Fluorescent Antibody Test (FAT).

The following procedures are for Renibacterium salmoninarum (bacterial kidney disease, BKD), Yersinia ruckeri (enteric redmouth, ERM), and Aeromonas salmonicida (furunculosis). In disease outbreaks involving small fish, 10 moribund or freshly dead fish per affected lot(s) shipped in plastic bags on ice (not frozen) are sufficient for a disease diagnosis. A presumptive diagnosis of A. salmonicida may not be confirmed by bacteriologic culture if samples are frozen. Sampling is according to that described earlier under bacteriology. An additional sample of 60 randomly selected normal appearing fish from the same lot(s) may be required at a later date to determine the prevalence of sub-clinical disease within a given group of fish before release is approved.

In situations where a disease history and/or broodstock screening is desired, a minimum sample size of 60 fish will be required. Family tracking for BKD will require screening of all parent fish involved in the egg take. Whole fish should be sent when sampling alevins, fry and fingerlings. In situations where large fish are to be examined, only kidney tissues are required. Sampling procedures are identical to those described for virology sampling of male kidney tissues.

Although fresh-on-ice samples are necessary for successful isolation of certain disease agents, freezing is the least desirable, but necessary, alternative if there will be excessive delay in getting the samples to the FPS.

In situations where it is more practical for field personnel to prepare the slides for FAT rather than mail tissues, the appropriate materials will be provided by the FPS. Briefly, after collection of kidney tissues the procedure requires:

1. Homogenization of the kidney sample from each fish by kneading within the plastic sample bag.
2. A sterile wooden applicator stick is touched to an individual homogenized kidney sample and then mixed with a drop of phosphate buffered saline (PBS) deposited in a single numbered well on a multiple well slide.
3. The samples are allowed to air dry at room temperature and the slides may be mailed to the FPS in slide boxes.

Each kidney sample requires a separate applicator stick and well. Slides are prepared in multiples for parallel testing if fish are to be screened for BKD, A. salmonicida and Y. ruckeri (2 types require duplicate slides). Homogenization of the kidney is important to break open BKD pustules and distribute the causative organism or any other target bacteria for easier detection. It is also important to not make kidney smears too thick within the depressions, which makes interpretation difficult. Also, such smears may wash off the slide during

processing.