FISH PATHOLOGY SECTION LABORATORY MANUAL - Alaska

251 Pages·2010·2.08 MB·English

by  Jenny Weir

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 to 6-22
I. Preparation of Tissue..................................................................................................6-1
II. Fixation and Decalcification........................................................................................6-9
III. Tissue Dehydration and Infiltration (all tissues)........................................................6-11
IV. Embedding Tissues into Paraffin Blocks..................................................................6-11
V. Cutting Paraffin Blocks and Mounting Sections on Glass Slides.............................6-12
VI. Routine Staining of Paraffin Sections – Hematoxylin and Eosin..............................6-14
VII. Special Staining of Paraffin Sections........................................................................6-15
VIII. References................................................................................................................6-18
7. Transmission Electron Microscopy........................................................................7-1 to 7-8
I. Fixation and Embedment of Tissues from Vertebrates..............................................7-1
II. Fixation and Embedment of Tissues from Marine Invertebrates...............................7-2
III. Retrieval and Embedment of Cut Sections from Histological Slides.........................7-2
IV. Negative Staining of Virus Particles............................................................................7-3
V. Staining Thick Sections for Light Microscopy.............................................................7-4
VI. Staining Thin Sections for TEM..................................................................................7-4
VII. Reagents.....................................................................................................................7-5
VIII. References..................................................................................................................7-7
8. Fluorescent Antibody Staining for Bacteria and Viruses..................................8-1 to 8-12
I. Fluorescent Antibody Methods for Bacteria................................................................8-1
II. Microwell Fluorescent Antibody Test for IHNV...........................................................8-7
III. Ovarian Fluid Filtration FAT........................................................................................8-8
IV. Reagents...................................................................................................................8-10
V. References................................................................................................................8-12
9. ELISA for the Detection of Antigen of Renibacterium salmoninarum ............9-1 to 9-30
I. Reagents.....................................................................................................................9-1
II. Reagent Preparation and Formulae...........................................................................9-1
III. ELISA Materials and Equipment ...............................................................................9-9
IV. Raw Sample Preparation..........................................................................................9-12
V. ELISA Preparation and Performance.......................................................................9-16
VI. Interpretation of ELISA Results ................................................................................9-21
VII. Miscellaneous – Protocol for acid washing glassware.............................................9-24
VIII. References................................................................................................................9-25
IX. ELISA Worksheets....................................................................................................9-25
10. Labeling Procedures for Laboratory Specimens 10-1 to 10-5
I. FAT. .......................................................................................................................10-1
II. Bacteriology...............................................................................................................10-2
III. Histology....................................................................................................................10-3
11. Detection of the Whirling Disease Agent...........................................................11-1 to 11-6
I. Myxobolus cerebralis Survey Procedure (Modification)...........................................11-1
II. Identification of Myxobolus cerebralis.......................................................................11-2

Rev. 11/09 ii