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研究生:周知林
研究生(外文):Chih-Lin Chou
論文名稱:建立分子生物診斷法檢測實驗動物囓齒類毛蟎並與傳統診斷法比較
論文名稱(外文):Development of a Molecular Assay for Laboratory Rodent Fur Mite Diagnosis and Comparison with the Traditional Diagnostic Methods
指導教授:萬灼華
指導教授(外文):Cho-Hua Wan
口試委員:蔡志偉廖欽峯
口試日期:2019-07-31
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:2019
畢業學年度:107
語文別:英文
論文頁數:42
中文關鍵詞:囓齒類毛蟎Myocoptes musculinusMyobia musculiRadfordia spp.臺灣新種囓齒類毛蟎多重引子聚合酶鏈鎖反應環境監測
DOI:10.6342/NTU201903951
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實驗鼠感染外寄生蟲毛蟎(fur mite)是實驗動物中心常見且難徹底解決的問題之一,乃主要受限於外寄生蟲診斷方法敏感度較差、檢測樣本不易具有代表性及常用的衛兵鼠健康監測系統並不適用於監控實驗鼠的外寄生蟲感染情形。為了提升毛蟎檢測的敏感性與效率,本研究建立了一個多重引子聚合酶鏈鎖反應檢測法(multiplex PCR),此檢測法能同時偵測並區別不同種類毛蟎,包括Myocoptes musculinus (COP)、Myobia musculi (MOB)/Radfordia spp. (RAD)以及於臺灣發現的新種毛蟎(species A;SPA),更可藉由同時檢測囓齒動物管家基因(housekeeping gene)以確定檢體的品質。當多種毛蟎等量感染時,此檢測法能同時偵測出僅10 copies的各種毛蟎。而當同時有多種不等量的毛蟎感染時,此檢測法仍能偵測到感染量相差10至100倍的不同毛蟎感染。為了比較此多重引子聚合酶鏈鎖反應與多種傳統診斷法(拔毛測試、膠帶測試、毛皮檢查),本研究分別以不同診斷法來檢測48隻囓齒動物與25個鼠籠的毛蟎汙染情形。於診斷個體動物毛蟎感染情形,此多重引子聚合酶鏈鎖反應檢測法的敏感度與準確度(86 %與95.1 %)明顯高於所有傳統診斷方法(敏感度:6 % - 46 %,準確度: 67.4 % - 81.3 %)。更值得一提的是,當應用於檢測鼠籠擦拭樣本(環境樣本)時,此毛蟎多重引子聚合酶鏈鎖反應法可完全正確地區別檢測出每個鼠籠的不同毛蟎的汙染情形,其敏感度與準確度均達100 %。本研究所建立的毛蟎多重引子聚合酶鏈鎖反應檢測法(COP/MOB-RAD/SPA/Actin multiplex PCR assay)是一可靠的替代診斷方法,能應用於實驗動物中心的例行性病原監測(不論動物或環境樣本),更可用於實驗動物中心疑似毛蟎感染的追蹤調查。
Rodent fur mite infestation is a persistent and intractable problem in laboratory rodent colonies, due to insensitive diagnostics, unrepresentative samples for testing, and improper sentinel system. To improve the sensitivity and efficiency of fur mite detection, a multiplex PCR assay was developed to simultaneously detect and differentiate different species of fur mites, including Myocoptes musculinus (COP), Myobia musculi (MOB) and/or Radfordia spp. (RAD), and species A (SPA; a novel rodent fur mite identified in Taiwan), with the existence of a rodent housekeeping gene. This multiplex PCR could specifically detect as low as 10 copies of each species in equal-amount triple infestation. Super-infestation with 10 to 100-fold differences in mite burdens could be also detected. In comparison of the multiple PCR and traditional methods (pluck test, tape test, and pelt exam) for fur mite diagnosis, 48 rodents and 25 cage environment samples were evaluated for the fur mite infestation. In screening the status of various fur mites on individual animals, the multiplex PCR assay showed distinctly higher in sensitivity and accuracy (86 % and 95.1 %) than that of traditional methods (sensitivity: 6 % - 46 %, accuracy: 67.4 % - 81.3 %). Interestingly, by using cage wipe environmental samples, the multiplex PCR assay exhibited 100 % in both sensitivity and accuracy on the fur mite detection and differentiation. The COP/MOB-RAD/SPA/Actin multiplex PCR assay developed in this study could be a reliable alternative method for routine pathogen monitoring (animal or environment) or for tracing the suspect fur mite outbreak in rodent colonies.
口試委員審定書 i
致謝 ii
中文摘要 iii
ABSTRACT iv
CONTENTS v
LIST OF FIGURES vii
LIST OF TABLES viii
Introduction 1
Methods and Materials 5
Animals and Sample Collection 5
DNA Extraction 6
PCR Analysis 6
DNA Sequencing 8
Traditional Diagnostic Methods 9
Results 10
Ectoparasite Identification 10
Specificity and Sensitivity of Single Specific PCR 10
Specificity and Sensitivity of the COP/MOB-RAD/SPA/Actin Multiplex PCR Assay 11
Detection of Different Amounts of Fur Mite Super-infestations by the Multiplex PCR Assay 12
Comparison of Fur mite Diagnostic Methods in Animals 13
Environment (cage wipe) Samples by the Multiplex PCR Assay for Fur Mite Detection 15
Discussion 16
References 39
1.Animal Protection Act. Council of Agriculture. ROC. 2018. https://law.moj.gov.tw/ENG/LawClass/LawAll.aspx?pcode=M0060027.
2.Baker DG. Arthropods. In: Fox J, Barthold SW, Davisson MT, et al. (eds) The mouse in biomedical research, vol 2: diseases. San Diego (CA): Academic Press, 2007, p.565-576.
3.Baker DG. Parasites of rats and mice. In: Baker DG (ed) Flynn''s parasites of laboratory animals. 2nd ed. Ames (IA): Blackwell Publishing, 2007, p.359-368.
4.Bauer BA, Besch-Williford C, Livingston RS, et al. Influence of rack design and disease prevalence on detection of rodent pathogens in exhaust debris samples from individually ventilated caging systems. J Am Assoc Lab Anim Sci 2016; 55: 782-788.
5.Bornstein DA, Scola J, Rath A, et al. Multimodal approach to treatment for control of fur mites. J Am Assoc Lab Anim Sci 2006; 45: 29-32.
6.Carty AJ. Opportunistic infections of mice and rats: Jacoby and Lindsey revisited. ILAR J 2008; 49: 272-276.
7.Clifford CB, Henderson KS and Chungu C. A guide to modern strategies for infection surveillance of rodent populations: beyond sentinels. Charles River Laboratories, 2014.
8.Council of Agriculture. A Guidebook for the Care and Use of Laboratory Animals. Taipei: Chinese-Taipei Society of Laboratory Animal Sciences. ROC. 2010.
9.Gerwin PM, Ricart Arbona RJ, Riedel ER, et al. PCR testing of IVC filter tops as a method for detecting murine pinworms and fur mites. J Am Assoc Lab Anim Sci 2017; 56: 752-761.
10.Grove KA, Smith PC, Booth CJ, et al. Age-associated variability in susceptibility of Swiss Webster mice to MPV and other excluded murine pathogens. J Am Assoc Lab Anim Sci 2012; 51: 789-796.
11.Henderson KS, Perkins CL, Havens RB, et al. Efficacy of direct detection of pathogens in naturally infected mice by using a high-density PCR array. J Am Assoc Lab Anim Sci 2013; 52: 763-772.
12.Iijima OT, Takeda H, Komatsu Y, et al. Atopic dermatitis in NC/Jic mice associated with Myobia musculi infestation. Comp Med 2000; 50: 225-228.
13.Jensen ES, Allen KP, Henderson KS, et al. PCR testing of a ventilated caging system to detect murine fur mites. J Am Assoc Lab Anim Sci 2013; 52: 28-33.
14.Johnston NA, Trammell RA, Ball-Kell S, et al. Assessment of immune activation in mice before and after eradication of mite infestation. J Am Assoc Lab Anim Sci 2009; 48: 371-377.
15.Jungmann P, Freitas A, Bandeira A, et al. Murine acariasis. II. Immunological dysfunction and evidence for chronic activation of Th-2 lymphocytes. Scand J Immunol 1996; 43: 604-612.
16.Jungmann P, Guenet JL, Cazenave PA, et al. Murine acariasis: I. Pathological and clinical evidence suggesting cutaneous allergy and wasting syndrome in BALB/c mouse. Res Immunol 1996; 147: 27-38.
17.Kapoor P, Hayes YO, Jarrell LT, et al. Evaluation of anthelmintic resistance and exhaust air dust PCR as a diagnostic tool in mice enzootically infected with Aspiculuris tetraptera. J Am Assoc Lab Anim Sci, 2017; 56: 273-289.
18.Karlsson EM, Pearson LM, Kuzma KM, et al. Combined evaluation of commonly used techniques, including PCR, for diagnosis of mouse fur mites. J Am Assoc Lab Anim Sci 2014; 53: 69-73.
19.Lindstrom KE, Carbone LG, Kellar DE, et al. Soiled bedding sentinels for the detection of fur mites in mice. J Am Assoc Lab Anim Sci 2011; 50: 54-60.
20.Macy JD, Paturzo FX, Ball-Goodrich LJ, et al. A PCR-based strategy for detection of mouse parvovirus. J Am Assoc Lab Anim Sci 2009; 48: 263-267.
21.Metcalf Pate KA, Rice KA, Wrighten R, et al. Effect of sampling strategy on the detection of fur mites within a naturally infested colony of mice (Mus musculus). J Am Assoc Lab Anim Sci 2011; 50: 337-343.
22.Miller M and Brielmeier M. Environmental samples make soiled bedding sentinels dispensable for hygienic monitoring of IVC-reared mouse colonies. Lab Anim 2018; 52: 233-239.
23.Miller M, Ritter B, Zorn J, et al. Exhaust air dust monitoring is superior to soiled bedding sentinels for the detection of Pasteurella pneumotropica in individually ventilated cage systems. J Am Assoc Lab Anim Sci 2016; 55: 775-781.
24.Morita E, Kaneko S, Hiragun T, et al. Fur mites induce dermatitis associated with IgE hyperproduction in an inbred strain of mice, NC/Kuj. J Dermatol Sci 1999; 19: 37-43.
25.Pochanke V, Hatak S, Hengartner H, et al. Induction of IgE and allergic-type responses in fur mite-infested mice. Eur J Immunol 2006; 36: 2434-2445.
26.Pritchett-Corning KR, and Clifford CB. Parasitic infections of laboratory mice. In: Hedrich HJ (ed) The laboratory mouse. 2nd ed. San Diego (CA): Academic Press, 2012, p.512-518.
27.Pritchett-Corning KR, Cosentino J, and Clifford CB. Contemporary prevalence of infectious agents in laboratory mice and rats. Lab Anim 2009; 43: 165-173.
28.Ricart Arbona RJ, Lipman NS and Wolf FR. Treatment and eradication of murine fur mites: II. Diagnostic considerations. J Am Assoc Lab Anim Sci 2010; 49: 583-587.
29.Rice KA, Albacarys LK, Metcalf Pate KA, et al. Evaluation of diagnostic methods for Myocoptes musculinus according to age and treatment status of mice (Mus musculus). J Am Assoc Lab Anim Sci 2013; 52: 773-781.
30.Sahinduran S, Ozmen O, Haligur M, et al. Severe Myocoptes musculinus infestation and treatment in laboratory mice. Ankara Univ Vet Fak 2010; 57: 73-75.
31.Wang KW, Chueh LL, Wang MH, et al. Multiplex polymerase chain reaction assay for the detection of minute virus of mice and mouse parvovirus infections in laboratory mice. Lab anim 2013; 47: 116-121.
32.Weiss EE, Evans KD and Griffey SM. Comparison of a fur mite PCR assay and the tape test for initial and posttreatment diagnosis during a natural infection. J Am Assoc Lab Anim Sci 2012; 51: 574-578.
33.Whary MT, Baumgarth N, Fox J, et al. Biology and diseases of mice. Otto GM, Franklin CL and Clifford CB. Biology and diseases of rats. In: Fox J, Anderson LC, Otto GM, et al. (eds) Laboratory animal medicine. 3rd ed. San Diego (CA): Academic Press, 2015, p.125-128, p.185-187.
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