Professional Documents
Culture Documents
11 November 2000
Performing Diagnostic
FOCAL POINT Procedures on
★A thorough physical examination,
observation of salmonids within
their environment, proper
Salmonid Fishes
collection of diagnostic samples,
and evaluation of water quality Purdue University
help to distinguish disease Melvin Randall White, DVM, PhD
processes unique to salmonids.
ABSTRACT: In the United States, salmonids are raised for exportation of eggs and fry as well as
KEY FACTS for meat production. In this rapidly growing segment of the private aquaculture business, the
primary role of veterinarians is to provide diagnostic services for health and disease evaluation.
■ Salmonids that lose their “fear” Diagnostic evaluation includes taking a proper history, performing a physical examination, iden-
or “fright” response will not seek tifying clinical signs of salmonid disease, and recognizing acute and chronic water-quality prob-
shelter from a shadow or a hand lems. Biopsy methods and procedures, venipuncture techniques, anesthesia, necropsy, eu-
thanasia, and proper tissue sample collection unique to salmonids are also examined.
waved slowly over the tank;
healthy fish will respond by
rapidly swimming away.
A
quaculture is a rapidly developing agribusiness. The culture and harvest
■ When fish are submitted to a of salmonids by private industries for food consumption and exportation
diagnostic laboratory, a separate represent a $79 million industry in the United States.1 This article pro-
water sample (other than the vides guidelines on how to perform diagnostic techniques and properly collect
water used to transport the fish) tissue samples from salmonids. In addition, various diagnostic techniques can
should be included to test water help distinguish disease processes unique to salmonids.
quality.
DISEASE EVALUATION
■ The corpuscle of Stannius is a Obtaining a Disease History
normal endocrine organ embedded Important information can be learned about an aquaculture facility by asking
in the kidney and should not be the proper questions while compiling a disease history. Although many ques-
misidentified as a granulomatous tions may be directly related to a particular disease, basic information regarding
or neoplastic lesion. the facility’s operating procedures should be obtained to reveal possible environ-
mental concerns or problems that are personnel related (see Questions to Ask
■ Renal biopsies can be performed When Compiling a Disease History).
on salmonids to detect such
bacteria as Yersinia ruckeri, the Environmental Criteria
causative agent of enteric red Water quality is the most important environmental parameter for salmonid
mouth disease, and producers. Environmental abnormalities can be categorized into acute and
Renibacterium salmoninarum, chronic problems. With acute water problems, at least one of the parameters is
which causes bacterial kidney severely affected and poses a life-threatening condition to the fish; high mortali-
disease. ty will result if the situation is not corrected immediately. A chronic water prob-
lem acts as a stressor and not as the initiating cause of mortality.
Compendium November 2000 Food Animal
that must be constantly monitored. Acute nitrite toxi- such abnormalities may first be noticed in fish that
cosis has been called brown blood disease because of the swim in a circular or “whirling” pattern.
rapid oxidation of hemoglobin, which occurs when ni- Practitioners should observe the movement of the
trite diffuses across the gill epithelium of fish. This oxi- operculum, which is the covering over the gills. Fish
dation results in methemoglobin formation, which with respiratory difficulties have more rapid operculum
causes brown discoloration of the blood.2 Although this movement (pumping) than do healthy fish. With se-
condition is observed in catfish raised for production, it vere respiratory compromise, fish may actually extend
is not a common problem in salmonids. heads out of the water and may be “piping.” Piping is
The pH as well as the hardness and alkalinity of wa- the term that characterizes the fish with flared opercula
ter should be monitored periodically to denote trends that actually appear to be gulping air at the water–air
or changes in parameters that may result in chronic wa- interface.2
ter problems. The pH of water is the measurement of Cutaneous lesions (e.g., fraying, loss of fins, ulcera-
the hydrogen ion content expressed as the negative log- tions, neoplasms) should also be noted. Because fish
arithm of the hydrogen ion concentration; thus the pH have several layers of skin pigments, changes in the col-
measures the acidity or alkalinity of water. The alkalini- or of the fish should be observed; sick fish may be dark-
ty is a determination of the buffering capacity of water er or paler than are healthy fish. Hemorrhages of the
as measured by the amount of bicarbonate (HCO3–) skin, especially around the fins, and accumulation of
and/or carbonate (CO3–) in milligrams per liter (mg/l) fluid within the coelomic cavity (ascites) are nonspecif-
in the water. The hardness of water is the measurement ic lesions commonly associated with bacterial sep-
of divalent metal cations (e.g., calcium, iron, zinc, mag- ticemia. Exophthalmos or “bug-eyes” can be unilateral
nesium). The majority of the cations are calcium. or bilateral and is commonly caused by osmotic regula-
Chronic poor water quality reportedly can diminish the tory failure or gas bubble disease; however, this condi-
growth, feed efficiency, and feed conversion rates of tion can occur with many different disease processes.
fish. After the fish have been observed in their aquatic en-
vironment, a small number of fish with lesions or clini-
DIAGNOSTIC TECHNIQUES cal findings representative of the current disease prob-
Observation, Physical Examination, lem should be removed from the water and examined.
and Laboratory Evaluation Latex gloves are recommended when handling the fish
Before a physical examination is performed, sal- because some bacterial pathogens of fish (e.g., Mycobac-
monids should be observed in their aquatic environ- terium species) may also cause diseases in humans. A
ment. Feeding response as well as swimming behavior closer examination of these fish can often reveal lesions
should be evaluated. Sick salmonids will usually not that were not detected while the fish were in the water.
eat; however, they may put the feed in their mouths If the fish have lesions, samples should be taken for
and then rapidly spit it back into the water. Therefore, biopsy (see Biopsy section) or the fish euthanized for
close observation of the fish when they are offered food necropsy (see Necropsy section).
is critical. If sick salmonids lose their “fear” or “fright” The ideal sample to submit to a diagnostic laboratory
response, they will not seek shelter from a shadow or a for evaluation is acutely affected, untreated live fish.
hand waved slowly over the tank. Healthy salmonids The number of fish to be submitted varies and depends
will usually respond to this stimulus by rapidly swim- on the size of the fish. If fry or fingerlings are submit-
ming away from shadows; conversely, fish that are ac- ted, then 20 to 30 fish should be adequate for diagnos-
customed to being fed by hand may actually surface in tic tests. If the specimens are adult fish, then three to
anticipation of being fed. six are usually sufficient. To transport the fish, it is rec-
Erratic swimming behaviors should be noted. In a ommended that they be placed in a large thick trans-
raceway system, sick fish can usually be found at the parent plastic bag filled one third with water. An “air-
end of the raceway nearest to the drainage outflow cap” of oxygen occupying approximately one third to
pipe, whereas healthy fish are usually swimming against one half of the plastic bag should be present immedi-
the current closer to the inflow water pipe. Sick salmo- ately above the water surface. The bag should be tied
nids may be deeper in the water column and not swim- and placed inside another bag to prevent leakage. This
ming vigorously. Flashing is a common clinical sign bag should be placed within a thick wax-coated card-
that fish have external parasites. Flashing occurs when board box or Styrofoam® cooler for overnight ship-
fish rub against the sides of the tank, making their un- ment. To evaluate water quality, a separate water sam-
derside visible2. Practitioners should also observe for ple should be shipped in addition to the fish samples
any physical abnormalities (e.g., curvature of the spine); (see Necropsy section).
Figure 1—Kidney tissue is the dark linear tissue along the Figure 2— Collection of renal biopsy aspirate in smaller
ventral aspect of the vertebrae. The probe denotes the cor- salmonids. Aspirated fluid can be used for bacteriologic cul-
puscle of Stannius, which is a normal endocrine organ. ture of such pathogens as Renibacterium salmoninarum and
Yersinia ruckeri.
Biopsy
Biopsies provide valuable information about the ments can be viewed immediately to detect bacterial
cause of diseases affecting salmonids. Samples should and parasitic pathogens. Skin scrapings can be obtained
be taken after the fish have been properly anesthetized. to detect the presence of skin parasites by lightly scrap-
The most common sites of biopsy samples are the gills, ing (in a cranial to caudal direction) the lesion with a
skin, and kidney. To rapidly anesthetize salmonid fish- microscope coverslip, which should be placed on a
es, a dose of 80 to 135 mg/L of tricaine methanesul- standard microscope slide that contains a few drops of
fonate or MS–222 (Finquel ®, Argent Laboratories, saline. The slide should be viewed immediately because
Redmond, WA) can be added to a separate container of drying will cause the saline solution to form salt crys-
adequately aerated water to be used as an “anesthesia tals. A small biopsy of the anal fin can also be obtained
tank.” If the fish are being used as a food source, practi- by clipping a 1- to 2-mm section. This tissue can then
tioners must remind producers to maintain the proper be placed on a microscope slide that contains saline so-
21-day withdrawal time when using tricaine methane- lution and viewed using a microscope.
sulfonate. When fish reach surgical anesthesia, they will Skin biopsies can be taken using a small dermal skin
roll over (“belly up”) and can then be removed from the punch, as it is used on dogs and cats. Usually one or
tank. The entire fish should be covered with a wet soft two interrupted sutures of a 3-0 nonabsorbable suture
towel to keep its surface moist. can be used to close the biopsy site. The sutures can be
Special consideration should be given to biopsy sam- removed in 10 to 14 days. A sterile swab of the biop-
ples obtained from the kidney, which is a unique sied lesion can be used for bacteriologic culture; the re-
anatomic feature of salmonids. A fibrous connective tis- maining tissue can be evaluated by histopathology or
sue capsule covers the kidney of salmonids, which lies immunohistochemistry.
just ventral to the vertebrae. The salmonid kidney ex- Renal biopsies are commonly performed to evaluate
tends the entire length of the vertebrae and is a dark the presence of disease caused by such bacterial infec-
black, friable parenchymatous organ. The corpuscle of tions as Yersinia ruckeri (the causative agent of enteric
Stannius should not be confused with a granulomatous red mouth disease) and Renibacterium salmoninarum
lesion or neoplasm of the kidney. This specialized en- (the cause of bacterial kidney disease). Kidney biopsies
docrine organ, located approximately midway of the can be performed in two ways. When taking samples
length of the kidney, is present as a single nodule or from small fish, the needle biopsy technique is the best
multiple, small, raised white nodules. The corpuscle of method.6 A needle should be placed into the kidney tis-
Stannius is embedded in the ventral aspect of the renal sue by directing it through the lateral pharyngeal region
tissue (Figure 1). lateral to the last branchial arch and medial to the clei-
Gill samples may be obtained by snipping a small thrum (the concave semicircular bone that supports
number (three to five maximum) of the primary gill fil- that portion of the pharynx). The needle should be
aments from the cartilage arch of the gill. Practitioners guided in a caudodorsal manner into the cranialmost
must use care not to transect the gill arch. An unstained portion of the kidney (Figure 2). Negative pressure
squash preparation with added saline of the gill fila- should be applied to the syringe and then the needle re-
Figure 3—Collection of renal biopsy samples in adult sal- Figure 4—Cardiac venipuncture of anesthetized adult sal-
monid broodstock uses equine endometrial biopsy forceps. A monid broodstock illustrating proper needle placement for
biopsy sample can be used for bacteriologic culture as well as blood collection.
ELISA testing and/or immunohistochemistry to detect Reni-
bacterium salmoninarum.
of clinical pathology parameters have been published8–10;
however, factors such as water temperature, nutrition,
moved. No sutures are needed using this procedure. reproductive status, age of fish, and species may make
Large fish (e.g., adult broodstock) can be surgically the reference ranges nonapplicable. Therefore, care
biopsied7 to obtain a larger sample for bacteriologic cul- should be exercised in extrapolating data from an affect-
ture, fluorescent antibody testing, or histopathology ed fish population to known reference ranges.
and immunohistochemistry (Figure 3).
Necropsy
Venipuncture Necropsy is commonly performed in salmonids to
Anesthetization is required before blood samples are determine disease processes. The necropsy procedure
taken via nonlethal venipuncture. Venipuncture of can be used to evaluate the organ systems as well as to
small salmonids should be performed by taking a blood collect samples for bacteriologic and virologic testing
sample from the caudal vein. Blood can be taken from and histopathology. Practitioners should also consider
this location either by placing the needle at a right an- collecting samples for toxicologic analysis. Fish can be
gle to the lateral surface of the fish and probing for the euthanized by an overdose of tricaine methanesul-
caudal vein between the hemal arches or by placing the fonate. After the fish have been euthanized, the exterior
needle through the ventral abdominal musculature per- of the fish should be thoroughly examined. All fish
pendicular to the long axis of the body, posterior to the necropsies should include gross examination of the
anal fin. In both instances, the needle should be insert- gills, skin scrapings, and fin clippings. Any lesions of
ed until resistance is encountered and then pulled back the integumentary system should be evaluated by per-
ventrally, approximately 1 to 3 mm, to allow blood to forming a skin scraping as well as by taking a sterile
flow into the syringe as the practitioner applies a small swab of the lesion for bacteriologic cultures. Swabs that
amount of negative pressure.3 In larger fish (e.g., adult contain transport medium should be used if the sample
broodstock), cardiac puncture can be used to obtain a is to be sent to a diagnostic laboratory for bacteriologic
blood sample. For this technique, the needle should be culture. The fins should be thoroughly examined for
placed at a 20° to 25° angle from the ventral midline of any evidence of fraying or overt necrosis as well as for
the fish across the anteriormost portion of the pectoral congestion. Small portions of the fin can be clipped
fins and guided cranially until the heart is penetrated and evaluated (see Biopsy section). Observation of the
(Figure 4). Approximately 3 to 5 ml of blood can be re- gills and opercula may provide insight into a disease or
moved from a 15- to 20-lb salmonid with no adverse problem. Flared opercula in fish that die naturally may
consequences. indicate a water-quality and/or respiratory problem.
Gills that have a thickening of the lamellae may have
Clinical Pathology gill epithelial hyperplasia secondary to gill parasites or
Blood from salmonid fishes is not usually submitted bacterial gill disease.
for clinical analysis. However, some clinical pathology After the external system has been evaluated, an inci-
data may be useful. Several guidelines for normal ranges sion should be made just cranial to the anal opening at