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IMMUNITY AND PERFORMANCE OF CHICKENS VACCINATED AGAINST INFECTIOUS BURSAL DISEASE A dissertation submitted to the University of Khartoum in partial fulfillment for the requirements of the degree of Master of Tropical Animal Health (M.T.A.H) By Sahar Mohmmed Osman Abdalla (B.V. Sc., University of Khartoum, 1994) Supervisor Dr. Abdelwahid Saeed Ali Department of Preventive Medicine and Public Health, Faculty of Veterinary Medicine, University of Khartoum January 2004 1 To all whom I love 2 TABLE OF CONTENTS Dedication ------------------------------------------------------------------------------iii List of tables ---------------------------------------------------------------------------iv List of figures --------------------------------------------------------------------------v Acknowledgements -------------------------------------------------------------------vi Summary--------------------------------------------------------------------------------vii Arabic summary -----------------------------------------------------------------------ix INTRODUCTION -------------------------------------------------------------------1 CHAPTER ONE: LITERATURE REVIEW 1.1. Infectious bursal disease (IBD) ------------------------------------------------3 1.1.1. Definition ------------------------------------------------------------------3 1.1.2. History----------------------------------------------------------------------3 1.1.3. Economic importance ----------------------------------------------------4 1.1.4. Clinical signs --------------------------------------------------------------5 1.1.5. Lesions ---------------------------------------------------------------------5 1.1.5.1. Macroscopic lesions-------------------------------------------------5 1.1.5.2. Microscopic lesions ------------------------------------------------6 1.1.6. Immunosuppression ------------------------------------------------------8 1.1.7. IBD in Sudan --------------------------------------------------------------10 1.2. Infectious bursal disease virus (IBDV) ---------------------------------------12 1.2.1. Classification of IBDV ---------------------------------------------------12 1.2.2. Serotypes and variants ----------------------------------------------------12 1.2.3. Very virulent strains ------------------------------------------------------14 1.2.4. Pathogenesis of IBDV ----------------------------------------------------15 1.2.5. Immunity to IBDV --------------------------------------------------------15 1.2.6. Diagnosis of IBDV--------------------------------------------------------16 1.3. Control of IBD infection -------------------------------------------------------19 1.3.1. Vaccination ---------------------------------------------------------------19 1.3.2. Types of vaccines --------------------------------------------------------21 1.3.2.1. Live vaccines ------------------------------------------------------------21 1.Mild strains ---------------------------------------------------------------21 2.Intermediate strains ------------------------------------------------------22 3 3.Hot Strains ----------------------------------------------------------------23 1.3.2.2. Inactivated vaccine ----------------------------------------------------23 1.3.2.3. Genetically engineered vaccines--------------------------------------23 1.3.3. Vaccination failure----------------------------------------------------------24 CHAPTER TWO: MATERIALS AND METHODS 2.1. Chicks -----------------------------------------------------------------------------26 2.2. IBD vaccines----------------------------------------------------------------------26 2.2.1. Intermediate D78----------------------------------------------------------26 2.2.2. Lz228E ---------------------------------------------------------------------26 2.3. Collection of blood---------------------------------------------------------------27 2.4. The agar gel precipitation test (AGPT) ---------------------------------------27 2.4.1. The antigen ----------------------------------------------------------------29 2.4.2. Procedure of the test ------------------------------------------------------27 2.5. Experiment 1----------------------------------------------------------------------28 2.5.1 Objectives-------------------------------------------------------------------28 2.5.2. Experimental plan and procedure --------------------------------------28 Experiment 2 ---------------------------------------------------------------------------29 2.6.1 Objectives-------------------------------------------------------------------29 2.6.2. Experimental plan and procedure---------------------------------------29 CHAPTER THREE: RESULTS 3.1. Experiment 1 ---------------------------------------------------------------------31 3.1.1. Maternal immunity -------------------------------------------------------31 3.1.2. Immune response to the vaccine ---------------------------------------31 3.2. Experiment2 ----------------------------------------------------------------------32 3.2.1. Maternal immunity -------------------------------------------------------32 3.2.2. Immune response to the vaccine ----------------------------------------32 3.2.3. Post mortem lesions in vaccinated chickens --------------------------33 CHAPTER FOUR: DISCUSSION -----------------------------------------------49 REFERENCES -----------------------------------------------------------------------53 APPENDIX----------------------------------------------------------------------------67 4 5 LIST OF TABLES Table -----------------------------------------------------------------------------------page 1. AGPT–Ab response in susceptible chicks vaccinated with an intermediate strain of IBDV at seven day-old (group A) ---------------34 2. AGPT Ab- response in susceptible chicks vaccinated with an intermediate strains at 14 day old (group B) ------------------------------35 3. AGPT Ab- response in susceptible chicks vaccinated with an intermediate strains at 21 day old (group C) ------------------------------36 4. AGPT, Ab- response in non-vaccinated chicks (group D) ------------------5. AGPT of day- old chicks ---------------------------------------------------------38 6. AGPT of chicks reared in closed pens at 13day-old (before vaccination)--39 7. AGPT of chicks reared in open pens at 13 day old (before vaccination). -------------------------------------------------------------------39 8. AGPT of chicks in closed pens at 20 day old (before vaccination with 228E).---------------------------------------------------------------------40 9. AGPT of chicks at open pens at 20 day-old (before vaccination with 2288). ---------------------------------------------------------------------40 10. AGPT of chicks reared in closed pens at five weeks age. ------------------41 11. AGPT of chicks reared in open pens at five weeks age.---------------------41 12. Post Morton observation at 42days-old for chicks reared in closed pens. --------------------------------------------------------------------42 13. Post Morton observation at 42 days old for chicks reared in open pens.--43 6 7 LIST OF FIGURES Fig 1. Slight hemorrhages on pectoral muscle of chick after three weeks of vaccination with 228E ---------------------------------------------------44 Fig 2. Enlarged bursa in chick after three weeks of vaccination with 228E --45 Fig3. Haemorrhages on thigh muscle of chick after three weeks of vaccination with 228E---------------------------------------------------------46 Fig 4. Haemorrages on thigh muscle of chick vaccinated with 228E ----------47 Fig 5. Bursae of chick after three weeks of vaccination with 228E. ----------48 8 ACKNOWLEDGMENTS First and foremost, my heart felt thanks to Almight Allah for giving me the strength and will power to complete this challenging task. I would like to express my gratefulness and gratitude to my supervisor Dr. Abdelwahid Saeed Ali for the tremendous help and guidance. My thanks are also due to Dr. Mahasin Elnur, Head Department of virology, Central Veterinary Research Laboratory (CVRL) for her help and advice in doing the experiments, as well as rendering all her laboratory facilities to accomplish this work. My thanks are also extended to the staff of Department of virology, (CVRL) for their help, throughout the study course. Thanks are extended to staff of the department of Preventive Medicine and Public Health. For their continuous encouragement and support. My gratitude is extended to all colleagues and friends who had been helpful whenever I ran into difficulties. Finally thanks to my family for their encouragement and great help. 9 SUMMARY This study was carried out to determine the immune response to infectious bursal disease (IBD) vaccination in maternally immuned chicks. The effect of vaccination on meat quality of broilers was also studied. IBD intermediate vaccines were given to maternally immune chicks at different ages so as to identify the best time for the vaccines to break through maternally derived antibodies (MDA). The antibody titres were determined after two weeks of vaccination using agar gel precipitation test (AGPT)and were found as follows: In chicks vaccinated at one week of age is 1: 2 in 43% of chicks and 1:4 in 34% of them. In chicks vaccinated at two weeks of age is 1: 2 in 43% of chicks and 1: 4 in 50% of them, while in chicks vaccinated at three weeks of age: 1: 2 in 31% and 1: 4 in 38% and 1: 8 in 31% of them. The results obtained also showed that vaccination of chicks at three weeks of age is recommended as very low of maternal antibodies were detected. Great variation (spread) of titres in just hatched chicks was also observed. This variation within off spring from one parent flock is attributed to titre variation between inividual mother hens. Booster dose with 228E give a slight increase in Ab response in chicks primary vaccinated with intermediate strain. In another experiment, it is concluded that vaccination of chicks using hot vaccine obviously affected the meat quality. Haemorrhages in 10 thigh and pectoral muscles and bursal lesion, were observed. There fore, hot vaccine should only be administered in severly affected areas, but not under normal conditions. The results obtained also revealed that better Ab responses to the vaccine virus were observed in chicks reared in open pens compared to those reared in closed pens. 11 ﻤﻠﺨﺹ ﺍﻷﻁﺭﻭﺤﺔ ﻫﺫﻩ ﺍﻟﺩﺭﺍﺴﺔ ﺃﺠﺭﻴﺕ ﻟﻤﻌﺭﻓﺔ ﺍﻻﺴﺘﺠﺎﺒﺔ ﺍﻟﻤﻨﺎﻋﻴﺔ ﻟﻠﺘﻁﻌـﻴﻡ ﻀـﺩ ﻤـﺭﺽ ﺍﻟﻘﻤﺒﻭﺭﻭ ﻓﻲ ﺍﻟﻜﺘﺎﻜﻴﺕ ﺫﺍﺕ ﺍﻟﻤﻨﺎﻋـﺔ ﺍﻷﻤﻴـﺔ ﻭﻟﻤﻌﺭﻓـﺔ ﺘـﺄﺜﻴﺭ ﻋﺘـﺭﺓ ﺍﻟﻠﻘـﺎﺡ ﻋﻠﻰ ﺍﻟﻠﺤﻡ ﻓﻲ ﺍﻟﺩﺠﺎﺝ ﺍﻟﻼﺤﻡ. ﺘﻡ ﺘﻁﻌﻴﻡ ﺍﻟﻜﺘﺎﻜﻴﺕ ﻓﻲ ﺃﻋﻤﺎﺭ ﻤﺨﺘﻠﻔﺔ ﺒﻠﻘﺎﺡ ﻤﺘﻭﺴﻁ ﺍﻟﻀـﺭﺍﻭﺓ ﻟﻤﻌﺭﻓـﺔ ﺍﻟﻭﻗﺕ ﺍﻟﻤﻨﺎﺴﺏ ﻻﺨﺘﺭﺍﻕ ﺍﻟﻤﻨﺎﻋﺔ ﺍﻷﻤﻴﺔ. ﺘﻡ ﻗﻴﺎﺱ ﻤﺘﻭﺴﻁ ﺍﻟﻤﻌﻴﺎﺭ ﺒﻌﺩ ﺃﺴﺒﻭﻋﻴﻥ ﻤـﻥ ﺇﻋﻁـﺎﺀ ﺍﻟﻠﻘـﺎﺡ ﺒﺎﺴـﺘﻌﻤﺎل ﺍﺨﺘﺒﺎﺭ AGPTﻭﻗﺩ ﻭﺠﺩﺕ ﻜﺎﻵﺘﻲ : ﻓﻲ ﺍﻟﻜﺘﺎﻜﻴﺕ ﺍﻟﺘﻲ ﺘﻡ ﺘﻁﻌﻴﻤﻬﺎ ﻓﻲ ﻋﻤـﺭ ﺃﺴـﺒﻭﻉ ½ ﻓـﻲ %43 ﻤﻥ ﺍﻟﻜﺘﺎﻜﻴﺕ ﻭ ¼ ﻓﻲ %34ﻤﻨﻬﺎ. ﻓﻲ ﺍﻟﻜﺘﺎﻜﻴﺕ ﺍﻟﺘﻲ ﺘﻡ ﺘﻁﻌﻴﻤﻬﺎ ﻓﻲ ﻋﻤﺭ ﺃﺴﺒﻭﻋﻴﻥ ½ ﻓﻲ %43 ﻤﻥ ﺍﻟﻜﺘﺎﻜﻴﺕ ﻭ ¼ ﻓﻲ % 50ﻤﻨﻬﺎ .ﻭﺍﻟﺘﻲ ﺘﻡ ﺘﻁﻌﻴﻤﻬﺎ ﻓﻲ ﻋﻤﺭ ﺜﻼﺜﺔ ﺃﺴﺎﺒﻴﻊ ½ ﻓﻲ %31ﻤﻨﻬﺎ. ﺃﻭﻀﺤﺕ ﺍﻟﻨﺘﺎﺌﺞ ﺘﻁﻌﻴﻡ ﺍﻟﻜﺘﺎﻜﻴﺕ ﻓﻲ ﻋﻤﺭ ﺜﻼﺜﺔ ﺃﺴـﺎﺒﻴﻊ ﻴﻌﻁـﻲ ﻨﺘـﺎﺌﺞ ﺃﻓﻀل ﺒﺤﻴﺙ ﺘﻨﺨﻔﺽ ﺍﻟﻤﻨﺎﻋﺔ ﺍﻷﻤﻴﺔ. ﻟﻭﺤﻅ ﺃﻴﻀﹰﺎ ﺍﺨﺘﻼﻑ ﻓﻲ ﻤﺘﻭﺴﻁ ﺍﻟﻤﻌﻴﺎﺭ ﻓﻲ ﺍﻟﻜﺘﺎﻜﻴـﺕ ﻭﻫـﺫﺍ ﺍﻻﺨـﺘﻼﻑ ﻗﺩ ﻴﻌﺯﻯ ﻻﺨﺘﻼﻑ ﻤﺘﻭﺴﻁ ﺍﻟﻤﻌﻴﺎﺭ ﺒﻴﻥ ﺍﻷﻤﻬﺎﺕ ﻓﻲ ﻨﻔﺱ ﺍﻟﻘﻁﻴﻊ. 12 ﺇﻋﻁﺎﺀ ﺠﺭﻋﺔ ﻤﻨﺸﻁﺔ ﺒﻠﻘﺎﺡ 228E ﺘﻌﻁـﻲ ﺯﻴـﺎﺩﺓ ﻓـﻲ ﺍﻻﺴـﺘﺠﺎﺒﺔ ﺍﻟﻤﻨﺎﻋﻴـﺔ ﻓﻲ ﺍﻟﻜﺘﺎﻜﻴﺕ ﺍﻟﺘﻲ ﺴﺒﻕ ﺘﻁﻌﻴﻤﻬﺎ ﺒﻠﻘﺎﺡ ﻤﺘﻭﺴﻁ ﺍﻟﻀﺭﺍﻭﺓ. ﻓﻲ ﺘﺠﺭﺒﺔ ﺃﺨﺭﻯ ﻨﺨﻠﺹ ﺇﻟﻰ ﺃﻥ ﺘﻁﻌﻴﻡ ﺍﻟﻜﺘﺎﻜﻴﺕ ﺒﺎﺴﺘﻌﻤﺎل ﻟﻘﺎﺡ ﺸـﺩﻴﺩ ﺍﻟﻀﺭﺍﻭﺓ ﻴﺅﺜﺭ ﻋﻠﻰ ﻨﻭﻉ ﺍﻟﻠﺤﻡ. ﻟﻬﺫﺍ ﺍﻟﺴﺒﺏ ﺍﺴﺘﻌﻤﺎل ﻟﻘﺎﺡ ﺸﺩﻴﺩ ﺍﻟﻀﺭﺍﻭﺓ ﻴﺠﺏ ﺃﻥ ﻴﻌﻁﻰ ﻓﻘﻁ ﻓﻲ ﺍﻟﻤﻨﺎﻁﻕ ﺍﻟﺸﺩﻴﺩﺓ ﺍﻹﺼﺎﺒﺔ ﻭﻟﻜﻥ ﻟﻴﺱ ﻓﻲ ﺍﻷﺤﻭﺍل ﺍﻟﻌﺎﺩﻴﺔ . ﺃﻭﻀﺤﺕ ﺍﻟﻨﺘﺎﺌﺞ ﺃﻴﻀﹰﺎ ﺘﻭﺠﺩ ﺍﺴﺘﺠﺎﺒﺔ ﻤﻨﺎﻋﻴﺔ ﺃﻓﻀل ﻓﻲ ﺍﻟﻜﺘﺎﻜﻴﺕ ﺘﺭﺒﻰ ﻓﻲ ﺤﻅﺎﺌﺭ ﻤﻔﺘﻭﺤﺔ ﻤﻘﺎﺭﻨﺔ ﺒﺎﻟﺘﻲ ﺘﺭﺒﻰ ﻓﻲ ﺤﻅﺎﺌﺭ ﻤﻐﻠﻘﺔ. 13 ﺍﻟﺘـﻲ TABLE OF CONTENTS Dedication ------------------------------------------------------------------------------iii List of tables ---------------------------------------------------------------------------iv List of figures --------------------------------------------------------------------------v Acknowledgements -------------------------------------------------------------------vi Summary--------------------------------------------------------------------------------vii Arabic summary -----------------------------------------------------------------------ix INTRODUCTION -------------------------------------------------------------------1 CHAPTER ONE: LITERATURE REVIEW 1.1. Infectious bursal disease (IBD) ------------------------------------------------3 1.1.1. Definition ------------------------------------------------------------------3 1.1.2. History----------------------------------------------------------------------3 1.1.3. Economic importance ----------------------------------------------------4 1.1.4. Clinical signs --------------------------------------------------------------5 1.1.5. Lesions ---------------------------------------------------------------------5 1.1.5.1. Macroscopic lesions-------------------------------------------------5 1.1.5.2. Microscopic lesions ------------------------------------------------6 1.1.6. Immunosuppression ------------------------------------------------------8 1.1.7. IBD in Sudan --------------------------------------------------------------10 1.2. Infectious bursal disease virus (IBDV) ---------------------------------------12 1.2.1. Classification of IBDV ---------------------------------------------------12 1.2.2. Serotypes and variants ----------------------------------------------------12 1.2.3. Very virulent strains ------------------------------------------------------14 1.2.4. Pathogenesis of IBDV ----------------------------------------------------15 1.2.5. Immunity to IBDV --------------------------------------------------------15 1.2.6. Diagnosis of IBDV--------------------------------------------------------16 1.3. Control of IBD infection -------------------------------------------------------19 1.3.1. Vaccination ---------------------------------------------------------------19 1.3.2. Types of vaccines --------------------------------------------------------21 1.3.2.1. Live vaccines ------------------------------------------------------------21 1.Mild strains ---------------------------------------------------------------21 2.Intermediate strains ------------------------------------------------------22 14 3.Hot Strains ----------------------------------------------------------------23 1.3.2.2. Inactivated vaccine ----------------------------------------------------23 1.3.2.3. Genetically engineered vaccines--------------------------------------23 1.3.3. Vaccination failure----------------------------------------------------------24 CHAPTER TWO: MATERIALS AND METHODS 2.1. Chicks -----------------------------------------------------------------------------26 2.2. IBD vaccines----------------------------------------------------------------------26 2.2.1. Intermediate D78----------------------------------------------------------26 2.2.2. Lz228E ---------------------------------------------------------------------26 2.3. Collection of blood---------------------------------------------------------------27 2.4. The agar gel precipitation test (AGPT) ---------------------------------------27 2.4.1. The antigen ----------------------------------------------------------------29 2.4.2. Procedure of the test ------------------------------------------------------27 2.5. Experiment 1----------------------------------------------------------------------28 2.5.1 Objectives-------------------------------------------------------------------28 2.5.2. Experimental plan and procedure --------------------------------------28 Experiment 2 ---------------------------------------------------------------------------29 2.6.1 Objectives-------------------------------------------------------------------29 2.6.2. Experimental plan and procedure---------------------------------------29 CHAPTER THREE: RESULTS 3.1. Experiment 1 ---------------------------------------------------------------------31 3.1.1. Maternal immunity -------------------------------------------------------31 3.1.2. Immune response to the vaccine ---------------------------------------31 3.2. Experiment2 ----------------------------------------------------------------------32 3.2.1. Maternal immunity -------------------------------------------------------32 3.2.2. Immune response to the vaccine ----------------------------------------32 3.2.3. Post mortem lesions in vaccinated chickens --------------------------33 CHAPTER FOUR: DISCUSSION -----------------------------------------------49 REFERENCES -----------------------------------------------------------------------53 APPENDIX----------------------------------------------------------------------------67 15 16 LIST OF TABLES Table -----------------------------------------------------------------------------------page 1. AGPT–Ab response in susceptible chicks vaccinated with an intermediate strain of IBDV at seven day-old (group A) ---------------34 2. AGPT Ab- response in susceptible chicks vaccinated with an intermediate strains at 14 day old (group B) ------------------------------35 3. AGPT Ab- response in susceptible chicks vaccinated with an intermediate strains at 21 day old (group C) ------------------------------36 4. AGPT, Ab- response in non-vaccinated chicks (group D) ------------------5. AGPT of day- old chicks ---------------------------------------------------------38 6. AGPT of chicks reared in closed pens at 13day-old (before vaccination)--39 7. AGPT of chicks reared in open pens at 13 day old (before vaccination). -------------------------------------------------------------------39 8. AGPT of chicks in closed pens at 20 day old (before vaccination with 228E).---------------------------------------------------------------------40 9. AGPT of chicks at open pens at 20 day-old (before vaccination with 2288). ---------------------------------------------------------------------40 10. AGPT of chicks reared in closed pens at five weeks age. ------------------41 11. AGPT of chicks reared in open pens at five weeks age.---------------------41 12. Post Morton observation at 42days-old for chicks reared in closed pens. --------------------------------------------------------------------42 13. Post Morton observation at 42 days old for chicks reared in open pens.--43 17 18 LIST OF FIGURES Fig 1. Slight hemorrhages on pectoral muscle of chick after three weeks of vaccination with 228E ---------------------------------------------------44 Fig 2. Enlarged bursa in chick after three weeks of vaccination with 228E --45 Fig3. Haemorrhages on thigh muscle of chick after three weeks of vaccination with 228E---------------------------------------------------------46 Fig 4. Haemorrages on thigh muscle of chick vaccinated with 228E ----------47 Fig 5. Bursae of chick after three weeks of vaccination with 228E. ----------48 19 ACKNOWLEDGMENTS First and foremost, my heart felt thanks to Almight Allah for giving me the strength and will power to complete this challenging task. I would like to express my gratefulness and gratitude to my supervisor Dr. Abdelwahid Saeed Ali for the tremendous help and guidance. My thanks are also due to Dr. Mahasin Elnur, Head Department of virology, Central Veterinary Research Laboratory (CVRL) for her help and advice in doing the experiments, as well as rendering all her laboratory facilities to accomplish this work. My thanks are also extended to the staff of Department of virology, (CVRL) for their help, throughout the study course. Thanks are extended to staff of the department of Preventive Medicine and Public Health. For their continuous encouragement and support. My gratitude is extended to all colleagues and friends who had been helpful whenever I ran into difficulties. Finally thanks to my family for their encouragement and great help. 20 SUMMARY This study was carried out to determine the immune response to infectious bursal disease (IBD) vaccination in maternally immuned chicks. The effect of vaccination on meat quality of broilers was also studied. IBD intermediate vaccines were given to maternally immune chicks at different ages so as to identify the best time for the vaccines to break through maternally derived antibodies (MDA). The antibody titres were determined after two weeks of vaccination using agar gel precipitation test (AGPT)and were found as follows: In chicks vaccinated at one week of age is 1: 2 in 43% of chicks and 1:4 in 34% of them. In chicks vaccinated at two weeks of age is 1: 2 in 43% of chicks and 1: 4 in 50% of them, while in chicks vaccinated at three weeks of age: 1: 2 in 31% and 1: 4 in 38% and 1: 8 in 31% of them. The results obtained also showed that vaccination of chicks at three weeks of age is recommended as very low of maternal antibodies were detected. Great variation (spread) of titres in just hatched chicks was also observed. This variation within off spring from one parent flock is attributed to titre variation between inividual mother hens. Booster dose with 228E give a slight increase in Ab response in chicks primary vaccinated with intermediate strain. In another experiment, it is concluded that vaccination of chicks using hot vaccine obviously affected the meat quality. Haemorrhages in 21 thigh and pectoral muscles and bursal lesion, were observed. There fore, hot vaccine should only be administered in severly affected areas, but not under normal conditions. The results obtained also revealed that better Ab responses to the vaccine virus were observed in chicks reared in open pens compared to those reared in closed pens. 22 ﻤﻠﺨﺹ ﺍﻷﻁﺭﻭﺤﺔ ﻫﺫﻩ ﺍﻟﺩﺭﺍﺴﺔ ﺃﺠﺭﻴﺕ ﻟﻤﻌﺭﻓﺔ ﺍﻻﺴﺘﺠﺎﺒﺔ ﺍﻟﻤﻨﺎﻋﻴﺔ ﻟﻠﺘﻁﻌـﻴﻡ ﻀـﺩ ﻤـﺭﺽ ﺍﻟﻘﻤﺒﻭﺭﻭ ﻓﻲ ﺍﻟﻜﺘﺎﻜﻴﺕ ﺫﺍﺕ ﺍﻟﻤﻨﺎﻋـﺔ ﺍﻷﻤﻴـﺔ ﻭﻟﻤﻌﺭﻓـﺔ ﺘـﺄﺜﻴﺭ ﻋﺘـﺭﺓ ﺍﻟﻠﻘـﺎﺡ ﻋﻠﻰ ﺍﻟﻠﺤﻡ ﻓﻲ ﺍﻟﺩﺠﺎﺝ ﺍﻟﻼﺤﻡ. ﺘﻡ ﺘﻁﻌﻴﻡ ﺍﻟﻜﺘﺎﻜﻴﺕ ﻓﻲ ﺃﻋﻤﺎﺭ ﻤﺨﺘﻠﻔﺔ ﺒﻠﻘﺎﺡ ﻤﺘﻭﺴﻁ ﺍﻟﻀـﺭﺍﻭﺓ ﻟﻤﻌﺭﻓـﺔ ﺍﻟﻭﻗﺕ ﺍﻟﻤﻨﺎﺴﺏ ﻻﺨﺘﺭﺍﻕ ﺍﻟﻤﻨﺎﻋﺔ ﺍﻷﻤﻴﺔ. ﺘﻡ ﻗﻴﺎﺱ ﻤﺘﻭﺴﻁ ﺍﻟﻤﻌﻴﺎﺭ ﺒﻌﺩ ﺃﺴﺒﻭﻋﻴﻥ ﻤـﻥ ﺇﻋﻁـﺎﺀ ﺍﻟﻠﻘـﺎﺡ ﺒﺎﺴـﺘﻌﻤﺎل ﻭﻗﺩ ﻭﺠﺩﺕ ﻜﺎﻵﺘﻲ AGPT:ﺍﺨﺘﺒﺎﺭ ﻓﻲ %43 ½ﻓﻲ ﺍﻟﻜﺘﺎﻜﻴﺕ ﺍﻟﺘﻲ ﺘﻡ ﺘﻁﻌﻴﻤﻬﺎ ﻓﻲ ﻋﻤﺭ ﺃﺴﺒﻭﻉ ﻓﻲ %34ﻤﻨﻬﺎ ¼.ﻤﻥ ﺍﻟﻜﺘﺎﻜﻴﺕ ﻭ ﻓﻲ ½ %43ﻓﻲ ﺍﻟﻜﺘﺎﻜﻴﺕ ﺍﻟﺘﻲ ﺘﻡ ﺘﻁﻌﻴﻤﻬﺎ ﻓﻲ ﻋﻤﺭ ﺃﺴﺒﻭﻋﻴﻥ ﻓﻲ % 50ﻤﻨﻬﺎ .ﻭﺍﻟﺘﻲ ﺘﻡ ﺘﻁﻌﻴﻤﻬﺎ ﻓﻲ ﻋﻤﺭ ﺜﻼﺜﺔ ¼ ﻤﻥ ﺍﻟﻜﺘﺎﻜﻴﺕ ﻭ ﻓﻲ %31ﻤﻨﻬﺎ ½ .ﺃﺴﺎﺒﻴﻊ ﺃﻭﻀﺤﺕ ﺍﻟﻨﺘﺎﺌﺞ ﺘﻁﻌﻴﻡ ﺍﻟﻜﺘﺎﻜﻴﺕ ﻓﻲ ﻋﻤﺭ ﺜﻼﺜﺔ ﺃﺴـﺎﺒﻴﻊ ﻴﻌﻁـﻲ ﻨﺘـﺎﺌﺞ ﺃﻓﻀل ﺒﺤﻴﺙ ﺘﻨﺨﻔﺽ ﺍﻟﻤﻨﺎﻋﺔ ﺍﻷﻤﻴﺔ. ﻟﻭﺤﻅ ﺃﻴﻀﹰﺎ ﺍﺨﺘﻼﻑ ﻓﻲ ﻤﺘﻭﺴﻁ ﺍﻟﻤﻌﻴﺎﺭ ﻓﻲ ﺍﻟﻜﺘﺎﻜﻴـﺕ ﻭﻫـﺫﺍ ﺍﻻﺨـﺘﻼﻑ ﻗﺩ ﻴﻌﺯﻯ ﻻﺨﺘﻼﻑ ﻤﺘﻭﺴﻁ ﺍﻟﻤﻌﻴﺎﺭ ﺒﻴﻥ ﺍﻷﻤﻬﺎﺕ ﻓﻲ ﻨﻔﺱ ﺍﻟﻘﻁﻴﻊ. 23 228Eﺇﻋﻁﺎﺀ ﺠﺭﻋﺔ ﻤﻨﺸﻁﺔ ﺒﻠﻘﺎﺡ ﺘﻌﻁﻲ ﺯﻴﺎﺩﺓ ﻓﻲ ﺍﻻﺴﺘﺠﺎﺒﺔ ﺍﻟﻤﻨﺎﻋﻴﺔ ﻓﻲ ﺍﻟﻜﺘﺎﻜﻴﺕ ﺍﻟﺘﻲ ﺴﺒﻕ ﺘﻁﻌﻴﻤﻬﺎ ﺒﻠﻘﺎﺡ ﻤﺘﻭﺴﻁ ﺍﻟﻀﺭﺍﻭﺓ. ﻓﻲ ﺘﺠﺭﺒﺔ ﺃﺨﺭﻯ ﻨﺨﻠﺹ ﺇﻟﻰ ﺃﻥ ﺘﻁﻌﻴﻡ ﺍﻟﻜﺘﺎﻜﻴﺕ ﺒﺎﺴﺘﻌﻤﺎل ﻟﻘﺎﺡ ﺸـﺩﻴﺩ ﺍﻟﻀﺭﺍﻭﺓ ﻴﺅﺜﺭ ﻋﻠﻰ ﻨﻭﻉ ﺍﻟﻠﺤﻡ. ﻟﻬﺫﺍ ﺍﻟﺴﺒﺏ ﺍﺴﺘﻌﻤﺎل ﻟﻘﺎﺡ ﺸﺩﻴﺩ ﺍﻟﻀﺭﺍﻭﺓ ﻴﺠﺏ ﺃﻥ ﻴﻌﻁﻰ ﻓﻘﻁ ﻓﻲ ﺍﻟﻤﻨﺎﻁﻕ ﺍﻟﺸﺩﻴﺩﺓ ﺍﻹﺼﺎﺒﺔ ﻭﻟﻜﻥ ﻟﻴﺱ ﻓﻲ ﺍﻷﺤﻭﺍل ﺍﻟﻌﺎﺩﻴﺔ . ﺃﻭﻀﺤﺕ ﺍﻟﻨﺘﺎﺌﺞ ﺃﻴﻀﹰﺎ ﺘﻭﺠﺩ ﺍﺴـﺘﺠﺎﺒﺔ ﻤﻨﺎﻋﻴـﺔ ﺃﻓﻀـل ﻓـﻲ ﺍﻟﻜﺘﺎﻜﻴـﺕ ﺍﻟﺘﻲ ﺘﺭﺒﻰ ﻓﻲ ﺤﻅﺎﺌﺭ ﻤﻔﺘﻭﺤﺔ ﻤﻘﺎﺭﻨﺔ ﺒﺎﻟﺘﻲ ﺘﺭﺒﻰ ﻓﻲ ﺤﻅﺎﺌﺭ ﻤﻐﻠﻘﺔ. 24 INTRODUCTION Infectious bursal disease (IBD) is an acute, highly contagious, viral disease of young chickens characterized by destruction of the lymphoid cells in the bursa of Fabricius and other lymphoid organs. The virus particularly infects the actively dividing and differentiating B-lymphocyte. Although the disease was diagnosed in older age of chickens but young chicks are more susceptible. The clinical disease is responsible for losses due to impaired growth and excessive condemnation of carcasses because of skeletal muscle hemorrhages. Infected chickens less than three weeks of age don’t exhibit clinical signs but have a sub-clinical infection characterized by macroscopic lesions in the bursa of Fabricius and immunosuppression. The greatest economic losses of the disease results from immunosuppression. This causes increased susceptibility to other diseases, and interfer with effective vaccination against other diseases such as Newcastle disease (ND), Marek’s disease (MD) and infectious bronchitis (IB). Therefore, IBD considered as one of the most important viral infections of commercial poultry. Following oral inoculation, initial viral replication occurs in gutassociated lymphoid cells and secondary replication occurs in the bursa of Fabricius. 25 The infectious bursal disease virus (IBDV) is very stable under hard environmental conditions. Houses of infected birds will remain infective for more than four months following the termination of the clinical disease. Water, feed, and dropping taken from infected pens were also remain infectious for long time. There is no evidence that IBDV is transmitted through the egg or that a true carrier state sxist in recovered birds. Immunization is the principal method used for the control of IBD in chickens especially important is the active immunization of breeder flocks so as to confer parental immunity to their progeny. Such maternal antibodies protect the chick for early immunosuppressive infections. Maternal antibody will normally protect chicks for 1-3 weeks, but by boosting the immunity in breeder flocks with oil adjuvant vaccines, passive immunity may be extended to four or five weeks. Objectives of the present work 1. To determine the break down in maternally immune chicks by using intermediate vaccine strain of IBDV. 2. To evaluate the immune response to vaccines mostly used in the Sudan (D78, 228E) and to study the effect of these vaccines on performance of broilers. 26 CHAPTER ONE LITERATURE REVIEW 1.1 . Infectious bursal disease (IBD) 1.1.1. Definition It is a highly contagious viral disease of chickens, which is recognized world-wide. The outcome of the disease was reported depending on the virulence of virus strain, age and immune status of the chickens chickens at time of infection (Weiss and Ilse Käufer, 1994). The incubation period is short, 2-3 days, after infection (Käufer, Ilse and Weiss, 1976). 1.1.2. History The disease was first described by Cosgrove (1962) and referred to as, avian nephrosis because of the extreme kidney damage found in infected birds. Later, Winterfield et al. (1962) isolated the virus and named it infectious bursal agent (IBA). Hitchner (1970) proposed the term infectious bursal disease (IBD) as the name of the disease causing specific pathognomic lesions of the cloacal bursa. Allan et al. (1972) reported that IBD virus infection at an early age were immunosuppressive. The existence of a second serotype was reported by Mc Ferran et al. (1980). Variant strains of serotype1 of IBDV were found in Delmarva poultry-producing area, USA (Saif, 1984). These strains were breaking through materanl immunity against “standard” strains, and they also differed from standard strains in their biological properties. 27 1.1.3. Economic importance The economic impact of infectious bursal disease is influnced by strain of the virus, susceptibility and breed of flock, inter current primary and secondary pathogen, and environmental and management factors (Shane et al., 1994) in recent study, it was also documented that the ecnomic impact of the IBD out break is influenced by season, floor, space/broiler, age of the bird, immunization schedule, interval between two batches, presence of coccidiosis in a flock and hygienic status of the farm (Farooq, et al., 2003). In addition to mortality, IBDV is immunosuppressive (Allan et al., 1972) which results in increased losses due to primary viral and secondary bacterial respiratory infections and result in a decline in egg production, hatchability, and shell quality. Simulation studies can predict the microeconomic cost of IBD infection in an integrated enterprise. Flocks liveability, weight gain, feed conversion, and reproductive efficiency are all adversely affected (Shane et al., 1994). In study performed between 1993 and 1997, IBDV antibody titres and performance data were recorded in a vertically integrated monitoring scheme in order to make a follow-up for day old parents down to the broilers at slaughter (DeHerdt et al., 2000). It appeared that high and/or uniform antibody titres in the parents were correlated with increased daily weight gain and decreased mortality and slaughter house condemnation in the broilers. 28 1.1.4. Clinical signs Cosgrove (1962) first described the clinical signs of IBD as soiled vent feather, trembling, severe prostration, and finally death. Affected birds became dehydrated and in terminal stage of the disease, had a subnormal temperature. Lukert and Saif (1991) reported that in fully susceptible flocks, the disease appears suddenly and there is a high morbidity rate, usually approaching 100%. Morality usually begins on the 3rd day post infection and will peak and recede in a period of 5-7 days. Actual mortality may be nil but can be as high as 20-30%. Striking feature of this disease are the sudden and high morbidity rates, spiking death curve, and rapid flock recovery. Initial outbreaks on a farm are usually the most acute. Recurrent outbreaks in succeeding broods are less severs and frequently go undetected. Many infections are silent, owing to age of birds (Less than 3 wk), infection with a virulent field strains, or infection in presence of maternal antibody. 1.1.5. Lesions 1.1.5.1. Macroscopic lesions Lesions normally appear in the bursa before the onset of clinical signs. Helmboldt and Garner (1964) detected histologic evidence of IBD infection in the cloacal bursa within 24hr. Bursa of Fabricius is the target organ for IBDV (Käufer, and Weiss, 1980). Weiss and Käufer (1994), using 29 immunofluorescent techniques, observed infected gut-associated macrophages and lymphoid cells within 4-5hr after an exposure to IBDV (organ of primary affinity). In the bursa of Fabricius, massive virus replication usually takes place (Strong specific fluorescence from 11hr post infection) and leads to the pronounced viremia. Gross lesions included dehydration with darkened discoloration of pectoral muscles. Frequently, hemorrhages are present in the thigh and pectoral musdes. There is increased mucus in the intestine, and renal changes may be prominent in birds that die or are in advanced stages of the disease (Cosgrove, 1962). Occasionally, hemorrhages are observed in the mucosa at the juncture of the proventriculus and gizzard on the 3rd day post infection and the bursa begins to increase in size and weight because of odema and hyperemia. By the 4th day, it usually is double its normal weight and then it begins to recede in size. By the 5th day it has returned to normal weight, but the bursa continues to atrophy and from the 8th day on its approximately one-third its original weight. Usually double its normal weight, but the bursa continues to atrophy and from the 8th day on it is approximately one third it is original weights s (Lukert and Saif, 1991). Variant isolates of IBDV were reported not to induce an inflammatory response (Sharma, Dohms and Metz, 1989). Hence initial enlargement of the bursa might not be observed. 30 1.1.5.2. Microscopic lesions Histopathologic lesions of IBD occur primarly in the lymphoid structures, cloacal bursa, splecn, thymus, harderian gland, and caecal tonsils. Histopathology at the level of light microscopy has been studied by Helmboldt and Garner (1964). As early as one-day post infection there was degeneration and necrosis of lymphocytes in the medullary area of the bursal follicle. Lymphocytes were soon replaced by heterophils, pyknotic debris and hyperplastic reticuloendothelial cells. Hemorrhages often appear but were not consistent (Lukert and Saif, 1991). All lymphoid follicles were affected by3 or 4 days post infection. As the inflammatory reaction declined, cystic cavities developed in medullary areas of follicles, necrosis and phagocytosis of heterophils and plasma cell occurred and there was a fibroplasia in interfollicular connective tissues (Lukert and Saif, 1991). Proliferation of the bursal epithelial layer produced a glandular structure of columnar epithelial cells containing globules of mucin. The spleen showed hyperplasia of reticulo endothetial cells around the adenoid sheath and arteries in early stages of infection. By the 3rd day, there was lymphoid necrosis in the germinal follicles and periarteriolar lymphoid sheath. The spleen recovered from the infection rather rapidly, with no sustained damage to the germinal follicles (Lukert and Saif, 1991). The thymus and caecal tonsil exhibited some cellular reaction in the lymphoid tissues in early stage of infection but was less extensive than in the bursa (Lukert and 31 Saif, 1991). The harderian gland was severely affected by infection of dayold chicks with IBDV, which prevented infiltration of the gland by plasma cells (Dohms et al., 1981). 1.1.6. Immunosuppression Allan et al. (1972) and Faragher et al. (1974) reported immunosuppressive effects of IBDV infections. Suppression of the antibody response to Newcastle (ND) virus was greatest in chicks infected with IBD at 1 day of age. There was moderate suppression when chicks were infected at 7 days and negligible effects when infection was at 14 or 21 days (Farragher et al., 1974). Li-Wei Jen and Cho (1980) demonstrated that when IBDV inoculated simultaneously with Turkey herpes virus (HVT) vaccination or at 3 weeks post vaccination, caused a significant immuno-suppression of the production of virus neutralizing (VN) antibodies in the vaccinated chickens. This would result in a reduced antiviral immunity against Marek’s disease virus (MDV) and possibly viral antigens. Not only was the immune response to vaccines suppressed but chicks infected early with IBDV were more susceptible to hemorrhagic aplastic anemia and gangrenous dermatitis (Rosenberger et al., 1975), salmonellosis and colibacillosis (Wyeth, 1975), inculusion body hepatitis (Fadley et al., 1976) coccidosis (Anderson et al., 1977), infectious laryngotracheitis (Rosenberger and Gelb, 1978) infectious bronchitis 32 (Pejkovski, Davelaar and Kouwenhoven, 1979) and chicken anemia agent (Yuasa et al.,1980). Giambrone et al. (1976) reported that chickens vaccinated with Turkey herpes virus develop gross Marek’s disease, lesion more frequently when they were exposed to IBDV. A paradox associated with IBDV infections of chickens is that while there is immunosuppression against many antigens, the response against IBD it self is normal, even in day-old susceptible chickens (Skeeles et al., 1979). There appears to be a stimulation of the proliferation of B-cells committed to anti-IBDV antibody production (Skeeles et al., 1979). Immunosuppression due to IBDV infection has been reported to depend on the age of birds ( Higashihara et al., 1991). It was established that immunosuppression is also correlated with the virulence of IBDV strains, Sivanandand and Maheswaran (1981) observed suppression of cellmediated immune (CMI) responsiveness, using the lymphoblast transformation assay, in IBD infected chickens. They found that maximal depression of cellular immunity occurred 6 weeks post infection. IBDV infection of 1-5 days old chicks produced a drastic reduction in plasma cell content of the harderian gland that persisted for up to 7 weeks (Dhoms et al., 1981). .Chickens infected with IBDV at 1 day of age were completely deficient in serum immunoglobulin G (IgG) and produced only 33 amonomenic IgM (Ivanyi, 1975). The number of B-cells in peripheral blood was decreased following infection with IBDV but T- cells were not appreciably affected (Sivanandan and Maheswaran, 1980). T-cells mediated and humoral immune responses were measured in chickens infected with standard and variant strains of IBDV in one-day old and 3 week-old chicken (Craft, et al., 1990). It was found that during the first week of infection, 1-day old and 3 week-old chickens had lower neutralizing antibody titres to the variant strain than to the standard strain. The lymphoblast transformation responses indicated that the variant strain was significantly more suppressive than the standard strain in one-day-old chickens. Three-week old-chickens had humoral immune suppression with the standard strain, but not with the variant strain and the lymphoblast transformation response was transiently suppressed at age by the variant strains only. 1.1.7. IBD in Sudan In December 1980 and early January 1981, the disease was observed in 6 -weeks old chickens at El Obied government poultry unit (Western Sudan). A morality rate of 36% was reported in that outbreak. Another outbreak in chicks introduced in the same premises in March 1981, resulted into 22% mortality. The virus was isolated by Mohammed and co-workers (1982) and since then the disease has been persistently reported in the country. In Khartoum, the disease was reported by Hajer et al. (1988). In 34 Kassala, the disease was reported by Elamin et al. (1988). In all the above mentioned reports, IBD occurred mostly in layer chicks at the age of 2-12 weeks with no previous history of IBD vaccination. The mortality ranged from 17.8 to 66.6%. In August 1986, an outbreak of IBD of high mortality was recorded in 40 day-old chicks imported from Holland to a private poultry farm at Sennar in the central region of the country. Morbidity and mortality rates were 15.06-14.1% receptively (Ginawi and Shuaib, 1988; Ginawi and Shuaib, 1993). In February 1990, an outbreak occurred in a large poultry project situated South East of Khartoum. The affected birds vaccinated against Newcastle disease (ND) and infectious broanchitis (IB) and their parents were vaccinated against IBD at 2,5 and 15 weeks of age using TAD Gumboro vaccine in drinking water. The data presented in this outbreak revealed that a low virulent strain of IBDV is present among broiler chicks in the Sudan (Kalafalla et al., 1990-1991). Poultry diseases diagnosed in El Damer province for the period of 1993-1997 revealed that the IBD infection is about 17% (Hussein, et al.,1998). Serological detection of IBD antibodies among non- vaccinated, non-previously infected flocks confirmed the existence of sub-clinical IBD in the Sudan (Mahasin, 1998). The vaccination failure to IBDV in the field was attributed to this subclinical IBD. The field viruses isolated after 1994 were similer to each other in the degree of the pathogenicity and were more pathogenic than those isolated 35 before. Incidence of the disease was recorded throughout the year but seasonality was obvious since continous outbreaks were recorded during the rainy season (Mahasin, 1998). 1.2. Infectious bursal disease virus (IBDV) 1.2.1. Classification of IBDV Infectious bursal disease virus (IBDV) is a member of the family Birnaviridae (Dobos et al., 1979). The family comprises three genera: Avibirnavirus, Aquabirnavirus, and Entombirnavirus. Infectious bursal disease virus is the sole member of the genus Avibirnavirus. 1.2.2. Serotypes and variants McFerran et al. (1980) were the first to report antigenic variations among IBDV isolates of European origin. They presented evidence for the presence of two serotypes designated 1 and 2. Similar results were reported in the USA by Jackwood et al. (1982). Mc Nulty and Saif (1988) indicated the relatedness of the European and the American isolates of the second serotype. The two serotypes are differentiated by virus neutralization (VN) test, although they share a common antigen (Saif, 1994). Serotype1 viruses are only pathogenic for chickens and differ markedly in their virulence, whereas serotype2 viruses infect chickens and turkeys but these infections are of unknown clinical significance (Ismial, Saif and Moorhead, 1988). Viruses of serotype2 have been isolated from chickens (Ismail et al., 1988) Immunization against serotype2 does not 36 protect against serotype1 and antibodies to serotype2 IBDV are common in both chickens and turkeys (Jackwood and Saif, 1983). In USA, Saif (1984) isolated a virus from broiler chicks that had bursal lesions inspite of the presence of high levels of maternal antibodies. This virus (MD strain) was shown to be antigenically different from strains isolated prior to that time hence, it was designated as a variant, whereas virus isolated before 1984 were identified as classic viruses. Jackwood and Saif (1987) conducted a cross neutralization study of eight serotype1 commercial vaccine strains, five serotype1 field strains, and two serotype2 field strains. Six subtypes were distinguished among the 13serotype1 strains studied, one of the subtype included all of the variant isolates. Synder et al. (1988) using monoclonal antibodies, suggested that a major antigenic shift in serotype1 viruses had occurred in the filed. Antigenicity and immunogencity studies conducted by Saif (1994) involving the use of classic and variant strains of the virus showed that, the variants as a group were significantly different from the classic viruses. These studies also showed that the classic and variant serotype1 viruses share protective and non protective antigens. Beside that, vaccines made of variant viruses protect against both classic and variant viruses whereas standard vaccines protect from homologus virus and are less effective against variant viruses. 37 1.2.3. Very virulent strains In Europe, “very virulent” (vv) strains of IBDV, which can cause up to 70% mortality in laying pullets, have emerged since 1986 (Chettle, Stuart, Wyeth, 1989). These strains cause lesions typical of IBD and are antigenically similar to the classical European strain, which have been prevalent for some decades, but can establish infection in the face of levels of maternal antibody that were protective against classical strain. Tanimura et al. (1994) showed that the lesions were typical of classical virulent strains of IBDV but they were more extensive and pronounced. In August 1990, outbreaks of an acute IBDV with high mortality occurred in broiler chicken flocks in western Japan (Nakamura et al. 1994). The same authors showed that all isolates of highly virulent IBDV were characterized as pathogenic variant, not as antigenic variant. Pathological changes caused by highly virulent European and Japanese strains were examined (Tanimura et al., 1994). The results obtained revealed that severe signs and high mortality, severe depletion of lymphoid cell not only in the bursa of Fabricius but also in the bursal lymphoid tissues, atrophy of thymus, severe depletion of hematopoietic cells in the bonemarrow, increased number of macrophages in various organs and increased frequency of viral antigen positive cells in spleen and bone marrow were observed. 38 1.2.4. Pathogenesis of IBDV It was hypothesized that the immune complex may play a role in the lesion formation for IBD (Ley and Yamamoto, 1979). Skeeles et al., (1979) found increased clotting time in IBBV infected chickens and suggested that such coagulopathies would contribute to the hemorrhagic lesions observed with the disease. It was found that T-cells modulate IBDV pathogenesis in two ways, firstly they limit viral replication in the bursa in the early phase of the disease at 5 days post infection, and secondly the intrabursal T-cells promote bursal tissue damage and delay tissue recovery possibly through the release of cytokines and cytotoxic effects (Rautenschlein et al., 2002). The potential role of apoptosis in the pathogenesis of Gumboro disease in the bursa of Fabricius was studied by Ojeda et al. (1997) and the results showed that 1-3 days after infection of young chickens with IBD, the number of apoptic cells increased and cellularity and proliferation decrease and there is concomitant increase of macrphages in infected bursae suggesting that an increase in apoptosis may be important cause of cell depletion. 1.2.5. Immunity to IBDV Winterfield (1969) described the immune response of chicks to IBDV. Birds that recovered from IBDV infection or vaccination showed serum-neutralizing activity against homologus and heterologus IBDV strains when assayed in chick embryos. Chicks exposed to IBDV at three 39 days of age did not develop as high a neutralizing titre as those exposed four weeks later (Winterfield, 1969). Maternal antibody will normally protect chicks for 1-3 weeks, but boostering breeder flocks immunity with oil adjuvant vaccines, passive immunity may be extended to 4-5week (Lucio & Hitchner, 1979; Zaheer and Saeed, 2003). Maternally derived antibodies (MDA) were found insufficient to protect broiler chicks against a highly pathogenic strain of IBDV during the growth period even if the parent flocks had been boostred at point of lay by using oil emulsion vaccine (Van Den Berg et al., 1991). Vaccines of low virulence breakdown the MDA in the fourth week of life, whereas vaccines with intermediates virulence are able to induce active immunity to chickens with MDA in the first two week of life (Carmen, 1994). Maternal immunity are able to protect the chicks against the disease but can also neutralize the vaccine virus (Vob, and Vielitz, 1994; Zaheer and Saeed, 2003). Variation in levels of MDA is one of the major foctors in vaccine breaks (Fadly, 1994). 1.2.6. Diagnosis of IBDV Clinical disease due to infection with the IBDV can usually be diagnosed by a combination of characteristic signs and post-mortem lesions. Differential diagnosis to other disease is important. Example of these diseases are coccidosis, nephrosis-causing condition, infectious bronchitis, hemorrhagic syndrome. Jakowski et al. (1969) reported bursal atrophy in experimentally induced infection with four isolates of Marek’s 40 disease. The atrophy was observed 12 day-post inoculation, but the histology response was distinctly different from that found in IBD. Diagnosis of the sub-clinical disease, can be carried out by demonstration of a humoral immune response in unvaccinated chickens or by detecting the presense of viral antigen in tissues using immunohistochemistry. In the absence of such tests, histologic examination of bursa may be helpful. Isolation and identification of the agent (IBDV) provide the most certain diagnosis, but are not usually attempted for routine diagnostic purposes (Lukert and Saif, 1991). In practice, laboratory diagnosis of IBD depends on detection of specific antibodies to the virus, or on detection of the virus in tissues, using immunological methods. Identification of the virus by direct immumoflurescent staining of affected organs or direct examination by electron microscopy have proven to be an adjunct to the isolation and identification of IBDV (Mc Ferran et al., 1980). Many serological assays are used for diagnosis of IBDV including an agar gel immuno diffusion test (AGID) which is the most useful for detection of specific antibodies in serum, or for detecting viral antigens or antibodies in bursal tissuse. It can also be used to measure antibody levels (Cullen and Wyeth, 1975) which are very useful for measuring maternal or vaccinal antibodies and for deciding the best time for vaccination. Virus neutralization test (VNT) was carried out in cell culture, the test is more 41 laborious and expensive than the AGID, but is more sensitive in detecting antibody. This sensitivity is not required for routine diagnostic purpose, but may be useful for evaluating vaccine responses or to differentiate between IBDV 1 and 2 serotypes (Skeel et al., 1979, Ismail and Saif, 1990). An enzyme linked immuno sorbent assay (ELISA) was developed for the detection of antibody to IBDV (Marquardt et al., 1980). It was used for serosurveyes of chicken flocks (Synder et al., 1986), and examination of the efficiency of vaccines (Solano, Giambrone Panagala, 1985). The ELISA procedure has the advantage of being a rapid test with the results easily entered in to computer soft ware programs. With these programs one can establish antibody profile on breeder flocks that will indicate the flock immunity level and provide information for developing proper immunization programs for both breeder flocks and their progeny. Immuno peroxcidase (IP) staining is also available tool for localization of antigens in both routine histopathology and research materials being sensitive and specific in detection of virus antigens (Mahani et al., 1999). It was found that, VPX-based ELISA is a good alternative to conventional ELISA, that use whole virions (Martinez et al., 2000). This is in accordance with Jack wood, Sommer and Odor (1999) who examined the potential utility of baculovirus-expressed IBDV proteins to act as antigens in the ELISA. The three IBDV protein antigens tested included a truncated VP2, whole VP2and polyprotein VP2, VP3 and VP4. The results of this 42 study indicated that predicting the percentage of protection against classic or variant IBDV strains in broiler from vaccinated breeder flocks can be improved when VP2 is used as the only antigen in the ELISA. Nucleic acid probes (Jackwood, 1988) and monoclonal antibodies are used for detection of IBDV and differentiate IBD viruses directly in tissues which is beneficial for rapid diagnosis and typing of field viruses (Synder et al., 1988). One step RT-PCR has been standardized to amplify the hyper variable region of the VP2 gene sequence of IBDV and the technique was successfully applied to the detection of the virus directly in clinical samples (Kataria, 2001). 1.3. Control of IBD infection Beside the vaccination progrommes, which were discussed in elaboration herein, other managemental measures to reduce the losses due to IBD should be considered. They include optimal utilization of floor space/ broiler, protection of birds from extreme climatic conditions, following recommended immunization schedule, maintenance of good hygienic conditions at the farm and a flock interval of at least more than one week (Farooq et al., 2003). 1.3.1. Vaccination Control of infectious bursal disease proved difficult for at least two reasons. First, because of common occurrence of sub-clinical infection, 43 (Lukert, and Hitchner, 1984). Second, because modified live-virus vaccines are commonly used in young birds (Winterfield, et al., 1972), and the efficacy of these vaccines depends on successful replication within the bird, assays for presence of field-origin virus can be confounded by the presence of vaccine viruses. There are two methods of preventing IBD damage to the immune system. Progeny can be protected either by vaccinating the parent stock, thereby providing passive immunity, or young birds can be immunized by vaccination with live IBD vaccine. Passive immunity is of critical importance, because chicks should be protected throughout the early period of their lives (O’ Brien, 1976), when they are subjected to the most immunosuppressive effects of the disease. In early 1970, only live vaccines were used to control IBD. The main two problems encountered at that time were, first, the large variation in the degree of the attenuation of vaccine strains. Secondly the effects of heterogeneous levels of maternal antibodies which hampered in determing the proper time to vaccinate (Wyeth, 1980). Wyeth and Cullen (1978) showed that the age of 100 percent susceptibility of progeny of live IBDvaccinated parents varied between flocks. To improve this, inactivated –oilemulsion vaccines have been developed (Wyeth and Cullen, 1979). 44 Maternal antibody will normally protect chicks for 1-3 weeks, but by boosting the immunity in breeder flock with oil adjuvant vaccine, it may be extended to 4or 5 weeks. (Lucio and Hitchner, 1979). From late 1980s up to now, the epidemiological situation had changed and IBDV has returned to prominence. Due to the spread of antigenic and pathotypic variants. Different vaccines were proposed to face the situation. 1.3.2. Types of vaccines Two types of vaccines have been used for the control of IBD, these are live attenuated vaccines and inactivated oil-emulsion adjuvanted vaccines. To date, IBD vaccines have been made from type1 IBDV only, although type2 virus has been detected in poultry. 1.3.2.1. Live vaccines According to the test Bursa/body weight ratios live vaccines strains can be categorized in to 3 groups (Michele Guittet et al., 1994). 1- Mild strains They never induce bursal lesion and used in parent chickens to produce primary response prior to vaccination near to point of lay using inactivated vaccine. It is susceptible to the effect of maternally derived antibody (MDA) so should be administered only after all MDA has waned. Application is by means of intramuscular injection, spray or drinking water, usually at 8 weeks age (Skeels et al., 1979). 45 2- Intermediate strains They induce temporary significant difference compared to mild strains. The size of the bursa never exceeds twice the normal birds, producing moderate microscopic lesions (Rosales et al., 1989). They used to protect broiler chickens and commercial layer replacements. They are also used in young parent chickens if there is a high risk of natural infection with virulent IBD. They have the capability of overcoming higher levels of MDA. Intermediate vaccines are sometimes administered at 1-day old, as a coarse spray, to protect any chicken in the flock that may have no or only minimal level of MDA (OIE, 2000). This also establishes a reservoir of vaccine virus within the flock that allows lateral transmission to other chicken when their MDA decays. Second and third doses are usually administered, especially when there is a high risk of exposure to virulent forms of the disease (OIE, 2000). The timing of these will depend on the antibody titre of the parent birds at the time the eggs were laid. The second dose is usually given at 10-14days of age when about 10% of the flock is susceptible to IBD, and the 3rd dose 7-10 days later. The route of administration is by means of spray or in drinking water. I/m injection is used rarely. Feed based IBDV vaccination was recently tried with good results (Hair-Bejo et al., 2004). 46 3. Hot Strains The size of bursa exceeding twice or 3 times than ones of normal birds. This is following injection of hot strains because the virulent virus can overcome higher maternal antibody levels than the intermediate vaccine and hence the virus causes sub-clinical IBD. In severly affected areas, this has led to the use of vaccine with more residual pathogencity .such as Intervet Lz228E and TADLC75. There is an evidence Lz228E is hotter than LC75, its use should be restricted to very severly affected areas, where no other means of control exist. In Europe, the use of Lz228E needs permission from the authorities (Löhren, 1994). 1.3.2.2. Inactivated vaccine Produce high, long lasting and uniform levels of antibodies in breeding hens have previously been primed by live vaccine (Wyeth and Cullen, 1978) or by natural exposure to filed virus during rearing period (Cullen and Wyeth, 1976). The usual program is to administer the live vaccine of about 8 weeks of age followed by the inactivated vaccine 6-20 wks of age. Oil adjuvant vaccines presently may contain both standard and variant of IBDV. 1.3.2.3. Genetically engineered vaccines A baculovirus expressed VP2 immunogen of IBDV induces excellent protection and offers a great potential for subunit vaccine against IBDV (Van Den Berg et al., 1994). Expression of the VP2 capsid protein of 47 IBDV in vaccine strains of fowl pox has produced an experimental recombinant vaccine, fp IBDI. Successful vaccination with it was dependent on the titre of challenge virus. For high titres of challenge virus were able to overcome protection induced by fp IBDI whereas challenge with a low titre of virus did not. The genotype of chicken also has an important effect on the outcome of challenge possibly as a result of the major histocompatibility complex (MHC) and its ability to present VP2derived peptide to the immune system (Shaw and Davison, 2000). 1.3.3. Vaccination failure The failure of vaccination against IBD can be concluded in the following: 1- A great variation (spread) of titres in just hatched chicks. It is clear that those chicks hatched with the lowest titres will be susceptible to the vaccine virus at a significant younger age than those hatched with the highest titre. This variation within offspring from one parent flock is due to titre variation between individual hens (Kouwenhoven and Van den bos, 1994). 2- Occasionally there were also a great titre variation between hatchers from different breeder flocks. This was most likely due to inadequate vaccination of breeder with oil emulsion vaccine. To overcome this disadvantage of this variation, 48 hatcheries were advised to avoid to as much as possible combination of hatches from different breeder flock and if necessary to combine preferably offspring from flocks with identical titre (Kouwenhoven and Van den bos, 1994). 3- In sufficient knowledge of the dynamic of the titre decline and hence a proper time of vaccination can be determined. A vital part of the success of vaccination depends on this timing based on a proper serological test system. They found ELISA an acceptable test to work with under field conditions (Kouwenhoven and Van den bos, 1994). 4- Virulent field virus can overcome residual maternal antibodies about one week earlier than the intermediate vaccine strains. In many cases some residual field virus will be circulating. Thus the vaccine virus is not given the chance to be the first to multiply in the host. Hygienic measurements are necessary to reduce and delay field exposure until immunity has been established in a flock. Cleaning and disinfections after removing of infected crop, all in/all out policy and maximum biosecurity measurements to avoid new infections from the environment (Kouwenhoven and Van den bos, 1994). 49 5- The vaccination failure may be attributed to the degradation of the vaccine quality during transportation or in a new environmental condition or due to antigenic dissimilarities among the local field virus and the imported vaccines. 50 CHAPTER TWO MATERIALS AND METHODS 2.1. Chicks Chicks used in the experiments were obtained from two sources: African poultry company (Khartoum, Sudan) and Koral commercial poultry farm (Khartoum, Sudan). They were obtained as one-day-old, broilers and reared in separated areas until the required age. 2.2. IBD vaccines: The vaccines used in the study were as follow: 2.2.1. Intermediate D78 Strain D78 is a live freeze-dried vaccine against IBD. It has a good immunagenicity that can protect against challenge with virulent IBDV. It is stable that it doesn’t revert to virulence after successive passages. It causes slight atrophy in the bursa of Fabricius and moderate microscopic bursal lesions, not immunodepressive to ND vaccination. Stability of the D78 in solution was investigated at +4°C and –20°C. At - 20°C the virus remained stable for 6 months, while at +4°C it lost 0.4 log TCID50 every month. It is highly immunogenic in both immune and susceptible chicks (Giambrone and Clay, 1986). 2.2.2. Lz228E It’s a live freeze-dried vaccine against IBD, grown on embryonated eggs. It is less attenuated than intermediate IBDV strain. It is capable to breakthrough maternal immunity at an earlier age and will spread better 51 through vaccinated flocks. The vaccine reduced dramatically the clinically virulent IBD especially in layer pullet flocks because this type of birds, which appears to be highly sensitive to IBD is also easy to vaccinate (Lohren, 1994). Lz 228E produce bursal lesions, but no direct mortality. Its use is restricted to very severely affected areas, where no other means of control exist (Löhren 1994). 2.3. Collection of blood The heart puncture method was employed for blood collection from chicks. One-millilter disposable syringes were used for this purpose. Collected blood was left for 2-4 hours at room temperature to clot, after which the clot was loosened. It was then kept at 4°C over-night, separated the following day by low speed centrifugation. The sera after separation were stored at –20°C till needed. 2.4. The agar gel precipitation test (AGPT) 2.4.1 The antigen The reference antigen used in this study was kindly denoted by Dr. Mahsin, head department of virology at CVRL (Khartoum, Sudan). 2.4.2. Procedure of the test Wells in the agar in the petridish were cut in circle shape containing six outer wells and one inner well. Two fold serial dilutions of the sera were prepared in PBS in 0.025ml quantities in the microtitre plates. Since the test 52 was perfromed to detect rise in antibody titres, so the first well contained the undiluted sera and second 4 wells contained the diluted sera, while the 6th outer well contained the positive control serum. The reference antigen was placed in the inner well. The agar gel was then incubated in humidified chamber at room temperature for 48 hours. The test was read visually. Clear precipitin lines were recorded as positive results. 2.5. Experiment 1 2.5.1. Objectives The purpose of this experiment was to detect sero conversion in maternally immuned chicks when vaccinated at different ages by intermediate strain. 2.5.2 Experimental plan and procedure Fifty-six, day-old broiler chicks were obtained from African poultrycompany. They were divided into four groups and treated as follows. Group (A): 14 chicks were vaccinated with intermediate strain at one week in drinking water. Group (B): 14 chicks were vaccinated with intermediate strain at two weeks in drinking water. Group (C): 13 chicks were vaccinated with intermediate strain at three weeks in drinking water. 53 Group (D): 15 chicks used as unvaccinated control group. All groups of chickens were kept in clean and disinfected metal cages, in confined area, each group separately. There were special workers for all groups to supply food and water. Chicks were fasted for three hours before vaccination, 0.1 ml of reconstituted vaccine diluted in 800 ml of tap water and skimmed milk (1:400) was added to minimize the effect of the chemicals used to purify drinking water. Blood for sera was collected one day before vaccination and at weekly intervals, three times after vaccination. The collected sera were tested to detect antibody titre by the quantitative AGPT. Experiment 2 2.6.1. Objectives 1- To detect immune response in broilers after vaccination with D78 and 228E. 2- To study the effect of the vaccines on performance of broilers. 2.6.2. Experimental plan and procedure Fifty, one day old broiler chicks were divided into two groups. In group one (G1), 25 chicks were put in closed pens whereas in group two (G2), 25 other chicks were put in open pens. Both groups were vaccinated with D78 at 14 day old with manufacture’s recommended dose and with 228E at 21 day old with manufacture’s recommended dose in drinking water. Sera were collected at one day-old to detect maternal antibody and 54 before first and second vaccination, then at 35 days old. The collected sera were tested for antibodies by the agar gel precipitation test. At 42-day-old all the chicks were killed and post mortem findings were observed. The observation of meat quality, bursal size and lesions were recorded. 55 CHAPTER THREE RESULTS 3.1. Experiment 1: The maternal immunity and immune responses in chicks vaccinated against IBD using the intermediate strain at different ages are as follows. 3.1.1. Maternal immunity: The maternal immunity of IBDV was determined for all chicks in these experiments before vaccination at 6 days, 13 days, and 20 days respectively. Group 1: In 14 chicks vaccinated at one week of age the titre was 1:4 in 7 chicks, and 1:2 in other 7 chicks at six day (Table 1). Group 2: 14 chicks vaccinated at two weeks of age, the titre was zero in 2 chicks, 1:2 in 8 chicks 1:4 in 4 chicks (Table 2). Group 3: 13 chicks vaccinated at three weeks of age, the titre was zero in five chicks, 1:2 in 8 chicks (Table 3) Group 4: The titre of control group at 6 days old was zero in 4 chicks, 1:2 in 11 chicks. At 13 day old the titre was zero for five chick. 1:2 in 10 chicks. At 20 day old the titre was zero for 12 chicks, 1:2 in 3 chicks. (Table 4) 3.1.2 Immune response to the vaccine Chicks vaccinated at one weeks of age showed that the titre was zero in 2 ,1:2 in 11, 1:4 in 1 , 1:4 in 2 after one week, .of vaccination. After 14 14 14 14 56 two weeks of vaccination the titre was zero 2 ,1:2 in , 1:4 in 6 6 14 14 14 After 3 weeks of vaccination, the titre was zero in 6 , 8 (Table 1). 14 14 chicks vaccinated at two weeks of age showed that the titre in one week post vaccination was zero in 2 1:2 in 5 , 1:4 in 3 After two weeks of 14 14 14 vaccination the titre was zero in 1 , , 1:2 in 6 1:4 in 7 After three weeks 14 14 14 of vaccination the titre was zero in 7 , 1:2 in 6, 1:4 in 1 . (Table 2). 14 14 14 chicks vaccinated at three weeks of age,the titre was zero 6 , 1:2 in 7 After 13 13 one week of vaccination the titre was 1:2 in 4 , 1:4 5 , 1:8 in 4 (Table 3) 13 13 13 3.2.1. Maternal immunity The maternal immunity of IBDV was determined for chicks at one day old and was found that only 13 out of 35 (37%) showed positive results of AGPT. (Table 5). 3.2.2. Immune response to the vaccines At 13 day old of age all the sera collected before first vaccination were found negative in both groups. (Table 6 and 7). The sera collected at the 20 days-old chicks before the second vaccination and after one week of first vaccination were found negative in both groups (Table 8 and 9). Sera collected at five weeks of age were found to be positive in 2 out of 15 chicks (13,33%) reared in closed pens and in 8 out of 23 chicks (34,78%) in chicks reared in open pens. 57 3.2.3. Post mortem lesions in vaccinated chickens Observations on post mortem lesions namely muscle haemorrhage on the thigh and pectoral muscle and enlargement in the size of bursa of Fabricius are demonstrated in Tables (12) and (13). 58 CHAPTER FOUR DISCUSSION Immuization is the principal method used for the control of IBD in chickens. Especially most important is the immunization of breeder flocks so as to confer parental immunity to their progeny (Lukert and Saif, 1991). Maternally derived antibodies (MDA) were found insufficient to protect broiler chicks against highly pathogenic strains of IBDV during the growth period even if the parent flocks had been boostered at point of lay by using oil emulsion vaccine (OEV) (Van Den Berg et al., 1991). The major problem with active immunization of young maternally immuned chicks is determing the proper time of vaccination. This varies with levels of maternal antibody, route of vaccination and virulence of the vaccine virus. Environmental stresses and management are also essential factors to be considered when developing a vaccination program. Monitoring of antibody levels in breeder flock or its progeny can aid in determing the proper time to vaccinate (Lukert and Saif, 1991). In experiment 1, of this study the results obtained showed that at least 3 weeks are required before the maternal immunity decreased so as not to interfere with vaccine and at the same time protect chickens from the disease during the early period of life. 59 A similar finding was previously documented by winterfield (1969). The interference of maternal antibodies with the active vaccination was recently confirmed by Zaheer and Saeed (2003). The variation in the presence of MDA among chicks were also noted. This stands as a great obstacle in front of implementing a universal vaccination programme substantiated by the variation in the managemental as well as operational conditions existing among the flocks. In experiment 2, the maternal derived antibody was found in 38.143% of chicks as measured by AGPT. This variation within the offspring from one parent flock is due to titre variation between individual hens, which constitutes one of the major factor in vaccination failure. The result obtained showed that at 13 days old of age (before first vaccination) the maternally derived antibody had been completely waned (sera were all negative according to AGPT). This is in agreement with Skeeles et al. (1979) who found that the half - life of maternal antibodies to IBD is between 3 and 5 days. Studies by Lucio and Hitchner (1979) indicated that oil emulsion IBD vaccines can stimulate adequate maternal immunity to protect chicks for 4-5 wks, while progeny from breeders vaccinated with live vaccines are protected only 1-3 weeks. The results showed that in 20 days old, there was no immune response observed according to AGPT. This is in agreement with Van Den Berg (1991) who found that a satisfactory vaccination with D78 vaccine was always followed 60 by an excellent seroconversion which occurred as soon as ten days after vaccination. In 35 days (after two weeks of vaccination with hot vaccine) there is a slight increase in immune response 13.33% in closed pens and 34.78% in open ones. This may be due to the fact that the AGPT may not be a sensitive test enough for detecting antibodies. Gagic (1996) found that the antigen prepared from an isolated strain of IBDV for AGPT revealed false negative reaction. This may be the case for our results. The results in Tables (12) and (13) showed that there was post mortem lesions including haemarrhages and bursal lesions when hot vaccine strain was used. There is an evidence that Lz 228E is a hot vaccine strain with residual pathogenicity and its use should be restricted to very severely affected areas, where no other means of control exist. Similar observation and recommendations were perilously stated (Löhren, 1994). In Europe, the use of Lz 228E therefore needs permission from the authorities. Bursal lesions and muscular lesions were noticed in chickens vaccinated with intermediate and hot strains of IBDV as they proved to have some residual pathogenicity and immunosuppression effects. However, no mortality or other visible signs were recorded (Bohra, 1996). In conclusion, the maternal immunity which was confirmed to interfere with active immunization to IBDV require at least 3 weeks to wane. Although, better antibody responses were noted in chicks sera 61 vaccinated with hot strains of the virus but some pathogenic effects were exerted by the vaccine virus affecting the carcass weight and quality. It was also observed that chicks reared in open pens stimulated better antibody responses to the vaccine virus as compared to those reared in closed pens. 62 Recommendations: From the data obtained in this study and in view of previous studies, the following recommendations can be addressed: a) At least 3 weeks are required for the maternal immunity to decrease to the level that cannot interfere with the active immunization against IBDV. b) Vaccination of chicks against IBDV using hot strains of the virus should no be adopted. That is only possible if other means do not exist. 63 Table (1) AGPT– antibody response in susceptible chicks vaccinated with an intermediate strain of IBDV at seven day-old (group A) Before vaccination at 6day old After one week of vaccination Undiluted 1 1 1 Undiluted 1 1 1 1 1 serum 2 4 8 16 serum 2 4 8 16 1 + 2 + 3 + 4 + 5 + 6 + 7 + 8 + 9 + 10 + 11 + 12 + 13 + 14 + Footnotes: + + + + + + + + + + + + + + + + + + + + + - - + + + + + + + + + + + + + + + + + + + + + + + + + + - - - 64 After tow weeks of vaccination Undiluted 1 1 1 1 serum 2 4 8 16 After three weeks of vaccination Undiluted 1 1 1 1 serum 2 4 8 16 + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + - - + + + + + + + + - - - + = precipitating lines observed - = precipitating lines not observed 65 Table (2) AGPT antibody - response in susceptible chicks vaccinated with an intermediate strains at 14 day old (group B) Before vaccination at 13day old After one week of vaccination Undiluted 1 1 1 Undiluted 1 1 1 1 1 serum 2 4 8 16 serum 2 4 8 16 1 + 2 + 3 + 4 + 5 + 6 + 7 + 8 + 9 + 10 + 11 + 12 + 13 + 14 + Footnotes: - + + + + + + + + + + + + + + + + - - - + + + + + + + + + + + + + + + + + + + + + + + + + - - + = Precipitating lines observed 66 After tow weeks of vaccination Undiluted 1 1 1 1 serum 2 4 8 16 After three weeks of vaccination Undiluted 1 1 1 1 serum 2 4 8 16 + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + - - + + + + + + + + - - - = Precipitating lines not observed 67 Table (3) AGPT antibody - response in susceptible chicks vaccinated with an intermediate strain at 21 day old (group C) Before vaccination at 20day old After one week of vaccination Undiluted 1 1 1 Undiluted 1 1 1 1 1 serum 2 4 8 16 serum 2 4 8 16 After tow weeks of vaccination Undiluted 1 1 1 1 serum 2 4 8 16 After three weeks of vaccination Undiluted 1 1 1 1 serum 2 4 8 16 1 2 3 4 5 6 7 8 9 10 11 12 13 + + + + + + + + + + + + + - + + + + + + + + + + + + + - + + + + + + + + + + + + + - + + + + + + + + - - - - + + + + + + + + + + + + + - + + + + + + + - - - - Footnotes: + = Precipitating lines observed 68 + + + + + + + + + + + + + - + + + + + + + + + - + + + + - - + + + + + + + + + + + + - + + + + + + - - - - = Precipitating lines not observed 69 Table (4) AGPT, antibody - response in non-vaccinated chicks (group D) First week Undiluted serum 1 + 2 + 3 + 4 + 5 + 6 + 7 + 8 + 9 + 10 + 11 + 12 + 13 + 14 + 15 + Footnotes: - 1 2 1 4 1 8 1 16 2nd week Undiluted serum + + + + + + + + + + + - - - + + + + + + + + + + + + + + + 1 2 1 4 1 8 + + + + + + + + + + - - + = Precipitating lines observed 70 1 16 Third week Undiluted serum 1 2 1 4 1 8 1 16 - + + + + + + + + + + + + + + + + + - - - - - = Precipitating lines not observed 71 Table (5) AGPT- antibody detection of day- old chicks used in experiment 2 No of sample antibody detected No of sample antibody detected 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 +ve +ve +ve +ve +ve +ve +ve +ve +ve +ve +ve - 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 +ve +ve Footnotes: +ve = Precipitating lines observed - = Precipitating lines not observed 72 - Table (6) AGPT- antibody detection in chicks reared in closed pens at 13day-old (before vaccination) No of sample 1 2 3 4 5 6 7 8 9 Footnotes: - antibody detected - No of sample 10 11 12 13 14 15 16 17 18 antibody detected - +ve = Precipitating lines observed - = Precipitating lines not observed Table (7) AGPT- antibody detection in chicks reared in open pens at 13 day old (before vaccination). No of sample 19 20 21 22 23 24 25 26 27 28 29 30 antibody detected - No of sample 31 32 33 34 35 36 37 38 39 40 41 Footnotes: - 73 antibody detected - +ve = Precipitating lines observed - = Precipitating lines not observed 74 Table (8) AGPT- Ab response of chicks in closed pens at 20 day old (before vaccination with 228E). No of sample 1 2 3 4 5 6 7 antibody detected - No of sample 8 9 10 11 12 13 14 15 antibody detected - Footnotes: +ve = Precipitating lines observed - = Precipitating lines not observed Table (9) AGPT- Ab response of chicks in open pens at 20 day-old (before vaccination with 228E). No of sample 16 17 18 19 20 21 22 23 24 25 26 27 Footnotes: - antibody detected - +ve = Precipitating lines observed 75 No of sample 28 29 30 31 32 33 34 33 36 37 38 39 antibody detected - - = Precipitating lines not observed 76 Table (10) AGPT- antibody response in chicks reared in closed pens at five weeks age. No of sample 1 2 3 4 5 6 7 - antibody detected +ve +ve - No of sample 8 9 10 11 12 13 14 15 antibody detected - Table (11) AGPT - antibody response in chicks reared in open pens at five weeks of age. 2 weeks post vaccination with 228E . No of sample 16 17 18 19 20 21 22 23 24 25 26 antibody detected +ve +ve +ve +ve +ve +ve - Footnotes: +ve = Precipitating lines observed - = Precipitating lines not observed 77 No of sample 27 28 29 30 31 32 33 34 35 36 37 38 antibody detected +ve +ve - 78 Table (12) Post mortem findings at 42 days-old for chicks reared in closed pens. No of chicks Carcass weight Haemorrhage Bursal size enlargement (gm) 1 900 +* + 2 1000 +* + 3 750 +* + 4 850 +* + 5 900 +* ++ 6 900 + ++ 7 600 + - 8 1000 + - 9 1.500 + - 10 1000 + - 11 1000 + - 12 900 - - 13 950 - - 14 900 - - 15 900 - Remarks: + Slight enlargement ++ Marked enlargement * Slight hemorrhage - Normal 79 Table (13) Post mortem findings at 42 days-old for chicks reared in open pens. No of chicks 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 Carcass weight Haemorrhage (gm) 1000 + 750 + 1000 + 1000 + 770 + 1000 + 900 + 1000 + 750 + 1000 + 900 + 800 + 950 + 750 750 1000 850 900 700 700 950 1000 1000 - Remarks: + Slight enlargement ++ Marked enlargement * Slight hemorrhage - Normal 80 Bursal size enlargement + + + + - Fig. 1 light haemorrhage on pectoral muscle after three weeks of vaccination with 228E Fig. 2. Enlarged bursa after three 81 weeks vaccination with 228E. Fig. 3. Haemorrhages on thigh muscle after three weeks of vaccination with 228E. 82 Fig. 4. haemorrhages on thigh uusde after three weeks of vaccination with 228E 83 Fig. 5. Bursae vaccinated after three weeks of vaccination with 228E. 84 REFERENCES Allan, W. H., Farragher, J. T. and Cullen. G.A. (1972). Immunosuppression by the infectious bursal agent in chickens immunized against Newcastle disease. Veterinary Record 99: 511512. Anderson, W.I., Reid, W.M. Lukert, P.D. and Fletcher, O.J. 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International Journal of Poultry Science 2 (4): 251-255. 100 APPENDIX Preparation of solutions and buffers used in AGPT 1) Preparation of 500 ml of AGPT buffer Nacl 40.0 gm Phenol 2.5gm DDW complete 500 ml 3 ml of l molar NaoH was added to adjust the pH to 7.2 2) Preparation of lmolar (Inormal) NaoH solution (100 ml) NaoH powder (Mwt 40) DDW 4g 100 ml They were mixed to gather 3) Preparation of 1.4% agar in AGPT buffer 1.4 g of purified agar was boiled in 100 ml of AGPT buffer for half an hour, then it was dispensed in petridishes in 17 ml quantities per dish and left to cool and solidify. 101
