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  • LOCALIZATION AND PARTIAL IMMUNOLOGICAL

    CHARACTERIZATION

    OF Fasciola hepatica THIOREDOXIN

    A Dissertation

    by

    RICHARD DWAYNE MCKOWN

    Submitted to the Office of Graduate Studies of Texas A&M University

    in partial fulfillment of the requirements for the degree of

    DOCTOR OF PHILOSOPHY

    December 2004

    Major Subject: Veterinary Microbiology

  • LOCALIZATION AND PARTIAL IMMUNOLOGICAL

    CHARACTERIZATION

    OF Fasciola hepatica THIOREDOXIN

    A Dissertation

    by

    RICHARD DWAYNE MCKOWN

    Submitted to Texas A&M University in partial fulfillment of the requirements

    for the degree of

    DOCTOR OF PHILOSOPHY

    Approved as to style and content by: ____________________________ ___________________________ Allison Rice-Ficht Thomas M. Craig (Co-Chair of Committee) (Co-Chair of Committee) ____________________________ ___________________________ Karen F. Snowden Pete D. Teel (Member) (Member) ____________________________ Ann B. Kier (Head of Department)

    December 2004

    Major Subject: Veterinary Microbiology

  • iii

    ABSTRACT

    Localization and Partial Immunological Characterization

    of Fasciola hepatica Thioredoxin. (December 2004)

    Richard Dwayne McKown, B.S., Kansas State University; D.V.M.,

    Kansas State University; M.S., Kansas State University

    Co-Chairs of Advisory Committee: Dr. Allison C. Rice-Ficht Dr. Thomas M. Craig

    This study reports the localization and partial characterization of thioredoxin from

    the parasitic trematode Fasciola hepatica. Snails (Pseudosuccinia columella) were raised

    in culture and infected with F. hepatica so that Western blotting and

    immunohistochemical techniques could be utilized to determine the presence of

    thioredoxin in different stages of the parasite’s development. The results of these

    experiments showed that thioredoxin was present in the tegument, gut epithelium,

    excretory canal epithelium and sperm, of the adult parasite as well as in the tegument and

    gut of the redia and cercaria intermediate stages. In situ hybridization was used to

    determine the localization and possible differential mRNA expression of two different F.

    hepatica thioredoxin isotypes (Fh2020.A and Fh2020.SL) in the adult parasite. The in

    situ hybridization results showed that both isotypes are expressed in the tegument and gut

    epithelium. Fh2020.A stains with a greater intensity possibly demonstrating a difference

    in the amount of expression between the two isotypes.

    Recombinant F. hepatica thioredoxin expressed in bacteria using the pMAL™

    Protein Fusion and Expression System was used to test its affects on the production of

    super oxide anion by murine peritoneal macrophages, bovine monocyte-derived

  • iv

    macrophages and bovine whole blood neutrophils, and nitric oxide production by mouse

    peritoneal macrophages and bovine monocyte-derived macrophages. The results of the

    cellular assays were not definitive due to the fact that the maltose binding protein (MBP)

    moiety of the recombinant thioredoxin, when tested alone, increased production of nitric

    oxide by bovine monocyte-derived macrophages. Consequently, since the MBP could

    not be effectively separated from the thioredoxin portion of the recombinant, allowing the

    thioredoxin affects to be tested independently, no true conclusions regarding its affects on

    the host immune cells tested could be drawn.

    This is the first report of the localization of thioredoxin in both the adult F.

    hepatica as well as in specific intermediate stages of the parasite. These studies

    demonstrate the possible affects that a protein tag can have on experimental results and

    demonstrate how such data may be interpreted when a non-cleaved recombinant protein

    is used in cellular or other assays when compared to native or cleaved recombinant

    proteins.

  • v

    DEDICATION

    This dissertation and all the work done to get to this point are due entirely to my

    family, especially my wife Carol and daughter Genny. Without their support and

    encouragement over the years this never would have been completed.

  • vi

    ACKNOWLEDGEMENTS

    I would like to thank the individuals at Texas A & M University who have

    provided support, guidance and friendship during my tenure there, particularly the

    administrative and support staff of the Departments of Veterinary Pathobiology and

    Medical Biochemistry and Genetics.

    Additionally, I would like to thank the administration and staff of the U.S. Meat

    Animal Research Center, and the Great Plains Veterinary Education Center, both in Clay

    Center, Nebraska for the generous use of their facilities.

    Much of the work described within this dissertation would have been much more

    difficult to achieve without the assistance of the technical, support, and secretarial staffs

    at both of these facilities – for their unselfish help and good humor, I am deeply indebted.

    Finally, I owe immeasurable thanks to Dr. Judy Ball for all of the effort and stress

    she dedicated to helping a graduate student she basically didn’t know, but fought for

    anyway. May you be truly blessed!

  • vii

    TABLE OF CONTENTS

    Page

    ABSTRACT.................................................................................................................. iii DEDICATION.............................................................................................................. v ACKNOWLEDGEMENTS ......................................................................................... vi TABLE OF CONTENTS.............................................................................................. vii LIST OF FIGURES ...................................................................................................... xi LIST OF TABLES........................................................................................................ xiv CHAPTER

    I INTRODUCTION AND LITERATURE REVIEW ............................ 1 History....................................................................................... 3

    Description................................................................................ 4 Life Cycle.................................................................................. 4 Tegument .................................................................................. 7 Veterinary Importance .............................................................. 9 Medical Importance .................................................................. 12 Protein Biochemistry ................................................................ 14

    II MATERIALS AND METHODS.......................................................... 21

    Fasciola hepatica Adult and Intermediate Stage Collection .... 21

    Adult Flukes and Eggs .................................................. 21 Miracidia ....................................................................... 22 Snail Culture ................................................................. 23

    Snail Infection............................................................... 24 Sporocysts ..................................................................... 25 Redia ............................................................................. 26 Cercaria ......................................................................... 26 Metacercaria.................................................................. 27 Mouse Infection ............................................................ 27 Juvenile Flukes.............................................................. 28

    Parasite Soluble Protein Preparation and Analysis................... 28 Preparation of Parasite Soluble Protein ........................ 28 Determination of Protein Concentration....................... 29

  • viii

    CHAPTER Page

    Sodium Dodecyl Sulfate – PolyacrylamideGel Electrophoresis (SDS-PAGE)........................... 29

    Coomassie® Brilliant Blue Staining of Polyacrylamide Gel................................................................................. 30 Polyacrylamide Gel Drying .......................................... 30 Electrophoretic Transfer of Protein from SDS-PAGE to Nitrocellulose............................................................ 31

    Protein Immunodetection.......................................................... 32 Western Blotting ........................................................... 32

    Sample Handling for Immunohistochemistry........................... 33 Fixation of Tissues........................................................ 33 Processing and Embedding of Tissues.......................... 33 Sialanization of Glass Slides......................................... 34

    Immunohistochemistry ..............................