臺灣博碩士論文加值系統

過敏一直以來在犬隻的皮膚症狀中扮演著重要的角色，而過敏性皮膚炎在犬隻間所發生的比例可高達百分之十五。此研究目的為了解在台灣地區造成犬隻過敏的重要過敏原種類及其重要性。我們針對了2006年六月至2008年二月間由凌越生醫公司所進行的62項蛋白質晶片過敏原分析結果進行分析。在這期間所蒐集到的505個病例中有高達80% (405/505)的犬隻對於62項中至少一項過敏原有陽性反應；54.06% (273/505)的犬隻則對至少一種塵螨有陽性反應。由此分析可見塵螨則是造成犬過敏性皮膚炎(Canine allergic dermatitis, CAD) 的主要元凶,其中歐洲塵螨陽性率高達29.50% (149/505)，腐蝕酪螨則高達24.55% (124/505)位居第二。其他佔有較高陽性率的重要過敏原還包括了: 煙色黴菌18.02% (91/505)、熱帶五爪螨 17.82% (90/505)、啤酒酵母17.43% (88/505)。在在台灣地區犬過敏性皮膚炎病例中，美洲塵螨的陽性率雖然也算是高(17.03%)，但卻顯著的低於歐洲塵螨的陽性率(p<0.0001)。除此之外，由於啤酒酵母常被使用於寵物飼料的維他命B來源添加物，甚至在一些低過敏配方飼料中也常出現，本研究亦針對啤酒酵母這一項過敏原進行了更進一步的分析。使用西方墨點轉漬法來找出主要造成過敏的啤酒酵母蛋白可能包括磷酸丙酮異構酶, 磷酸甘油酸變位酶1, 烯醇化酶 2, 烯醇化酶 1 and alpha-glucosidase，這些分析出的蛋白質可能有助於日後在設計低過敏性配方飼料時，或是飼主在為寵物選擇適當的飼料時作為參考。

Allergy has always played an important role in canine skin related diseases. The prevalence of canine allergic dermatitis (CAD) in the canine population was also estimated to be around 3-15%. The purpose of this study is to analyze and understand the type of allergens causing CAD in Taiwan. We analyzed 505 results from Excelsior bio-system’s protein chip microarray 62 item canine allergy test collected from May of 2006 to February of 2008. The result showed 80% of the cases had at least one positive result to the 62 items tested, and 54.06% of the dogs had at least one positive result in the mites group. Our finding shows that mites are the most important group of allergens responsible for CAD in Taiwan. Dermatophagoides pteronyssinus is the top allergen associated with CAD in Taiwan, having a positive rate of 29.50% (149/505), followed by Tyrophagus putrescentiae (24.55%, 124/505)、Aspergillus fumigates (18.02%, 91/505)、Blomia tropicalis (17.82%, 90/505)、and brewer’s yeast (Saccharomyces cerevisiae 17.43%, 88/505). In most countries, Dermatophagoides farinae is more commonly associated with CAD than Dermatophagoides pteronyssinus; however, our protein chip microarray showed that although Dermatophagoides farinae had a high positive rate (17.3%, 86/505), it is still significantly lower than that of Dermatophagoides pteronyssinus (p<0.0001). Other than identifying important allergens causing CAD in Taiwan, we also took brewer’s yeast, the only food allergen that made it to the top five lists further for western blot analysis. Brewer’s yeast is a common food supplement in which it serves rich vitamin B source; its presence is even common in hypoallergenic diets. With western blot analysis, we were able to identify the major proteins responsible for the positive results obtained from protein chip microarray such as triosephosphate isomerase, phosphoglycerate mutase 1, enolase 2, enolase 1 and alpha-glucosidase. Such information may be provided as valuable reference when pet owners choose the right diet for their pets, or manufacturers selecting ingredients for hypoallergenic diets.

ENGLISH TITLE PAGE………………………………………………………………..i
CHINESE TITLE PAGE………………………………………………………………...ii
ACKNOWLEDGEMENT……………………………………………………………...iii
CHINESE ABSTRACTI………………………………………………………………..iv
ENGLISH ABSTRACT…………………………………...…………………………….v
ABBREVIATIONS USED………………………………………………………….....vii
TABLE OF CONTENS………………………………………………………………..viii
CHAPTER 1 INTRODUCTION………………………………………………………1
CHAPTER 2 LITERATURE REVIEW………………………………………………4
2-1 Types of allergy……………………………………………...…………………4
2-1.1 Asthma………………………………………………………………….4
2-1.2 Adverse food reaction…………………………………………………..5
2-1.3 Allergicc dermatitis..................................................................................8
2-2 Introduction on Canine Allergic Dermatitis (CAD)..........................................10
2-2.1 Clinical symptoms of CAD……………………………………………10
2-2.2 Pathogenesis of CAD………………………………………………….11
2-2.3 Common allergens……………………………………………………..12
2-3 Detection methods of CAD…………………………………………………...16
2-3.1 Clinical history of symptoms – Willemse major and minor criteria…..16
2-3.2 Clinical history of symptoms – CADESI scoring…………………….16
2-3.3 Serum sIgE ELISA test………………………………………………..17
2-3.4 Protein chip microarray………………………………………………..17
2-3.5 Intradermal skin test (IDST)…………………………………………..20
2-4 Controlling CAD……………………………………………………………...21
2-4.1 Steroids use.............................................................................................21
2-4.2 Antihistamine.........................................................................................21
2-4.3 Hypo-allergenic diets............................................................................22
2-4.4 Avoidance of known allergens………………………………………...22
2-4.5 Regulating immune responses with probiotics………………………..22
2-4.6 Immunotherapy………………………………………………………..23
CHAPTER 3 MATERIALS AND METHODS……………………………………..25
3-1 Sample source………………………………………………………………...25
3-1.1 505 retrospective data analysis………………………………………..25
3-1.2 Collection of 30 control normal dog sera and 31 CAD sera samples…25
3-2 Sample microarray……………………………………………………………27
3-3 62 items tested using sIgE protein chip microarray…………………………..28
3-3.1 Mites (5 different species)…………………………………………….28
3-3.2 Insects (6 different species)……………………………………………28
3-3.3 Hair/skin scrapes (from 5 different species)…………………………...28
3-3.4 Moulds (5 different species)…………………………………………...28
3-3.5 Pollen (7 different species)…………………………………………….28
3-3.6 Milk/egg (4 different sources)…………………………………………29
3-3.7 Meat (6 different sources)……………………………………………..29
3-3.8 Seafood (7 different sources)………………………………………….29
3-3.9 Nuts/grains (4 different sources)………………………………………30
3-3.10 Fruits (7 different sources)...................................................................30
3-3.11 Vegetables (3 different sources)...........................................................30
3-3.12 Yeasts (2 different sources)…………………………………………..30
3-3.13 Others (flaxseed)……………………………………………………..30
3-4 Statistical analysis…………………………………………………………….32
3-5 Western blot analysis………………………………………………………….33
3-5.1 Brewer’s yeast preparation…………………………………………….33
3-5.2 Sodium dodecyl sulfate polyacrylamide gel electrophoresis………….33
3-5.3 Coomassie blue staining……………………………………………….34
3-5.4 Polyvinylidene difluoride (PVDF) membrane transfer………………..35
3-5.5 Ponceau S membrane staining…………………………………………35
3-5.6 Blocking……………………………………………………………….35
3-5.7 Serum hybridization…………………………………………………...36
3-5.8 Reagent hybridization…………………………………………………36
3-5.9 Secondary developing reagent hybridization………………………….36
3-5.10 Substrate addition and detection……………………………………..37
3-6 Protein identification analysis……………………………………………...…38
3-6.1 LC-MS/MS…………………………………………………………….38
CHAPTER 4 RESULTS………………………………………………………………39
4-1 505 Retrospective protein chip microarray analysis………………………….39
4-1.1 Study group information analysis……………………………………...39
4-1.2 Control group information analysis……………………………………39
4-1.3 Positive rate among different allergen groups…………………………40
4-1.4 Seasons as risk factor………………………………………………….40
4-1.5 Age as risk factor………………………………………………………41
4-2 31 new cases protein chip microarray analysis……………………….………43
4-2.1 Case group information analysis………………………………………43
4-2.2 Positive rate among different allergen groups…………………………43
4-3 Major proteins identified as causative allergens in brewer’s yeast………...…45
CHAPTER 5 DISCUSSION.........................................................................................46
REFERENCES..............................................................................................................56
TABLES AND FIGURES………………………………………………….………….64
APPENDIX………………………………………………………………….…………73

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