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MOLECULAR EPIDEMIOLOGY OF HOUSE DUST MITES IN POTHWAR, PAKISTAN RUBABA SHAFIQUE 10-arid-1986 Department of Zoology/Biology Faculty of Sciences Pir Mehr Ali Shah Arid Agriculture University Rawalpindi Pakistan 2018 MOLECULAR EPIDEMIOLOGY OF HOUSE DUST MITES IN POTHWAR, PAKISTAN by RUBABA SHAFIQUE (10-arid-1986) A thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Zoology Department of Zoology/Biology Faculty of Sciences Pir Mehr Ali Shah Arid Agriculture University Rawalpindi Pakistan 2018 CERTIFICATION I hereby undertake that this research is an original and no part of this thesis fall under plagiarism. If found otherwise, at any stage, I will be responsible for the consequences. Name: Rubaba Shafique Signature: ______________________ Registration No: 10-arid-1986 Date: __________________________ Certified that, the contents and form of thesis entitled “Molecular Epidemiology of House Dust Mites in Pothwar, Pakistan” submitted by Ms. Rubaba Shafique have been found satisfactory for the requirement of degree. Supervisor: ______________________ (Dr. Shamim Akhter) Co-Supervisor: ______________________ (Dr. Muhammad Ismail) Member: ______________________ (Prof. Dr. Mazhar Qayyum) Member: ______________________ (Dr. Farhana Riaz Ch.) Member: _____________________ (Prof. Dr. Azra Khanum) Chairman: ________________ Dean: ____________________ Director Advanced Studies: _________________________ ii Dedicated to my beloved mother and my sweet family who stood by me throughout my PhD iii CONTENTS Page List of Tables vii List of Figures viii List of Abbreviations ix List of Publications and Presentations xii Acknowledgments xiii ABSTRACT xv 1 INTRODUCTION 1 2 REVIEW OF LITERATURE 10 2.1 HYPERSENSITIVITY 10 2.2 ALLERGY (TYPE I HYPERSENSITIVITY) 11 2.3 EPIDEMIOLOGY OF ALLERGIC DISEASES 13 2.3.1 Physiology of Allergic Response 13 2.4 EPIDEMIOLOGY OF HDM ALLERGIES 18 2.5 HDM ASSOCIATED ALLERGY DISEASES 20 2.5.1 Asthma 21 2.5.2 Allergic Rhinitis 22 2.5.3 Atopic Dermatitis 23 2.5.4 Allergic Conjunctivitis 24 2.5.5 Anaphylaxis 25 2.5.6 Gastrointestinal Allergy 27 2.6 MORPHOLOGY OF PYROGLYPHIDS 27 2.6.1 The Mite Body 28 2.6.2 Water Balance 32 2.6.3 Life Cycle 33 2.6.4 Differential Anatomy of D. farinae and D. pteronyssinus 36 2.7 IMPORTANCE OF MOLECULAR CHARACTERIZATION 37 2.8 ENVIRONMENTAL FACTORS EFFECTING MITE 39 POPULATION 2.9 GEOGRAPHICAL DISTRIBUTION OF HDM 40 2.10 HDM ALLERGENS 47 2.10.1 The Peptidases 48 2.10.2 The Glycosidases 52 iv 2.10.3 The Transferases 54 2.10.4 The Lipid-binding Proteins 55 2.10.5 The Muscle Proteins 59 2.10.6 Proteins of the Cytoskeleton 61 2.10.7 Other Allergens 63 2.11 POLYMORPHISM IN HDM ALLERGENS 65 3 MATERIALS AND METHODS 69 3.1 EPIDEMIOLOGICAL STATUS OF HDM ALLERGIES 69 3.1.1 Data Collection 69 3.1.2 Skin Prick Test (SPT) 69 3.2 SPECIES DIVERSITY OF HDM 69 3.2.1 Study Site 70 3.2.2 Study Design 70 3.2.3 Calculation of Sample size 70 3.2.4 Sampling Protocol 76 3.2.5 Isolation of HDM in the Dust Sample 76 3.2.6 Morphological Characterization 78 3.2.7 Molecular Characterization 78 3.2.7.1 DNA extraction from single mites 78 3.2.7.2 PCR for RFLP 79 3.2.7.3 Gel electrophoresis 80 3.3 MOLECULAR CHARACTERIZATION OF HDM ALLERGENS 80 3.3.1 Quantification of Group 1 Allergens in the Environment 80 3.3.2 ELISA for Der p 1 and Der f 1 levels 82 3.3.3 Polymorphism in Group 1 Allergens 82 3.3.4 HDM Samples 82 3.3.5 Amplification by Nested PCR 83 3.3.6 Sequencing and Analysis 83 4 RESULTS AND DISCUSSION 86 4.1 EPIDEMIOLOGICAL STATUS OF HDM ALLERGIES 86 4.2 IDENTIFICATION AND PREVALENCE OF HDM 93 4.2.1 Species Diversity 93 4.2.2 Comparison of Mite Counts between Random and Patient houses 100 4.2.3 Seasonal Variation 103 v 4.3 MOLECULAR CHARACTERIZATION OF HDM ALLERGENS 103 4.3.1 Group 1 Allergen Levels 103 4.3.2 Group 1 Allergen Polymorphism 108 4.3.2.1 Der f1 gene polymorphism 114 4.3.2.2 Der p1 gene polymorphism 114 4.3.2.3 Polypeptide analysis 120 4.4 CONCLUSION 130 SUMMARY 133 LITERATURE CITED 137 APPENDICES 212 vi List of Tables Table No. Page 3.1 Sampling Sites 72 3.2 Overall sampling strategy 75 3.3 Expected product size and fragment length of mite species 81 3.4 Primers used in nested PCR 84 4.1 Year wise percent sensitization to HDM allergens, pollens and food 87 allergens 4.2 Pairwise comparison of sensitivity to HDM, Pollen and food 89 allergens 4.3 Gender bias in allergen sensitization. 90 4.4 Mite counts based on morphological examination 98 4.5 Pairwise comparison of mean mite counts 99 4.6 Mean mite counts from random vs. patients’ houses 101 4.7 Seasonal variation in pyroglyphid mites 104 4.8 Der f 1 and Der p1 allergen levels in dust from selected samples 105 4.9 Seasonal variation in Der p1 and Der f 1 levels in dust samples 110 4.10 Exons, introns, and sequence polymorphism in the group 1 115 allergen-encoding gene in two house dust mite species 4.11 Geographical polymorphism in the Der p 1 allergen 121 4.12 Percent Identity Matrix of Aligned Group1 Allergens 123 4.13 Comparison of conserved amino acid residues involved in 4C1mAb 127 and Ca+ binding epitopes in aligned group 1 allergens vii List of Figures Figure No. Page 1.1 Phylogram showing recent classification of HDM 4 2.1 Relationship between hypersensitivity and immune response to 12 disease 2.2 Steps of allergic immune response 14 2.3 Segmentation of pyroglyphid body 29 2.4 Life cycle of HDM 35 3.1 Map of sampling sites (Pothwar) 71 3.2 Online sampling tool used to calculate the number of households 74 per cluster in the study area 3.3 Alteration/modification in the vacuum cleaner pipe for the 77 collection of dust samples 4.1 Year wise percent sensitization to HDM allergens, pollens and 88 food allergens 4.2 Gender bias in allergen sensitization 91 4.3 Comparative anatomy of pyroglyphid mites 94 4.4 Morphologically identified HDM species 95 4.5 ITS2 gene amplification 96 4.6 Restriction fragments of ITS 2 rDNA from D. farinae and D. 97 pteronyssinus 4.7 Mean mite counts from random vs. patients’ houses 102 4.8 Fluctuations in D. farinae count around the year 106 4.9 Fluctuations in D. pteronyssinus count around the year 107 4.10 Correlation between mite counts and allergen levels 109 4.11 Seasonal variations in Der p1 and Der f1 levels 111 4.12 Amplification of Der f 1 gene by nested PCR 112 4.13 Amplification of Der p1 gene by nested PCR 113 4.14 Gene map of Group 1 allergen 117 4.15 Alignment of group 1 allergens of selected mite species 118 4.16 I-TASSER result showing tertiary structure predictions for RS33 119 and RS31 4.17 Maximum likelihood tree of the group 1 allergen protein of 124 acariform mites 4.18 Alignment map of cysteine proteases from selected mite species 125 viii List of Abbreviations A. siro Acarus siro AC Allergic conjunctivitis AD Atopic dermatitis AE Atopic eczema AKC Atopic keratoconjunctivitis APCs Antigen presenting cells AR Allergic rhinitis B cells B lymphocytes B. tropicalis Blomia tropicalis CD4 Cluster of differentiation 4 CEA Critical equilibrium activity CEH Critical equilibrium humidity CLC Contact lens conjunctivitis COX 1 or CO1 Cytochrome c oxidase 1 D Aspartic acid D. farinae Dermatophagoides farinae D. microcerus Dermatophagoides microcerus D. pteronyssinus Dermatophagoides pteronyssinus D. siboney Dermatophagoides siboney DC Dendritic cells E Glutamic acid E. maynei Euroglyphus maynei EC number Enzyme Commission number ECP Eosinophilic cationic protein FcεR High affinity IgE receptor G Glycine G. domesticus Glycyphagus domesticus H. domicola Hirstia domicola HDM House dust mites I Isoleucine ix IFN-γ Interferon gamma IgE Immunoglobin E IgG Immunoglobin G IgM Immunoglobin M IL-13 Interleukin-13 IL-4 Interleukin-4 I-TASSER Iterative Threading ASSEmbly Refinement ITS Internally transcribed spacer region L Leucine L. destructor Lepidoglyphus destructor LOD Limit of detection M. intermedius Malayoglyphus intermedius mAb monoclonal antibodies MBP Major basic protein MHC II Major histocompatibility complex II OD Optical density P. ovis Psoroptes ovis PAC Persistent or perennial allergic conjunctivitis PM Peritrophic membrane PPIase Peptidylprolyl cis-trans isomerase Q Glutamine R Arginine RH Relative humidity RT-PCR Reverse transcriptase-polymerase chain reaction S Serine S. scabiei Sarcoptes scabiei SAC Seasonal allergic conjunctivitis Serpins Serine protease inhibitors SPT Skin prick test T Threonine T. putrescentiae Tyrophagus putrescentiae TBE Tris-Borate-EDTA T-cells T-lymphocytes x TCR T-cell receptors Th0 Naïve T-helper cells Th1 T-helper cells 1 Th2 T-helper cells 2 TPI Triosephosphate isomerases VKC Vernal keratoconjunctivitis W Tryptophan WAO World Allergy Organization Y Tyrosine xi List of Publications and Presentations International peer-reviewed publications Shafique et al., 2014. Group 1 Allergen Genes in Two Species of House Dust Mites, Dermatophagoides farinae and D. pteronyssinus (Acari: Pyroglyphidae): Direct Sequencing, Characterization and Polymorphism. PLOS ONE. 9(12): e114636. doi:10.1371/journal.pone.0114636. USA. Shafique et al., 2012. Group 10 allergens (Tropomyosin) from House Dust Mites may be a cause of covariation of sensitization to allergens from other invertebrates. Journal of Allergy and Rhinology, USA. GenBank Shafique et al., 2014. Dermatophagoides farinae isolate RS17 cysteine proteinase-1 preproenzyme gene, partial cds GenBank: KJ542065.1 http://www.ncbi.nlm.nih.gov/nuccore/KJ542065 Shafique et al., 2014.