Home   Contact Us
Summer 2001 Newsletter

Pancreatic Eurytremiasis
Procedure for Salmonid Fishes
Porto Systemic Shunts
Foot and Mouth Disease
Serology Samples
Staff News
Printable Version


Enter Keywords:

Editor’s note:  This is the conclusion of a two-part article that is being reprinted with the written permission of Compendium on Continuing Education For the Practicing Veterinarian.  This article was originally printed in this journal on November, 2000, Vol 22(11), pages S160-166.

Performing Diagnostic Procedures on Salmonid Fishes

Melvin Randall White, DVM, PhD



Observation, Physical Examination, and Laboratory Evaluation

            Before a physical examination is performed, salmonids should be observed in their aquatic environment.  Feeding response as well as swimming behavior should be evaluated.  Sick salmonids will usually not eat; however, they may put the feed in their mouths and then rapidly spit it back into the water.  Therefore, close observation of the fish when they are offered food is critical.  If sick salmonids lose their “fear” or “fright” response, they will not seek shelter from a shadow or a hand waved slowly over the tank.  Healthy salmonids will usually respond to this stimulus by rapidly swimming away from shadows; conversely, fish that are accustomed to being fed by hand may actually surface in anticipation of being fed.

            Erratic swimming behaviors should be noted.  In a raceway, sick fish can usually be found at the end of the raceway nearest to the drainage outflow pipe, whereas healthy fish are usually swimming against the current closer to the inflow water pipe.  Sick salmonids may be deeper in the water column and not swimming vigorously.  Flashing is a common clinical sign that fish have external parasites.  Flashing occurs when fish rub against the sides of the tank, making their underside visible2.  Practitioners should also observe for any physical abnormalities (e.g., curvature of the spine); such abnormalities may first be noticed in fish that swim in a circular or “whirling” pattern.

            Practitioners should observe the movement of the operculum, which is the covering over the gills.  Fish with respiratory difficulties have more rapid operculum movement (pumping) than do healthy fish.  With severe respiratory compromise, fish may actually extend heads out of the water and may be “piping.”  Piping is the term that characterizes the fish with flared opercula that actually appear to be gulping air at the water-air interface.2

             Cutaneous lesions (e.g., fraying, loss of fins, ulcerations, neoplasms) should also be noted.  Because fish have several layers of skin pigments, changes in the color of the fish should be observed; sick fish may be darker or paler than are healthy fish.  Hemorrhages of the skin, especially around the fins, and accumulation of fluid within the coelomic cavity (ascites) are nonspecific lesions commonly associated with bacterial septicemia.  Exophthalmos or “bug-eyes” can be unilateral or bilateral and is commonly caused by osmotic regulatory failure or gas bubble disease; however, this condition can occur with many different disease processes.

            After the fish have been observed in their aquatic environment, a small number of fish with lesions or clinical findings representative of the current disease problem should be removed from the water and examined.  Latex gloves are recommended when handling the fish because some bacterial pathogens of fish (e.g., Mycobacterium species) may also cause diseases in humans.  A closer examination of these fish can often reveal lesions that were not detected while the fish were in the water.  If the fish have lesions, samples should be taken for biopsy (see Biopsy section) or the fish euthanized for necropsy (see Necropsy section).

            The ideal sample to submit to a diagnostic laboratory for evaluation is acutely affected, untreated live fish.  The number of fish to be submitted varies and depends on the size of the fish.  If fry or fingerlings are submitted, then 20 to 30 fish should be adequate for diagnostic tests.  If the specimens are adult fish, then three to six are usually sufficient.  To transport the fish, it is recommended that they be placed in a large thick transparent plastic bag filled one third with water.  An “air-cap” of oxygen occupying approximately one third to one half of the plastic bag should be present immediately above the water surface.  The bag should be tied and placed inside another bag to prevent leakage.  This bag should be placed within a thick wax-coated cardboard box or Styrofoamâ cooler for overnight shipment.  To evaluate water quality, a separate water sample should be shipped in addition to the fish samples (see Necropsy section).


            Biopsies provide valuable information about the cause of diseases affecting salmonids.  Samples should be taken after the fish have been properly anesthetized.  The most common sites of biopsy samples are the gills, skin, and kidney.  To rapidly anesthetize salmonid fishes a dose of 80 to 135 mg/L of tricaine methanesulfonate or MS-222 (Finquelâ, Argent Laboratories, Redmond, WA) can be added to a separate container of adequately aerated water to be used as an “anesthesia tank.”  If the fish are being used as a food source, practitioners must remind producers to maintain the proper 21-day withdrawal time when using tricaine methanesulfonate.  When fish reach surgical anesthesia, they will roll over (“belly up”) and can then be removed from the tank.  The entire fish should be covered with a wet soft towel to keep its surface moist.

            Special consideration should be given to biopsy samples obtained from the kidney, which is a unique anatomic feature of salmonids.  A fibrous connective tissue capsule covers the kidney of salmonids, which lies just ventral to the vertebrae.  The salmonid kidney extends the entire length of the vertebrae and is a dark black, friable parenchymatous organ.  The corpuscle of Stannius should not be confused with a  granulomatous lesion or neoplasm of the kidney.  This specialized endocrine organ, located approximately midway of the length of the kidney, is present as a single nodule or multiple, small, raised white nodules.  The corpuscle of Stannius is embedded in the ventral aspect of the renal tissue.

              Gill samples may be obtained by snipping a small number (three to five maximum) of the primary gill filaments from the cartilage arch of the gill.  Practitioners must use care not to transect the gill arch.  An unstained squash preparation with added saline of the gill filaments can be viewed immediately to detect bacterial and parasitic pathogens.  Skin scrapings can be obtained to detect the presence of skin parasites by lightly scraping (in a cranial to caudal direction) the lesion with a microscope coverslip, which should be placed on a standard microscope slide that contains a few drops of saline.  The slide should be viewed immediately because drying will cause the saline solution to form salt crystals.  A small biopsy of the anal fin can also be obtained by clipping a 1- to 2-mm section.  This tissue can then be placed on a microscope slide that contains saline solution and viewed using a microscope.

            Skin biopsies can be taken using a small dermal skin punch, as it is used on dogs and cats.  Usually one or two interrupted sutures of a 3-0 nonabsorbable suture can be used to close the biopsy site.  The sutures can be removed in 10 to 14 days.  A sterile swab of the biopsied lesion can be used for bacteriologic culture; the remaining tissue can be evaluated by histopathology or immunohistochemistry.

            Renal biopsies are commonly performed to evaluate the presence of disease caused by such bacterial infections as Yersinia ruckeri (the causative agent of enteric red mouth disease) and Renibacterium salmoninarum (the cause of bacterial kidney disease).  Kidney biopsies can be performed in two ways.  When taking samples from small fish, the needle biopsy technique is the best method.6 A needle should be placed into the kidney tissue by directing it through the lateral pharyngeal region lateral to the last branchial arch and medial to the cleithrum (the concave semicircular bone that supports that portion of the pharynx).  The needle should be guided in a caudodorsal manner into the cranialmost portion of the kidney.  Negative pressure should be applied to the syringe and then the needle removed.  No sutures are needed using this procedure.  Large fish (e.g., adult broodstock) can be surgically biopsied7 to obtain a larger sample for bacteriologic culture, fluorescent antibody testing, or histopathology and immunohistochemistry.


            Anesthetization is required before blood samples are taken via nonlethal venipuncture.  Venipuncture of small salmonids should be performed by taking a blood sample from the caudal vein.  Blood can be taken from this location either by placing the needle at a right angle to the lateral surface of the fish and probing for the caudal vein between the hemal arches or by placing the needle through the ventral abdominal musculature perpendicular to the long axis of the body, posterior to the anal fin.  In both instances, the needle should be inserted until resistance is encountered and then pulled back ventrally, approximately 1 to 3 mm, to allow blood to flow into the syringe as the practitioner applies a small amount of negative pressure.3  In larger fish (e.g., adult broodstock), cardiac puncture can be used to obtain a blood sample.  For this technique, the needle should be placed at a 20° to 25° angle from the ventral midline of the fish across the anteriormost portion of the pectoral fins and guided cranially until the heart is penetrated.  Approximately 3 to 5 ml of blood can be removed from a 15- to 20-lb salmonid with no adverse consequences.

Clinical Pathology

            Blood from salmonid fishes is not usually submitted for clinical analysis.  However, some clinical pathology data may be useful.  Several guidelines for normal ranges of clinical pathology parameters have been published8-10; however, factors such as water temperature, nutrition, reproductive status, age of fish, and species may make the reference ranges nonapplicable.  Therefore, care should be exercised in extrapolating data from an affected fish population to known reference ranges.


            Necropsy is commonly performed in salmonids to determine disease processes.  The necropsy procedure can be used to evaluate the organ systems as well as to collect samples for bacteriologic and virologic testing and histopathology.  Practitioners should also consider collecting samples for toxicologic analysis.  Fish can be euthanized by an overdose of tricaine methanesulfonate.  After the fish have been euthanized, the exterior of the fish should be thoroughly examined.  All fish necropsies should include gross examination of the gills, skin scrapings, and fin clippings.  Any lesions of the integumentary system should be evaluated by performing a skin scraping as well as by taking a sterile swab of the lesion for bacteriologic cultures.  Swabs that contain transport medium should be used if the sample is to be sent to a diagnostic laboratory for bacteriologic culture.  The fins should be thoroughly examined for any evidence of fraying or overt necrosis as well as for congestion.  Small portions of the fins can be clipped and evaluated (see Biopsy section).  Observation of the gills and opercula may provide insight into a disease or problem.  Flared opercula in fish that die naturally may indicate a water-quality and/or respiratory problem.  Gills that have a thickening of the lamellae may have gill epithelial hyperplasia secondary to gill parasites or bacterial gill disease.

            After the external system has been evaluated, an incision should be made just cranial to the anal opening at the ventral midline and extended up to the heart.  An incision should then be made from the heart to the dorsal midline.  Most of the skeletal muscle can then be reflected and removed for access to the viscera.  The operculum should also be clipped away to expose the gills.

            Pyloric ceca consist of numerous blind sac-like structures that extend just distal to the pylorus of the stomach.11,12  Histologically, the pyloric ceca are morphologically compatible with tissue of the small intestine.  The pancreatic tissue surrounding numerous pyloric ceca should be closely examined for necrosis, atrophy, or hyperplasia, possibly caused by infectious pancreatic necrosis virus.  If this disease is suspected, pancreatic tissue should be collected for histopathology.

            The appearance of the reproductive tracts of salmonids varies according to the stage of sexual maturity.  In sexually immature fish, the testes and ovaries appear very similar.  Both reproductive tracts are paired organs, which extend from the caudal to the cranial portion of the coelomic cavity.  The ovaries are slightly more transparent and triangular-shaped, especially toward their cranial poles.13 In sexually mature salmonids, the male and female reproductive tracts can be easily distinguished.  The testes are diffusely pale white, whereas the ovaries commonly contain numerous amber eggs.  For virology sampling, a cannula with a blunt end or an oral gavage needle can be used to aspirate ovarian fluid.  This fluid should be placed in Dulbeccos’ phosphate buffered saline in a sterile container to be transported to a diagnostic laboratory for fluorescent antibody and viral isolation testing.  Before collecting these samples, practitioners should check with the diagnostic laboratory regarding the preferred transport medium.

            If bacterial septicemia is suspected, tissue samples should be collected from the spleen and kidney for bacteriologic culture.  The spleen is easily located and identified within the coelomic cavity.  This dark red to mahogany-colored organ, which varies in size and shape, generally is ellipsoid shaped and appears grossly similar to the spleen of mammals.

            Practitioners should flame the renal parenchyma before collecting kidney tissue at necropsy to culture for Renibacterium salmoninarum.  Flaming will destroy any surface contaminants, thus allowing a sterile sample to be collected.  If fluorescent antibody testing is to be conducted, a sterile disposable loop or swab can be inserted into the renal parenchyma.  The swab can then be quickly removed and a smear made on a 10-well fluorescent antibody microscope slide.  For bacterial kidney disease evaluation using ELISA, a small amount (approximately 1 cm2) of renal tissue from small salmonids can be obtained by scraping the tissue in a lateral motion to remove the tissue from its location.  In large fish, the tissue can be snipped using sterile tissue forceps and scissors.  The kidney sample should be placed in a sterile tube with a cap; the tube should be sealed before sending the tissue to a diagnostic laboratory.

            The brain can be easily removed in small fish by using rongeurs to remove the skull over the dorsal midline just caudal to the eye sockets.  Brain tissue can be cultured for evidence of bacterial meningitis, although this condition is uncommon in salmonids.  After brain tissue has been collected for bacteriologic culture, the remaining tissue can be placed in formalin for histopathology.

            Tissue collection is necessary for the evaluation of whirling disease.  Myxobolus cerebralis, the causative agent of whirling disease, is detected by various digestion and centrifugation methods coupled with histopathology.  In addition, polymerase chain reaction technology is becoming commonly used to evaluate fish samples for whirling disease.  Because of the extreme sensitivity of polymerase chain reaction, practitioners should properly disinfect all instruments used for sample collection after each fish is sampled.  For smaller salmonids, the entire head may be removed from the dead fish at the time of necropsy and submitted to the laboratory.  The heads of the fish can be placed in properly sealed plastic bags, each containing a five-fish pool of the samples.

            Although toxicologic problems are not commonly encountered in a private salmonid aquaculture setting, samples should be taken occasionally at necropsy to be evaluated for potential causes of disease.  All water samples taken for water-quality and/or toxicologic analysis should be placed in a clean, acid-washed, triple-rinsed quart glass jar and shipped chilled to a diagnostic laboratory.  Additional samples needed for toxicologic analysis include a large fillet of muscle (at least 200 g), 50 to 100 g of liver tissue, and bile aspirated in a sterile syringe.  Before they are transported to a diagnostic laboratory, muscle and liver samples should be wrapped separately in aluminum foil, properly labeled, and frozen.  Tests commonly conducted by toxicology laboratories include screening water and fish tissue for herbicides and pesticides as well as heavy metal analysis.



            Veterinary practitioners can aid salmonid producers by obtaining a proper history of disease, evaluating the water quality of the facility, and performing physical examinations as well as other diagnostic tests on salmonids.  Practitioners can obtain additional information about potential disease problems by performing such techniques as venipuncture, biopsy, and necropsy of affected fish.



1.      U.S. Department of Agriculture: Aqua-culture Outlook, publication Aquaculture 8. Rockville, MD, Economic Research Service, 1998.

2.      Noga EJ: Fish Disease: Diagnosis and    Treatment. St. Louis, Mosby, 1996, pp 3-75.

3.      Collins R: Principles of disease diag-nosis, in Brown L (ed): Aquaculture for Veterinarians, Fish Husbandry and     Medicine. New York, Pergamon Press, 1993, pp 69-89.

4.      U.S. Department of the Interior: Fish     Manual for the Investigation of Fish Kills. Springfield, VA, Fish and Wildlife Service, National Technical Information Service, 1990, p 41.

5.      Osweiler GD, Carson TL, Buck WB, Van Gelder GA (eds): Urea and nonprotein nitrogen, in: Clinical and Diagnostic Veterinary Toxicology (ed 3). Dubuque, IA, Kendall/Hunt Publishing Co, 1985, pp 160-166.

6.      Noga EJ, Levine JF, Townsend K, et al: Kidney biopsy: A non-lethal method for      diagnosing Yersinia ruckeri infection (enteric red mouth disease) in rainbow trout (Salmo gairdneri). Am J Vet Res 49:363-365, 1988.

7.      White MR, Albregts SR, Wu CC, et al: The use of kidney biopsy of broodstock steelhead trout (Oncorhyncus mykiss) to determine the status of bacterial kidney       disease infection. J Vet Diagn Invest 8:519-522, 1996.

8.      Hille S: A literature review of the blood chemistry of rainbow trout, Salmo gairdneri   Richardson. J Fish Biol 20:535-569, 1982.

9.      Hoffman R, Lommel  R: Haema-tological studies in proliferative kidney disease of rainbow trout, Salmo gairdneri. Richardson. J Fish Dis 7:323-326, 1984.

10.   Miller WR, Hendricks AC, Cairns Jr J: Normal ranges for diagnostically important hematological and blood chemistry characteristics of rainbow trout (Salmo gairdneri) Can J Fish Aquatic Sci 40:420-425, 1983.

11.   Smith LS, Bell GR: A Practical Guide to the Anatomy and Physiology of Pacific     Salmon.  Ottawa Department of the Environment, Fisheries and Marine Service. 1976, p 11.

12.   Yasutake WT, Wales JH: Microscopic Anatomy of Salmonids: An Atlas. Research publication 150, Washington, DC, U.S. Depatment of the Interior, 1983, p 44.

13.   Sundararaj BI: Reproductive Phy-siology of Teleost Fishes. A Review of Present Knowledge and Needs for Future Research. Rome, United Nations Development Programme, 1981, p 15.





ADDL-West Lafayette:
406 S. University
West Lafayette, IN 47907
Phone: 765-494-7440
Fax: 765-494-9181

11367 E. Purdue Farm Road
Dubois, IN 47527
Phone: (812) 678-3401
Fax: (812) 678-3412

Home Users Guide Fee Schedule Online Case Reports Intranet


Annual Reports Home Users Guide Fees Newsletters Online Reports Intranet