臺灣博碩士論文加值系統

本篇論文之目的在於建立非侵入式、簡易、有效偵測實驗小鼠病原的方法，並能在不影響使用者實驗的情況下，簡單有效地清除病原。
2007 年國家實驗動物中心北區將 Mouse parvovirus (MPV) 列入 Specific pathogens free (SPF) list 及健康監測服務項目。衛兵鼠送檢結果顯示動物房有 MPV 感染，在 2007 年底即以淘汰方式去除MPV 感染小鼠，至今未再感染。2009 年國家實驗動物中心的南科中心將 MNV (Murine norovirus) 與 Helicobacter spp. 列入 SPF list 中，並且開始提供高品質實驗小鼠，而 MNV 與 Helicobacter 尚未列在北區國家實驗動物中心的 SPF list 以及健康監測服務的項目中。故我們建立 MNV 及 Helicobacter 偵測方法後，開始監測檢疫房以及抽檢代養區的動物，確認了本動物房這兩種病原存在。
我們使用交叉撫養的方式來清除病原：使用確定沒有感染 MNV 及 Helicobacter 的乾淨母鼠作為奶媽，將來自感染母鼠的仔鼠在出生 24 小時內，交與奶媽哺育。施行交叉撫養前，仔鼠先短暫浸泡過 Hypochloric acid (HOCl 50ppm) 消毒液數次，去除體表可能沾有的病原後，再交由奶媽哺育。本動物房內設置有 HOCl 消毒系統，代養房內年平均環境落菌量小於 1，故清除病原的過程嘗試在同一個代養房內完成，感染母鼠與乾淨母鼠放置同一個代養房內的不同層架上。小鼠斷奶後，分別使用糞便 PCR 及 RT-PCR，鑑定小鼠 Helicobacter 與 MNV 的感染情形。目前已完成7次代養清除 MNV，以及 11 次清除 Helicobacter，其中有 6 次是同時清除 MNV與 Helicobacter。
本動物房中 MPV 與 MNV 之基因體序列全長，皆與目前已經發表的序列不同。將 MPV命名為 MPV-1f；而 MNV 基因體序列則與Norovirus mouse/TW2006/TWN 與 Norovirus mouse/TW2007/TWN 相近，命名為 MNV/Taipei/2009/TWN。

The purpose of this investigation was to establish a non-invasive, simple and efficient method to detect mouse pathogens, and to eliminate pathogens without interrupting users’ experiments.
In 2007, the mouse parvovirus (MPV) was included in the specific pathogen free (SPF) list and health monitoring services by the National Laboratory Animal Center (NLAC). The MPV infection in the animal facility was first detected in the sentinel mice by our routine health monitoring program in May, 2007. The infected mice were then identified cage by cage by ourselves by ELISA and then depopulated at the end of 2007. In 2009, the NLAC Tainan began to provide high quality laboratory mice which are MNV and Helicobacter free. However, the SPF list and health monitoring services of NLAC Taipei still did not comprise MNV and Helicobacter. Therefore, we established a fecal PCR-based method to monitor the MNV and Helicobacter in the animal facility. We found that some strains of mice in the quarantine room and holding room were infected with Helicobacter and some mice were infected with MNV in the holding room.
We used neonatal transfer in the same room for rederivation. The cages for foster dams and the infectious dams were kept on different shelves during the cleaning procedure. Within 24 hours of birth, the contaminated litters were rinsed with hypochloric acid solution (HOCl 50 ppm) several times and then transferred to the foster dams. After 3 weeks of fostering, the mice were tested for the presence of Helicobacter spp. and MNV by fecal PCR and fecal RT-PCR, respectively. Until now, neonatal transfer cleaned MNV in 7 successful transfers, and eliminated Helicobacter infection in 11 transfers, and 5 of neonatal transfer cleaned both MNV and Helicobacter at the same time.
The MPV and MNV genomic sequence are different from these being the published. The MPV is named MPV-1f and the MNV murine nororvirus Taipei/2009/TWN.

中文摘要 ................................................. i
英文摘要 ................................................ ii
目錄 .................................................... iv
表目錄 .................................................. vi
圖目錄 .................................................. vi
第一章、文獻回顧 ......................................... 1
第一節、緒論 ............................................ 1
第二節、Mouse parvovirus 簡介 ........................... 1
第三節、Mouse Norovirus 簡介 ............................ 3
第四節、Helicobacter 簡介 ............................... 5
第五節、常見的病源清除方法............................... 5
第六節、次氯酸水溶液 (Hypochlorous acid，HOCl) 簡介...... 6
第二章、研究動機與實驗設計 ............................... 7
第三章、材料與方法 ...................................... 10
第一節、動物飼養環境與健康狀況 ......................... 10
1. 動物房飼養環境 ..................................... 10
2. HOCl 給水系統 ...................................... 10
3. 動物健康狀況 ....................................... 11
第二節、方法 ........................................... 11
1. 引子設計 ........................................... 11
2. 交叉撫養操作 ....................................... 12
3. 小鼠體表細菌之培養.................................. 13
4. 單一菌落的 Helicobacter之培養 ...................... 13
5. 組織 DNA之製備 ..................................... 14
6. 總量組織 RNA 之製備................................. 14
7. 糞便DNA 之製備...................................... 15
8. 糞便總量RNA 之製備.................................. 15
9. 反轉錄-聚合酶連鎖反應 (RT-PCR) ......................16
10.聚合酶連鎖反應 (PCR) ............................... 17
11. DNA 膠體回收 (DNA extraction) ..................... 18
12. 質體接合 (T-A cloning) ............................ 19
13. 質體轉型 (Transformation) ......................... 19
14. DNA 序列分析 ...................................... 20
15. 胺基酸序列分析 .................................... 20
16. 親緣關係樹狀圖分析方法 ............................ 20
第四章、結果............................................. 22
第一節、動物房感染情形.................................. 22
1. Mouse parvovirus ................................... 22
2. Mouse norovisus .................................... 22
3. Helicobacter ....................................... 22
第二節、偵測新進動物 Helicobacter 感染情形.............. 23
第三節、清除小鼠體表細菌................................ 23
第四節、交互撫養結果.................................... 24
第五節、病原菌分子特徵.................................. 25
1. Mouse parvovirus ................................... 25
2. Mouse norovisus .................................... 26
第五章、討論 ............................................ 28
第一節、無特定病原菌清單 (SPF list) .................... 28
第二節、病原偵測與交互撫養 ............................. 28

參考文獻 ................................................ 29


表目錄

表一、引子序列 ...........................................35
表二、交叉撫養紀錄 .......................................37
表三、新生小鼠由感染母鼠移往代養母鼠之紀錄 ...............38
表四、比較MPV的核苷酸及胺基酸序列 ........................39

圖目錄

圖一、動物房小鼠感染 MPV、MNV 與Helicobacter之證據....... 40
圖二、糞便 PCR 膠圖結果證明交叉撫養可有效的清除Helicobacter .. 42
圖三、糞便RT-PCR 結果證明將交叉撫養可有效的清除MNV ...... 43
圖四、比較 HOCl 與碘液清除小鼠體表細菌之效力 ............ 44
圖五、MPV-1f 定序的策略以及其基因體結構 ................. 45
圖六、MPV 基因體全長之親緣關係樹狀圖 .................... 46
圖七、MPV-1f 核苷酸序列與 MVMi 及 MPV-1a 之比較 ......... 47
圖八、MNV/2009/Taipei/TWN 定序策略以及基因體結構 ........ 51
圖九、MNV/2009/Taipei/TWN 預測的胺基酸序列與 MNV-1 序列的不同處.. 52
圖十、MNV 的親緣關係樹狀圖 .............................. 53

附錄一、小鼠無特定病原清單比較 .......................... 54

慈濟大學實驗動物中心. (2006). 慈濟大學實驗動物中心使用手冊. 第六節 實驗動物傳染性疾病; Available from: http://www.lac.tcu.edu.tw/detail.php?recordID=47. Accessed August 3, 2009.
統安生技股份有限公司. (2009). HSP超次亞水生成機. Available at: http://www.topangle.com.tw/b-3-a.html. Accessed August 2, 2009
Artwohl JE, Purcell JE & Fortman JD. (2008). The use of cross-foster rederivation to eliminate murine norovirus, Helicobacter spp., and murine hepatitis virus from a mouse colony. J Am Assoc Lab Anim Sci 47, 19-24.
Bauer BA & Riley LK. (2006). Antemortem detection of mouse parvovirus and mice minute virus by polymerase chain reaction (PCR) of faecal samples. Lab Anim 40, 144-152.
Ball-Goodrich LJ & Johnson E. (1994). Molecular characterization of a newly recognized mouse parvovirus. J Virol 68, 6476-6486.
Belliot G, Lavaux A, Souihel D, Agnello D & Pothier P. (2008). Use of Murine Norovirus as a surrogate to evaluate resistance of human norovirus to disinfectants. Appl Environ Microbiol 74, 3315-3318.
Berns KI. (1990). Parvovirus replication. Microbiol Rev 54, 316-329.
Besselsen DG, Pintel DJ, Purdy GA, Besch-Williford CL, Franklin CL, Hook RR, Jr. & Riley LK. (1996). Molecular characterization of newly recognized rodent parvoviruses. J Gen Virol 77 ( Pt 5), 899-911.
Besselsen DG, Romero-Aleshire MJ, Munger SJ, Marcus EC, Henderson KS & Wagner AM. (2008). Embryo transfer rederivation of C.B-17/Icr-Prkdc(scid) mice experimentally infected with mouse parvovirus 1. Comp Med 58, 353-359.
Besselsen DG, Romero MJ, Wagner AM, Henderson KS & Livingston RS. (2006). Identification of novel murine parvovirus strains by epidemiological analysis of naturally infected mice. J Gen Virol 87, 1543-1556.
Cannon JL, Papafragkou E, Park GW, Osborne J, Jaykus LA & Vinje J. (2006). Surrogates for the study of norovirus stability and inactivation in the environment: a comparison of murine norovirus and feline calicivirus. J Food Prot 69, 2761-2765.
Chang SL. (1971). Modern concept of disinfection. J Sanitation Eng Div 97, 689-707.
Christensen J, Cotmore SF & Tattersall P. (1995). Minute virus of mice transcriptional activator protein NS1 binds directly to the transactivation region of the viral P38 promoter in a strictly ATP-dependent manner. J Virol 69, 5422-5430.
Clemens KE & Pintel D. (1987). Minute virus of mice (MVM) mRNAs predominantly polyadenylate at a single site. Virology 160, 511-514.
Compton SR. (2008). Prevention of murine norovirus infection in neonatal mice by fostering. J Am Assoc Lab Anim Sci 47, 25-30.
Cotmore SF & Tattersall P. (1987). The autonomously replicating parvoviruses of vertebrates. Adv Virus Res 33, 91-174.
Feng S, Ku K, Hodzic E, Lorenzana E, Freet K & Barthold SW. (2005). Differential detection of five mouse-infecting Helicobacter species by multiplex PCR. Clin Diagn Lab Immunol 12, 531-536.
Foltz CJ, Fox JG, Yan L & Shames B. (1996). Evaluation of various oral antimicrobial formulations for eradication of Helicobacter hepaticus. Lab Anim Sci 46, 193-197.
Fox, Barthold, Newcomer, Smith, Quimby & Davisson. (2007). The mouse in biomedical research. Elsevier Inc.
Fox JG, Dewhirst FE, Tully JG, Paster BJ, Yan L, Taylor NS, Collins MJ, Jr., Gorelick PL & Ward JM. (1994). Helicobacter hepaticus sp. nov., a microaerophilic bacterium isolated from livers and intestinal mucosal scrapings from mice. J Clin Microbiol 32, 1238-1245.
Franklin CL, Riley LK, Livingston RS, Beckwith CS, Besch-Williford CL & Hook RR, Jr. (1998). Enterohepatic lesions in SCID mice infected with Helicobacter bilis. Lab Anim Sci 48, 334-339.
Goto K, Hayashimoto N, Yasuda M, Ishida T, Kameda S, Takakura A & Itoh T. (2009). Molecular detection of murine norovirus from experimentally and spontaneously infected mice. Exp Anim 58, 135-140.
Hailey JR, Haseman JK, Bucher JR, Radovsky AE, Malarkey DE, Miller RT, Nyska A & Maronpot RR. (1998). Impact of Helicobacter hepaticus infection in B6C3F1 mice from twelve national toxicology program two-year carcinogenesis studies. Toxicol Pathol 26, 602-611.
Hampton MB, Kettle AJ & Winterbourn CC. (1998). Inside the neutrophil phagosome: oxidants, myeloperoxidase, and bacterial killing. Blood 92, 3007-3017.
Hardy ME. (2005). Norovirus protein structure and function. FEMS Microbiol Lett 253, 1-8.
Jacoby RO, Ball-Goodrich LJ, Besselsen DG, McKisic MD, Riley LK & Smith AL. (1996). Rodent parvovirus infections. Lab Anim Sci 46, 370-380.
Jacoby RO, Johnson EA, Ball-Goodrich L, Smith AL & McKisic MD. (1995). Characterization of mouse parvovirus infection by in situ hybridization. J Virol 69, 3915-3919.
Karst SM, Wobus CE, Lay M, Davidson J & Virgin HWt. (2003). STAT1-dependent innate immunity to a Norwalk-like virus. Science 299, 1575-1578.
Kastenmayer RJ, Perdue KA & Elkins WR. (2008). Eradication of murine norovirus from a mouse barrier facility. J Am Assoc Lab Anim Sci 47, 26-30.
Kostomitsopoulos N, Donnelly H, Kostavasili I, Paronis E, Alexakos P & Karayannacos P. (2007). Eradication of Helicobacter bilis and H. hepaticus from infected mice by using a medicated diet. Lab Anim (NY) 36, 37-40.
Labieniec-Pintel L & Pintel D. (1986). The minute virus of mice P39 transcription unit can encode both capsid proteins. J Virol 57, 1163-1167.
Letterman, R.D. (1999) Water Quality and Treatment - A Handbook of Community Water Supplies 5ed. Charpter 14. Disinfection, McGraw-Hill.
Lipman NS, Newcomer CE & Fox JG. (1987). Rederivation of MHV and MEV antibody positive mice by cross-fostering and use of the microisolator caging system. Lab Anim Sci 37, 195-199.
Mahler M & Kohl W. (2009). A serological survey to evaluate contemporary prevalence of viral agents and Mycoplasma pulmonis in laboratory mice and rats in western Europe. Lab Anim (NY) 38, 161-165.
Mainnemare A, Megarbane B, Soueidan A, Daniel A & Chapple IL. (2004). Hypochlorous acid and taurine-N-monochloramine in periodontal diseases. J Dent Res 83, 823-831.
McKisic MD, Lancki DW, Otto G, Padrid P, Snook S, Cronin DC, 2nd, Lohmar PD, Wong T & Fitch FW. (1993). Identification and propagation of a putative immunosuppressive orphan parvovirus in cloned T cells. J Immunol 150, 419-428.
McKisic MD, Macy JD, Jr., Delano ML, Jacoby RO, Paturzo FX & Smith AL. (1998). Mouse parvovirus infection potentiates allogeneic skin graft rejection and induces syngeneic graft rejection. Transplantation 65, 1436-1446.
McKisic MD, Paturzo FX & Smith AL. (1996). Mouse parvovirus infection potentiates rejection of tumor allografts and modulates T cell effector functions. Transplantation 61, 292-299.
Mohammadi Z. (2008). Sodium hypochlorite in endodontics: an update review. Int Dent J 58, 329-341.
Muller B, Klemm U, Mas Marques A & Schreier E. (2007). Genetic diversity and recombination of murine noroviruses in immunocompromised mice. Arch Virol 152, 1709-1719.
Mumphrey SM, Changotra H, Moore TN, Heimann-Nichols ER, Wobus CE, Reilly MJ, Moghadamfalahi M, Shukla D & Karst SM. (2007). Murine norovirus 1 infection is associated with histopathological changes in immunocompetent hosts, but clinical disease is prevented by STAT1-dependent interferon responses. J Virol 81, 3251-3263.
Park GW, Boston DM, Kase JA, Sampson MN & Sobsey MD. (2007). Evaluation of liquid- and fog-based application of Sterilox hypochlorous acid solution for surface inactivation of human norovirus. Appl Environ Microbiol 73, 4463-4468.
Pritchett-Corning KR, Cosentino J & Clifford CB. (2009). Contemporary prevalence of infectious agents in laboratory mice and rats. Lab Anim 43, 165-173.
Russell RJ, Haines DC, Anver MR, Battles JK, Gorelick PL, Blumenauer LL, Gonda MA & Ward JM. (1995). Use of antibiotics to prevent hepatitis and typhlitis in male scid mice spontaneously infected with Helicobacter hepaticus. Lab Anim Sci 45, 373-378.
Rzhetsky A & Nei M. (1992). Statistical properties of the ordinary least-squares, generalized least-squares, and minimum-evolution methods of phylogenetic inference. J Mol Evol 35, 367-375.
Smith AL, Jacoby RO, Johnson EA, Paturzo F & Bhatt PN. (1993). In vivo studies with an "orphan" parvovirus of mice. Lab Anim Sci 43, 175-182.
Sosnovtsev SV, Belliot G, Chang KO, Prikhodko VG, Thackray LB, Wobus CE, Karst SM, Virgin HW & Green KY. (2006). Cleavage map and proteolytic processing of the murine norovirus nonstructural polyprotein in infected cells. J Virol 80, 7816-7831.
Tamura K, Dudley J, Nei M & Kumar S. (2007). MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 24, 1596-1599.
Tattersall P, Shatkin AJ & Ward DC. (1977). Sequence homology between the structural polypeptides of minute virus of mice. J Mol Biol 111, 375-394.
Thompson JD, Higgins DG & Gibson TJ. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22, 4673-4680.
Truett GE, Walker JA & Baker DG. (2000). Eradication of infection with Helicobacter spp. by use of neonatal transfer. Comp Med 50, 444-451.
Van Keuren ML & Saunders TL. (2004). Rederivation of transgenic and gene-targeted mice by embryo transfer. Transgenic Res 13, 363-371.
Ward JM, Fox JG, Anver MR, Haines DC, George CV, Collins MJ, Jr., Gorelick PL, Nagashima K, Gonda MA, Gilden RV & et al. (1994). Chronic active hepatitis and associated liver tumors in mice caused by a persistent bacterial infection with a novel Helicobacter species. J Natl Cancer Inst 86, 1222-1227.
Ward JM, Wobus CE, Thackray LB, Erexson CR, Faucette LJ, Belliot G, Barron EL, Sosnovtsev SV & Green KY. (2006). Pathology of immunodeficient mice with naturally occurring murine norovirus infection. Toxicol Pathol 34, 708-715.
Watson J, Thompson KN & Feldman SH. (2005). Successful rederivation of contaminated immunocompetent mice using neonatal transfer with iodine immersion. Comp Med 55, 465-469.
Whary MT & Fox JG. (2006). Detection, eradication, and research implications of Helicobacter infections in laboratory rodents. Lab Anim (NY) 35, 25-27, 30-36.
Wobus CE, Karst SM, Thackray LB, Chang KO, Sosnovtsev SV, Belliot G, Krug A, Mackenzie JM, Green KY & Virgin HW. (2004). Replication of Norovirus in cell culture reveals a tropism for dendritic cells and macrophages. PLoS Biol 2, e432.