Virology

• Clinical Disease: In clinical disease outbreaks of suspected IHNV in sockeye salmon, 10 moribund or freshly dead fish are sufficient to isolate the virus for a confirmed diagnosis. In other salmonid species, 60 moribund fish may be required to establish an etiology. For alevins, fry, and fingerlings, whole fish should be sent by following instructions given above under finfish bacteriology. Also, enclose 10 additional moribund fry per lot, 5 per bag or the equivalent number to equal 1 g if very small fish. Do not add water.
• Broodstock and Disease History Examination: For establishing a disease history in adult fish or in broodstock screening, 60 samples from adult fish will be required. Samples of choice are from spawning or postspawning female fish consisting of ovarian fluids collected from each fish and shipped in separate disposable centrifuge tubes with snap caps. When required, samples from spawning or postspawning males should consist of 0.5 g each anterior and posterior portions of kidney and whole spleen from each fish, aseptically removed and pooled in individual sealed 2 ounce white-labeled plastic Whirl- Pak Tissues from more than one fish should not be combined in one bag. All tissues and fluids for virus assays should be shipped to the FPS on blue ice (4 degree C) but never frozen. Freezing at low temperatures and subsequent thawing can inactivate IHNV, producing lower titers, which in some samples may be too low to detect routinely. Virus samples on blue ice should be sent to the FPS lab as soon as possible within 72 hours of collection.

These sampling procedures are applicable to assays for other finfish viruses should the need arise.

1. Ovarian fluids for virology testing: Obtain instructions from the lab staff regarding whether you should take ovarian fluids from ripe fish used in the egg take or from postspawning fish. Disinfect the external ventral surface and wipe dry with paper towels. For postspawners, partially strip a single fish's ovarian fluids into a paper cup (recommend 4 oz pleated cups but paper drink cups can be used), avoiding the extrusion of blood, fecal material or nematode worms if present. For ripe fish, you may either extrude a small amount of fluid prior to taking eggs or pour fluid off the eggs. Two ml of fluid are adequate for ripe fish, but 3-5 ml should be obtained if sampling postspawners in case there is a need to filter the samples. Crimp edges of the cup to form a spout and pour fluid into a 10 ml centrifuge tube with cap, "straining out" any eggs. Avoid contaminating the rim with your hands. Discard the cup after each fish. Do not provide more than 5 ml of ovarian fluid. Cap the tube tightly making sure that the cap is not improperly seated. Place tubes in a rack in a plastic Ziploc bag labeled with stock of fish and species, sampling location, date, fish life stage, and number of samples. Place upright in a cooler with cold packs. Do not freeze. If samples cannot be shipped within 72 hours they may be kept on ice up to 6 days if not grossly contaminated. Otherwise they must be frozen.
2. Tissue samples from males for virology testing: Disinfect the external ventral surface and either rinse with clean, pathogen-free water or wipe dry with paper towels. Carefully cut open fish, taking care not to cut the gastrointestinal tract which would contaminate tissues with bacterial flora. Aseptically remove the spleen and anterior and posterior portions of the kidney, each about 0.5 g in size (size of thumbnail) and place into a single 2 oz white-labeled plastic Whirl-Pak bag using a spoon, knife or forceps. Seal each bag and keep cool (4 degree C).  Between the sampling of each fish, clean dissecting utensils with 3% iodine and dry with a clean paper towel. Organic matter will affect the working ability of the disinfectant, so any tissue should be wiped off utensils with a separate paper towel prior to disinfection. Wooden tongue depressors, discarded between samples, eliminate the need for disinfection. Disinfect hands between the sampling of each fish. Rubber gloves should be worn to reduce human contact with disinfectant. When sampling is done, place all sealed sample bags in a large plastic bag. Label the bag with the number of samples, stock of fish and species, sample location, fish life stage and date. Place in a cooler on ice or a cold pack and transport within 3 days. Do not freeze. Make sure you include a Sample Submission Form with each shipment.

Finfish Bacteriology

Small fish must be received either alive or freshly dead (within 1-2 hours) on blue ice in a cooler. Fish must not be frozen. Bagged fish should not be in direct contact with blue ice or they will freeze.
Live fish are preferred for diagnostic samples. At least 10 moribund fish should be placed in one or more large leak-proof plastic bags containing hatchery water. Seal the bags so space for air remains and leakage will not occur. Label bags with fish status (moribund or healthy), incubator or raceway number, stock and species and enclose a Sample Submission Form (see page 1-8) with each shipment. If oxygen is available, add to bags before sealing. Addition of an oxygen tablet to each bag is recommended, particularly for samples that must be shipped. Make a similar bag containing 10 healthy fish. Again, if the fish are large fingerlings or smolts, the amount of fish per bag should be adjusted accordingly.
In addition, enclose 10 moribund fish with a damp paper towel in a smaller dry plastic bag. Do not add water. If the live fish do not survive transport, then the "dry" fish, which will have undergone less deterioration and contamination from the water and its bacterial flora, will be processed instead. In a disease outbreak, 30 fish per lot of affected fish will be required for shipment (10 moribund, 10 healthy and 10 moribund, but dry).

Finfish Diagnostics

The prioritization of the basic user diagnostic needs are as follows, in descending order of
importance:

• Disease outbreaks or finfish/shellfish mortality
• Broodstock screening for Family Tracking of bacterial kidney disease (BKD) (finfish)
• Broodstock screening for shellfish (oyster) certification and importation of Crassostrea gigas spat
• Screening of broodstock or resident animals to establish a disease history, generally to satisfy a Fish Transport Permit (FTP) for finfish and shellfish
• Required pre-release inspection of apparently healthy fish or shellfish

The major purpose of this section is to clarify to laboratory staff and user groups the proper sampling procedures to be carried out by clients when finfish or shellfish disease problems arise. This is an absolute necessity to insure that samples received by the pathology labs are adequate for allowing a definitive disease diagnosis.

• Disease Recognition and Action – Whenever abnormal behavior patterns, external abnormalities, or high mortality occur at a hatchery, an immediate response from the hatchery staff in charge is imperative. Assistance should be requested from the Fish Pathology Section (FPS) of ADF&G whenever mortality appears excessive and is not related to known handling or mechanical malfunction of the physical plant. An epizootic is occurring when mortality reaches 1.5% per day. This requires immediate attention. A total commitment of the facility staff and appropriate personnel is needed to save the remaining fish. Mortality less than 1.5% down to 0.5% indicates that a fish health problem is present and the FPS should be notified for consultation. Mortality of less than 0.5% per day but greater than 0.3% should be investigated. Hatchery personnel should attempt to remedy the situation by modifications of environment or feeding and notify the FPS. The percentages given above are for total mortality. It is no less a matter of concern, however, if one lot of fish or shellfish is dying at 1.5% per day while the others remain healthy. The sick animals should be isolated as much as possible to prevent transmission of the disease to other lots. In order to reduce the spread of disease, dead fish and shellfish should be incinerated or soaked in a solution of 200 ppm of chlorine or iodine (active ingredient) for 12 hours before disposal.

• Sample Collection and Shipment – Prior to collecting any samples, the FPS must be contacted to discuss whether samples are necessary, and if so, the appropriate type of sample and numbers of fish or shellfish needed. Advance notice of sample submission by at least one week is preferred. Obviously, serious disease outbreaks will merit an exception. If advance notice is not given, samples may not be processed if other samples have priority or if appropriate lab personnel are not alerted and therefore unavailable to process the samples. The following instructions are general guidelines but some samples need special treatment and the pathology personnel will provide details. Samples that are not in an adequate condition (either substandard or improperly packaged) upon arrival may not be processed. All proposals for sampling (Southeast Region, Southcentral Region and AYK-Westward Regions) should be cleared through pathology staff by contacting the appropriate lab personnel.

• Preparing Samples – Different procedures are followed in sampling for bacteriological, virological, parasitological, ELISA, FAT or histological analyses. Further details regarding the procedures below will be provided to hatchery personnel upon initial contact with the FPS.

• In clinical cases of disease ( 0.5% mortality/day) 10 moribund fish or shellfish are generally a sufficient sample size to make a diagnosis. In situations where no excessive mortality or clinical disease is apparent, a larger sample size of 60 animals may be necessary. However, depending upon individual circumstances, sample sizes may vary between 10 and 60. Samples should be examined from each affected lot, incubator, or rearing container. Consult with the FPS for specific sampling requirements in each situation.