|
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
DIAGNOSTIC TECHNIQUES
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).
Biopsy
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.
Venipuncture
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
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.
SUMMARY
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.
REFERENCES
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.
|
