CHARACTERIZATION OF MANGE MITES AND DERMATOPHYTES IN DOMESTIC ANIMALS OF DISTRICT CHITRAL KHALID HAMEED 06-arid-601 Department of Zoology/Biology Faculty of Sciences Pir Mehr Ali Shah Arid Agriculture University Rawalpindi Pakistan 2017 CHARACTERIZATION OF MANGE MITES AND DERMATOPHTES IN DOMESTIC ANIMALS OF DISTRICT CHITRAL by KHALID HAMEED (06-arid-601) 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 2017 ii CONTENTS Page List of Tables vii List of Figures ix List of Plates xi List of Abbreviations xv Acknowledgments xvi Abstract xviii 1 INTRODUCTION 1 2 REVIEW OF LITERATURE 09 2.1 ETIOLOGY OF MANGE IN DOMESTIC ANIMALS 09 2.1.1 Morphology and Taxonomy of Demodex Mites 10 2.1.2 Morphology and Taxonomy of Chorioptes Mites 10 2.1.3 Morphology and Taxonomy of Psoroptes Mites 12 2.1.4 Morphology and Taxonomy of Sarcoptes Mites 13 2.2 EPIDEMIOLOGY OF MANGE IN DOMESTIC 16 ANIMALS 2.2.1 Prevalence of Mange in Domestic Animals of Pakistan 19 2.3 ETIOLOGY OF DERMATOPHYTOSIS 20 2.3.1 Epidemiology of Dermatophytes in Domestic Animals 21 2.3.2 Prevalence of Trichophyton verrucosum in Cattle and 22 Transmission to Animal Attendants 3 MATERIALS AND METHODS 24 3.1 STUDY AREA 24 iii 3.1.1 Epidemiology of Mange in Domestic Animals 28 3.2 SURVEYS AND SAMPLING 28 3.2.1 Criteria for Skin Scrapings 28 3.3 DIAGNOSIS OF MANGE AND IDENTIFICATION OF 29 MITES 3.4 MOLECULAR CHARACTERIZATION OF Sarcoptes 29 scabiei 3.4.1 Preparation of Sarcoptes DNA 29 3.4.1.1 Procedure for HotSHOT plus Thermal SHOCK 30 3.4.2 PCR Amplification 30 3.4.3 Microsatellite Analysis: 31 3.5 CHARACTERIZATION OF DERMATOPHYTES IN 35 DOMESTIC ANIMALS 3.5.1 Microscopic Examination of Fungal Hyphae and Spores 35 3.5.2 Real Time PCR for Diagnosis of Dermatophytes 35 3.5.3 DNA Extraction of Dermatophytes 35 3.5.4 Real Time PCR for the confirmation of Dermatophytes: 36 3.6 STATISTICAL ANALYSIS 36 4 RESULTS AND DISCUSSION 38 4.1 PREVALENCE OF SKIN DISEASES IN LIVESTOCK 38 4.1.1 Geographical Variations in the Prevalence of Skin Diseases 38 in Livestock 4.1.2 Seasonal Variations in the Prevalence of Skin Diseases in 39 Livestock iv 4.2 PREVALENCE OF MANGE IN LIVESTOCK 39 4.2.1 Prevalence of Psoroptic and Sarcoptic Mange in Small 40 Ruminants 4.2.2 Geographical Variations in the Prevalence of Mange in 40 small Ruminants 4.2.3 Gender Based Comparison of Mange in Small Ruminants: 52 4.2.4 Prevalence of Mange between different Age groups: 52 4.2.5 Seasonal Variations in the Prevalence of Mange: 52 4.2.6 Relationship between Mange Prevalence and Herd Size: 52 4.2.7 Prevalence of Mange in Separate v/s Mixed Herds: 59 4.2.8 Generalized Linear Model with Binomial Distribution 59 for the Quantification of Risk Factors for Mange: 4.3 PREVALENCE OF SKIN DISEASES IN DOGS 60 4.3.1 Seasonal Prevalence of Skin Diseases in Dogs 60 4.3.2 Prevalence of Mange Infestation in Dogs 64 4.3.3 Seasonal Variations in Prevalence of Mange Infestation in 64 Dogs 4.3.4 Prevalence of Mange in Different Age Groups of Dogs 64 4.3.5 Gender Based Comparison of Mange in Dogs 65 4.4 MOLECULAR CHARACTERIZATION OF Sarcoptes 72 scabiei 4.4.1 Allelic Frequencies of Nine Microsatellite Markers in Dog 73 Derived Sarcoptes scabiei from District Chitral, Pakistan 4.4.2 Allelic Frequencies of Nine Microsatellite Markers in Lynx 74 v Derived Sarcoptes scabiei from District Chitral, Pakistan 4.4.3 Bayesian Cluster Analysis in STRUCTURE 75 4.5 DERMATOPHYTES IN DOMESTIC ANIMALS: 101 4.5.1 Prevalence of Trichophyton in Livestock 101 4.5.2 Seasonal Prevalence of Trichophyton verrucosum in 109 Livestock 4.5.3 Geographical Distribution of Trichophyton verrucosum in 109 Livestock 4.5.4 Gender Based Comparison of Trichophyton verrucosum 109 4.5.5 Prevalence of Trichophyton verrucosum in Age Groups 110 4.5.6 Relationship between Trichophyton verrucosum and Herd 110 Size: 4.5.7 Prevalence of Trichophyton verrucosum in separate v/s 110 Mixed Herds 4.5.8 Generalized Linear Model with Binomial Distribution 119 for Quantification of Risk Factors for Trichophyton verrucosum in Livestock: CONCLUSION 127 SUMMARY 129 LITERATURE CITED 131 APPENDICES 158 Annexure 158 vi List of Tables Table No. Page 3.1 Reagents for HotSHOT plus Thermal SHOCK 32 3.2 Primer Specifications for the 9 Sarcoptes mite Microsatellite DNA 33 loci in the Multiplex PCR 3.3 Reagents and Solutions for DNA Extraction 34 4.1 Chi Square Analysis for Mange Infestation in sheep and goats of 58 District Chitral 4.2 Generalized Linear Model with binomial distribution for Risk 61 Factor Assessment for Mange Infestation in Livestock of District Chitral 4.3 Total Number of Alleles Detected at the Nine Microsatellite Loci of 95 the Dog and Lynx Derived Sarcoptes scabiei Population from District Chitral, Pakistan together with their frequencies 4.4 Private Alleles Detected at the Nine Microsatellite Loci of the Dog 96 and Lynx Derived Mite Population from District Chitral, Pakistan together with their frequencies 4.5 Total number of shared alleles detected at the nine microsatellite 97 loci of the dog derived Sarcoptes scabiei population from district Chitral, Pakistan together with their frequencies 4.6 Total number of shared alleles detected at the nine microsatellite 98 loci of the lynx derived Sarcoptes scabiei population from district Chitral, Pakistan together with their frequencies 4.7 Chi square analysis for Trichophyton verrucosum infection in 117 vii livestock of district Chitral 4.8 Generalized Linear Model for risk factor assessment for T. 118 verrucosum infection in livestock of district Chitral viii List of Figures Figure No. Page 4.1 Prevalence (95% C.I.) of skin diseases in livestock species of district 41 Chitral during entire Study 4.2 Prevalence of skin diseases in livestock species from studied 42 villages of district Chitral during entire study 4.3 Prevalence (95% C.I.) of skin diseases in livestock species of district 43 Chitral during summer 4.4 Prevalence of skin diseases in livestock species from studied 44 villages of district Chitral during summer 4.5 Prevalence (95% C.I.) of skin diseases in livestock species of district 45 Chitral during winter 4.6 Prevalence (95% C.I.) of skin diseases in livestock species in 46 studied villages of district Chitral during winter 4.7 Prevalence of mange in sheep and goats of district Chitral 50 4.8 Prevalence of mange in studied village of district Chitral 51 4.9 Gender Based Comparison of mange in small ruminants of district 53 Chitral 4.10 Age wise distribution of mange in small ruminants of district Chitral 54 4.11 Seasonal comparison of mange in small ruminants of district Chitral 55 4.12 Herd based comparison of mange in small ruminants of district 56 Chitral 4.13 Distribution of mange in different herds in small ruminants of 57 district Chitral ix 4.14 Seasonal prevalence of skin diseases in dogs of district Chitral 62 4.15 Results of negative log likelihoods [(ln P(D)] versus K in 99 STRUCTURE Harvestor indicating 5 clusters (K=5) suitable for mites under study 4.16 Results of the delta K in STRUCTURE Harvestor indicating 5 99 clusters (K=5) suitable for mites under study. 4.17 Bar plot developed in STRUCTURE software showing degree of 100 individual variations among Sarcoptes scabiei mites from different hosts and different geographical regions in four clusters (K=4) and five clusters (K=5). Five clusters (K=5) best suited to represent above mentioned mite populations. 4.18 Prevalence of T. verrucosum in livestock species of district Chitral 108 4.19 Seasonal prevalence of T. verrucosum in livestock of district Chitral 111 4.20 Village based comparison of T. verrucosum in livestock of district 112 Chitral 4.21 Gender based distribution of T. verrucosum in livestock of district 113 Chitral 4.22 Prevalence of T. verrucosum in age groups of livestock of district 114 Chitral 4.23 Prevalence of T. verrucosumi in different herd sizes of livestock in 115 district Chitral 4.24 Prevalence of T. verrucosum in different types of herds in district 116 Chitral x List of Plates Plate No. Page 3.1 Map of district Chitral showing the locations of studied villages 25 3.2 Temporary herds and grazing at high altitude pastures 26 3.3 Animals with Visible sign of Sign Diseases 27 4.1 Psoroptes ovis mites in skin scrapings from sheep after digestion 47 with 10% KOH at 40X magnification 4.2 Psoroptes ovis mites in skin scrapings from sheep after digestion 47 with 10% KOH at 40X magnification 4.3 Psoroptes ovis nymph in skin scrapings from sheep after digestion 48 with 10% KOH at 10X magnification 4.4 Psoroptes ovis nymph in skin scrapings from sheep after digestion 48 with 10% KOH at 40X magnification 4.5 Sarcoptes scabiei mites in skin scrapings after digestion from sheep 49 at 40x magnification 4.6 Sarcoptes scabiei mites along with nymph in skin scrapings after 49 digestion from sheep at 10x magnification 4.7 Dog with alopacea and crust lesions on legs 63 4.8 Sarcoptes scabiei isolated from skin scrapings of dog after 63 digestion with 10% KOH at 40X 4.9 Alleles of dog derived Sarcoptes scabiei specific microsatellite 77 Sarm 33. 4.10 Alleles of lynx derived Sarcoptes scabiei specific microsatellite 78 Sarm 33.