 96205.*These two authors contributed equally in this work.

VECTOR-BORNE AND ZOONOTIC DISEASESVolume 11, Number 9, 2011ª Mary Ann Liebert, Inc.DOI: 10.1089/vbz.2010.0261

1243

After the systematic identification of ectoparasites and hostrelationships, zoonotic pathogens were identified in rodentsand associated hosts (Kim et al. 2003, 2005, 2006, Chae et al.2008, Kim et al. 2010b, O’Guinn et al. 2010, Sames et al. 2010).The development of rodent-borne disease surveillance pro-grams, which identify the potential for human exposure tovector-borne disease agents observed in feral and domestic

animals as well as ectoparasite diversity, provide a betterunderstanding of disease maintenance cycles. In turn, thisinformation can be used to identify and predict diseaseemergence events (Nieto et al. 2007).

Flea species, and associated hosts and pathogens wereidentified to determine the prevalence of the Rickettsia felis andR. typhi in fleas collected from small mammals in the ROK. In

FIG. 1. Geographical locations of small mammal collection sites at 15 U.S. and ROK–operated training sites (B), 4 U.S. militaryinstallations (,), and 1 Communications Post (6), Gyeonggi Province, ROK: Camp Bonifas (A) and Warrior Base (B), Gunnae-myeon, Paju-si; Camp Casey (C), Bosan-dong, Dongducheon-si; Osan AB (D), Osan-si; KC-39 (D), Pocheon-gun; Monkey Range 7(a)and Story Range(b), Jindong-myeon, Paju-si; Dagmar North Training Area (c), Jeokseong-myeon, Paju-si; Twin Bridge TrainingArea (d), Jikcheon-ri, Beobwon-eup, Paju-si; Local Training Area [LTA 130 (e), 320 (f), 36(g), 37(h)], and Chaparral Training Area(i), Misan-myeon, Yoencheon-gun; Santa Barbara Range (j), Yeoncheon-gun; Firing Point (FP) 10(k), 60 (l) and 66 (m), Yeoncheon-gun; Rodriguez Range (n), Youngjung-myeon, Pocheon-gun; and Nightmare Range (o), E-dong, Pocheon-si, Gyeonggi Province,ROK.

Table 1. Number Collected, Infestation Rates, and Indices for Fleas from Small Mammals Captured

at 4 U.S. Military Installations, 15 U.S. and Republic of Korea–Operated Military Training Sites,

and 1 U.S. Communications Post in Gyeonggi Province, Republic of Korea, 2005–2007

Host species Total captured (%) No. with fleasa Infestation rate (%)b No. of fleas Flea index (FI)c

Apodemus agrarius 2,637 (93.1) 694 26.3 1,189 1.71Apodemus peninsulae 1 (< 0.1) 0 0.0 0 0Mus musculus 36 (1.3) 3 8.3 3 1.00Micromys minutus 15 (0.5) 0 0.0 0 0Rattus norvegicus 12 (0.4) 2 16.7 5 2.50Rattus rattus 1 (< 0.1) 0 0.0 0 0Microtus fortis 19 (0.7) 1 5.3 1 1.00Tscherskia triton 3 (0.1) 0 0.0 0 0Myodes regulus 19 (0.7) 5 26.3 6 1.20Crocidura lasiura 89 (3.1) 20 22.5 44 2.20Mogera robusta 1 (< 0.1) 0 0.0 0 0Total 2,833 (100.0) 725 25.6 1,248 1.72

aPercent of small mammals captured with fleas.bAverage number of fleas per infested animal for each species.cFlea index (FI) = Numbers of fleas/total numbers of infested rodents.

1244 KO ET AL.

addition, the relative seasonal abundance of fleas collectedfrom wild-caught small mammals at military training siteswas determined. These data are useful for compiling vector-borne disease risk assessments for pathogens transmitted byfleas at military training sites and installations in the ROK.

Materials and Methods

Samples

Fleas were collected from small mammals that were live-trapped at 4 U.S. military installations, 15 U.S. and ROK–operated

Table 2. Seasonal Abundance of Fleas and Flea Indices for Small Mammals Captured

at 4 U.S. Military Installations, 15 U.S. and Republic of Korea–Operated Military Training Sites,

and 1 U.S. Communications Post in Gyeonggi Province, Republic of Korea, 2005–2007

No. of fleas collected (flea infestation rate)

Collection sitesaNo. of small

mammalsNo. (%)infested

No. offleas

Fleaindexb

MAR(n = 651)c

JUN(n = 507)c

SEP(n = 273)c

DEC(n = 1402)c

Dagmar North Training Area 525 151 (28.8) 240 1.59 25 (10.4) 129 (53.8) 71 (29.6) 15 (6.3)Rodriguez Range 136 50 (36.8) 101 2.02 26 (25.7) 7 (6.9) 5 (5.0) 63 (62.4)Firing Point 10 158 19 (12.0) 23 1.21 2 (8.7) 10 (43.5) 1 (4.3) 10 (43.5)Firing Point 60 234 48 (20.5) 71 1.48 0 31 (43.7) 0 40 (56.3)Chaparral Range 112 1 (0.9) 2 2.00 0 0 0 2 (100.0)Local Training Area 130 93 11 (11.8) 15 1.36 1 (6.7) 7 (46.7) 1 (6.7) 6 (40.0)Warrior Base 17 1 (5.9) 1 1.00 0 0 0 1 (100.0)Monkey Range 7 95 4 (4.2) 6 1.50 2 (33.3) 3 (50.0) 1 (16.7) 0Story Range 65 3 (4.6) 7 2.33 5 (71.4) 2 (28.6) 0 0Camp Bonifas 12 0 (0.0) 0 0.00 0 0 ns 0Local Training Area 36 13 2 (15.4) 2 1.00 ns ns ns 2Local Training Area 37 19 0 (0.0) 0 0.00 ns ns ns 0Local Training Area 320 31 12 (38.7) 19 1.58 ns ns ns 19KC 39 4 0 (0.0) 0 0.00 ns ns ns 0Firing Point 66 13 2 (15.4) 2 1.00 ns ns ns 2Twin Bridge Training Area 876 337 (18.5) 603 1.79 126 (20.9) ns ns 477 (79.1)d

Osan Air Base 83 20 (24.1) 28 1.40 ns ns ns 28Nightmare Range 128 3 (2.3) 3 1.0 ns ns ns 3Santa Barbara Range 158 36 (22.8) 62 1.72 ns ns ns 62Camp Casey 61 25 (41.0) 63 2.52 ns ns ns 63Total 2,833 725 (25.6) 1248 1.72 187 (15.0) 189 (15.1) 79 (6.3) 793 (63.5)

aDagmar North Training Area: Jeokseong-myeon, Paju-si, Gyeonggi province; Camp Bonifas, and Warrior Base: Gunnae-myeon, Paju-si,Gyeonggi province; Monkey Range 7, and Story Range: Jindong-myeon, Paju-si, Gyeonggi province; Firing Point (FP) 10, 60 and 66:Yeoncheon-gun, Gyeonggi province; Chaparral Training Area, and Local Training Area (LTA) 36,37,130, 320: Misan-myeon, Yoencheon-gun,Gyeonggi province; Rodriguez Range: Youngjung-myeon, Pocheon-gun, Gyeonggi province; Twin Bridge Training Area: Jikcheon-ri,Beobwon-eup, Paju-si, Gyeonggi province; Osan AB: Osan-si, Gyeonggi province; KC 39: Pocheon-gun, Gyeonggi province; Camp Casey:Bosan-dong, Dongducheon-si, Gyeonggi province; Nightmare Range: E-dong, Pocheon-si, Gyeonggi province; Santa Barbara Range:Yeoncheon-gun, Gyeonggi province, ROK.

bFlea index = Number of fleas/total number of infested rodents.cNumber of small mammals captured.dThis sample was collected during the winter seasons (Dec) of 2005 and 2006.ns = not surveyed.

FIG. 2. Mean number of the two most frequently collected species of fleas of infested small mammals and percent ofinfested for all flea species captured during all four seasons at nine U.S. and ROK–operated military training sites in northernGyeonggi Province, ROK, in 2005.

DETECTION OF Rickettsia felis AND Rickettsia typhi IN KOREA 1245

military training sites, and 1 communications post, in GyeonggiProvince, ROK (Fig. 1). Surveys were conducted seasonally from2005 to 2007 during the spring (March–April), summer (June), fall(late August–September), and winter (late November–Decem-ber). Sherman traps (7.7 · 9 · 23 cm aluminum collapsible live-traps (H.B. Sherman, Tallahassee, FL), baited with peanut butterplaced between two saltine crackers, were set out during daylighthours and collected the following morning. Since nighttimetemperatures often fell below 0�C, nonabsorbent cotton ballswere placed in each trap during the winter and spring trap-ping periods so that the trapped animals would retain heatuntil processed. Live small mammals were transported toKorea University and euthanized under an approved animaluse protocol as described by O’Guinn et al. (2010). Fleas wereremoved, identified to species using conventional taxonomickeys, then placed in 1.0 mL cryovials, and stored individuallyat - 70�C until assayed for selected pathogens (Hopkins andRothschild 1953, 1956, Hong 1994). Flea infestation rates, in-dices, and minimum field infection rates (MFIR) were deter-mined by the following formulas:

Flea Infestation Rate = Number of Captured Small Mammalswith Fleas/Total Number of Small Mammals Captured

Flea Index = Number of Fleas Collected from Small Mam-mals/Number of Small Mammals Infested

MFIR* = Number of Positive Pools of Fleas/Total Numberof Fleas

*Based on a maximum of one positive (infected) flea perpositive pool, although there may be more than one positiveflea in the pool.

DNA extraction

DNA was extracted from 300 pools (600 fleas, 2 fleas/pool)for detection of R. felis and R. typhi. Fleas were pooled ac-cording to collection dates and sites, flea species, and hostanimal species. Fleas were homogenized mechanically using aBeadbeater TissueLyser II (QIAGEN, Hilden, Germany) with180 lL lysis buffer, 20 lL proteinase K (600 AU/mL), and5 mm stainless steel beads at 30 frequencies/s for 5 min, fol-lowed by incubation at 56�C overnight and centrifugation at12,000 g for 15 min at room temperature. After centrifugation,the supernatant was used for genomic DNA extraction per-formed with DNeasy� Tissue Kits (QIAGEN) according to themanufacturer’s instructions.

Polymerase chain reaction detection

Extracted DNA was amplified in two separate nested-polymerase chain reaction (PCR) runs with different targetgenes to confirm results and differentiate Rickettsia species. Allsamples were first screened using genus-specific primers(RpCS.877p: 5¢-GGGGACCTGCTCACGGCGG; RpCS.1258n:ATTGCAAAAAGTACAGTGAACA; RpCS.896p: GGCTAATGAAGCAGTGATAA; RpCS.1233n: GCGACGGTATACCCATAGC), derived from the citrate synthase–encoding(gltA) gene for Rickettsia (Roux et al. 1997). Screened samplesthat were positive were selected for a second PCR assay withspecies-specific primers targeting the ompB gene (rompB OF:GTAACCGGAAGTAATCGTTTCGTAA, rompB OR: GCTTT

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1246 KO ET AL.

AATACGTGCTGCTAACCAA, rompB SFG IF: GTTTAATACGTGCTGCTAACCAA, rompB SFG/TG IR: GGTTTGGCCCATATACCATAAG) (Choi et al. 2005).

Positive controls for R. typhi (strain Wilmington) and R. felis(strain URRWXCal2) DNA were provided by the Division ofZoonosis, Center for Immunology and Pathology, KoreaNational Institute of Health, and the Viral and RickettsialDiseases Department, Naval Medical Research Center, re-spectively. To minimize the possibility of nested productcontamination, two separated rooms, which had dedicatedPCR equipment, supplies, and solutions, were used. Negativecontrols, including a water control, negative wild rodent tis-sues, and flea DNA, were included for each of the nested PCR

assays. No detectable bands representing R. typhi or R. feliswere observed from negative controls.

DNA sequencing and phylogenetic analysis

The PCR products were purified with QIAquick Gel Ex-traction kits (QIAGEN). After purification, all 16 gltA-positivePCR products were cloned with pGEM�-T Easy Vectors(Promega Corporation, Madison, WI), followed by transfor-mation into Escherichia coli JM109, and then plated onto LBagar containing 50 lg/mL of ampicillin. Three clones for eachof the amplicon plasmid DNA for sequencing were purifiedusing the Wizard� Plus SV Minipreps DNA Purification

Table 4. Rickettsia Species Identified by gltA Gene Targeting Polymerase Chain Reaction in Selected

Flea Species from Rodents at 4 U.S. Military Installations, 15 U.S. and Republic of Korea–Operated

Military Training Sites, and 1 U.S. Communications Post, Gyeonggi Province, Republic of Korea, 2005–2007

Year Species No. of fleas No. of poolsa No. of positive pools (MFIR)b

2005 Ctenophthalmus congeneroides 78 39 1 (1.28)Stenoponia sidimi 120 60 3 (2.5)Rhadinopsylla insolita 2 1 0

Subtotal 200 100 4 (2)2006 Ctenophthalmus congeneroides 46 23 0 (0)

Stenoponia sidimi 120 60 5 (4.16)Rhadinopsylla insolita 34 17 3 (8.82)

Subtotal 200 100 8 (4)2007 Ctenophthalmus congeneroides 66 33 2 (3.03)

Stenoponia sidimi 100 50 0 (0)Rhadinopsylla insolita 26 13 0Doratopsylla coreana 4 2 2 (50)Rhadinopsylla concava 4 2 0

Subtotal 200 100 4 (2)Total 600 300 16 (2.67)

a2 fleas per pool (total 600 fleas).bMFIR (Minimum field infection rates) = Number positive pools/Total number fleas assayed.

Table 5. Detection of Rickettsia felis and Rickettsia typhi ompB and gltA Partial Gene from

Fleas from Rodents Collected at 4 U.S. Military Installations, 15 U.S. and Republic

of Korea–Operated Military Training Sites, and 1 U.S. Communications Post, Gyeonggi Province,

Republic of Korea, 2005–2007

Pathogens Year Collection siteHost

speciesFlea vector

speciesgltAgene

ompBgene Strain

Rickettsia felis 2005 LTA 130/Yeoncheon A. agrarius C. congeneroides + + + + KRS42005 Warrior Base/Paju A. agrarius S. sidimi + + - KRS12006 Twin Bridge Training Area/Paju A. agrarius R. insolita + + - KRS72006 Twin Bridge Training Area/Paju A. agrarius S. sidimi + + - KRS112006 Twin Bridge Training Area/Paju A. agrarius S. sidimi + + - KRS122007 Camp Casey/Dongducheon A. agrarius C. congeneroides + + + + KRS14

Rickettsia typhi 2005 Rodriguez Range/Pocheon A. agrarius S. sidimi + + + KRS22005 Twin Bridge Training Area/Paju A. agrarius S. sidimi + + + + KRS32006 Twin Bridge Training Area/Paju A. agrarius R. insolita + + + + KRS52006 Twin Bridge Training Area/Paju A. agrarius R. insolita + + + KRS62006 Twin Bridge Training Area/Paju A. agrarius S. sidimi + + + KRS82006 Twin Bridge Training Area/Paju A. agrarius S. sidimi + + + KRS92006 Twin Bridge Training Area/Paju A. agrarius S. sidimi + + + KRS102007 Twin Bridge Training Area/Paju A. agrarius C. congeneroides + + + KRS132007 Santa Barbara Range/Yeoncheon C. lasiura D. coreana + + + KRS152007 Santa Barbara Range/Yeoncheon C. lasiura D. coreana + + + KRS16

- , not amplified; + , amplified and not sequenced; + + , amplified and sequenced.

DETECTION OF Rickettsia felis AND Rickettsia typhi IN KOREA 1247

System (Promega Corporation) according to the manufac-turer’s instructions. Purified recombinant plasmid DNA wassequenced using a T7 and SP6 promoter primer set by di-deoxy termination with an automatic sequencer (ABI 3730xlcapillary DNA sequencer, NY). Comparative analyses of thenucleotide sequences were completed using Rickettsia gltAgene sequences in the GenBank database. Otherwise, all 12positive amplicons of ompB gene fragments were sequenceddirectly. Phylogenetic analyses of the gltA and ompB genefragments were constructed using the ClustalX 1.60 programand a phylogenetic tree deduced by the neighbor-joining methodwith MEGA 4.0 software. Numbers on the branches representbootstrap support ( > 50%) generated from 500 replications.

Results

From March 2005 through December 2007, a total of 2833small mammals belonging to seven genera of rodents, oneshrew genus, and one mole genus were collected (Table 1,Fig. 1). Apodemus agrarius, the striped field mouse, accountedfor 93.1% (2,637) of the small mammals captured, followed byCrocidura lasiura (3.1%), Mus musculus (1.3%), Microtus fortis(0.7%), Myodes regulus (0.7%), Micromys minutus (0.5%), Rattus

norvegicus (0.4%), and Tscherskia triton (0.1%). An additionalthree species (accounted for < 1.0% of the total number ofsmall mammals captured (Table 1).

A total of 1251 fleas (869 females and 382 males) werecollected from 725/2,833 (25.6%) small mammals. Smallmammal infestation rates for 6/11 species of small mammalsinfested with fleas ranged from a low of 5.3% (M. fortis) to ahigh of 26.3% (A. agrarius and M. regulus). The overall fleaindex for all flea infested mammals was 1.72. Flea indiceswere 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) (Table 1).

Seasonal surveys were conducted during 2005, whereassurveys were only conducted during the winter and springseasons during 2006 and 2007. For sites that were surveyedduring all four seasons during 2005, the overall flea infes-tation rates were lowest during the spring (13.1%) andhighest during the summer season (40.6%). The highestnumbers of rodents infested with fleas were found atRodriguez Range (36.8%), and Dagmar North TrainingArea (28.8%) (Table 2). For all sites surveyed, the mostcommon flea species recovered was Stenoponia sidimi(56.5%), followed by Ctenophthalmus congeneroides (38.3%),

FIG. 3. Phylogenetic tree for members of the genus Rickettsia inferred from comparison of ompB sequences using theneighbor-joining method. The phylogenetic tree shows the position of Rickettsia felis (KRS 4, HQ236989) and Rickettsia typhistrains (KRS 13, HQ236990) identified in Gyeonggi Province, ROK, 2005–2007. Bootstrap values for the nodes are indicated.

1248 KO ET AL.

Rhadinopsylla insolita (3.9%), and Neopsylla bidentatiformis(0.4%; Table 3). An additional five species accounted for< 1.0% of all fleas collected. Doratopsylla coreana was infre-quently encountered and only collected from the Ussuriwhite-toothed shrew, C. lasiura. While C. congeneroides wascollected during all seasons surveyed, the highest popula-tions were observed during the spring through fall seasons.Conversely, S. sidimi was most commonly collected duringthe winter survey period (Fig. 2). Species R. insolita and S.sidimi were collected only during the spring and wintersurvey periods, whereas Hystrichopsylla microti and D. cor-eana were collected only during the winter survey period.Local Training Area 320 and Camp Casey, which were notsurveyed seasonally, demonstrated high infestation ratesduring the December collection period.

A total of 10/300 pools (600 fleas) were positive for R.typhi (3.3%) with an MFIR of 1.7%, whereas six of the poolswere positive for R. felis (2.0%) with an MFIR of 1.0% (Table4). Sixteen and 12 samples were positive using gltA andompB gene detecting primers, respectively. Eight gltA nu-cleotide sequences (2 R. typhi and 6 R. felis) were obtained,whereas sequencing for eight amplicons failed. In the secondPCR, 4/16 gltA-positive DNAs were not amplified withompB gene targeting primers (Table 5). R. felis–positive poolswere observed for C. congeneroides, R. insolita, and S. sidimicollected from A. agrarius; from LTA 130 (Yeoncheon),Warrior base (Paju-si), Twin bridge training area (Paju-si),and Camp Casey (Dongducheon-si), Gyeonggi Province(Table 5). A total of 8/10 R. typhi–positive pools were fromfleas collected from A. agrarius, whereas the other two pos-

itive pools were identified from C. lasiura. The MFIRs for R.typhi were 1.5% for S. sidimi (5/340), 3.2% for R. insolita (2/62), 0.5% for C. congeneroides (1/190), and 50.0% for D. cor-eana (2/4) (Tables 4 and 5). Acquired ompB oligonucleotidesequences of the PCR products demonstrated 98.7% and99.3% identity to reference sequences of R. typhi, strainWilmington (L04661), and R. felis, strain California 2(AF210695), respectively.

Sequences reported in this article have been deposited inthe GenBank database. The phylogenetic tree based on ompBgene sequences [accession numbers: R. felis strain KRS 4(HQ236989); R. typhi strain KRS 13 (HQ236990)] (Fig. 3) andgltA gene sequences [R. felis strain KRS 1 ( JF 448468), KRS 7( JF 448469), KRS 12 ( JF 448470), KRS 11 ( JF 448471), KRS 4 ( JF448472), KRS 14 ( JF 448473); R. typhi strain KRS 3 ( JF 448474),KRS 5 ( JF 448475)] (Fig. 4) illustrates the positions of strainsidentified in this study.

Discussion

Adult fleas are mobile obligate parasites that will oftenabandon their natural host(s), especially after death of thehost, and infest alternate hosts, including humans, to obtainblood meals (Azad et al. 1977, Schriefer et al. 1994, Higginset al. 1996). During 2005, when training sites were surveyedduring all seasons, flea infestations were generally higherduring the mid-winter months, similar to that observed byWalton and Hong (1976). The exception was C. congeneroides,which was collected more frequently during the spring,summer, and fall survey periods. The biology of these species

FIG. 4. Phylogenetic tree for members of the genus Rickettsia inferred from comparison of gltA sequences using theneighbor-joining method. The phylogenetic tree shows the position of R. felis [KRS 1 ( JF 448468), KRS 7 ( JF 448469), KRS 12( JF 448470), KRS 11 ( JF 448471), KRS 4 ( JF 448472), and KRS 14 ( JF 448473)] and R. typhi [KRS 5 ( JF 448475) and KRS 3 ( JF448474)] strains identified in Gyeonggi Province, ROK, 2005–2007. Bootstrap values for the nodes are indicated.

DETECTION OF Rickettsia felis AND Rickettsia typhi IN KOREA 1249

is not well known in Korea and may reflect host and/or ec-toparasite reproductive seasonal differences.

R. felis was identified as a flea-borne rickettsial pathogen in1990, with C. congeneroides as the primary zoonotic vectorplaying an important role in human transmission (Adamset al. 1990). Recently, noticeable increases have been observedworldwide in the number of human R. felis infections reportedbased on PCR or serological tests (Nogueras et al. 2006, Oteoet al. 2006). The associated illness is typically mild and char-acterized by fever, headache, myalgia, and cutaneous mani-festations (rash, eschar, and ulcer), but lymphadenopathy andneurological expressions (photophobia, hearing loss), ab-dominal symptoms (nausea, diarrhea), and pneumonia mayalso occur. Evidence of spotted fever group rickettsiosis wassupported by serologic surveys and DNA analysis in the ROK(Lee et al. 1994, Jang et al. 2004, Choi et al. 2005), with humancases of infections reported in 2005 (Chung et al. 2006). In aprevious study, we identified R. typhi in fleas captured in2003–2004 (Kim et al. 2010b). While the fleas tested in theprevious study had blood meals in their guts after removalfrom R. typhi–negative hosts, this does not preclude themfrom having taken recent blood meals from R. typhi–positivehosts.

A total of three species of fleas (C. congeneroides, R. insolita,and S. sidimi) collected from A. agrarius were found infectedseparately with R. typhi and R. felis. Doratopsylla coreana col-lected from C. lasiura was infected only with R. typhi. Similarto results by Noden et al. (1998), co-infections with both R.typhi and R. felis among individual fleas were not observed.

Human infections of murine typhus among Korean popu-lations from 2005 to 2007 were more prevalent from Octoberto November (46.1% and 36.7%, respectively), whereas for theother months, infection rates ranged from 0.0% to 4.1%(K-CDC 2009). Unfortunately, small mammal surveys werenot conducted during October to determine flea indices forC. congeneroides, which was most frequently collected duringAugust–September, and S. sidimi, which was very abundantduring November–December (Kim et al. 2010b). Although itis not conclusive, data suggest that while S. sidimi appears tobe the principal vector for R. typhi in the ROK, C. congeneroides,R. insolita, and D. coreana are also vectors. Data for spottedfever group Rickettsia, that is, R. felis, are not reportable eventsand therefore data for human cases are lacking.

Transmission of R. felis and R. typhi through fecal con-tamination of bite wounds or skin abrasions by these fleaspecies that were PCR positive has not been verified. How-ever, we detected R. felis and R. typhi DNA in spleen tissues ofA. agrarius captured during a 2009 rodent survey using thesame PCR method (data not shown). These results indicatethat fleas that were positive for rickettsial parasites may haveonly contained parasites from recent blood meals taken fromhost animals. It is clear that the data on R. felis and R. typhi inA. agrarius are diverse in terms of the species implicated ashosts. Infections with R. felis and R. typhi have generally beenconsidered to be host specific. Therefore, additional efforts todefine the spectrum of host susceptibility in domestic andwild animals are needed. Although there are numerous re-ports throughout the world on the epidemiology of flea-bornepathogens and reservoir host/vector associations, this is thefirst report of R. felis from the ROK.

These studies provide further information on the epide-miology of flea-associated pathogens and the potential for

transmission to humans in the ROK. Further studies are re-quired for a better understanding of potential emerging zoo-notic flea-borne pathogens in the ROK, which will enablehealth professionals to be more proactive regarding envi-ronmental changes and activities that impact the health ofboth civilian and military populations.

Acknowledgments

We thank the commanders and personnel of the 5th and38th Medical Detachments, 168th Multifunctional MedicalBattalion, for their support in conducting small mammalsurveillance. We especially thank Dr. Joel Gaydos, GlobalEmerging Infections Surveillance and Response System, Sil-ver Spring, MD, for his support and constructive criticism.Funding for portions of this work was provided by the ArmedForces Health Surveillance Center, Global Emerging Infec-tions Surveillance and Response System, Silver Spring, MD,the National Center for Military Intelligence, Ft. Detrick, MD,and through the BK21 Program for Veterinary Science, SeoulNational University, Seoul, Korea.

Disclosure Statement

The opinions expressed in this article are those of the au-thors and do not reflect official policy or positions of the U.S.Department of the Army, the U.S. Department of Defense, orthe U.S. Government.

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Address correspondence to:Joon-Seok Chae

Department of Veterinary Internal MedicineCollege of Veterinary Medicine

Seoul National UniversitySeoul 151-742

Korea

E-mail: jschae@snu.ac.kr

DETECTION OF Rickettsia felis AND Rickettsia typhi IN KOREA 1251