IMMUNITY AND TRANSMISSION OF HISTOMONAS MELEAGRIDIS IN TURKEYS by PEGGY L. ARMSTRONG (Under the Direction of Larry R. McDougald) ABSTRACT The immune response to Histomonas meleagridis and dynamics of lateral transmission was investigated in turkeys. The inoculation of birds 2-16 weeks old produced no evidence of age differences in susceptibility. Commingling of uninoculated poults with infected poults in battery cages showed that litter type was not important in transmission, but infection rates were higher on bedding compared with wire floors. Repeated infection and treatment with dimetridazole produced birds resistant to reinfection, shown by reduced liver and cecal lesions. Immune protection was investigated by inoculation of poults with live or dead Histomonas from cultures. Vaccination by subcutaneous or intramuscular routes produced a protective response as shown by a reduction in the number of infected birds and the severity of lesions after commingling with infected birds. An ELISA test was devised to analyze the antibody response. Turkeys produced IgG antibodies in response to infection and after vaccination with killed histomonads. INDEX WORDS: Histomonas, Immunity, Antibodies, Enzyme-linked Immunosorbent Assay, Transmission IMMUNITY AND TRANSMISSION OF HISTOMONAS MELEAGRIDIS IN TURKEYS by PEGGY L. ARMSTRONG B.S.A. University of Georgia, 2004 A Thesis Submitted to the Graduate Faculty of The University of Georgia in Partial Fulfillment of the Requirements for the Degree MASTER OF SCIENCE ATHENS, GEORGIA 2006 © 2006 PEGGY L. ARMSTRONG All Rights Reserved IMMUNITY AND TRANSMISSION OF HISTOMONAS MELEAGRIDIS IN TURKEYS by PEGGY L. ARMSTRONG Major Professor: Larry R. McDougald Committee: Mark M. Compton Adam J. Davis Scott M. Russell Electronic Version Approved: Maureen Grasso Dean of the Graduate School The University of Georgia December 2006 iv DEDICATION To the birds, who fascinate and inspire me, fuel my passion and enrich my life v ACKNOWLEDGEMENTS I take great pleasure in thanking the many people who have helped make this thesis possible. I would like to thank Larry R. McDougald, my major professor, for allowing me to find my way while being guided by his wisdom. His encouragement and faith are most appreciated. I am grateful to my committee, Mark Compton, Adam Davis and Scott Russell for their support and guidance with my research. They have always made themselves available to me whether I needed to talk about science, research or life. I am forever indebted to Michael Lacy, head of our department, for believing in me and pushing me forward. I wish to thank all of our support staff, they are invaluable. Without research farm workers, secretaries and technicians, our work would be impossible. I thank my children, Nicholas, Timothy and Anthony for their patience and understanding. I am especially grateful to Michael Kattner, who is there at the end of my day, bringing balance to my life. I appreciate his love, support and encouragement. And last, but certainly not least, I would like to thank Kira Moresco. Her friendship is precious and I am honored to be her friend. vi TABLE OF CONTENTS Page ACKNOWLEDGEMENTS.............................................................................................................v LIST OF TABLES........................................................................................................................ vii CHAPTER 1 Introduction and Literature Review ..........................................................................1 2 Immune Protection Against Histomonas meleagridis in Young Turkeys ............19 3 Influence of Bedding Material on the Transmission of H. meleagridis in Turkeys ......................................................................................................................48 4 Conclusion ..................................................................................................................59 vii LIST OF TABLES Page Table 2.1: Susceptibility of turkeys of various ages to infection with Histomonas meleagridis...35 Table 2.2: Resistance to reinfection with Histomonas meleagridis infected 1 or 2 times and treated with dimetridazole.....................................................................................36 Table 2.3: Vaccination of turkeys against H. meleagridis: Comparison of live vs killed preparations and route of inoculation ................................................................38 Table 2.4: The effect of vaccination on direct challenge of Histomonas meleagridis in turkeys......................................................................................................................39 Table 2.5: The effect of vaccination on indirect challenge of Histomonas meleagridis in turkeys ..................................................................................................41 Table 2.6: IgG antibodies produced by turkeys against Histomonas meleagridis cultured in vitro. ..........................................................................................................................43 Table 3.1: Infections of turkey poults with Histomonas meleagridis by direct and indirect contact in battery cages .................................................................................................60 Table 3.2: Influence of bedding on transmission of Histomonas meleagridis from directly inoculated to uninoculated turkeys by comingling. .........................................61 1 CHAPTER 1 Introduction and Literature Review Histomoniasis, also known as blackhead disease or entero-hepatitis, is an infectious disease in gallinaceous birds caused by the parasitic protozoan Histomonas meleagridis. Histomoniasis can cause 90-100% mortality in turkeys but is less severe in chickens. Only one compound is effective in prevention (nitarsone) and there is no effective treatment for the disease since regulatory authorities banned the use of nitromidazole products in the early 1990's in the U.S. and later in Europe. Attempts to provide active immunity against histomoniasis have been considered unsuccessful or impractical overall. Studies have shown thart some degree of immune protection may occur after infection and treatment with antihistomonal drugs. Precipitating antibodies have been detected in recovered birds. Immunization of birds has not been adequately investigated, particularly with regard to the administration of cultured Histomonas. The cecal worm (Heterakis gallinarum) plays an important role in the transmission of histomoniasis and Histomonas is known to survive for a long period of time in cecal worm eggs providing a reservoir of infection year after year. Previous research has shown that the disease can spread through a flock of turkeys in the absence of vectors by cloacal drinking. It is unclear whether the infection spreads by direct contact with infected birds or by contact with contaminated bedding. 2 The objectives were to investigate the immune response in turkeys to H. meleagridis and to determine what role bedding serves in the indirect transmission of histomoniasis within turkey flocks. Histomonas meleagridis and histomoniasis Histomoniasis, also known as enterohepatitis or blackhead, is a severe disease of gallinaceous birds caused by the parasitic protozoan Histomonas meleagridis (8, 48). Histomonas is in the phylum Parabasala, the class Trichomonadae, the family Monoceromonadidae in Cavalier-Smith's six kingdom system (5). Dwyer (13) determined Histomonas to be related to Dientamoeba, Trichomonas and Entamoeba based on antigenic analysis. Dientamoeba and Histomonas have been shown to cross-react strongly in fluorescent antibody studies, indicating many shared antigens. Histomonas contains a few antigens which cross-reacted with E. invadens and E. histolytica. The nucleotide sequence analysis of a small subunit rRNA of the organism showed a close relationship between Dientamoeba and Histomonas (20). The rRNA subunit of both organisms have reduced G-C content and increased G-C chain length in relation to other members of the Parabasalidae family. A comparison of the nucleotide sequence among all identified parabasalid gene sequences and the phylogenetic trees constructed from the host genes showed 100% maximum likelihood, distance, and parsimony between Histomonas and D. fragilis. In 1893, Cushman first described histomoniasis as the cause of the decline of turkey production in Rhode Island and called it blackhead disease (8). Two years later, Smith of the U.S.D.A. described the disease as infectious enterohepatitis and named the 3 responsible organism Amoeba meleagridis (48). The author found the protozoan in fresh cecal preparations and described the organism as round or oval and 8-15 μm in diameter. In tissues fixed and stained, they ranged from 6-10 um. in diameter. The organism was described as having a defined outline with a group of minute granules that probably represented a nuclear structure (48). Liver lesions associated with histomoniasis were first described by Cushman in 1893 but the primary site of the infection is the ceca. The cecal lesions were described as a mass of yellow, hardened lumps which developed into thickened cecal tissue and contained a fibrinous or cheesy textured core. Necrosis of the ceca may occur and birds then succumb to peritonitis. Durant (12) showed that histomoniasis could be controlled in turkeys by cecal abligation. He surgically ligated the cecal necks to restrict passage of material into the ceca. Even though post-surgical losses were