Detection of Erythrocyte Bound Globulin in Plasmodium Gallinaceum Infected Chickens
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This dissertation has been microfilmed exactly as received 67-10,892 GAUTAM, Om Parkash, 1931- DETECTION OF ERYTHROCYTE BOUND GLOBULIN IN PLASMODIUM GALLINACEUM INFECTED CHICKENS. The Ohio State University, Ph.D., 1967 Agriculture, animal pathology University Microfilms, Inc., Ann Arbor, Michigan DETECTION OF ERYTHROCYTE BOUND GLOBULIN IN PLASMODIUM GALLINACEUM INFECTED CHICKENS DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Om Parkash Gautam, B.V.SC., M.SC. ****** The Ohio State University 1967 Approved by 4a) ft« Adviser'' Department of Veterinary Medicine and Clinic f t m Adviser Department of Microbiology and Veterinary Preventive Medicine .ACKNOWLEDGMENTS During the course of this work I have been fortunate to have obtained constant help and guidance from my advisers Dr. W. G. Venzke and Dr. J. P. Kreier— true scholars and out standing teachers in the field of veterinary medicine. In no less way the encouragement and help of Dr. V. L. Tharp, Dr. P. W. Murdick, and Dr. W. F. Loeb from the Department of Veterinary Medicine and Clinics is sincerely appreciated. To all these gentlement I am highly indebted for their efforts and time spent for making this piece of work a success. Mr. Bill Green of the Abbott Laboratories, Chicago, who invited me to visit his laboratory and helped me in label ing globulin, deserves my thanks and deepest regards. My thanks are also due to Mrs. Ruth Kreier for collect ing references and editing this thesis. Miss Diana Walker for drawing the charts and graphs, and Mrs. Ruth Reed for typing this manuscript. Last, but not least, my parents, wife, and children deserve great appreciation for their interest in this work. This work was done under a Rockefeller Foundation ii Fellowship and I am grateful to the Foundation for financial assistance and to Mr. J. P. Perry, Jr., and Mr. Robert Fis- chelis for all the assistance and facilities provided. I am also thankful to the Punjab Agricultural uni versity for providing me an opportunity to undertake this work. The investigation was supported in part by a research grant to Dr. J. P. Kreier (AI06108) from the Tropical Medicine and Parasitology Section of the National institute of Allergy and Infectious Diseases, U.S. Public Health Service. VITA April 1, 1931 Born - Nawana, India 1 9 5 1 ........ B.V. Sc. (medalist), The Punjab Veterinary College, Hissar, India 1951-1952 . Veterinary Assistant Surgeon, Rohtak, India 1952-1958 . Demonstrator (Veterinary Medicine and Clinic) Punjab Veterinary College, Hissar, India 1959 . M.Sc., The Ohio State University, Columbus, Ohio— Sponsored by the Punjab Government 1960-1963 . , Assistant Professor (Veterinary Parasitology) College of Veterinary and Animal Science, Punjab Agricultural University, Hissar 1963-1964 . , Associate Professor (Veterinary Medicine) College of Veterinary and Animal Science, Punjab Agricultural University, Hissar 1964-1967 The Rockefeller Foundation Scholar Department of Veterinary Medicine and Clinic, The Ohio State University, Columbus, Ohio PUBLICATIONS 1. Laboratory Manual of Clinical Veterinary Medicine. Veter inary College Hissar. First printed in 1958. 2. Tetanus in a mule. Indian Vet. J. 35(1958):234-235. 3. Modern teaching methods. Indian J. Agr. Vet. Ed. 3(1960)-.37-41. 4. Treatment of mastitis. Indian J. Vet. 37(1960)481-482. IV VITA— Continued 5. Ascites in dogs and its treatment with a new diuretic— bendrofluazide. Punjab Vet. 2(1962):76-80. 6. Glanders in mules. Indian Vet. J. 39(1962):588-593. 7. Traumatic pericarditis in a bullock. Punjab Vet. (1962): 115-119. 8. How U.S. Veterinarians are employed. Indian J. Agr. Vet. -Ed. 6(1963) .-18-21. 9. Sarcoptic mange in dogs and its treatment. Punjab Vet. 3(1963)i22-26. 10. Haematological Norms in goats. Indian J. Vet. Sc. A.H. 35(1965):173-177. 11. Teaching and practice of Veterinary Medicine in the United States. Punjab Vet. 4(1965):l-7. 12. Normal haematological values in buffaloes. J. Res. Pb. Agr. Univ. 3(1966):76-83. FIELDS OF STUDY Major Field: Veterinary Medicine and Clinic Studies in Veterinary Medicine and Clinic: Veterinary Medicine - Professors H. E. Amstutz and E. F. Donovan Veterinary Clinic - Professors V. L. Tharp, J. C. Donham and P. W. Murdick Veterinary Preventive Medicine - Professor R. D. Henthorne v FIELDS OF STUDY— Continued Endocrinology - Professor W. 6. Venzke Ophthalmology - Professors E.F. Donovan and M. Wyman Studies in Microbiology; General and Pathogenic Microbiology - Professors M. S. Rheins and G. L. Stahly Serology and immunology - Professors M. S. Rheins and J. Kreier Veterinary Microbiology and Protozoology - Professor J. Kreier Studies in Veterinary Clinical Pathology: Professors W. L. Loeb, and G. E. McKissick Studies in Veterinary Parasitology: Professors F. R. Koutz and H. F. Groves Studies in Chemistry/Physiological Chemistry: Professors M. P. Cava and D. G. Cornwell Studies in Technical Photography/Radiation Biophysics Professors H. G. Binau and W. G. Myers ILLUSTRATIONS Figure Page 1. Standard curve for protein determination by colorimetric m e t h o d . .................... 81 2. Titration of rabbit origin anti-chicken globulin globulin by capillary precipita tion reaction. ....................... 85 3. Graph showing half-life of iodine-125........ 93 4. Tubes showing positive and negative Coombs' reactions......................... 103 125 5. Plate test to detect with I labeled antiglo bulin globulin bound in vivo to erythrocytes of chickens with P. gallinaceum infection. 105 125 6. Uptake of I labeled antiglobulin by iso - antibody sensitized chicken erythrocytes. 121 7. Blood smear from a chicken showing P.. crallinaceum infected erythrocytes. A laras number of parasitized erythro cytes are seen................ 127 8. Blood smear from a chicken showing P. gallinaceum infection. Only a few para sitized and a large number of basophilic erythrocytes are seen.......................... 129 9. Detection with I labeled antiglobulin of globulin bound in vivo to erythrocytes of chickens with P. gallinaceum infection. 130 10. Production of anemia and basophilia in chickens by cardiac and venous bleeding. 133 vii ILLUSTRATIONS —Continued Figure Page 11. Blood smear from an anemic chicken. Anemia was produced by phenylhydrazine hydrochloride injection................................... ... 12. Production of anemia and basophilia in chick ens by phenylhydrazine hydrochloride in jection..................................... ... 13. Uptake of •L2^i labeled antiglobulin by baso philic erythrocytes produced by phenyl hydrazine injection superimposed upon cardiac bleeding.......................... 138 14. Effect of absorption with basophilic ery throcytes upon uptake of -*-25j labeled anti globulin by basophilic and normal erythro cytes................................... 139 125 15. The effect of absorption of I labeled anti- globulin with basophilic erythrocytes upon its ability to agglutinate normal and basophilic erythrocytes. ........................... 1 4 0 125 16. Detection with I labeled antiglobulm of globulin bound in vivo to erythrocytes of a chicken with P. gallinaceum infection. .... 146 125 17. Detection with I labeled antiglobulm of globulin bound in vivo to erythrocytes of a second chicken with P. gallinaceum infection. 149 125 18. Uptake of I labeled antiglobulin (absorbed with basophilic erythrocytes sensitized with plasma from P. gallinaceum infected chickens. 152 viii TABLE OF CONTENTS Page ACKNOWLEDGMENT ..................................... ii VITA ............................................... iv ILLUSTRATIONS ..................................... vii INTRODUCTION................................. 1 LITERATURE REVIEW . ......................... 6 I. Avian Malaria in General Mechanisms for Development of Anemia Use of Iodine Labeling in Immunology MATERIALS AND M E T H O D S ....................... 72 I. General Techniques II. Radioisotopic Techniques III. Serological Techniques IV. Production of Anemia and Basophilia in Chickens V. Serological Techniques with Basophilic Erythrocytes and Erythrocytes from Chickens with Patent Parasitemias VI. Trypsinized Erythrocyte Tests VII. Elution Experiments VIII. Specificity of the Anti-Trypsinized Erythrocyte Antibody IX. Reductive Cleavage by Mercaptoethanol of the Anti-Trypsinized Erythrocyte Antibody X. Effect of Absorption of Plasma from Z- gallinaceum Infected Chickens with Trypsinized Non-Infected Erythrocytes Upon Sensitization of Trypsinized Non-Infected and Infected Erythrocytes RESULTS 120 TABLE OF CONTENTS— Continued Page DISCUSSION........... 156 CONCLUSIONS ............. 172 SUMMARY ..................................... 175 APPENDIXES .................................... • 178 LITERATURE CITED . ....................... 246 INTRODUCTION Malaria, still a dreaded disease of the tropic and subtropic lands, has played an important role in the shaping of the history of mankind. It is considered to have been a major factor in the fall of some ancient civilizations. Ma laria has been known to man since antiquity. The oldest men tion of it is found in the ancient Hindu medical literature based on the Atharva Veda (Neuberger and Froude, 1910), where it has been recognized as a distinct disease entity. Pres ently, malaria has been eliminated from most parts of the temperate world, but it still remains a major disease problem in some tropical countries. The importance of bird malarias lies chiefly in their use as