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研究生:陳謙豪
研究生(外文):Chien-Hao Chen
論文名稱:囓齒類實驗動物內寄生蟲多重引子聚合酶鏈鎖反應之建立與傳統診斷方法之比較
論文名稱(外文):Development of the Multiplex PCR Assay for the detection of endoparasites in laboratory rodent feces and comparison of the multiplex PCR assay and traditional diagnostic methods
指導教授:萬灼華
指導教授(外文):Cho-Hua Wan
口試委員:王明升廖欽峯
口試日期:2018-07-06
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:分子暨比較病理生物學研究所
學門:獸醫學門
學類:獸醫學類
論文種類:學術論文
論文出版年:2018
畢業學年度:106
語文別:英文
論文頁數:57
中文關鍵詞:蟯蟲鼠螺旋核原蟲鼠三毛滴蟲內寄生蟲多重引子聚合酶鏈鎖反應診斷法實驗鼠內寄生蟲健康監測法
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大小鼠的蟯蟲(Pinworm)、鼠三毛滴蟲(Tritrichomonas muris)以及鼠螺旋核原蟲(Spironucleus muris)是國內外各單位實驗動物須定期監測、預防與控制的內寄生蟲疾病。然而,目前傳統方法不但在內寄生蟲診斷上有效率與準確性的問題,且需犧牲動物,因此不符合動物福利原則。此研究針對實驗大小鼠的五種內寄生蟲,包含三種蟯蟲 (Syphacia obvelata 、 Syphacia muris 、 Aspiculuris tetraptera) 、鼠 三 毛 滴 蟲 (Tritrichomonas muris)及鼠螺旋核原蟲(Spironucleus muris),以及大小鼠的管家基因(housekeeping gene),建立了一個內寄生蟲多重引子聚合酶鏈鎖反應診斷法(Pinworm/Spironucleus/Tritrichomonas/Actin Multiplex PCR Assay)。此診斷法可特異性的增幅出目標基因,並能針對所有目標寄生蟲之偵測極限達到10 copies 的高敏感性。當模擬多種寄生蟲不等量共同感染的情況下,當兩種寄生蟲基因量相差高達100倍的情況下(104 vs. 102 copies),或是三種寄生蟲量相差10倍的情況下(104 vs. 103 copies),此診斷法仍能維持穩定的偵測效果。為比較此多重引子聚合酶鏈鎖反應與其他傳統診斷方法,收集來自11個單位共65個小鼠樣本與11大鼠樣本,以及來自5個單位共21個小鼠糞便樣本以及6個大鼠糞便樣本進行診斷。研究結果顯示,本多重引子聚合酶鏈鎖反應對於各寄生蟲皆具有高敏感性(97.2%-100%)與正確性(99%-100%),優於其他的生前檢查法(敏感性:83.5%-100%;正確性:96.6%-100%)與死後檢查法(敏感性:75%-100%;正確性:92.1%-100%)。同時,此一試劑亦成功診斷出一件疑似蟯蟲感染的初期病例。本研究新開發的內寄生蟲多重引子聚合酶鏈鎖反應診斷法在不需要犧牲與干擾動物的前提下,可同時診斷五種內寄生蟲的感染情形,應為實驗動物疾病監控與管理上一個相當有用的工具。
Rodent pinworms, Spironucleus muris and Tritrichomonas muris are the endoparasites routinely monitored and excluded from laboratory animal colonies. Nevertheless, traditional diagnostic methods may not efficiently detect and accurately demonstrate the endoparasite infestation status even the animals are sacrificed and screened with postmortem diagnostic methods. In this study, a multiplex PCR assay was developed to target the rRNA genes to simultaneously detect and differentiate five endoparasites, including Syphacia obvelata, Syphacia muris, Aspiculuris tetraptera, Spironucleus muris, and Tritrichomonas muris, as well as a housekeeping gene (α-actin) in feces. Each primer set applied in this multiplex PCR is specific for each parasite target or rodent housekeeping gene. The multiplex PCR could diagnose an equivalent co-infection of pinworm, Spironucleus muris, and Tritrichomonas muris with a detection limit as low as 10 copies. Furthermore, dual infection with up to 100-fold differences in parasite loads (104 vs. 102 copies) and triple infection with 10-fold differences (104 vs. 103 copies) can also be detected. In comparison of traditional methods and the multiplex PCR assay, 65 mice and 11 rats from 11 colonies and another 21 mouse fecal samples and 6 rat fecal samples from 5 research facilities were screened for infestation of these endoparasites. The multiplex PCR exhibited the highest sensitivity and accuracy of 97.2%-100% and 99%-100%, respectively, compared to the traditional antemortem (sensitivity: 83.5%-100%; accuracy: 96.6%-100%) and postmortem methods (sensitivity: 75%-100%; accuracy: 92.1%-100%). In this study, a limited early outbreak of Syphacia obvelata in a SPF mouse colony was also diagnosed by this multiplex PCR assay. The Pinworm/Spironucleus/Tritrichomonas/Actin multiplex PCR assay developed in this study should be a powerful tool for diagnosing endoparasite infestations without sacrificing animals in routine health monitoring in the future.
ACKNOLEGEMENTS..……………………………………………………………….i
ABSTRACT IN CHINESE……………………………………………………………ii
ABSTRACT IN ENGLISH………………………….…………………………….….iii
TABLE OF CONTENTS……………………………………………………..……….iv
LIST OF FIGURES………………………………………………...………………….v
LIST OF TABLES…………………………………………………………….………vi
INTRODUCTION……………………………………………………………………..1
METHODS AND MATERIALS………………………………………....……………7
Animals……………………………………………………………………………..7
Parasitic-positive samples…………………………………………………………..8
Comparison of the multiplex PCR and traditional diagnostic methods…………….8
DNA extraction……………………………………………………………………..9
PCR analysis……………………………………………………………………….10
DNA sequencing…………………………………………………………………...11
Perianal tape test………………………………………..……………..………...…12
Fecal flotation.……………………………………………………………………..12
Subgross examination of cecocolon contents…………………………………...…12
Direct smear of cecocolon contents..………………………………………....……13
Histopathologic examination………………………………………………….…...13
RESULTS………………………………………………………………………….…14
Endoparasite identification………………………………………………….……..14
Specificity and sensitivity of single specific PCR…………………….…….…..…14
Specificity and sensitivity of the multiplex PCR…………………….………...…..16
Detection of different amounts of pinworms, Tritrichomonas and Spironucleus
by the multiplex PCR assay…………………………………………………….….17
Comparison of endoparasite diagnostic methods…………………………………18
DISCUSSION………………………………………………………………………..20
REFERENCES…………………………………………………………………….…49
APPENDIX…..………………………………………………………………………55
Baker, D. G. (1998). Natural pathogens of laboratory mice, rats, and rabbits and their effects on research. Clinical microbiology reviews, 11(2), 231-266.
Baker, D. G. (2008). Parasites of rats and mice. In Baker, D. G. (Eds.) Flynn''s parasites of laboratory animals. (pp303-399). Iowa, IA: Blackwell
Behnke, J. M. (1975). Immune expulsion of the nematode Aspiculuris tetraptera from mice given primary and challenge infections. International journal for parasitology, 5(5), 511-515.
Bugarski, D., Jovčić, G., Katić-Radivojević, S., Petakov, M., Krstić, A., Stojanović, N., & Milenković, P. (2006). Hematopoietic changes and altered reactivity to IL-17 in Syphacia obvelata-infected mice. Parasitology International, 55(2), 91-97.
Bauer, B. A., Besch-Williford, C., Livingston, R. S., Crim, M. J., Riley, L. K., & Myles, M. H. (2016). Influence of rack design and disease prevalence on detection of rodent pathogens in exhaust debris samples from individually ventilated caging systems. Journal of the American Association for Laboratory Animal Science, 55(6), 782-788.
Carty, A. J. (2008). Opportunistic infections of mice and rats: Jacoby and Lindsey revisited. ILAR journal, 49(3), 272-276.
Clarke, C. L., & Perdue, K. A. (2004). Detection and clearance of Syphacia obvelata infection in Swiss Webster and athymic nude mice. Journal of the American Association for Laboratory Animal Science, 43(3), 9-13.
Clifford, C. B., & Watson, J. (2008). Old enemies, still with us after all these years. ILAR journal, 49(3), 291-302.
Clifford, C. B., Henderson, K. S., & Chungu, C. (2014). A Guide to Modern Strategies for Infection Surveillance of Rodent Populations: Beyond Sentinels. MA: Charles River Laboratories, 14-15
Dole, V. S., Zaias, J., Kyricopoulos-Cleasby, D. M., Banu, L. A., Waterman, L. L., Sanders, K., & Henderson, K. S. (2011). Comparison of traditional and PCR methods during screening for and confirmation of Aspiculuris tetraptera in a mouse facility. Journal of the American Association for Laboratory Animal Science, 50(6), 904-909.
Elnifro, E. M., Ashshi, A. M., Cooper, R. J., & Klapper, P. E. (2000). Multiplex PCR: optimization and application in diagnostic virology. Clinical microbiology reviews, 13(4), 559-570.
Fain, M. A., Karjala, Z., Perdue, K. A., Copeland, M. K., Cheng, L. I., & Elkins, W. R. (2008). Detection of Spironucleus muris in unpreserved mouse tissue and fecal samples by using a PCR assay. Journal of the American Association for Laboratory Animal Science, 47(5), 39-43.
Feng, S., Ku, K., Hodzic, E., Lorenzana, E., Freet, K., & Barthold, S. W. (2005). Differential detection of five mouse-infecting helicobacter species by multiplex PCR. Clinical and diagnostic laboratory immunology, 12(4), 531-536.
Gerwin, P. M., Ricart Arbona, R. J., Riedel, E. R., Lepherd, M. L., Henderson, K. S., & Lipman, N. S. (2017). Evaluation of traditional and contemporary methods for detecting Syphacia obvelata and Aspiculuris tetraptera in laboratory mice. Journal of the American Association for Laboratory Animal Science, 56(1), 32-41.
Hawash, Y., DNA extraction from protozoan oocysts/cysts in feces for diagnostic PCR (2014). The Korean journal of parasitology, 52(3), 263-271.
Hayashimoto, N., Morita, H., Ishida, T., Yasuda, M., Kameda, S., Uchida, R., & Itoh, T. (2013). Current microbiological status of laboratory mice and rats in experimental facilities in Japan. Experimental animals, 62(1), 41-48.
Henderson, K. S., Perkins, C. L., Havens, R. B., Kelly, M. J. E., Francis, B. C., Dole, V. S., & Shek, W. R. (2013). Efficacy of direct detection of pathogens in naturally infected mice by using a high-density PCR array. Journal of the American Association for Laboratory Animal Science, 52(6), 763-772.
Hu, W., Wu, S., Yu, X., Abullahi, A. Y., Song, M., Tan, L., & Li, G. (2015). A multiplex PCR for simultaneous detection of three zoonotic parasites Ancylostoma ceylanicum, A. caninum, and Giardia lamblia assemblage A. BioMed research international, 2015.
Jensen, E. S., Allen, K. P., Henderson, K. S., Szabo, A., & Thulin, J. D. (2013). PCR testing of a ventilated caging system to detect murine fur mites. Journal of the American Association for Laboratory Animal Science, 52(1), 28-33.
Jackson, G. A., Livingston, R. S., Riley, L. K., Livingston, B. A., & Franklin, C. L. (2013). Development of a PCR Assay for the Detection of Spironucleus muris. Journal of the American Association for Laboratory Animal Science, 52(2), 165-170.
Kashiwagi, A., Kurosaki, H., Luo, H., Yamamoto, H., Oshimura, M., & Shibahara, T. (2009). Effects of Tritrichomonas muris on the mouse intestine: a proteomic analysis. Experimental animals, 58(5), 537-542.
Lachaud, L., Marchergui-Hammami, S., Chabbert, E., Dereure, J., Dedet, J. P., & Bastien, P. (2002). Comparison of six PCR methods using peripheral blood for detection of canine visceral leishmaniasis. Journal of clinical microbiology, 40(1), 210-215.
Leblanc, M., Berry, K., Graciano, S., Becker, B., & Reuter, J. D. (2014). False-positive results after environmental pinworm PCR testing due to rhabditid nematodes in corncob bedding. Journal of the American Association for Laboratory Animal Science, 53(6), 717-724.
Lipman, N. S., Lampen, N., & Nguyen, H. T. (1999). Identification of pseudocysts of Tritrichomonas muris in Armenian hamsters and their transmission to mice. Comparative Medicine, 49(3), 313-315.
Lübcke, R., Hutcheson, F. A. R., & Barbezat, G. O. (1992). Impaired intestinal electrolyte transport in rats infested with the common parasite Syphacia muris. Digestive diseases and sciences, 37(1), 60-64.
McNair, D. M., & Timmons, E. H. (1977). Effects of Aspiculuris tetraptera and Syphacia obvelata on exploratory behavior of an inbred mouse strain. Laboratory animal science, 27(1), 38-42.
Michels, C., Goyal, P., Nieuwenhuizen, N., & Brombacher, F. (2006). Infection with Syphacia obvelata (pinworm) induces protective Th2 immune responses and influences ovalbumin-induced allergic reactions. Infection and immunity, 74(10), 5926-5932.
Mohn, G., & Philipp, E. M. (1981). Effects of Syphacia muris and the anthelmintic fenbendazole on the microsomal monooxygenase system in mouse liver. Laboratory animals, 15(2), 89-95.
Müller, N., Zimmermann, V., Forster, U., Bienz, M., Gottstein, B., & Welle, M. (2003). PCR-based detection of canine Leishmania infections in formalin-fixed and paraffin-embedded skin biopsies: elaboration of a protocol for quality assessment of the diagnostic amplification reaction. Veterinary parasitology, 114(3), 223-229.
National Research Council. (1991). Infectious diseases of mice and rats. National Academies Press, Washington, D.C.
Parel, J. D. C., Galula, J. U., & Ooi, H. K. (2008). Characterization of rDNA sequences from Syphacia obvelata, Syphacia muris, and Aspiculuris tetraptera and development of a PCR-based method for identification. Veterinary parasitology, 153(3-4), 379-383.
Perdue, K. A., Copeland, M. K., Karjala, Z., Cheng, L. I., Ward, J. M., & Elkins, W. R. (2008). Suboptimal ability of dirty-bedding sentinels to detect Spironucleus muris in a colony of mice with genetic manipulations of the adaptive immune system. Journal of the American Association for Laboratory Animal Science, 47(5), 10-17.
Pritchett-Corning, K. R. (2007). Helminth parasites of laboratory mice. In: Fox, J. G., Barthold, S., Davisson, M., Newcomer, C. E., Quimby, F. W., & Smith, A. (2007). The mouse in biomedical research: normative biology, husbandry, and models (Vol. 3). Elsevier. (pp. 551-564).
Pritchett-Corning, K. R., Cosentino, J., & Clifford, C. B. (2009). Contemporary prevalence of infectious agents in laboratory mice and rats. Laboratory animals, 43(2), 165-173.
Sato, Y., Ooi, H. K., Nonaka, N., Oku, Y., & Kamiya, M. (1995). Antibody production in Syphacia obvelata infected mice. The Journal of parasitology, 559-562.
Sebesteny, A. (1969). Pathogenicity of intestinal flagellates in mice. Laboratory Animals, 3(1), 71-77.
Sebesteny, A. (1974). The transmission of intestinal flagellates between mice and rats. Laboratory animals, 8(1), 79-81.
Sint, D., Raso, L., & Traugott, M. (2012). Advances in multiplex PCR: balancing primer efficiencies and improving detection success. Methods in Ecology and Evolution, 3(5), 898-905.
Taffs, L. F. (1976). Pinworm infections in laboratory rodents: a review. Laboratory animals, 10(1), 1-13.
Wang, K. W., Chueh, L. L., Wang, M. H., Huang, Y. T., Fang, B. H., Chang, C. Y., Fang, M. C., Chou, J. Y., & Wan, C. H. (2013). Multiplex polymerase chain reaction assay for the detection of minute virus of mice and mouse parvovirus infections in laboratory mice. Laboratory animals, 47(2), 116-121.
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