臺灣博碩士論文加值系統

本研究於台北、嘉義及高雄之動物醫院共採集19個疑似感染艾利希體之犬隻血液樣本，並於台灣南部地區共隨機採集107個流浪犬之血液樣本，以血液塗抹片、間接免疫螢光抗體試驗（IFA）、酵素連結免疫吸附試驗（ELISA）及聚合酶鏈反應（PCR）等方法檢測艾利希體。本研究之目的為利用不同之實驗室診斷方法檢測台灣地區犬隻感染艾利希體之種別，並比較各種檢測方法之優缺點及其在臨床診斷上的實用價值。以血液塗抹片檢查法檢測艾利希體，結果顯示於單核細胞之細胞質內發現桑椹體之陽性率為1.6 %（2/126）；於血小板內發現桑椹體之陽性率為4.0 %（5/126）；而在顆粒性白血球之細胞質中，並無發現桑椹體之存在。以商業化之IFA kit檢測Anaplasma phagocytophilum（A. phagocytophilum）之抗體，結果顯示陽性率為50.0 %（55/110）；以商業化之ELISA kit檢測Ehrlichia canis（E. canis）之抗體，結果顯示陽性率為14.8 %（18/122）；以PCR檢測A. phagocytophilum及A. platys之核酸，結果顯示陽性率分別為11.1 %（14/126）及7.9 %（10/126）；以nested PCR檢測Ehrlichia canis（E. canis）之核酸，結果顯示於primary PCR為陰性反應，而nested PCR之陽性率為4.0 %（5/126）。將所有PCR陽性之產物進行序列分析，結果顯示以ehr521-ehr790增幅A. phagocytophilum 16S rRNA片段之PCR陽性產物之序列與GenBank發表之A. platys以色列分離株（AY530806）之序列相似度略高於A. phagocytophilum標準株（AY055469）；以EC3-EC4增幅E. canis 16S rRNA片段之5個PCR陽性產物之序列與GenBank發表之E. canis台灣分離株（DQ228501）、日本分離株（AF536827）及中國分離株（AF162860）完全一致；以EHR16SR-PLATYS增幅A. platys 16S rRNA片段之10個PCR陽性產物之序列與AY530806相似度達97.6 %以上。以IFA kit檢測為A. phagocytophilum陽性結果的55個樣本中，同時為PCR陽性的樣本分別只佔了18.2 %（10/55）；以ELISA檢測為E. canis陽性結果的18個樣本中，同時為nested PCR陽性的樣本只佔了11.1 %（2/18）。根據結果顯示，實驗室診斷艾利希體之方法中，雖然IFA及ELISA具有操作簡便快速之優點，但抗體陽性反應可能是因帶菌期抗原刺激所殘留下來，或因不同種別之艾利希體感染造成之血清交叉反應。因此，要檢測台灣犬隻艾利希體之感染，PCR檢測為準確性及特異性均甚高的一種方法

Nineteen canine blood samples that was suspected with ehrlichiosis were collected from animal hospitals in Taipei, Chiayi and Kaohsiung. And 107 canine blood samples from stray dogs were also collected randomly in southern Taiwan. Diagnostic methods for Ehrlichia spp. infection such as blood smear, indirect immunofluorescent antibody assay (IFA), enzyme- linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR) were used in this study. The purpose of this study was to detecte Ehrlichia species of canine in Taiwan by using these laboratory diagnostic methods, and to compare their usage on clinical diagnosis. Ehrlichial morula were detected by blood smear, the positive rate of morular observed in mononuclear leukocytes was 1.6 % (2/126). The positive rate of morular observed in platelet was 4.0 % (5/126), and no morula were observed in granulocyte. Antibodies of Anaplasma phagocytophilum (A. phagocytophilum ) were detected by commercial IFA kit, the positive rate was 50.0 % (55/110). Antibodies of Ehrlichia canis (E. canis) were detected by commercial ELISA kit, the positive rate was 14.8 % (18/122). We also detected A. phagocytophilum and A. platys with PCR, the positive rates were 11.1 % (14/126) and 7.9 % (10/126), respectively. E. canis were also detected by nested PCR, all samples were negative in primary PCR, and the positive rates were 4.0 % (5/126) in nested PCR. All positive PCR products were sequenced, the results suggested that all sequences of positive PCR products that were amplified by primer ehr521-ehr790 were more similar to A. platys Israeli isolated strain (AY530806) than to A. phagocytophilum standard strain (AY055469). The sequences of 5 positive PCR products that were amplified by primer EC3-EC4 were identical to E. canis Taiwanese isolated strain (DQ228501), Japanese isolated strain (AF536827) and Chinese isolated strain (AF162860). The sequences of 10 positive PCR products that were amplified by primer EHR16SR-PLATYS were similar to AY530806 above 97.6 %. In 55 A. phagocytophilum positive samples which were detected with IFA kit, only 18.2 % (10/55) were also positive with PCR. In 18 E. canis positive samples which were detected with ELISA kit, only 11.1 % (2/18) were also positive with nested PCR. These results suggested that different diagnostic methods for ehrlichiosis, even though IFA and ELISA allowed advantages of less time consuming and easy to use. But positive results of antibodies maybe were remained by antigens stimulation in infected phase, or serologic cross-reactivity of other species of Ehrlichia. In conclusion, antigen detection with PCR might be a more accurate and specific method to diagnose ehrlichial infection in dogs in Taiwan.

目次

中文摘要．．．．．．．．．．．．．．．．．．．．．．．．． i
英文摘要．．．．．．．．．．．．．．．．．．．．．．．．． iii
目次．．．．．．．．．．．．．．．．．．．．．．．．．．． v
圖次．．．．．．．．．．．．．．．．．．．．．．．．．．． viii
表次．．．．．．．．．．．．．．．．．．．．．．．．．．． xi
第一章 緒論．．．．．．．．．．．．．．．．．．．．．．． 1
第二章 文獻探討．．．．．．．．．．．．．．．．．．．．． 4
第一節 歷史背景．．．．．．．．．．．．．．．．．．． 4
第二節 病原體．．．．．．．．．．．．．．．．．．．． 5
第三節 傳播途徑．．．．．．．．．．．．．．．．．．． 7
第四節 臨床症狀．．．．．．．．．．．．．．．．．．． 9
第五節 血液學變化．．．．．．．．．．．．．．．．．． 10
第六節 診斷方法．．．．．．．．．．．．．．．．．．． 10
第七節 治療．．．．．．．．．．．．．．．．．．．．． 13
第三章 材料與方法．．．．．．．．．．．．．．．．．．．． 14
第一節 實驗材料．．．．．．．．．．．．．．．．．．． 14
第二節 血液學檢查．．．．．．．．．．．．．．．．．． 17
第三節 血液化學檢查．．．．．．．．．．．．．．．．． 18
第四節 間接免疫螢光抗體試驗．．．．．．．．．．．．． 18
第五節 酵素連結免疫吸附試驗．．．．．．．．．．．．． 19
第六節 核酸之萃取．．．．．．．．．．．．．．．．．． 20
第七節 聚合酶鏈反應．．．．．．．．．．．．．．．．． 21
第八節 巢式聚合酶鏈反應．．．．．．．．．．．．．．． 24
第九節 電泳分析．．．．．．．．．．．．．．．．．．． 25
第十節 DNA定序及分析．．．．．．．．．．．．．．． 25
第四章 結果．．．．．．．．．．．．．．．．．．．．．．． 26
第一節 血液學檢查．．．．．．．．．．．．．．．．．． 26
第二節 血液化學檢查．．．．．．．．．．．．．．．．． 28
第三節 間接免疫螢光抗體試驗．．．．．．．．．．．．． 28
第四節 酵素連結免疫吸附試驗．．．．．．．．．．．．． 29
第五節 聚合酶鏈反應．．．．．．．．．．．．．．．．． 29
第六節 巢式聚合酶鏈反應．．．．．．．．．．．．．．． 30
第七節 DNA定序及分析．．．．．．．．．．．．．．． 30
第八節 以血液塗抹片檢查法及PCR檢測A. platys之結果比較．．．．．．．．．．．．．．．．．．．．．．
31
第九節 以血液塗抹片檢查法及PCR檢測E. canis之結果比較．．．．．．．．．．．．．．．．．．．．．．
32
第十節 以血液塗抹片檢查法及ELISA檢測E. canis之結果比較．．．．．．．．．．．．．．．．．．．．．
32
第十一節 以IFA及PCR檢測A. phagocytophilum之結果比較．．．．．．．．．．．．．．．．．．．．．
33
第十二節 以ELISA及nested PCR檢測E. canis之結果比較．．．．．．．．．．．．．．．．．．．．．
34
第五章 討論．．．．．．．．．．．．．．．．．．．．．．． 35
參考文獻．．．．．．．．．．．．．．．．．．．．．．．．． 68
附錄．．．．．．．．．．．．．．．．．．．．．．．．．．． 77

沈永紹。犬艾利希體症。動物醫學雜誌。14：56-60，1981。
殷嘉諄。犬貓艾利希體症之研究。屏東科技大學獸醫學研究所碩士論文。屏東。2000。
Aguero-Rosenfeld ME, Kalantarpour F, Baluch M, Horowitz HW, McKenna DF, Raffalli JT, Hsieh TZ, Wu J, Dumler JS, Wormser GP. Serology of culture-confirmed cases of human granulocytic ehrlichiosis. J Clin Microbiol 38: 635-638, 2000.
Alberti A, Zobba R, Chessa B, Addis MF, Sparagano O, Pinna Parpaglia ML, Cubeddu T, Pintori G, Pittau M. Equine and canine Anaplasma phagocytophilum strains isolated on the island of Sardinia (Italy) are phylogenetically related to pathogenic strains from the United States. Appl Environ Microbiol 71: 6418-6422, 2005.
Belongia EA, Reed KD, Mitchell PD, Kolbert CP, Persing DH, Gill JS, Kazmierczak JJ. Prevalence of granulocytic Ehrlichia infection among white-tailed deer in Wisconsin. J Clin Microbiol 35: 1465-1468, 1997.
Bjöersdorff A, Bagert B, Massung RF, Gusa A, Eliasson I. Isolation and characterization of two European strains of Ehrlichia phagocytophila of equine origin. Clin Diagn Lab Immunol 9: 341-343, 2002.
Bjöersdorff A, Svendenius L, Owens JH, Massung RF. Feline granulocytic ehrlichiosis－a report of a new clinical entity and characterization of the infectious agent. J Small Anim Pract 40: 20-24, 1999.
Bool PH, Sutmoller P. Ehrlichia canis infections in dogs on Aruba. J Am Vet Med Assoc 130: 418-420, 1957.
Cao WC, Zhao QM, Zhang PH, Dumler JS, Zhang XT, Fang LQ, Yang H. Granulocytic Ehrlichiae in Ixodes persulcatus ticks from an area in China where Lyme disease is endemic. J Clin Microbiol 38: 4208-4210, 2000.
Cao WC, Zhao QM, Zhang PH, Yang H, Wu XM, Wen BH, Zanh XT, Habbema JDF. Prevalence of Anaplasma phagocytophila and Borrelia burgdorferi in Ixodes persulcatus ticks from northeastern China. Am J Trop Med Hyg 68: 547-550, 2003.
Chang AC, Chang WL, Lin CT, Pan MJ, Lee SC. Canine infectious cyclic thrombocytopenia found in Taiwan. J Vet Med Sci 58: 473-476, 1996.
Chen SM, Dumler JS, Bakken JS, Walker DH. Identification of a granulocytotropic Ehrlichia species as the etiologic agent of human disease. J. Clin. Microbiol 32: 589-595, 1994.
Codner EC, Farris-Smith LL. Characterization of the subclinical phase of canine ehrlichiosis. J Am Vet Med Assoc 189: 47-50, 1986.
de la Fuente J, Torina A, Naranjo V, Caracappa S, Di Marco V, Alongi A, Russo M, Maggio AR, Kocan KM. Infection with Anaplasma phagocytophilum in a seronegative patient in Sicily, Italy: Case Report. Ann Clin Microbiol Antimicrob 4: 15, 2005.
Donatien A, Lestoquard F. Existence en Algerie d’une Rickettsia du chien. Bull Soc Pathol Exot 28: 418-419, 1935.
Dreher UM, de la Fuente J, Hofmann-Lehmann R, Meli ML, Pusterla N, Kocan KM, Woldehiwet Z, Braun U, Regula G, Staerk KD, Lutz H. Serologic cross-reactivity between Anaplasma marginale and Anaplasma phagocytophilum. Clin Diagn Lab Immunol 12: 1177-1183, 2005.
Dumler JS, Barbet AF, Bekker CPJ, Dasch GA, Palmer GH, Ray SC, Rikihisa Y, Rurangirwa FR. Reorganization of genera in the families Rickettsiaceae and Anaplasmataceae in the order Rickettsiales: unification of some species of Ehrlichia with Anaplasma, Cowdria with Ehrlichia and Ehrlichia with Neorickettsia, descriptions of six new species combinations and designation of Ehrlichia equi and “HGE agent” as subjective synonyms of Ehrlichia phagocytophilum. Int. J Syst Evol Microbiol 51: 2145-2165, 2001.
Engvall EO, Pettersson B, Persson M, Artursson K, Johansson KE. A 16S rRNA-based PCR assay for detection and identification of granulocytic Ehrlichia species in dogs, horses, and cattle. J Clin Microbiol 34: 2170-2174, 1996.
Enright M, Zawadski P, Pickerill P, Dowson CG. Molecular evolution of rifampicin resistance in Streptcoccus pneumoniae. Microb Drug Resist 4: 65-70, 1998.
Ewing SA. Canine ehrlichiosis. Adv Vet Sci Comp Med 13: 331-353, 1969.
García-Pérez AL, Mandaluniz N, Barral M, Juste RA. Microscopic and PCR finding in sheep after experimental infection with Ehrlichia phagocytophila. Small Rumin Res 37: 19-25, 2000.
Goodman JL, Nelson C, Vitale B, Madigan JE, Dumler JS, Kurtti TJ, Munderloh UG. Direct cultivation of the causative agent of human granulocytic ehrlichiosis. N Engl J Med 334: 209-215, 1996.
Gug E, Tasker S, Joynson DHM. Detection of the agent of human granulocytic ehrlichiosis (HGE) in UK ticks using polymerase chain reaction. Epidemiol Infect 21: 681-683, 1998.
Hancock SI, Breitschwerdt EB, Pitulle C. Differentiation of Ehrlichia platys and E. equi infections in dogs by using 16S ribosomal DNA-based PCR. J Clin Microbiol 39: 4577-4578, 2001.
Harrus S, Kass PH, Klement E, Waner T. Canine monocytic ehrlichiosis: a retrospective study of 100 cases, and an epidemiological investigation of prognostic indicators for the disease. Vet Rec 141: 360-360, 1997.
Harrus S, Waner T, Aizenberg I, Foley JE, Poland AM, Bark H. Amplification of ehrlichial DNA from dogs 34 months after infection with Ehrlichia canis. J Clin Microbiol 36: 73-76, 1998.
Heep M, Beck D, Bayerdorffer E, Lehn N. Rifampin and rifabutin resistance mechanism in Helicobacter pylori. Antimicrob Agents Chemother 43: 1497-1499, 1999.
Heo EJ, Park JH, Koo JR, Park MS, Park MY, Dumler JS, Chae JS. Serologic and molecular detection of Ehrlichia chaffeensis and Anaplasma phagocytophila (human granulocytic ehrlichiosis agent) in Korean patients. J Clin Microbiol 40: 3082-3085, 2002.
Hudson JR. The recognition of tick-borne fever as a disease of cattle. Br Vet J 106: 9-17, 1950.
Huxsoll DL, Hildebrandt PK, Nims RM, Ferguson JA, Walker JS. Ehrlichia canis－the causative agent of a haemorrhagic disease of dogs? Vet Rec 85: 587, 1969.
Inokuma H, Beppu T, Okuda M, Shimada Y, Sakata Y. Epidemiological survey of Anaplasma platys and Ehrlichia canis using ticks collected from dogs in Japan. Vet Parasitol 115: 343-348, 2003.
Inokuma H, Oyamada M, Kelly PJ, Jacobson LA, Fournier PE, Itamoto K, Okuda M, Brouqui P. Molecular detection of a new Anaplasma species closely related to Anaplasma phagocytophilum in canine blood from South Africa. J Clin Microbiol 43: 2934-2937, 2005.
Inokuma H, Raoult D, Brouqui P. Detection of Ehrlichia platys DNA in brown dog ticks (Rhipicephalus sanguineus) in Okinawa Island, Japan. J Clin Microbiol 38: 4219-4221, 2000.
Jackson CA, Lovrich SD, Agger WA, Callister SM. Reassessment of a Midwestern lyme disease focus for Borrelia burgdorferi and the human granulocytic ehrlichiosis agent. J Clin Microbiol 40: 2070-2079, 2002.
Kakoma I., Hansen RD, Anderson BE, Hanley TA, Sims KG, Liu L, Bellamy C, Long MT, Baek BK. Cultural, molecular and immunological characterisation of the etiologic agent for atypical canine ehrlichiosis. J Clin Microbiol 32: 170-175, 1994.
Keysary A, Amram L, Keren G, Sthoeger Z, Potasman I, Jacob A, Strenger C, Dawson JE, Waner T. Serologic evidence of human monocytic and granulocytic ehrlichiosis in Israel. Emerg Infect Dis 5: 775-778, 1999.
Kolbert CP. Detection of the agent of human granulocytic ehrlichiosis by PCR. In: Persing DH, ed. Protocols for emerging infectious disease. American Society for Microbiology, Washington D.C., 106-111, 1996.
Kuehn NF, Gaunt SD. Clinical and hematologic findings in canine ehrlichiosis. J Am Vet Med Assoc 186: 355-358, 1985.
Lester SJ, Breitschwerdt EB, Collis CD, Hegarty BC. Anaplasma phagocytophilum infection (granulocytic anaplasmosis) in a dog from Vancouver Island. Can Vet J 46: 825-827, 2005.
Macleod J, Gordon WS. Studies on tick-borne fever in sheep I: transmission by the tick Ixodes ricinus and description of the disease produced. Parasitology 25: 273-283, 1933.
Madewell BR, Gribble DH. Infection in two dogs with an agent resembling Ehrlichia equi. J Am Vet Med Assoc 180: 512-514, 1982.
Massung RF, Owens JH, Ross D, Reed KD, Petrovec M, Bjoersdorff A, Coughlin RT, Beltz GA, Murphy CI. Sequence analysis of the ank gene of granulocytic ehrlichiae. J Clin Microbiol 38: 2917-2922, 2000.
Massung RF, Slater KG. Comparison of PCR assay for detection of the agent of human granulocytic ehrlichiosis, Anaplasma phagocytophium. J Clin Microbiol 41:717-722, 2003.
Massung RF, Slater KG, Owens JH, Nicholson WL, Mather TN, Solberg VB, Olson JG. Nested PCR assay for detection of granulocytic ehrlichiae. J Clin Microbiol 36: 1090-1095, 1998.
Maurin M, Bakken JS, Dumler JS. Antibiotic susceptibilities of Anaplasma (Ehrlichia) phagocytophilum strains from various geographic areas in the United States. Antimicrob Agents Chemother 47: 413-415, 2003.
McDade JE. Ehrlichiosis—A disease of animals and humans. J Infect Dis 161: 609-617, 1990.
Nicholson, William L, Comer JA, Sumner JW, Gingrich-Baker C, Coughlin RT, Magnarelli LA, Olson JG, Childs JE. An indirect immunofluorscence assay using a cell culture-derived antigen for detection of antibodies to the agent of human granulocytic ehrlichiosis. J Clin Microbiol 35: 1510-1516, 1997.
Poitout FM, Shinozaki JK, Stockwell PJ, Holland CJ, Shukla SK. Genetic variants of Anaplasma phagocytophilum infecting dogs in western Washington State. J Clin Microbiol 43: 796-801, 2005.
Polin H, Hufnagl P, Haunschmid R, Gruber F, Ladurner G. Molecular evidence of Anaplasma phagocytophilum in Ixodes ricinus ticks and wild animals in Austria. J Clin Microbiol 42: 2285-2286, 2004.
Pusterla N, Huder J, Wolfensberger C, Litschi B, Parvis A, Lutz H. Granulocytic ehrlichiosis in two dogs in Switzerland. J Clin Microbiol 35: 2307-2309, 1997.
Pusterla N, Pusterla JB, Braun U, Lutz H. Serological, hematologic, and PCR studies of cattle in an area of Switzerland in which tick-borne fever (caused by Ehrlichia phagocytophila) is endemic. Clin Diagn Lab Immunol 5: 325-327, 1998.
Pusterla N, Leutenegger CM, Huder JB, Weber R, Braun U, Lutz H. Evidence of the human granulocytic ehrlichiosis agent in Ixodes ricinus ticks in Switzerland. J Clin Microbiol 37: 1332-1334, 1999.
Richter PJ, Kimsey RB, Madigan JE, Barlough JE, Dumler JS, Brooks DL. Ixodes pacificus（Acari: Ixodidae）as a vector of Ehrlichia equi（Rickettsiales: Ehrlichieae）. J Med Entomol 33: 1-5, 1996.
Rikihisa Y. The tribe Ehrlichieae and Ehrlichial diseases. Clin Microbiol Rev 4: 286-308, 1991a.
Rikihisa Y. Cross-reaction antigens between Neorickettsia helminthoeca and Ehrlichia species, shown by immunofluorescence and western immunoblotting. J Clin Microbiol 29: 2024-2029, 1991b.
Ristic M, Huxsoll DL. Ehrlichieae. In: Kreig NR, Holt JG, eds. Bergey’s manual of systematic bacteriology. Williams & Wilkins, Baltimore, 704-709, 1984.
Santos AS, Santos-Silva MM, Almedia VC, Bacellar F, Dumler JS. Detection of Anaplasma phagocytophilum DNA in Ixodes ticks (Acari: Ixodidae) from Madeira Island and Setúbal District, mainland Portugal. Emerg Infect Dis 10: 1643-1648, 2004.
Schouls LM, Van De Pol I, Rijpkema SG, Schot CS. Detection and identification of Ehrlichia, Borrelia burgdorferi sensu lato and Bartonella species in Dutch Ixodes ricinus ticks. J Clin Microbiol 37: 2215-2222, 1999.
Shaw SE, Day MJ, Birtles RJ, Breitschwerdt. Tick-borne infectious disease of dogs. Trends Parasitol 17: 74-80, 2001.
Stuen S, Van De Pol I, Bergström, Schouals LM. Identification of Anaplasma phagocytophila（formerly Ehrlichia phagocytophila）variants in blood from sheep in Norway. J Clin Microbiol 40:3192-3197, 2002.
Suksawat J, Pitulle C, Arraga-Alvarado C, Madrigal K, Hancock SI, Breitschwerdt EB. Coinfection with three Ehrlichia species in dogs from Thailand and Venezuela with emphasis on consideration of 16S ribosomal DNA secondary structure. J Clin Microbiol 39: 90-93, 2001.
Tarello W. Canine granulocytic ehrlichiosis (CGE) in Italy. Acta Vet Hung 51: 73-90, 2003.
Telford SR, Dawson JE, Katavlos P, Warner CK, Kolbert CP, Perding DH. Perpetuation of the agent of human granulocytic ehrlichiosis in a deer tick-rodent cycle. Proc Nat Acad USA 93: 6209-6214, 1996.
Whist SK, Storset AK, Larsen HJS. Functions of neutrophils in sheep experimentally infected with Ehrlichia phagocytophila. Vet Immunol Immunopathol 86: 183-193, 2002.
Woldehiwet Z, Scott GR. Tick-borne（pasture）fever. In: Woldehiwet Z, Ristic M, ed. Rickettsial and chlamydial disease of domestic animals. Oxford, Pergamon Press, 233-254, 1993.
Zhi N, Ohashi N, Rikihisa Y. Multiple p44 gene encoding major outer membrane proteins are expressed in the human granulocytic ehrlichiosis agent. J Biol Chem 274: 17828-17836, 1999.