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Fish Pathology Section Laboratory Manual

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Fish Pathology Section Laboratory Manual FISH PATHOLOGY SECTION LABORATORY MANUAL Edited by Theodore R. Meyers, Ph.D. Special Publication No. 12 3rd Edition Alaska Department of Fish and Game Division of Commercial Fisheries P.O. Box 115526 Juneau, Alaska 99811-5526 December 2009 The Special Publication Series was established in 1989 for the publication of departmental symposium or workshop proceedings, strategic fishery management plans, manuals, and other atypical publications of the Commercial Fisheries Management and Development Division. In general, the series is intended for fishery professionals and technically oriented fishing industry representatives. Distribution will be to selected libraries and fishery-related agencies in the Pacific Northwest and interested departmental staff. Copies may also be requested from the Alaska Department of Fish and Game at the address on the title page. Because articles in this series may not have undergone full editorial processing and complete peer review and may be published elsewhere in the formal literature, they should not be cited without approval of the author or the division. The Alaska Department of Fish and Game printed this publication at a cost of $12.42 in Anchorage, Alaska, USA. PREFACE There are many published sources for laboratory procedures used in the diagnosis of finfish diseases and less so with shellfish. This laboratory manual is not intended to be comprehensive in its treatment of this large subject area. Many of the finfish diseases found elsewhere in the United States and the world have not been found in the state of Alaska. Consequently, this manual addresses only those agents known to occur in Alaska (Myxobolus cerebralis has yet to be confirmed) while still providing a general scheme of approach to the disciplines of virology, bacteriology, histology, etc., to allow detection of potentially new or exotic agents as well. The procedures herein follow the AFS Fish Health Section Bluebook standards for the detection of fish pathogens where appropriate and in several instances protocols cited from other investigators in the published literature have been included as well. However, the real purpose of this manual is to provide a working document of very detailed information for ADFG pathology staff and clients regarding the daily routine in which we conduct finfish and shellfish diagnostics. As with most such manuals, this one will be continually updated as new and other procedures become necessary in our everyday use. NOTE: Mention of brand names or trademarks in the text of this manual is not an endorsement of any such product by ADF&G but rather serves as a descriptive model for the reader. EDITOR Theodore R. Meyers is the Principal Fish Pathologist managing the statewide fish pathology program for the Alaska Department of Fish and Game, Commercial Fisheries Division, P.O. Box 115526, Juneau, AK 99811-5526. ACKNOWLEDGEMENTS This manual could not have been completed without the tireless help and diligence of January Seitz who typed and formatted the first of many early drafts of the manuscript. We dedicate this manual in memory of Jenny Weir, our Pathology Section office clerk, who volunteered to complete this project and continued on with numerous revisions that finally evolved into the document presented here. TABLE OF CONTENTS CHAPTER/SECTION...................................................................................................................Page 1. Sample Collection and Submission........................................................................ 1-1 to 1-7 I. Finfish Diagnostics...................................................................................................... 1-1 II. Finfish Bacteriology..................................................................................................... 1-2 III. Virology........................................................................................................................ 1-3 IV. Fluorescent Antibody Test (FAT)................................................................................ 1-4 V. ELISA Sampling of Kidneys for the BKD Agent (see ELISA Chapter 9) ................... 1-5 VI. Parasitology and General Necropsy ........................................................................... 1-5 VII. Histology...................................................................................................................... 1-5 VIII. Sample Shipment Instructions .................................................................................... 1-6 2. Materials List for Sample Submission.................................................................... 2-1 to 2-2 3. Standard Necropsy Procedures for Finfish ........................................................... 3-1 to 3-6 I. General Necropsy Procedure ..................................................................................... 3-1 II. Staining Procedures.................................................................................................... 3-4 4. Bacteriology 4-1 to 4-26 I. Media Preparation....................................................................................................... 4-1 II. Media Formulations..................................................................................................... 4-2 III. Stains and Reagents................................................................................................... 4-7 IV. Test Descriptions......................................................................................................... 4-9 V. Summary/Storage of Quality Control Organisms for Bacteriology Test Media ....... 4-17 VI. Commercial Identification Systems........................................................................... 4-18 VII. Biochemical Characteristics of Common Bacterial Fish Pathogens........................ 4-19 VIII. Bacterial Fish Diseases: Causative Agents and Signs ........................................... 4-20 IX. References................................................................................................................ 4-21 X. Appendices................................................................................................................ 4-23 5. Virology and Cell Culture .......................................................................................5-1 to 5-44 I. Suggested Tissue Types and Sample Sizes.............................................................. 5-1 II. Collecting Ovarian Fluid, Whole Fish, and Tissue Samples in the Field ................... 5-2 III. Maintenance of Stock Cell Lines-Passage of Confluent Cell Monolayers................. 5-3 IV. Cell Counting Using the Hemocytometer ................................................................... 5-5 V. Incubating Cell Lines ................................................................................................... 5-7 VI. Storing Tissue Culture Cells........................................................................................ 5-7 VII. Detecting and Avoiding Tissue Culture Contaminants............................................... 5-8 VIII. Mycoplasma Screening of Continuous Cell Lines...................................................... 5-9 IX. Processing Ovarian Fluid, Whole Fish, and Tissue Samples .................................. 5-10 X. Cytopathic Effects (CPE) of Virus Infection in Tissue Culture Cells ........................ 5-12 XI. Plaque Assay............................................................................................................. 5-13 XII. Quantal Assay`.......................................................................................................... 5-17 XIII. Storing: Freezing and Thawing Virus Isolates.......................................................... 5-20 XIV. IHNV Concentration in Water Samples .................................................................... 5-21 XV. Alkaline Phosphatase Immunohistochemical Procedure (APIH)............................. 5-22 XVI. Biotinylated DNA Probe............................................................................................. 5-24 XVII. Plaque Reduction Serum Neutralization Assay........................................................ 5-30 XVIII. Fluorescent Antibody Staining .................................................................................. 5-32 Rev. 11/09 i CHAPTER/SECTION...................................................................................................................Page 5. Virology and Cell Culture (continued) XIX. PCR for IHNV and VHSV.......................................................................................... 5-32 XX. Virus Characterization Tests..................................................................................... 5-32 XXI. Washing Glassware.................................................................................................. 5-34 XXII. Media ....................................................................................................................... 5-34 XXIII. Appendix.................................................................................................................... 5-40 XXIV. References................................................................................................................ 5-40 XXV. Glossary..................................................................................................................... 5-42 6. Histology for Finfish and Shellfish........................................................................6-1
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