CLINICO-EPIDEMIOLOGICAL AND THERAPEUTIC STUDY ON BABESIOSIS IN DIFFERENT BREEDS OF CATTLE IN BALOCHISTAN By MUHAMMAD ESSA KAKAR 2005-va-229 A THESIS SUBMITTED IN THE PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE DEGREE OF THE DOCTOR OF PHILOSOPHY IN CLINICAL MEDICINE UNIVERSITY OF VETERINARY AND ANIMAL SCIENCES LAHORE 2013 77 To, The Controller of Examinations, University of Veterinary and Animal Sciences, Lahore. We, the Supervisory Committee, certify that the contents and form of the thesis, submitted by Muhammad Essa Kakar, Reg. No. 2005-VA-229, have been found satisfactory and recommend that it be processed for the evaluation by the External Examination (s) for award of the Degree. Supervisory Committee: Supervisor: Prof. Dr. Muhammad Arif Khan Co-Supervisor: Prof. Dr. Muhammad Azam Kakar Member: Prof. Dr. Muhammad Sarwar Khan Member: Prof. Dr. Kamran Ashraf 78 (i) 79 ACKNOWLEDGEMENTS In the name of Allah, Most Gracious, Most Merciful. All thanks and precise are to Allah and peace and blessing be upon His Messenger. There are lots of respected personalities I would like to thank for a huge variety of reasons. Foremost, I would like to thank my worthy supervisor Prof. Dr. Muhammad Arif Khan, Department of Clinical Medicine and Surgery, University of Veterinary and Animal Sciences (UVAS), Lahore, because this work would not have been possible without his support and encouragement. He deeply monitored my research work and has supported me at every step with friendly atmosphere. My supervisor is special because he is an example of hard work and dedication can achieve. I have the honor to express my deep sense of gratitude and indebtedness to ever- affectionate Prof. Dr. Muhammad Azam Kakar, Dean, Faculty of Veterinary and Animals Sciences, Lasbela University of Agriculture, Water and Marine Sciences, Balochistan, for his technical guidance, encouragement and support during the course of study. He was always available when I needed his help. I would like to say a big “thank you” to Prof. Dr. Muhammad Sarwar Khan, Professor of Medicine and Dean, Faculty of Veterinary Sciences, University of Veterinary and Animal Sciences, Lahore. I could not have imagined having a better advisor for my Ph.D. and without his guidance, knowledge, and perceptiveness I would never have finished. I also express my deep sense of appreciation to Prof. Dr. Kamran Ashraf, Department of Parasitology, (UVAS), Lahore, for providing numerous ideas, useful discussions, valuable instructions and suggestion throughout the study period. Special thanks to Provincial Secretary, and Director General, Livestock and Dairy Development Department, Balochistan for their kind permission and support to complete my study. I would like to thanks to Dr. Nazeer Ahmed, Assistant Prof. BUITEMS, Quetta & Mr. Asif for their full cooperation & utilization of laboratory during the period of my research work. (ii) 80 I wish to express my heartiest thanks to my close friends, Zia khan kakar, Abdul Hadi, Sadam, Asif, Idrees, Adnan, Azam, Choudhri Akram (Ajam), Umair pathoki and Ustad Boota for their kindness and affection, and for never letting me feel that I am away from my home. Special gratitude to all my colleagues, Dr. Abdul Razzaq, Dr. Masood-ul-Haq Kakar, Dr. Saadullah Jan, Dr. Ziauddin, Dr. Ejaz Ahmed (Assistant Prof. Department of CMS, UVAS, Lahore), Dr. Asad (UDL), Dr. Mir Ahmed, Dr Ihsanullah kakar, Dr. Ramzan, Dr. Hamdullah Khan, Dr. Saeed and Dr. Imtaiz Rawlakot for their good company during the course of my study, and also thankful to all the students of UVAS, particularly, Tariq khan, Salman Arshad, Muhammad Faiz Rasool and Noman Shah, and also Mr. Abdul Khaliq (DI Lab, Quetta, Balochistan) and Mr. Shahid, Laboratory Assistant, Department of Clinical Medicine and Surgery, UVAS, Lahore, who voluntarily worked with me during the collection and processing of the samples. I cannot end without thanking my respected teachers, Dr. Aneela Zameer Durani, Dr. Jaweria Ali khan and Dr. Muhammad Awais, Department of Clinical Medicine and Surgery, UVAS, Lahore, for their constructive and valuable suggestions during the whole period of my study. Finally, my special appreciation goes to my elder brothers, Muhammad Farooq, Muhammad Rauf and Dr. Naseebullah and all family members, particularly my Mother who always kept me away from family responsibilities and encouraged me to concentrate only on study. MUHAMMAD ESSA KAKAR 81 (iii) TABLE OF CONTENTS DEDICAT ION (i) ACKNOWLEDGEMENTS (ii) LIST OF TABLES (v) LIST OF FIGURES (xii) LIST OF ANNEXURE (xviii) S. N0. CHAPTERS PAGE NO. 1. INTRODUCTION……………………………………………………….1 2. REVIEW OF LITERATURE……………………………….…............10 3. MATERIALS AND METHODS………………………………………56 4. RESULTS……………………………………………………….............77 5. DISCUSSION………………………………………………………….162 6. SUMMARY……………………………………………………………176 7. LITERATURE CITED……………………………………………….178 82 (iv) LIST OF TABLES TABLE NO. TITLE PAGE NO. Table 2.1.1.1. Prevalence % of hemoprotozoans in different groups of cattle and buffaloes at ASI and BLPRI……………….…………………………….11 Table 2.1.1.2. Prevalence % of hemoprotozoans in different breeds of cattle at BLPRI……………………………………………………………………11 Table 2.1.1.3. Prevalence % of hemoprotozoans species in different breeds of cattle and buffaloes at ASI and BLPRI……………………………………………..11 Table 2.1.1.4. District wise prevalence of b. bovis in cattle and buffaloes……………..18 Table 2.1.4. Prevalence of hemoprotozoan in Jersey Holstein Friesian cattle………...23 Table 3.5.1.1. Breed and age wise distribution of Bhag Nari, Holstein Friesian and Crossbred Cattle in each District……………………...............................64 Table 3.5.1.2. Sex wise distribution of Bhag Nari, Holstein Friesian and Crossbred Cattle in each District……………………………..............................................65 Table 3.5.1.3. Season wise distribution of BhagNari, Holstein Friesian and Crossbred Cattle in each District…............................................................................65 Table 3.5.2. Distribution of experimental animal groups………………………..……66 Table 3.9.2.1. Sets of primers used to amplify Babesia bigemina and Babesia bovis SSU rRNA gene sequences…………………………...………………….71 (v) 83 Table 4.1.1. Overall Prevalence of babesiosis in District Quetta…………………..…78 Table 4.1.1.1. Breed wise prevalence of babesiosis in District Quetta…………….……79 Table 4.1.1.2. Overall Age wise prevalence of babesiosis in BhagNari, Holstein Friesian and Crossbred in District Quetta………………………………………....81 Table 4.1.1.3. Age wise prevalence of babesiosis in BhagNari in District Quetta…......82 Table 4.1.1.4 Age wise prevalence of babesiosis in Holstein Friesian in District Quetta…………………………………………………………………….84 Table 4.1.1.5. Age wise prevalence of babesiosis in Crossbred in District Quetta……..86 Table 4.1.1.6. Overall sex wise Prevalence of babesiosis in BhagNari, Holstein Friesian and Crossbred in District Quetta…………………………………………87 Table 4.1.1.7. Sex wise prevalence of babesiosis in BhagNari Cattle in District Quetta…………………………………………………………………….88 Table 4.1.1.8. Sex wise prevalence of babesiosis in Holstein Friesian in District Quetta…………………………………………………………………….89 Table 4.1.1.9. Sex wise prevalence of babesiosis in Crossbred cattle in District Quetta…………………………………………………………………….90 Table 4.1.1.10. Overall Season wise prevalence of babesiosis in BhagNari, Holstein Friesian and Crossbred in District Quetta………………………………..92 (vi) 84 Table 4.1.1.11 Season wise prevalence of babesiosis in BhagNari Cattle in District Quetta…………………………………………………………………….93 Table 4.1.1.12. Season wise prevalence of babesiosis in Holstein Friesian in District Quetta…………………………………………………………………….95 Table 4.1.1.13. Season wise prevalence of babesiosis in Crossbred in District Quetta…..96 Table 4.1.2. Overall prevalence of babesiosis in District Sibi………………………...97 Table 4.1.2.1. Breed wise prevalence of babesiosis in District Sibi………………….....98 Table 4.1.2.2. Overall age wise prevalence of Babesiosis in District Sibi…………….100 Table 4.1.2.3. Age wise prevalence of babesiosis in BhagNari in District Sibi…….…101 Table 4.1.2.4. Age wise prevalence of babesiosis in Holstein Friesian in District Sibi……………………………………………………………………...103 Table 4.1.2.5. Age wise prevalence of babesiosis in Crossbred in District Sibi…….…104 Table 4.1.2.6. Overall sex wise prevalence of babesiosis in BhagNari, Holstein Friesian and Crossbred in District Sibi…………………………………………..105 Table 4.1.2.7. Sex wise prevalence of babesiosis in BhagNari Cattle in District Sibi……………………………………………………………………...106 Table 4.1.2.8. Sex wise prevalence of babesiosis in Holstein Friesian in District Sibi……………………………………………………………………...107 (vii) 85 Table 4.1.2.9. Sex wise prevalence of babesiosis in Crossbred cattle in District Sibi……………………………………………………………………...108 Table 4.1.2.10. Overall season wise prevalence of babesiosis in District Sibi…………109 Table 4.1.2.11. Season wise prevalence of babesiosis in BhagNari cattle in District Sibi……………………………………………………………………...110 Table 4.1.2.12. Season wise prevalence of babesiosis in Holstein Friesian in District Sibi……………………………………………………………………...111 Table 4.1.2.13. Season wise prevalence of babesiosis in Crossbred in District in Sibi…112 Table 4.1.2.14 Cumulative prevalence of Babesiosis in both Districts Quetta and Sibi……………………………………………………………………...114 Table 4.1.3.1 Overall prevalence of mixed hemoprotozoan infection in both Districts Quetta and Sibi………………………………………………………….117 Table 4.1.3.2.1. Prevalence of mixed hemoprotozoan infection in District Quetta……...118 Table 4.1.3.2.2. Prevalence of mixed hemoprotozoan