Survey of antibody on Orientia Tsutsugamushi among wild rodents in Gyeongnam area and detection by nested polymerase chain reaction

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(1)³¤¥Èó. * F* a. (2004) 44 2 Korea J Vet Res(2004) 44(2) : 241~249. ãÎæ¢ vöB0SJFOUJBUTVUTVHBNVTIJö&ÚÒ5 1$3ö~¦ï. ~& Áf'î Á;;ë Á;Ò7 ÁfÏ Á~Òv Á;«^ Áî;^ ÁB«ã « ÁÂ Áf SÁf~ãÁf«>* . . . . . . . . . . . ãç&v >~"& ³ë«"ö ãÎ~ãö ê"Öë&v ÿb«"" ã>~"¦ö ¦Öæö ãÎ»Öê² Î¦æ² (²Òß: 2004j 4ú 21¢) 1. 2. 3 4. Survey of antibody on Orientia Tsutsugamushi among wild rodents in Gyeongnam area and detection by nested polymerase chain reaction Dae-Sik Hah1, Young-Hoon Kim1, Jung-Ung Park1, Jae-Kap Park1, Chung-Hui Kim2, Jae-Doo Ryu3, Myung-Ho Jong4, Jung-Ho Heo4, Jong-Lip Shu4, Myung-Heui Cho4, Kuk-Cheon Lee4, Gon-Sup Kim, Eui-Kyung Kim, and Jong-Shu Kim* Agriculture & Life Science, College of Veterinary Medicine, Gyeongsang National University, Jinju 660-701, Korea 1 Gyeongnam Provincial Government Institute of Health and Environment, Changwoon 641-825, Korea 2 Department of Animal Science and Biotechnology, Jinju National University, Jinju 660-758, Korea 3 National Veterinary Research and Quarantine service Busan Regional Office, Busan 602-030, Korea 4 Gyeongnam Livestock Promotion Institute South-branch, TongYoung 650-817, Korea (Accepteda April 21, 2004) Abstract : As a part of epidemiologic investigation of tsutsugamushi disease, the wild rodents which were captured in Gyeongnam area were diagnosed with indirect immunofluorescent antibody assay (IFA) and Polymerase Chain Reaction (PCR) to find if they have an antibody against Orientia tsutsugamushi. The conclusion was drawn as followings. (1) The captured 58 wild rodents showed that the subspecies distribution of Apodemus agrarius was 86.2%, Microtus fortis was 8.6% and Crocidura lasiura was 5.2%. (2) The antibody positive rate of O. tsutsugamushi Gilliam, Karp, Karto and Boryong by IFA method was 32.0% in Apodemus agrarius among 50 wild rodents and 40.0% in Microtus fortis among 5 wild rodents, respectively. It was negative in the case of all the 3 Crocidura lasiura. (3) The antibody titers on Apodemus agrarius, Microtus fortis and Crocidura lasiura against Gilliam, Karp, Karto and Boryong were measured between 1:20 and 1:640. The antibody titer against each antigen was in the order Boryong>Gilliam>Karp. (4) O. tsutsugamushi was detected from the blood, spleen and kidney from the artificially infected mice by IFA and PCR. IFA showed the positive response between 3 and 18 days after inoculation. On the other hand, positive response was found from all the samples by PCR. (5) From PCR of the genomic DNA extracted from the blood, spleen and kidney samples of the captured wild rodents, Boryong-specific amplification product with size of 210 bp, which is particular in Boryong, was detected from spleen and kidney samples, but not detected in the blood. (6) Boryong-specific amplification product was detected from spleen and kidney samples which were obtained at 3, 6, 12, 18 and 24 days after the infection with Boryong.. *Corresponding author: Jong-Shu Kim Agriculture & Life Science, College of Veterinary Medicine, Gyeongsang National University, Jinju 660-701, Korea [Tel: 82-55-751-5821, Fax: 82-55-751-5803, E-mail: jskim@gsnu.ac.kr]. 241.

(2) 242. ~&Áf'îÁ;;ëÁ;Ò7ÁfÏÁ~ÒvÁ;«^Áî;^ÁB«ãÁ«ÁÂÁf SÁf~ãÁf«> But, it wasn’t detected from the uninfected samples. (7) From PCR of spleen and kidney samples of the captured wild rodents, it was found that positive rate of O. tsutsugamushi in Apodemus agrarius and Microtus fortis were 25.0% (4/16) and 20.0% (1/5), respectively. From the above results, it can be concluded that Apodemus agrarius resided in Gyeongnam area carried O. tsutsugamushi and PCR method might be a simple, precise, rapid and useful diagnostic tool than IFA for the diagnosis of O. tsutsugamushi. Key words : Orientia, thutsugamushi, PCR, Gilliam, Boryong. B . «¾ ®Jr~ Orientia º 0.3Ü1.2 µm ;ê~ ÞW ^ VW *bB ^ êV~ FÏ Ò² ~ çj « r 6">, ², vÛ ~ * Ãç ê "GÛ, â*.«c, b¦Bê j ¢bÎ . Rickettsia ³ f ?" ÿbö î÷j ¢bÊº ·ç, Ò j *2~º BÏ~ «~ 5 b' Wç ö V¢ typhus, spotted fever, scrub typhusb ª ~B [7, 31]. Scrub typhusöº ¢ « O. tsutsugamushi& ³ ®b, .Ó; ß ö B Gilliam, Kato 5 Karp~ ^&æ ö; " ª~> [29], *Ò . 3ö; "º êö B·" ö & " ÒÏ> ® [13, 15]. 3 .Ó; ö jj æ öBº Karp .Ó;" WÏ jÝ RA686, RA716, RA763, TH1817 5 TA678~ 5&æ .Ó; ®º ©b rJ^ ® [25]. " ¢öBê ^ &æ ö; " .Ó' >w·ç îÚ .Ó;b Kawasaki, Kuroki 5 Shimokoshi >î [28]. ÚöBº Gilliam" Karp .Ó; ö Vö rJê ö; "fº .Ó' >w·ç Boryong "& ªÒ> ® [14]. úú&Z÷~ êöº ~¶~ .b¦V O. tsutsugamushi ¢ ªÒ~º O» [8]" ~¶~ .Ó Úö B Ú¢ Ã«~º O» [9-11] 5 7 Î²ê>w (polymerase chain reaction, PCR)j Ï~ ~¶~ . ÚöB DNA¢ ¦Ò~º O» ® [16, 20, 21, 24]. 7 Ú¢ V·~ î÷j ê~º O»f V· * 4" ç º>æ B ~¶~ Vê Ï'bº ¦'~, .Ó' ê»f ¦ÒO »ö V¢ VF' r æ, *rW (pseudo negative), *·W (pseudo positive), Ò*W 5 ß> Ë~~ jº W ~ ^B6 ® . 7Î²ê>wf Ò9~j .Ãj B Ú& ;W>æ pº 6".Vöê ~¶~ .Úö ²ï~ Úò ®Úê ~ Ò¢ ; {® ê > ®º Ë6 ® [20]. O. tsutsugamushi öº æÚ¾ Ú& ìbæ &. Rickettsia tsutsugamushi tsutsugamushi (O. tsutsugamushi). ¦ª~ ö Wªf Wî ©b C&^ ® [5, 12, 18, 23]. .Ó;~ ·Wj Ö;ùº O. tsutsugamushi ~ ^ï Wööº « ßö" .Ó; ß ö ®b, O. tsutsugamushi¢ ;B öj polyacrylamideJ *V'ÿ" immunoblottingb ªC ~ 110-120, 58-60, 54-56, 46-47, 20-22 kDa Wî~ "º ö Ò . 7 22 kDa, 46-47 kDa, 54-56 kDa, 120 kDa~ Wî öWj &æ ®b, 5456 kDa Wîf .Ó; ßöb rJ^ ® [17, 27]. Þ O. tsutsugamushi Ú¢ trypsinb ¾Ò~ lymphocyteö R~º ËK ² B º Ò öB ^ ö" O. tsutsugamushi~ ÷öW Òöº &7 &ê& ®º ©b rJ^ ®b, O. tsutsugamushiö & OÚ .Ó;ö ß 6b j 57-56 kDa Wîf OÚ~ FBöê 7º j ~º ©b º;~ ® [30]. ÖÒ¾¢öB úú&Z÷f ³2j 7b ôf ~¶& B~ ÃêÂ., ?ÆÊb¢Ã, Bê" þ &jö F¯~º &' /WBWî~~ ~¾ "Ïj r ®º î÷ . Ë" ; [8], Ë [911]f ~¶~ .ÓöB úú&Z~ ªÒ 5 .Ó Ò¢ ~&, [5], [2], V [1], [6], ; [3]f ³2æöB ê.ê æÒ' B ê V~ ª¢ Ò ~&b, W [4]f ãVêf ;öêöB ¢ v~ Ò9~j 6" ¢ Ò : ®b¾ ~ Òº " 7 .§¦ æOöB Úr, ãÎ¢&ö & º ~ ìº ; . öBº úú&Z÷ö & ' ßWj Ï'b ãçÎê ¢&öB ³ v~ O. tsutsugamushi ö & ÚF çj Ò~, .Ó " ËVöB Indirect immunofluorescent antibody assay (IFA)»" PCR»b jv ¦Ò~& .. Òò 5 O». úú&Z÷ B ¢ v~ ª Ò ¢ vº 2001j" 2002j 10ú¦V 12úræ 2² ö ö ãÎæO~ úú&Z÷ Bæj 7b Chemal Wooden trap (20 cmÜ8 cmÜ7 cm)j [30] æ.

(3) ãÎæ ¢ vöB Orientia tsutsugamushi ö & Ú Ò 5 PCRö ~ ¦ï. ;~ B· Êv.Ê trapj ÒÏ~ Új ®º ç ³~& . vf v& Ú&B _¢ Òº B* « ^ öê>B v& -®ê nB trapb & æV* ¢Ö, , ¦X ¢ v& B ò Ë²ö J~~&b, ³B vº rÆ jö Új ®º ç þ Tâ . ¢ v~ *'" »ö ~ ö& Ú Ò Ú Òº *7;7Ú»ö V¢ ~&. [1]. O. tsutsugamushi Karp, Kato, Gilliam 5 Boryong" ¢ öb ÒÏ~ öÒ¢¢ acetonb ; ~ V 7öB ê U-bottom 96 well microtitier platerö PBS 1:10V C .Ó 25 µl¢ &~ 37 C öB 30ª* >wÎ r PBS, Ã~> Bb Ê#² ^¿ ~& . ê 1:32 C FITCconjugated goat anti-mouse IgG (ICN Co., USA)¢ ' well ö 25 µl O &~ ÿ¢ O»b 2N >wÊ ~ O»" ? ^¿, ~ mounting media (Sigma, USA) mounting~ ;7*ãb &V~& . Ú ·W 6;f Gilliam, Karp, Karto, Boryong 7öB 1" ç ·Wj º ©b ~&, >w ª«~² &V>º & .ÓC V>~ >¢ Ú& ;~ &, 1:10 ç~ &¢ º ©j ·Wb 6;~ & . ~ 6" þ ãööB ª· Af úú&Z~ Boryong" ¢ ãç&vöB Ö~º 4"_ ç ICR mouse¢ z> ªì ª·Aj Ú7ö V j~Òï(Sublethal dose, 5Ü10 infected cell units [ICU])j êÖ~ ; Ú 7«~&, Òòf bf ¶F² _ê ~&. . 7« ê ., jË, Ëj 3, 6, 12, 18 5 24¢ö j~&b, 1 3îÒ¢ Ï~& . &b Ò">¢ ;Ú R ©j ÒÏ~& . ËV'Â 5 .ÓªÒ. þ TÎê ¢ vf 6"Î ICR mouse¢ ether î~ Ë j. r Ë" j Ëj Z'b 'Â~& . .f DNA ¦ÂÏ" . ÓªÒÏb ¾*î, Ë" jËf PBS(0.1 M; pH 7.2) } ^¿~ -70 Cö &~& . ¢ v 5 6" NPVTF ËV¦V %/" ºÂ ¢ v 5 6" mouseö & O. tsutsugamushi 0 UTVUTVHBNVTIJ. o. 0. UTVUTVHBNVTIJ. 4. o. 243. DNA~ ¦ïf V [1]~ PCR » [1, 2, 4, 6, 19, 26] " Furuya [16] BB PCR»j æ;~ ÒÏ~ & . 58îÒ~ ¢ v¦V ., jË 5 Ëj j~ DNA¢ ºÂ~&b, r &¦b*~ vN J"j Oæ~V *~ Î £ 5 Vº 1²Ï" ¾ÒB ©j ÒÏ~& . DNA ºÂf spin column method (QIAamp Tissue kit, Blood Mini kit, QIAGen Inc. Germany)¢ ÒÏ~ -70 Cö &B çj 25 mg ; ê~ ·f 'b ¾¢B microcentrifuge tubeö I Buffer ATL 180 µlf Proteinase K¢ 20 µl Î& ê, vortexing~ ç j*® [F rræ 55 C shaking water bathö ;~~& . [& Â Ïj 15.* vortexing ~ Buffer AL 200 µl Î&~ vortexing ê 70 C shaking water bathöB 10ª* ;~~&, ethanol 200 µl¢ Î&~ vortexing ~& . DNeasy spin columnj 2 ml collection tubeö ¹ *~ bÏj &~ 8,000 rpmöB 1ª* öªÒ~ columnj Û " Ï" collection tube¢ ªÒ DNA& ³B DNeasy spin columnj îÚ 2 ml collection tubeö r Buffer AW1 500 µlj Î& ê 1ª* öª Ò~ columnj Û" Ï" collection tube¢ ª^. . DNeasy spin columnj îÚ 2 ml collection tube ö ~ Buffer AW2 500 µl¢ Î& ê 3ª* ö ªÒ~ DNeasy membranej V . r DNeasy spin columnj 1.5 ml microcentrifuge tubeö TV Buffer AE 200 µl¢ DNeasy membraneö I çNö B 1ª* ;~ ê 1ª* öªÒ~ ºÂ DNA ¢ template DNA ÒÏ~& . 1$3 ªC PCR oligonucleotide primerº Furuya [16]ö ~ nB nested PCR O»j £* æ;~ ÒÏ~&. (Table 1). ¢N PCR ªCf 10ÜPCR buffer 5 µl [100 mM Tris-HCl(pH 9.0), 400 mM KCl, 15 mM MgCl ], 1 ng~ template DNA, 200 µM dNTPs, ' .Ó; Û' o. o. o. 2. Table 1. Primer sequences used for nested PCR First PCR forward (R56f) : 5'-ATT-GCT-AGT-GCA-ATG-TCT-GC-3' reverse (R56r) : 5'-CTT-CTT-GCG-CTG-TAG-CTT-GA-3' Second PCR forward (R56c) : 5'-CAG-CCT-ACT-ATG-AGT-CCT-AT-3' reverse : B/K(Boryong) : 5'-CAC-CGG-ATT-TAC-CAT-CAT-AT-3' KP(Karp) : 5'-ACA-ATA-TCG-GAT-TTA-TAA-CC-3' KT(Karto) : 5'-GGA-ATA-TTT-AAT-AGC-ACT-GG-3' GM(Gilliam): 5'-CTT-TAT-ATC-ACT-ATA-TAT-CTT-3'.

(4) 244. ~&Áf'îÁ;;ëÁ;Ò7ÁfÏÁ~ÒvÁ;«^Áî;^ÁB«ãÁ«ÁÂÁf SÁf~ãÁf«>. 3' (R56r) 5 5' ö primer (R56f)¢ 'V 10 pmol O &~, Taq DNA polymerase (Bioneer Co., Korea)¢ 2 unit I « >w¦b¢ 50 µl ~ thermal cycler (Perkin-Elmer, GeneAmp PCR System 2400, USA) ò DNA¢ Ã~& . r ÃÅ>º C 30 cycle ~&b, 95 CöB 5ª* æWÎ ê 94 CöB 1ª* denaturation, 55 CöB 1ª 30.* annealing, 72 CöB 2ª 30.* extentionBb >wVb, ê >w ê 72 CöB 7ª* >wB « PCRÖb~ Ëj Fê ~& . N PCRf template DNA ¢N PCRöB á f Öbj 3 µl, 10ÜPCR buffer 5 µl, 200 µM dNTPs, ' .Ó;ö Û' 5'ö primer (R56c)f 3'ö primerº ' .Ó;ö ß primer j 'V 20 pmol O, Taqº 2 unit I « >w¦b¢ 50 µl ~ PCRj ~& . 95 CöB 5ª* æWÎ ê 94 C öB 1ª, 60 CöB 2ª 30., 72 CöB 3ª 30. 25 cycle >wÊ, ê >w ê 72 CöB 7ª* >w V . ' >w î rW &b template DNA & Ã~>¢ ÒÏ~&b, vN J"j .O~V * aerosol resistant micropipet tipj ÒÏ~& . *V'ÿ ÃB DNA 5 µlö 6V ³ê~ loading bufffer (0.03% bromophenol blue, 30% glycerol, 30 mM EDTA, 0.03% xylene cyanol) 1 µlj b Ê 0.002% ethidium bromide ¢ Î&~ B· 1% horizontal agarose minigel * V'ÿ ~& . standard molecular weight marker (Takara Co., Japan) 100 bp, 200 bp 5 20 bp DNA Ladderf þ ÃB DNA .Þj {~& . o. o. o. o. o. o. o. o. o. o. Ö ". ¢ v~ «ê ª 2001j" 2002j 10ú¦V 12úræ v®ö ö ã ÎæOöB ³ ¢ v~ «ê ªº Table 2f ? C 58îÒ 7 *v(Apodemus agrarius)& 50î Ò(86.5%) &Ë ô~, iv(Crocidura lasiura)& 3î Ò(5.2%), .Sv(Microtus fortis)& 5îÒ(8.6)î . ãÎæO ¢ v~ ö & Ú ·WN O. tsutsugamushi Gilliam, Karp, Karto, Boryong"ö & IFA»ö ~ Ú ·WNf Table 3" ? *v öBº 50îÒ 7 16.öB Ú& ¦Â>Ú 32.0%~ ·WNj ¾æÚî, æê &·öB 46.2%, ~_ 40.0%, c{ 41.7%, · 50.0%î . . 0. UTVUTVHBNVTIJ. Table 2. Species of wild rodents trapped in Gyeongnam area from 2001 to 2002 Locality. No. of rodents trapped (%) No. of tested Apodemus Crocidura Microtus rodents agrarius lasiura fortis. Miryang Uiryeong Changnyeong Goseong Hamyang Hapcheon. 15 6 15 8 7 7. 13(86.7) 5(83.3) 12(80.0) 8(100) 6(85.7) 6(85.7). 1(6.7) 0 1(6.7) 0 0 1(14.3). 1(6.7) 1(16.7) 2(13.3) 0 1(14.3) 0. Total. 58. 50(86.2). 3(5.2). 5(8.6). Table 3. Detection rates of antibody positive on O. tsutsugamushi Gilliam, Karp, Kato, Boryong by IFA Miryang. Apodemus agrarius. Crocidura lasiura. Microtus fortis. Miryang Uiryeong Changnyeong Goseong Hamyang Hapcheon. 6/13(46.2)a 2/5(40.0) 5/12(41.7) 0/8 3/6(50.0) 0/6. 0/1 0/0 0/1 0/0 0/0 0/1. 1/1 0/1 1/2 0/0 0/1 0/0. 16/50(32.0). 0/3. 2/5(40.0%). Total. a. No. of positive rodents / No. of tested rodents(%). SvöBº &·f c{öB 5îÒ 7 2îÒ öB ·Wj &, ivöBº 3îÒ Îv r W >wj ¾æÚî . ¢ v~ ö & Ú& *v, iv, .Sv~ Gilliam, Karp, Karto, Boryong "ö & immunofluorescent antibody assay(IFA)»ö ~ Ú&º Table 4f ? 1:20öB¦V 1:640ræ. ·~² ¾æÒb, Ú&º Boryong "öB &Ú ¸ ² ¾æ¾º >, Gilliam"öBº 7* ;ê, Ò Karp"º Ô² ¾æÒ . 6" îÖÊöB ¦ÂNö & *'" »" 1$3~ jv O. tsutsugamushi¢ ¦Â~V *~ Boryong"¢ î ÖÊ~ ;Ú R ê . 5 ËV Òò¦V IFA O»" PCR »b O. tsutsugamushi¢ ¦Â Ö " IFA»f 7« ê 3¢ö 3îÒ 7 2îÒöB ¦Â >V ·~ 6, 12¢öº '' 3îÒ ÎvöB ¦Â. (40.0%). 0. UTVUTVHBNVTIJ. 0. UTVUTVHBNVTIJ.

(5) ãÎæ ¢ vöB Orientia tsutsugamushi ö & Ú Ò 5 PCRö ~ ¦ï. 245. Table 4. Antibody titer of serum by against O. tsutsugamushi of wild rodents captured in Gongnam area Rodents No.. IFA antibody titer. Collection area. M-A. agrarius-2 M-A. agrarius-4 M-A. agrarius-7 M-A. agrarius-12 M-A. agrarius-13 M-A. agrarius-15 U-A. agrarius-1 U-A. agrarius-3 C-A. agrarius-3 C-A. agrarius-5 C-A. agrarius-8 C-A. agrarius-10 C-A. agrarius-12 HM-A. agrarius-1 HM-A. agrarius-3 HM-A. agrarius-6 M-M. fortis-14 C-M. fortis-15. Gilliam. Miryang Miryang Miryang Miryang* Miryang Miryang Uiryeong Uiryeong* Changnyeong Changnyeong Changnyeong Changnyeong* Changnyeong Hamyang* Hamyang Hamyang Miryang* Changnyeong. 20 0 40 80 0 0 0 80 20 0 40 160 0 80 40 40 80 0. Karp 0 0 20 0 20 0 0 0 20 0 0 0 40 0 0 0 0 20. Karto. Boryong. 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0. 0 20 0 640 0 40 80 160 0 20 0 640 0 320 0 0 320 40. *Antibody positive. Table 5. Comparison of detection rates of IFA and PCR for detecting O. tsutsugamushi in artificial infected mouse Detection method. Organ b. Clinical manifestation IFAc PCR PCR PCR. − Serum Blood Spleen kidney. Days after inoculation 3. 6. 0/3 2/3a 3/3 3/3 3/3. 3/3 3/3 3/3 3/3 3/3. 12 18. 24. 3/3 3/3 3/3 3/3 3/3. 1/3 0/3 3/3 3/3 3/3. 2/3 2/3 3/3 3/3 3/3. a : No. of positive samples / No. of tested samples. b : Clinical manifestation actually developed from 5 days after inoculation. c : Indirect immunofluorescent antibody assay.. >îb¾ 18¢¦V 6²~V ·~ 24¢öº ¦Â> æ p~ . ö >~ PCR»f 7« ê 3¢¦V 24¢ræ * BÚ~ Î òöB ¦Â>î (Table 5). . 5 ËV Òò¦V 1$3 ÃÖb~ { ¢ v 58îÒöB j ., jË 5 Ëb ¦V ºÂ ' genomic DNA¢ PCR ÃÎ r 1% agarose gelöB *V'ÿ Ö"º Fig. 1" ? . jË Òò 4 5 Ë 1öB 210 bp V~ Boryong. Fig. 1. Agarose gel electrophoresis of rickettsial DNAs amplified by polymerase chain reaction for the various specimen of wild rodents. [Lane 1, 2, 3: 100, 200, 20 bp DNA ladder, Lane 4: negative control, Lane 5: M-A 12, Lane 6: U-A 3, Lane 7: C-A-10, Lane 8: HM-A 1, Lane 9: M-M 14 PCR products from the spleen, and kidney specimen of wild rodents, Lane 10, 11: non-infected ICR mouse control spleen, kidney, Lane 12, 13: Gilliam, and Boryong strain].. " ß~ ÃÖbj ¾æÚîb¾, . 5 öBº ?f V~ ÃÖb ¾æ¾æ p~ . Þ Boryong"¢ 6"Î ê 3, 6, 12, 18 5 24¢ö j jË" Ë Òò¢ PCR Ö" Boryong" ß ~ DNA ÃÖb &V>îb¾, 6"Êæ pf &~ ËV ÒòöBº ÚÆ ÃÖbê ¾æ¾æ p~ (Fig. 2). PCRö ~ O. tsutsugamushi~ ¦ÂNj ¦Æ~V *.

(6) 246. ~&Áf'îÁ;;ëÁ;Ò7ÁfÏÁ~ÒvÁ;«^Áî;^ÁB«ãÁ«ÁÂÁf SÁf~ãÁf«>. Fig. 2. Products of rickettsial DNAs amplified by polymerase chain reaction for mouse specimen infected with Boryong strain. [Lane 1, 2, 3: 100, 200, 20 bp DNA ladder, Lane 4: negative control, Lane 5~9: PCR products from the infected ICR mouse 3, 6, 12, 18, 24 day after inoculation. Lane 10, 11: non-infected ICR mouse control spleen, kidney, Lane 12, 13: Gilliam, and Boryong strain]. Table 6. Detection rate of rickettsial DNAs from the spleen and kidney samples of wild field rodents by nested polymerase chain reaction Locality Miryang Uiryeong Changnyeong Hamyang Total(%). Detection rate of rickettsial DNA Apodemus agrarius a. Microtus fortis. 1/6 1/2 1/5 1/3. 1/1a 0/1 0/2 0/1. 4/16(25.0). 1/5(20.0). a. Total no. of detected samples. / No. of tested samples.. ~ &·, ~_, c{" · J æöB ³B v ~ jË" ËöB PCR»b rickettsial DNAs¢ ¦Â Ö" *vöBº 16îÒ 7 4îÒ(25.0%)öB Boryong" ß~ ÃÖb ¦Â>î, .Svº 5 îÒ 7 1îÒ(20.0%)öB ¦Â>î (Table 6).. V. úú&Z÷f j j ¢, 7 5 ÿÎj jö ª~º "º W î~bB , vÛ, B êj ßûb ~º êV& B~º Ò9~jW î ÷ . öÚ O. tsutsugamushiº ^Ú Vb B ?"^~ ö;îöB Ã~, ^º 0.3 - 0.5 µm, 0.2 - 0.4 µm ;ê~ VB ^ï~ ßWç ²rWb ª~B [7]. O. tsutsugamushiº «¾ b' ßWö V¢ Rickettsia ³b ª~>îb¾ ^ã~ & R. prowazekii, R. typhi, R. rickettsiif º Ò9~jf övN>wj ¢bÊæ. p, F*¶~ çÿW 'Ú Orientia ³b Òª~> î [11, 24]. úú&Z÷j B~º ^êVº r, FÏ, ®V 5 WÏ~ J ê~ Ò¢ ~º, ®V î:î~º FÏ êöB ÿb~ .j . . Ò²f Ö?"(incidental host)B O. tsutsugamushiö 6"B ^êV~ FÏ Ò²~ ç j « r Ú& . Ú "«>B úú&Z ÷ö 6"B [5]. ÖÒ¾¢ö B~º ^êV 7 Leptotrombidium pallidum, Leptotrombidium scutellare, Leptotrombidium palpale~ 3&æ « O. tsutsugamushi ¢ B~º © [3]b rJ^ ® . O. tsutsugamushi º B~ «b ª~>¾ · .Ó; ®b, 7 Gilliam, Karp, Kato "& &" C&^ ® b¾ ' ¾¢î. .Ó; >î [3, 22]. öB ªÒ>º "º .Ó;f Gilliam, Karp, Boryong , 7 Boryong "& &Ë ô ªÒB >Ú ® . ãÎæOöB ³ ¢ v~ «j ª~ : C 58îÒ 7 *v& 86.5% &Ë ¸f ª ¢ ¾æÚî rb .Sv(8.3%) 5 iv(5.2%) Bb ¾æÒ (Table 2). º ; [3]" W [4] Ö"f FÒ Ö"¢ ¾æÚî . f ?f Ö " Ú " r ãÎæOö B~º vê ÖÒ¾ ¢ 7, Î¦æOö B~º ¢ vf ?f *v& "«j ®º ©b r > ®î . ãÎ¢&öB ³ *v, iv, .Sv~ O. tsutsugamushi Gilliam, Karp, Karto, Boryong"ö & æ ê Ú FNj ¦Ò Ö" 50îÒ~ *vöB º ï 32.0%¢ ¾æÚî (Table 3). æê & ·& 46.2%, ~_f 40.0%, c{f 41.7%, · 50.0%~ ·WNj &, &·f c{~ . Sv 2îÒöBê ·Wj ¾æÚîº Ö"º W [4]" FÒ Ö"¢ ¾æÚî . f ? ãÎ æOöB B~º ¢ v 7öB *v& Ò9~j ÷j B~º &Ë FK ÿb ?" ©b ºG > ® . Ú&¢ G; Ö" Boryong "öB &Ú Ú& & ¸² ¾æ¾º >, Gilliam"öBº 7*;ê~ Ú&¢ &, Karp"öBº &Ú'b Ú&& Ô ² ¾æÒ . Boryong, Gilliam " 7öBê ¢¦ Ú & Ôf ©f öb ÒÏ 4" 7 2" ~öò > w~º ©ê ®îV r^ö &¦ª Boryong"öB ;~ ² ¾æÚî . Ö"º Ë [8-11], [5], W [4] Ö"f FÒ~² ¾æÎbB ãÎ ¢&öBê Gilliam"f Boryong"& " 6"> ® º ©b " > ® . ICR îÖÊö O. tsutsugamushi¢ 6"Ê.

(7) ãÎæ ¢ vöB Orientia tsutsugamushi ö & Ú Ò 5 PCRö ~ ¦ï. O»" PCR»j Ï~ O. tsutsugamushi¢ Ò Ö" IFA»f 7« ê 6¢" 12¢öº * BÚö B ¦Â>îb¾ 18¢ ê¦Vº ¦ÂN 6²~º ãËj º >, PCR»f blood, spleen, kidney Î òöB 7« ê 3¢¦V 24¢ræ ¦Â>î. (Table 5). Ö"º Kee [20] Ö"f FÒ Ö"¢ & . 58îÒ ¢ vöB j ., jË, Ë Òòö B ºÂ genomic DNA¢ PCR Ö" .öBº Ã Öb ¦Â>æ p~æò, jË 4" Ë 1öB Boryong" ß~ ÃÖbj &V > ®î(Table 4, 6), 6 6" ê 3, 6, 12, 18 5 24¢~ jË" ËöB ÿ¢ ÃÖbj &V > ®î . f ? f Ö"º &b Ï j6" îÖÊ~ jË" ËöB ÃÖb ¦Â>æ p~, 6" ËV ÒòöB 7« ê 3¢¦V 24¢ræ DNA ¦Â & Ë~&~ 6b Ú " r PCR»~ Ö>j «Ã > ®îb, PCR ¦Ò ÒòBº . jË 5 Ë Òò& FK ©b ÒòB . 6 ãÎæöB ³B v~ jË" ËöB PCR »b rickettsial DNAs¢ ¦Â Ö"öBê 4B æ 5B ¦Ú~ jË" ËöB ÃÖbj &V > ®î . Ö"º IFA»ö ~ Ú ·WN ¦ ÒöB Ú&& ¸j ãÖf 2« ç Ú&& ·W j º · Ú&¢ ¾æÞ ©" &'b PCR»~ ;{Wj . «Ã~º © . Kee [20] ö ~~ b' ò~ ¦Âê& . 1 ml Ú ö Ò9~j >& 10B ç Ú ®Ú¢ º 6b Ú " r º Ú ·W¢æ¢ê . þ2ö B ¦Â>æ pf ©f Ú& ¦ÂF 6öº .ö ÷öÚ& ² >, .Ú BÚ>& ~ ì¾ 6º J¾ *ö Ú& ² B ©b " > ® . Kee [20]f jËf . Ò9~j DNA¢ ¦ï~º ® ÚB ÎNW ÎÚæ jËöB ß DNA band& & VB ©f jËÚö &ï~ ^& Ò~V r^¢ ~& . þöBº f ?f *ç ¾æ¾æ p~ . º jËöB DNA ¦ïN ¸² ¾æ Â ©b Ú " r PCR»f Ò9~jW î÷~ ê ö FÏ ê» F ©b ÒòB .. IFA. Ö . úú&Z÷ö & ' ~ ¢~b ãÎæ OöB B~ ®º ¢ v¢ ³~ Orientia tsutsugamushiö & Ú F ç¢ IFA»b Ò ~ PCR»b 6ê ê Ö" r" ?f Öj. 247. áî . (1) ãÎæöB ³ ¢ v~ «ê ª¢ Ò : 58îÒ 7 86.5%(50îÒ)& *v&, .Sv& 8.6%(5îÒ), iv& 5.2%(3îÒ)& . (2) O. tsutsugamushi Gilliam, Karp, Karto 5 Boryong "ö & IFA»ö ~ Ú ·WNf *vº 50îÒ 7 32.0%(16îÒ)&, .Svº 5îÒ 7 40.0%(2îÒ)&b, ivº 3îÒ Îv r Wî . (3) *v, iv 5 .Sv~ Gilliam, Karp, Karto 5 Boryong"ö & Ú&º 1:20öB 1:640ræ. ·~&, &Ú'b Boryong"öB ¸f Ú&¢ ¾æÚî, Gilliam"º 7*;ê~ Ú &¢, Ò Karp"öBº Ô² ¾æÒ . (4) 6" îÖÊ~ ., jË 5 Ë Òò¦ V IFA»" PCR»b O. tsutsugamushi¢ ¦Â Ö" IFA»f 7« ê 3¢¦V 18¢ræ · Wj ¾æÚîb¾, PCR»f 24¢ ræ * ò öB ¦Â>î . (5) ³ ¢ v~ ., jË 5 Ë Òò¦ V ºÂ genomic DNA¢ PCR Ö" jË 5 Ë ÒòöBº 210 bp V~ Boryong" ß ~ ÃÖb ¦Â>îb¾ .öBº ¦Â> æ p~ . (6) Boryong"¢ 6"Î ê 3, 6, 12, 18 5 24 ¢ö áf jË 5 Ë Òò¦V Boryong" ß ~ ÃÖbj &V > ®îb¾ j6" Ò òöBº &V>æ p~ . (7) ³ v~ jË 5 Ë Òò¢ PCR Ö" O. tsutsugamushi ·WNf *vöB 25.0%(4/16 îÒ), .Svº 20.0%(1/5îÒ)& . ç~ Ö" Ú " r ãÎæOö B~º. v &Ú O. tsutsugamushi¢ B~º vº &¦ª *v ©b {>îb, êO»öº PCR » IFA»ö j~ Ö> ©b {>î .. ^^ò VF^, Ç*B, ËÖ*. Orientia tsutsugamushi F* ; Karp~ 56kDa F*¶æ. &b²æ. 1997, 32, 35-40. 2. "', VF^, Ç&, f>Î, '", ÇV&, f Ö", sß". 7 7Î² ê>wj Ï Orientia tsutsugamushi~ .Ó;~ ÿ;. Infection. 1997, 29, 138-189. 3. ;Ò7, ÎVê, Ë«^, ~", ÂK, fs", * ç>, ;, . ³2æ ·Ï÷ BêV~ 1..

(8) 248. ~&Áf'îÁ;;ëÁ;Ò7ÁfÏÁ~ÒvÁ;«^Áî;^ÁB«ãÁ«ÁÂÁf SÁf~ãÁf«>. ê.' ²Ëö & Ò(III). ãÎ~ã ö. 1998, 5, 9-32. 4. W£", Çêö, ÇV&, f', ;VÎ, Ï". ã Vêf ;öê ¢ v~ Ò9~j 6"ö & . Ó' Ò. Infection. 1998, 30, 443-449. 5. , f, Ë'. úú&Z÷ö & .Ó ' . ãö. 1989b, 26, 136-156. 6. Ï. Ú ^êV(Acarina, trombiculidae)~ ê .' ²Ë 5 æÒ' ª Ò. 1992, Â&v & ö CÒ*¢^. 7. ËÖ*. ~ úú&Z÷. p. 1-153, BÂ6Ò. 1994. 8. ËÖ*, ;Òß. ~¶öB Rickettsia tsutsugamushi~ ªÒ. &~7²æ. 1987, 30, 99-1008. 9. ËÖ*, fç, VF^. 1990jö öB B úú&Z÷~ .ÓÒ. &b²æ. 1991, 26, 273-277. 10. ËÖ*, fç, «. 1992jö öB B úú&Z÷~ .Ó Ò. &b²æ. 1994, 29, 153-160. 11. ËÖ*, «, VF^. 1989jö öB B úú&Z÷~ .Ó Ò. &b²æ.. 1990, 25, 227-235. 12. Amano, K., Tamura, A., Ohashi, N. and Urakami, K. Deficiency of peptidoglycan and lipopolysaccharide components in Rickettsia tsutsugamushi. Infect Immnu. 1987, 55, 2290-2292. 13. Bozeman, F. M. and Elisberg, B. L. Serologic diagnosis of scrub typhus by indirect immunofluorescence. Proc. Soc. Exp. Biol. 1963, 112, 568-573. 14. Chang, W. H., Kang, J. S., Lee, W. K., Choi, M. S. and Lee, J. H. Serological classification by immunoclonal antibodies of Rickettsia tsutsugamushi isolated in Korea. J. Clinical Microbiol. 1990, 28, 685688. 15. Eisenmann, C. S. and Osterman, J. V. Idenfitication on strain specific and group-reactive antigenic determinants on the Karp, Gilliam and Kato strains of Rickettsia tsutsugamushi. Am. J. Trop. Med. Hyg. 1985, 34, 1173-1178. 16. Furuya, Y., Yoshida. Y., Katayama, T., Kawamory, F., Yamamoto, S., Ohashi, N., Tamura, A. and Kawamura, A. Specific amplication of Rickettsia tsutsugamushi DNA from clinical specimens by polymerase chain reaction. J. Clin. Microbiol. 1991, 29, 2628-2630. 17. Hanson, B. Identification and partial characterization of Rickettsia tsutsugamushi major protein immunogens. Infect. Immun. 1985, 50, 603-609. 18. Jackson, E. B., Danauskas, J. X., Smadel, M. E. and Sekikawa, H. Occurrence of Rickettsia tsutsugamushi. 19.. 20.. 21.. 22.. 23.. 24.. 25.. 26.. 27.. 28.. 29.. in Korea rodents and chiggers. Am. J. Hyg. 1957, 66, 309-320. Kee, S. H., Choi, I. H., Choi, M. S., Kim, I. S. and Chang, W. H. Detection of Rickettisia tsutsugamushi in experimentally infected mice by PCR. J. Clinic. Microbiol. 1994, 43, 1435-1439. Kee, S. H., Jin, W. S., Lee, J. E., Kim, S. H., Park, K. S. Baek, L. J. and Song, K. J. Seroepidemiologic analysis of acute febrile illness in Korea during 19971998. J. Bacteriol. Virol. 2002, 32, 263-267. Kelly, D. J., Marana, D. P., Stover, C. K., Oaks, E. V. and Carl, M. Detection of Rickettsia tsutsugamushi by gene amplification using polymerase chain reaction techniques. Ann. NY. Acad. Sci. 1990, 590, 564-571. Rapmund, G., Gohany, A. L. and Manikumran, C. Transovarial transmission of Rickettsia tsutsugamushi in Leptotrombidium (Leptotrombidium) arenicola Traub (Acarina : Trombiculidae). J. Med. Entomol. 1972, 9, 71-72. Ree, H. I., Lee, H. S., Lee, I. Y. and Yoshida, Y. Epidemiological studies on host animals of tsutsugamushi disease in Korea. Kor. J. Parasitol. 1991b, 29, 181-188. Roberts, L. W. and Robinson, D. M. Efficiency of transovaran transmission Rickettsia sutsugamushi in Leptotrombidium arenicola (Acari: Trombiculidae). J. Med. Entomol. 1977, 13, 493-496. Shirai, A., Coolbaugh, J. C. and Gan, E. Serologic analysis of scrub typhus isolates from the Pescardores and Philippine islands. Japan J. Med. Scil. Biol. 1982, 35, 255-259. Song, H. J., Seong, S. Y., Huh, M. S., Park, S. G., Jang, W. J, Kee, S. H., Kim, K. H., Kim, S. C., Choi, M. S., Kim, I. S. and Chang, W. H. Molecular and serologic survey of Orientia tsutsugamushi infection among field rodents in southern cholla province of Korea. Am. J. Trop. Med. Hyg. 1997, 13, 56-61. Tamura, A., Ohashi, N., Urakami, H., Takahashi, K. and Oyanagi, M. Analysis of polypeptide composition and antigenic components of Rickettsia tsutsugamushi by polyacrylamide gel electrophoresis and immunoblotting. Infect. Immnun. 1985, 48, 671-675. Tamura, A., Takahashi, K. and Tsuruhara, T. Isolation of Rickettsia tsutsugamushi antigenically different from Kato, Karp and Gilliam strain from patients. Microbiol. Mmunol. 1984, 26, 87-882. Traub, R. and Wisserman, C., L. The ecology of.

(9) ãÎæ ¢ vöB Orientia tsutsugamushi ö & Ú Ò 5 PCRö ~ ¦ï chigger-borne rickettsiosis (Scrub typhus). J. Med. Ent. 1974, 11, 237-303. 30. Weiss, E. The biology of rickettsiae. Annu. Rev. Microbiol. 1982, 36, 345-370.. 249. 31. Weiss, E., Moulder, J. W. and Order, I. Rickettsiales in Bergeys manual of systemic bacteriology. p. 687698, Williams Wilkins, Baltimore. 1984..

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