Iowa State University Capstones, Theses and Retrospective Theses and Dissertations Dissertations 1987 Characterization of an immunogenic component of type A Pasteurella multocida Hyoik Ryu Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/rtd Part of the Microbiology Commons Recommended Citation Ryu, Hyoik, "Characterization of an immunogenic component of type A Pasteurella multocida " (1987). Retrospective Theses and Dissertations. 8583. https://lib.dr.iastate.edu/rtd/8583 This Dissertation is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Retrospective Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. 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For the Major Department Signature was redacted for privacy. For the Graduate College Iowa State University Ames, Iowa 1987 11 TABLE OF CONTENTS Page GENERAL INTRODUCTION 1 LITERATURE REVIEW 4 SECTION I. ANALYSIS OF LIPOPOLYSACCHARIDES OF PASTEURELLA MULTOCIDA AND SEVERAL GRAM-NEGATIVE BACTERIA BY GAS CHROMATOGRAPHY ON A CAPILLARY COLUMN 33 SUMMARY 34 INTRODUCTION 35 MATERIALS AND METHODS 37 RESULTS 42 DISCUSSION 55 LITERATURE CITED 60 SECTION II. CHARACTERIZATION OF A LIPOPOLYSACCHARIDE-PROTEIN COMPLEX OF TYPE A PASTEURELLA MULTOCIDA 63 SUMMARY • 64 INTRODUCTION 66 I-IATERIALS AbD METHODS 68 RESULTS 75 DISCUSSION 86 LITERATURE CITED 91 SECTION III. II-irCNOLOGIC REACTIVITY OF A LIPOPOLYSACCHARIDE- PROTEIN COMPLEX OF TYPE A PASTEURELLA MULTOCIDA IN MICE 94 SUMMARY 95 INTRODUCTION 96 iii Page MATERIALS AND METHODS 98 RESULTS 107 DISCUSSION 123 LITERATURE CITED 129 GENERAL SUMMARY 132 LITERATURE CITED 136 ACKNOWLEDGMENTS 148 1 ŒNEEIAL INTRODUCTION Pasteurella multocida (P. multocida) is a Gram-negative bacterium capable of infecting most animal species including man. Capsular type A and scmatic type 3 strains of the microorganism are known to be a major etiological factor of pneumonic pasteurellosis of cattle, and econcmic loss to the U.S. cattle industry from the disease exceeds a half billion dollars annually. The scientific and economic importance of £. multocida has led to the development and use of various bacterins for prevention; however, the efficacy of these products has been questioned. In fact, immunization of cattle with Pasteurella bacterins has been demonstrated to be detrimental to the health of the animal. The questionable efficacy and safety of bacterins has led to experimentation with various subcellular fractions of the microorganism in studies of bacterial pathogenesis and in pursuit of a better immunogen. Lipopolysaccharides (LPS, endotoxin) present in the outer membrane of Gram-negative bacteria have been demonstrated to play important roles in bacterial infection of animals. The £. multocida LPS extracted by conventional methods (phenol-water, phenol-chloroform-petroleum ether or trichloroacetic acid) has been observed to evoke a variety of endotoxic activities in a host including pyrogenicity, depression, diarrhea and death in mice, rabbits and chickens, and lethality for chicken anbryos. Although LPS extracted by the phenol-water method was poorly immunogenic, many researchers suggested that £. multocida fractions with demonstrated immunogenicity contained the LPS as a part of their constituents. 2 Previously, we have reported that a fraction isolated from a potassium thiocyanate (KSCN) extract of a P. multocida organism (strain P-2383; capsular type A and somatic type 3) induced resistance in mice against a challenge with the homologous bacterium. The fraction, called P-2383-1, was determined to be a LPS-protein complex since it contained 27% protein and serologically crossreacted with the LPS extracted by the phenol-water method. However, 2-keto-3-hydroxy-octonate (KDO), which is an important constituent of Gram-negative bacterial LPS, was not detected in P-2383-1. The outer surface of Gram-negative bacteria contains several distinctive structures including the outêr membrane, capsule, glycocalyx, fimbriae and flagella. Exopolysaccharides, the outer membrane and the cell wall may be extremely complex and. variant among bacterial species. This complexity contributes to a major problan in the extraction and characterization of individual structural components. Also, different extraction methods yield variant products; the basic components may be the same but the nature of complexes can be diverse. This variation extends to previous experimentation on P. multocida and may account for the diversity of products reported by individual investigators. Our previous experimentation on P-2383-1 failed to demonstrate the presence of KDO in this complex. However, we were convinced that this was probably due to a limited quantity of this material and an insensitivity of the assay procedure. This problan is not unique to our experimentation and similar problems have been observed in other immunogenic fractions of P. multocida such as a free endotoxin, a 3 particulate material and a LPS-protein complex. The potential for LPS to serve in immunogenic capacities such as the protective determinant or as an adjuvant necessitated further characterization of this immunogenic complex. The objective of this experimentation was to further characterize the chemical composition and biological activities of the LPS-protein complex. Specific aims were; 1) to determine the chemical composition of P. multocida LPS in comparison with the chemical composition of LPS of several Gram-negative bacterial pathogens, 2) to characterize the LPS- protein complex isolated from the KSCN extract of P. multocida and 3) to examine the immunologic reactivity of the LPS-protein complex in mice. An alternative format is used for this dissertation. Literature cited in each manuscript is listed at the end of each manuscript while references cited both in the literature review and individual manuscripts are included in "Literature Cited" at the end of the dissertation. 4 LITERATURE REVIEW Genus Pasteurella multocida History A disease process now known as pasteurellosis was reported long before the isolation of the causative microorganisms. During the 18th century, large epornitics in poultry were reported throughout European countries and were first studied in France by Chabert in 1782 (47). Maillet in 1836 termed the disease fowl cholera in connection with severe losses of fowl (47). Renault and Delafond, in the middle of the 19th century, presented the first experimental evidence of the transmissibility of the disease by the