0 MOLECULAR CHARACTERIZATION AND PATHOLOGICAL STUDIES OF BRUCELLA SPECIES IN NATURALLY INFECTED ANIMALS By Asim Shahzad M. Phil. Pathology Thesis submitted in partial fulfillment of the requirements for the award of the degree of DOCTOR OF PHILOSOPHY In PATHOLOGY DEPARTMENT OF PATHOLOGY FACULTY OF VETERINARY SCIENCE UNIVERSITY OF AGRICULTURE FAISALABAD PAKISTAN 2017 i DECLARATION I hereby declare that the contents of the Thesis ―Molecular characterization and pathological studies of Brucella species in naturally infected animals‖ are a product of my own research and no part has been copied from any published source (except the references, standard mathematical or genetic models/ equations/ formulate/ protocols etc.). I further declare that this work has not been submitted for the award of any other diploma/degree. The University may take action if the information provided is found inaccurate at any stage. (In case of any default the scholar will be proceeded against as per HEC plagiarism policy). Asim Shahzad Reg. No. 2004-ag-1642 Department of Pathology, University of Agriculture, Faisalabad. ii iii Dedication I would love to dedicate this manuscript to my loved ones: my loving family, mentors, special friends and loves of my life. All are truly the bright stars in my life. iv ACKNOWLEDGEMENTS All praises and humble thanks to ALMIGHTY ALLAH, The Creator of the universe, the most Generous, the most Compassionate whose blessings and exaltations flourished my thoughts, hose praise cannot be expressed even if oceans turn into ink and all of the trees become pawns. "He, 'Who is (Beneficent and 'Merciful 'Whose Blessings enabled me to complete this study. Countless salutations to the Holy Prophet Hazrat Muhammad (PBUH), "the city of knowledge", 'Who is forever a model of guidance and knowledge for humanity. 'Who has guided his "Ummah" to seek the knowledge from the cradle to grave". I feel great pleasure to express my gratitude to my honorable research supervisor Prof. Dr. Ahrar Khan, Department of Pathology for His able guidance, dynamic supervision, enlightening views for this study, inventive teaching, polite behavior and valuable suggestions throughout the course of my studies and research work. I do not have words in my command to thank the members of my supervisory committee Prof. Dr. M. Zargham Khan, Department of Pathology and Dr. Muhammad Saqib, Department of Clinical Medicine and Surgery for their kind, sympathetic behavior, valuable suggestions, technical guidance and keen interest during the course of my research work. I express my profound sense of appreciation to Prof. Dr. Muhammad Tariq Javed, Professor Department of Pathology for his technical guidance and constructive criticism during the course of my study. I am so thankful to Dr. Shafia Tehseen Gul Assistant Professor, Dr. M. Kashif Saleemi Assistant Professor and Dr. Farzana Rizivi Associate Professor for their kind support and encouragement throughout the research. Friends are asset of life, especially when they work together. A sense of special and sincere thanks and appreciation is own to my friends for their support, encouragement and untiring affectionate behavior in my research and thesis work. MY special gratitude to Senior and Junior students for their cooperation and contribution to my research work. I also record my thanks to Lab assistants for providing cooperation and assistance. Last but not least a gooey tribute to my beloved and ever affectionate Family for their moral support and countless prayers throughout the course of my study. My word's fail to give credit to my Parents whose prayers, Cove, patience, sacrifice, encouragement and spiritual inspiration was a great source of motivation for the quest of knowledge at every stage of my education. My success is reality fruit of my parent’s derailed-prayers. I am greatly indebted and submit my earnest thanks to them, they had potentially tolerated agony and all miseries. Asim Shahzad v vi LIST OF TABLES Table TITLE PAGE No No 2.1 Prevalence of brucellosis in livestock in neighboring countries 23 3.1 Sampling plan to detect Brucella species in different animal species from 47 different districts of Punjab, Pakistan 3.2 List of primer pairs for the detection and differentiation of Brucella 54 species 3.3 Tissue processing protocol for histopathology 55 3.4 Detailed procedures of H&E staining protocols for histopathology 56 4.1 Overall prevalence of brucellosis in livestock species of Punjab Pakistan 61 4.2 Prevalence of brucellosis in cattle in different regions of Punjab Pakistan 61 4.3 Prevalence of brucellosis in relation to their sex in cattle of Punjab 62 Pakistan 4.4 Prevalence of brucellosis in relation to their age in cattle of Punjab 62 Pakistan 4.5 Prevalence of brucellosis in relation to their history of reproductive 62 disorders in cows of Punjab Pakistan 4.6 Prevalence of brucellosis in relation to their pregnancy status in cows of 65 Punjab Pakistan 4.7 Prevalence of brucellosis in relation to their parity number in cows of 65 Punjab Pakistan 4.8 Prevalence of brucellosis in relation to their regions in buffalo of Punjab 67 Pakistan 4.9 Prevalence of brucellosis in relation to their sex in buffalo of Punjab 67 Pakistan 4.10 Prevalence of brucellosis in relation to their age in buffalo of Punjab 69 Pakistan 4.11 Prevalence of brucellosis in relation to their history of reproductive 69 disorders in buffalo of Punjab Pakistan 4.12 Prevalence of brucellosis in relation to their pregnancy status in buffalo of 72 Punjab Pakistan 4.13 Prevalence of brucellosis in relation to their parity number in buffalo of 72 Punjab Pakistan 4.14 Prevalence of brucellosis in relation to their regions in camel of Punjab 77 Pakistan 4.15 Prevalence of brucellosis in relation to their sex in camel of Punjab 77 Pakistan 4.16 Prevalence of brucellosis in relation to their age in camel of Punjab 78 Pakistan 4.17 Prevalence of brucellosis in relation to their Health in camel of Punjab 78 vii Pakistan 4.18 Prevalence of brucellosis in relation to their pregnancy status in camel of 79 Punjab Pakistan 4.19 Prevalence of brucellosis in relation to their parity number in camel of 79 Punjab Pakistan 4.20 Summary of results of conventional PCR for brucellosis 81 4.21 Summary of results of Real time PCR for brucellosis 82 viii LIST OF PLATES Plate TITLE PAGE No No 2.1 Global distribution of Brucella abortus (OIE, 2015) 24 2.2 Global distribution of Brucella melitensis (OIE, 2015) 24 3.1 Map of Punjab, Pakistan showing sampling plan to detect Brucella 48 species in different animal species from different districts 4.1 Photograph of selected samples positive for Brucella 81 4.2 Photograph of selected samples positive for Brucella species 81 4.3 Amplification Plots of RT-PCR for Brucella genus 82 4.4 Amplification Plots of RT-PCR for Brucella abortus 82 4.5 The evolutionary trends of Brucella isolates (PAK-CAMEL, Brucella 84 PAK-CATTLE1, PAK-CATTLE2, PAK-BUFFALO1 and PAK- BUFFALO2) are represented by the Neighbor-Joining method (Saitou and Nei, 1987) conducted with MEGA7 (Kumar et al., 2016). The evolutionary distance is estimated with Tamura 3-parameter method (Tamura, 1992). 4.6 Multiple sequence alignment of BCSP31 performed with the ClustalW 85 program (http://www.ebi.ac.uk). The identical sequences are shown by asterisks at the top 4.7 I-TASSER based predicted structure of BCSP31 protein. 86 4.8 Detection of the organism from the blood of experimental goats 88 4.9 Detection of the organism from the different organs in experimental 88 goats 4.10 Photomicrograph of lungs of goat infected with B. melitensis showing 89 thickened alveolar walls (arrow), emphysema (E) and alveoli‘s filled with fibrinous exudate (arrow head) 4.11 Photomicrograph of lungs of goat infected with B. melitensis showing 89 Emphysema (E), fibrinous exudate (asterisk) and infiltration of inflammatory cells (arrow) 4.12 Photomicrograph of lungs of goat infected with B. melitensis showing 90 Fibrinous exudate (asterisk), alveoli filled with RBCs and polymorpho- nuclear neutrophil (Arrow) 4.13 Photomicrograph of lungs of goat infected with B. melitensis showing 90 thickened and hemorrhagic pleural walls 4.14 Photomicrograph of liver of goat infected with B. melitensis showing 91 cloudy swelling (arrow), collapsed sinusoidal spaces and individual cell necrosis of hepatocytes (arrow head) 4.15 Photomicrograph of liver of goat infected with B. melitensis showing 91 Hemorrhages around the central vein (C) and mild mononuclear cell infiltration (I) 4.16 Photomicrograph of liver of goat infected with B. melitensis showing 92 areas of necrosis (N), hemorrhages and mild mononuclear cell ix infiltration (I) 4.17 Photomicrograph of liver of goat infected with B. melitensis showing 92 multiple degenerating areas and active von kupffer cells 4.18 Photomicrograph of kidneys of goat infected with B. melitensis 93 showing Necrosis with infiltration of granular and multilobed cells in glomeruli (arrow) and tubules with pretentious material (P) 4.19 Photomicrograph of kidneys of goat infected with B. melitensis 93 showing mild to moderate congestion (C) of medullary area 4.20 Photomicrograph of spleen of goat infected with B. melitensis showing 94 red pulp filled with lymphocytes, macrophages and plenty of RBC‘s 4.21 Photomicrograph of spleen of goat infected with B. melitensis showing 94 hyperplasia of the many germinal follicles, proliferation of the cells with lightly stained cytoplasm and increased population of macrophages 4.22 Photomicrograph of uterus of goat infected with B. melitensis showing 95 Necrotic debris comprised of intense inflammatory infiltrate (arrow), dead tissue 4.23 Photomicrograph of uterus of goat infected with B. melitensis showing 95 multiple foci of degenerating areas specifically below the epithelium x ABSTRACT Brucellosis is a globally distributed zoonotic problem mostly spreads by ingestion of unpasteurized dairy products of farm animals and human.