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U·M·I Urllverslty Microfilms International a Bell & Howell Information Company 300 North Zeeb Road Development of in vitro primary cell cultures from the penaeid shrimp, Penaeus stylirostris and Penaeus vannamei and evaluation of a potential application. Item Type text; Thesis-Reproduction (electronic) Authors Luedeman, Rene Annette. Publisher The University of Arizona. Rights Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author. Download date 05/10/2021 07:01:25 Link to Item http://hdl.handle.net/10150/144635 INFORMATION TO USERS The most advanced technology has been used to photograph and reproduce this manuscript from the microfilm master. UMI films the .text directly from the original or copy submitted. Thus, some thesis and dissertation copies are. in typewriter face, while others may be from any type of computer printer. The quality of this reproduction is depend,!nt upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleed through, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send UMI a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. Oversize materials (e.g., maps, drawings, charts) are reproduced by sectioning the original, beginning at the upper left-hand corner and continuing from left to right in equal sections with small overlaps. Each original is also photographed in one exposure and is included in reduced form at the back of the book. Photographs included in the original manuscript have been reproduced xerographically in this copy. Higher quality 611 x 911 black and white photographic prints are available for any photographs or illustrations appearing in this copy for an additional charge. Contact UMI directly to order. U·M·I UrllVerslty Microfilms International A Bell & Howell Information Company 300 North Zeeb Road. Ann Arbor. M148106-1346 USA 313/761-4700 800/521-0600 Order Number 1339902 Development of in vitro primary cell cultures from the penaeid shrimp, Penaeus stylirostris and Penaeus vannamei and evaluation of a potential application Luedeman, Rene Annette, M.S. The University of Arizona, 1990 U·M·I 300 N. Zeeb Rd. Ann Arbor, MI 48106 DEVELOPMENT OF lHVIIRO PRIMARY CELL CULTURES FROM THE PENAEID SHRIMP, lENAEUS STYlJROSmIS AND PENAEUS VANNAMEI AND EVALUATION OF A POTENTIAL APPLICATION by Ren~ Annette Luedeman A Thesis Submitted to the Faculty of the DEPARTMENT OF NUTRITION AND FOOD SCIENCE In Partial Fulfillment of the Requirements For the Degree of MASTER OF SCIENCE WITH A MAJOR IN FOOD SCIENCE In the Graduate College TIlE UNIVERSITY OF ARIZONA 1 990 2 STATEMENT BY AUTHOR This thesis has been submitted in partial fulfillment of requirements for an advanced degree at The University of Arizona and is deposited in the University Library to be made available to borrowers under rules of the Library. Brief quotations from this thesis are allowable without special permission, provided that accurate acknowledgment of the source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the head of the major department or the Dean of the Graduate College when in his or her judgment the proposed use of the material is in the interests of scholarship. In all other instances, however, permission must be obtained from the author. APPROVAL BY THESIS CO-DIRECTORS This thesis has been approved on the date shown below: '1po j'Q6 Dr. Charles P. Gerba Date Dr. Donald V. Lightner Date Assoc. Professor of Veterinary Science -------------------- ----- - -- 3 ACKNOWLEDGMENTS This work was supported by grants from the SeaGrant, United States Department of Commerce and from the U.S. Department of Agriculture through the U.S. Marine Shrimp Farming Program. I wish to thank Dr. Donald V. Lightner for his support, patience, and willingness to share his knowledge. I would like to express my appreciation to Leone Mohney for sharing her knowledge of tissue culture and to Thomas Bell for his computer assistance. I would also like to thank Rita Lightner for her help in preparing the histology slides and ultrathin sections for TEM. Also, thank you Rita for your advice and moral support. My thanks to David Bentley for his assistance on the TEM. I would like to express my appreciation to my family for their support and who made completing this thesis possible. ---_._------------_._-- - -- 4 TABLE OF CONTENTS LIST OF ILLUSTRATIONS .............................................................................................................................. 6 LIST OFTABLES ................................................................................................................................................ 7 ABSTRACT ........................................................................................................................................................... 8 INTRODUCTION................................................................................................................................................ 9 Commercial Shrimp Production ........................................................................................................... 9 Diseases ofPenaeid Shrimp ................................................................................................................ 11 Infectious hypodermal and hematopoietic necrosis disease ............................................ 12 Diagnosis ofIHHN disease .................................................................................................. 12 Tissue Culture ....................................................................................................................................... 13 History of Invertebrate Tissue Culture-Terrestrial Animals ........................................................... 14 History of Invertebrate Tissue Culture-Aquatic Animals ............................................................... 19 Mollusks .................................................................................................................................. 20 Crustaceans ............................................................................................................................ 23 MATERIALS AND METHODS ..................................................................................................................... 28 Development of a Primary Cell Culture System for Penaeid Shrimp ............................................ 28 Experimental Animals .......................................................................................................... 28 Tissue Culture Materials ...................................................................................................... 28 Assay Procedures .................................................................................................................. 28 Viral infectivity studies of IHHNV to f. stylirostris primary ovarian cell cultures ...................... 37 RESULTS ........................................................................................................................................................... .42 Deveiopment of a Primary Cell Culture System for Penaeid Shrimp ........................................... .42 Experimental Media .............................................................................................................. 44 Osmolality ................................................................................................................ 50 Culture Vessels ...................................................................................................... .52 Exposure to UV Light ........................................................................................... .52 Subsequent Tests using Grace's Insect medium ................................................. 52 Temperature and Gaseous Environment ............................................ .52 -------------------------- - -- --_. 5 TABLE OF CONTENTS (continued) Growth Factors ....................................................................................... .55 Alternate Species ..................................................................................... 55 Sub-culturing ............................................................................................ 55 IHHNV Exposure to Shrimp Ovary Cells ........................................................................................ .58 Detection of IHHNV using H & E staining ....................................................................... 58 Acridine orange staining of ovarian epithelioid cells exposed to IHHNV .................... .58 The use of transmission electron microscopy (TEM) for virus detection ...................... 59 BGM Infection with Poliovirus Model.. .............................................................. .59 IHHNV exposure to shrimp cell monolayers ...................................................... 59 DISCUSSION ...................................................................................................................................................... 61 Tissue Culture ......................................................................................................................................
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