AN ABSTRACT of the THESIS of Attractive Method of Vaccine
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AN ABSTRACT OF THE THESIS OF Jon D. Piganellifor the degree ofDoctor of Philosophyin Microbiology presented on June 6. 1994 Title:Development of Enteric Protected Vaccines for Aauaculture. Abstract approved: Stephen L. Kaattari At present all vaccines for fish are primarily delivered either by injection or immersion which introduces added stress and labor. A more attractive method of vaccine delivery is oral administration using an enteric protection system, Enteric Coated Antigen Microspheres (ECAMs), which can be utilized for a variety of antigenic forms. Relative efficacy of ECAMs was compared by inducing an antibody response in fish via injection (ip), immersion (im) or by ECAMs. Results showed that ECAMs were as effective as ip and im in inducing an antibody response to lipopolysaccharde and protein antigens. ECAMs were able to protect the protein antigen from gastric degradation. The ECAMs were employed to deliver a prototype vaccine for Renibacterium salmoninarum(Rs), the etiological agent of bacterial kidney disease. Upon characterization of the Rs antigens, one predominant cell- associated and extracellular protein (ECP), p57 was identified. The p57 molecule is elaborated in high concentrations in infected fish and exhibits pathogenic activitiesin vitro whichappear to suppress the immune response. Our studies have revealed that a 370C incubation of R. salmoninarum cells decreased the amount of p57 by the inductionof an autoproteolytic activity. This activity was exploited to producea prototype vaccine that was delivered by intraperitoneal injection (ip)and demonstrated a significant increase inmean time of death upon challenge by injection. A second experimentwas conducted using a natural exposure (bath challenge) and the heat treated, p57 -Rs cellswere delivered using ECAMs and ip administration. The results proved thatthe route of immunization was critical with respect to naturalexposure, as animals that received ECAMs with heat-treated p57 minus As cells demonstrated statistically significant reduction in the amount of ECPdetected versus control. The decrease in ECP concentration is indicativeof reduced Rs infection. Those animals vaccinated by ip showedno difference. Finally, in order to assess immunologicalmemory induced by antigenic stimulation, an assay was developed thatmeasured the production of cytokines induced by in vivo primingand subsequent in vitro exposure with the specific antigen. Development of Enteric Protected Vaccinesfor Aquaculture by Jon D. Piganelli A THESIS Submitted to Oregon State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Completed June 6, 1994 Commencement June 1995 APPROVED: 4 Professor of Microbiology in charge of major He d of the Department of Microbiology Dean of the Grad aSchool Date thesis presented June 6. 1994 Typed for researcher by Jon D. Piganelli ACKNOWLEDGEMENTS I would first like to thank my major professor Dr. Steve Kaattari, for giving me the opportunity to work in his laboratory and also for allowing me to finish my work in his laboratory even after he departed O. S. U. for a new appointment.I have benefited greatly from his un-selfish sharing of knowledge and for this I am indebted. Additionally I would like to thank my committee members Dr. Don Mattson, Dr. J. Mark Christensen, and Dr. Jerry Heidel for helpful comments on this thesis.I especially would like to thank Dr. Jo-Ann Leong and Dr. John Rohovec for looking out for the well being of the lab after Steve left. I would like to thank my fellow lab mates that had to put up with me all these years including Leslie "The Gilkster" Gilkey, Janel"Janelo" Bishop, Dan "The Rockhead"Rockey, Jenefer" The Queen" DeKoning-Loo, Mark "King Spamon" Adkison, John"The Hippie" Hansen, Hank "Hankenstien" Ortega, Patty "Woody" Wood, Dave"The Bigman" Shapiro, oh! lets not forget the ageless wonder Don Chen. Greg Wiens, I can't thank you enough for allowing me to work with you on these projects..... THANKS! To the members of the Leong Lab and especially Barb Drolet for her artistic ability. Dan "Dano" Mourich who taught me almost as much Immunology as Steve. Thanks for being a great friend. Greg "Oh what are those Raymond" Rutherford another great friend. The Rohovec lab ODFW, Harriet. The professional women in the office Carlene, Joy, Jenny, Marylin, Linda, and of course Bonnie, I owe most of my Scientific life to you guys. To my colleague and friend in the Pharmacy department Jia Allen Zhang thanks for all of your help and collaboration. I would like to thank my parents Lucille and Rocco for, always believing in me and letting me express myself. Also, for instilling in me the gift of faith in myself and in God and for their many prayers that I truly know have blessed me throughout my entire life.I Love you both very much. To Timmy, Chrissy and Rocco, my older brothers and Roxanne my older sister thank you for all your prayers and support throughout my years in school. Thanks to Dick and Susie my in-laws for their support. To my friends back home Joe Ed, Russ, Mitch, Mike, Dweeze, Kevin, Sam, Daddy "G". Specifically, I would like to thank my best friend, my wife Cheryl for her unconditional support and love without which I would not be complete. Finally, I would like to thank the one and only and the one and only knows who that is. To be given the gift to do Science is only the tip. For whom you choose to do it for is the iceberg............THANKS LORDI CONTRIBUTION OF AUTHORS Dr. S. L. Kaattari was the major advisor on all manuscripts. Dr's. J. M. Christensen and J. C. Leong were minor advisors on those manuscripts that their names appeared on. Jia Allen Zhang was responsible for preparation and feeding of the ECAMs to fish described in Chapter 3 and 5 and also participated in collecting samples. Dr. Greg Wiens was responsible for Tables 4.1, 4.2 and some of the SDS-PAGE work from Chapter 4. He also prepared some of antigen for vaccination in Chapter 5 and helped vaccinate some of the animals. Janel Bishop, Dr. R. Stevenson and L. Gilkey participated in collecting samples in Chapter 5. Dan Mourich was responsible for the fractionation and radiolabeling experiments in Chapter 6, as well as for helping with the oxidative burst assays. Dr. Linda Bootland was responsible for the anti-viral assays in Chapter 6. TABLE OF CONTENTS CHAPTER Page 1- INTRODUCTION 1 2- LITERATURE REVIEW 3 Aquaculture and Associated Disease Problems Vaccines Primary Cells of the Specific Immune Response The Macrophage / Antigen Presenting Cell B-Cells T-Cells Gut Associated Lymphoid Tissue Viral Vaccines Infectious Pancreatic Necrosis Virus Viral Hemorrhagic Septicemia Virus Infectious Hematopoietic Necrosis Virus Bacterial Vaccines/ Vibrio anguillarum Yersina ruckeri Aermonas salmonicida Renibacterium salmoninarum Vaccination Methods / Delivery Systems 3- Enteric Coated Microspheres As An Oral Method for Antigen Delivery to Salmonids 47 ABSTRACT 48 INTRODUCTION 49 MATERIALS AND METHODS 51 RESULTS 55 DISCUSSION 56 ACKNOWLEDGMENTS 60 4- Activation of an endogenous serine protease as a novel method for removal of p57 from the Renibacterium salmoninarum cell surface 66 ABSTRACT 67 INTRODUCTION 68 MATERIALS AND METHODS 71 RESULTS 73 DISCUSSION 75 ACKNOWLEDGMENTS 77 CHAPTER 5- Evaluation of a protease-modified, Renibacterium salmoninarum whole cell vaccine, deliveredorally and intraperitoneally 84 ABSTRACT 85 INTRODUCTION 87 MATERIALS AND METHODS 90 RESULTS 96 DISCUSSION 98 ACKNOWLEDGMENTS 102 6- Assessment of immunologicalmemory to a T-dependent antigen in rainbow trout (Oncorhynchus mykiss) by thein vitro elicitation of a respiratory burst enhancing cytokine 108 ABSTRACT 109 INTRODUCTION 110 MATERIALS AND METHODS 112 RESULTS 118 DISCUSSION 121 ACKNOWLEDGMENTS 125 7- CONCLUSIONS 131 BIBLIOGRAPHY 135 LIST OF FIGURES CHAPTER 2 Page 2.1. Diagram of the gastrointestinal tract of salmonid species. 16 2.2. Enteric Coated Antigen Microspheres (ECAMs). 45 CHAPTER 3 3.1. Serum antibody titers expressed in units / ml ofserum from coho salmon immunized with TNP-LPS in the form of. 61 3.2. Serum antibody titers expressed in units / ml ofserum from coho salmon immunized with TNP-KLH in the form of. 63 CHAPTER 4 4.1. Total protein stains of R. salmoninarumcells (A)or supernatants (B) after 16 hour treatment at370C(lane2), 17°C(lane3), 40C (lane 4), or -200C (lane 5), molecular weight markers (lane 1). 78 4.2. Total protein stain (A) and western blot using the anti p57 monoclonal antibody 4D3. (B) of R. salmoninarum cells after co-incubation with PMSF and 370C treatment. 79 4.3. Western blot using the anti p57 monoclonal antibody 4D3 of 370C treated R. salmoninarum cells with culture supernatant (ECP). 80 CHAPTER 5 5.1. Total protein stain(A)and western blot (using the monoclonal anti p57 antibody 4D3)(B)of R. salmoninarum after 370C treatment for 48h followed by 0.3% formalin incubation at 170C for 10 h: molecular weight marker (lane1),untreatedR.salmoninarum cells (lane 2), 370C treated R. salmoninarum cells (lane 3). 103 5.2. Percent survival of coho salmon immunized with saline / FIA(A), ECP / FIA (B), CSE / FIA (C) and p57- cells / FIA (D). 104 CHAPTER 6 6.1. Fluorescence measurement of oxidation of DCFH by rainbow trout pronephric macrophages (PM) in the presence of supernatant factors and the triggering agent (1 ug/ml PMA) after 24 hour incubation with supernatants derived from TNP-KLH ( ),ovalbumin (®), and non-antigen conditioned supernatants (®)cells. 126 6.2. Fluorescence measurement of oxidation of DCFH by rainbow trout HK macrophages in the presence of 1 ug / ml PMA after 24 hour incubation with centricon fractionated supernatants. 128 6.3. Fluorescence measurement of oxidation of DCFH by rainbow trout HK macrophages in the presence of 1 ug / ml PMA after 24 hour incubation with TNP-KLH derived supernatants from enriched cultures. 129 6.4. SDS-PAGE fluoregraph of soluble S35 methionine labeled proteins.