b9476ec703492483612a9734f6e37d7d

Annual Rev. of Fish Diseases, pp.
41-62, 1991
Printed in the USA. All rights reserved.
0959.8030/91 $3.00 + .@I
1991 Pergamon Press plc
THE ROLE OF THE DIAGNOSTIC LABORATORY
IN FISH DISEASE CONTROL
Douglas P. Anderson
U.S. Fish and Wildlife Service, National Fish Health Research Laboratory,
Box 700, Kearneysville, West Virginia 25430 USA
Phyllis J . Barney
U.S. Fish and Wildlife Service, Fish Disease Control Center, P.O. Box 1595,
La Crosse, Wisconsin 54602-1595 USA
Abstract. The diagnostic laboratory is becoming increasingly important to the productivity and
profitability of hatcheries, fish farms, and aquaculture stations. The basic function of the labora-
tory personnel is to isolate and identify viral, bacterial, protozoan, and other fish pathogens present
in cultured and feral fish stocks. New, rapid and accurate methods for the detection and identifi-
cation of fish disease agents based on immunological, biochemical, and physiological assays are be-
coming commonly used. Nearly every North American state or province and many foreign countries
have fish health regulations that require inspection of stocks for certain disease agents before the
fish are shipped into their areas. Decisions from the diagnostic laboratory on identification, treat-
ment, guaranteed isolation, immunization, and disposal of fish populations affect administrative
directives, hatchery placement, and national and international transportation of fish and fish prod-
ucts. This paper reviews concepts and describes the equipment, supplies, biologics, and media needed
for the basic diagnostic laboratory. Information management, including training of staff, certifi-
cation procedures, and quality control are also discussed.
Keywords. Fish, Disease, Diagnostic Laboratory, Pathology
INTRODUCTION TO THE FISH HEALTH
LABORATORY
The profession of fish health and disease di-
agnosis is experiencing rapid growth (l-4). New
techniques, often based on immunological or bio-
chemical analyses, are reported frequently in scien-
tific publications (5-8). To relate the dizzying array
of changes while maintaining a stable, respectable
daily-functioning laboratory is a challenge. Fish
disease diagnostic laboratories cannot emulate the
staffing or financing of some of the modern clin-
ical medical and veterinary laboratories; however,
we can draw on their scientific experience and
management design.
The functions of the fish disease diagnostic lab-
oratory are varied (Fig. 1). The laboratory is a cen-
ter for receiving and processing samples of fish
tissues and microorganisms from hatchery and
field locations. Analyses are done according to
standard methods. The laboratory recommenda-
tions may influence administrative decisions and
operational procedures. Research laboratories de-
velop new methods that can facilitate and increase
the accuracy of the diagnosis and the efficacy of
prophylactic and control measures. Also, complex
41
+
Research ,, Diagnostic Tralning
hb0r-w
Labomtory * Facilities
$
Verdicts
Recommendations
Fig. 1. The fish health diagnostic laboratory is a center
of communication where samples from cultured and feral
fish are received and processed. Results are given to the
biologists with recommendations for treatment or culture
modifications. Administrative actions involve immediate
decisions for short-term remedies and long-term planning
for physical hatchery modifications, including changes
for the improvement of water quality.
42 D. P. Anderson and P. J. Barney
or unusual clinical cases may be referred to research
personnel for diagnosis. Continuing education and
technical training are necessary to introduce and
update laboratory technical staff. Staff size of the
diagnostic laboratory may range from one biolo-
gist working at a geographically isolated location
to five or more people in a larger diagnostic or de-
velopment center that monitors several hatcheries.
Most laboratories are staffed by individuals with
various skills. Limits on level and diversity of lab-
oratory functions are mainly due to physical loca-
tion, training and expertise of staff, materials,
equipment, and travel funding. Indeed, a major
constraint is insufficient time for doing diagnoses
as substantial time may be spent advising pathol-
ogists on specific problems and performing exten-
sion information service. Some laboratories employ
students for performing some sample processing,
which in turn, gives valuable experience to the
trainees.
Biologists of state and federal salmon and trout
hatcheries in North America developed the first
models for fish disease diagnosis and control. In
the past few years commercial and government
hatcheries have begun employing pathologists to
monitor, treat, and research problems at these in-
creasingly large facilities. This is the authors back-
ground; the following discussions may reflect that
bias. Catfish culture now exceeds that of other spe-
cies. Warm water (e.g. channel catfish, Zctalurus
punctatus, and tilapia, Tilapia sp.) and cool water
(e.g. Northern pike, Esox Lucius, and muskellunge,
Esox musquinongy) fish disease diagnosis models
have similarities to the salmonid ones. Fish culture
for the aquarium trade also has a strong economic
base, however, models for control of fish disease
in nonfood fish are quite different. Drugs and
chemicals can be used for aquarium fish that may
not be allowed for food fish. In addition, because
of the relatively small-scale aquarium setting there
is less concern about release of chemicals into the
environment.
HISTORY OF THE FISH DISEASE LABORATORY
Historical perspective of fish disease diagnosis
Diagnostic laboratories as separate facilities for
fish disease control are a recent development. Early
fish-culturists recognized that fishes have patho-
gens that are similar to those of human and other
animals. In European carp farms most diagnoses
were done pondside by describing signs of the dis-
ease. Large protozoans could be easily observed as
early texts reflect this in their emphasis of the more
obvious pathogens (9-12). Bacterial isolation and
identification techniques followed the development
of modern medical and veterinary microbiology di-
agnostic methods. Sampling and confirming iden-
tification for viral infections were later events
(13,14). Davis (15) and Snieszko (16) detailed fish
diseases and methods for treatment in the United
States.
Avoidance of treating hatchery fish with drugs
or antibiotics is desirable. Indeed the physical stress
of handling and treating fish exacerbates mortalities,
thus aquaculturists have to carefully justify their
procedures (17). In ideal situations, pathologists
have opportunities to monitor their fish by taking
and examining tissues from healthy animals. Hemat-
ocrits, leukocrits, and neutrophil counts are health
parameters giving clues to certain physiological and
immunological disorders. Also, periodic examina-
tions of fish tissues can reveal the presence of vi-
ral, bacterial, or protozoan pathogens.
Occasionally, diagnosticians from laboratories
are called to the field for preliminary diagnoses of
fish disease cases. Field samples are collected, pro-
cessed, and brought back to the laboratory where
further investigations can be made (Fig. 2). In
other cases, fish tissues are delivered to the labo-
ratory, and preserved in a condition that permits
further biological analysis.
Zden tifable diseases
Fish health programs involve many disciplines.
A diagnostic laboratory should be able to identify
signs of physical and environmental stress as well
as viral, bacterial, and parasitic diseases for warm-,
cool-, and cold-water fishes (Table 1). Pathogenic
microorganisms are in aquatic environments and
fish tissues much of the time. Pathogens, including
Renibacterium salmoninarum, infectious pancre-
atic necrosis virus, and Yersinia ruckeri, can se-
quester in healthy-appearing,fish (18). This carrier
condition causes a precarious balance. When fish
are under.stress this balance between the protective
mechanisms of the fish and the growth pressure of
the pathogen may tip, favoring the pathogen and
resulting in a disease outbreak.
Diagnostic work is sought by fish culturists for
hatchery or a wild population of fishes when mor-
talities rise above normal. For diagnosis, often the
laboratory requires samples from moribund fish
showing signs of the disease. If possible, the diag-
nostician travels to the site to observe and measure
parameters of interest such as temperature and dis-
solved oxygen levels. The pattern of mortality is
helpful in charting the progress of the disease. For
instance, its helpful to note the pond units in a
QNslE
Examination of fresh materials from
healthy, moribund and dead fish
Collection of fish tissue samples
Measurement of environmental parameters
(temperature, oxygen)
Investigation of physical factors,
hatchery condiions
Gathering information on time-course
of mortalities
l l l t
IN THE LABORATORY
Presumptive identification of pathogens
(viral, bacterial, parasitic)
Positive identification and confirmation
Drug sensitiies and effectiveness
prior to treatment
Recommenckations and reports of
additional analysis
(histopathology, toxicology)
Fig. 2. General procedures to be followed in the inves-
tigation of a fish disease outbreak.
hatchery that were affected first when trying to de-
scribe the spread of a particular disease through a
rearing station. Furthermore, the biologist may re-
quire samples from fish residing in originating wa-
ter sources to detect the presence of carrier fish
seeding pathogens into the hatchery (Fig. 3). Elec-
troshocking fish in streams for obtaining informa-
tion about the occurrence of pathogens in wild fish
may be necessary (Fig. 4).
The diagnostic laboratory should be contacted by
the hatchery before mortalities have reached a crit-
ical level. If treatments are feasible at the facility,
they need to be undertaken before the entire hatch-
ery population is involved in a major epizootic.
The early detection is especially acute when fight-
ing internal bacterial diseases, because the most
common and approved means of treatment is mix-
ing an antibiotic into feed. Treatment cannot be
administered if fishes have stopped eating, which
is often the case with bacterial septicemias.
DIAGNOSIS OF INFECTIOUS AGENTS
Collecting information and samples
If an infectious agent is the suspected cause of
Table 1. A diagnostic laboratory should be able to
identify the following diseases
Diagnostic laboratory in fish diseases 43
Salmonids
Viral diseases
Infectious pancreatic necrosis
Infectious hematopoietic necrosis
Viral hemorrhagic septicemia (Egtved disease)
Erythrocytic inclusion body syndrome
Bacterial diseases
Bacterial Gill Disease
Furunculosis
Motile Aeromonad Septicemia
Columnaris
Enteric Redmouth
Vibriosis
Bacterial Kidney Disease
Coldwater disease
Varied gram negative septicemias
Parasitic diseases
Ciliate and Flagellate infestations
Monogenic and Digenetic Trematode infestations
Cestode, Acanthocephalan and Nematode worms
Ceratomyxosis
Whirling disease
Signs of physical and environmental stressors
Low dissolved oxygen
Excess nitrogen
Excess ammonia
Handling stress
Nutritional diseases
Chemical toxicity
Anemia
Catfish and other warmwater fish species
Viral diseases
Channel catfish virus disease
Lymphocystis
Golden shiner virus
Spring Viremia of carp
Bacterial diseases
Motile Aeromonad Septicemia
Columnaris
Enteric Septicemia of catfish
Other gram negative septicemias
Parasitic diseases
Ciliate and Flagellate infestations
Monogenic and Digenetic Trematode infestations
Cestode, Acathacephalan and Nematode worms
Henneguya sp.
Nitrite poisoning
Algal blooms
Cooiwater fishes
Columnaris
External parasites
Conditions such as uninflated swim bladders
(striped bass)
a fish mortality, the diagnostician collects samples
Nitrogen supersaturation
Soft water (striped bass)
Fig. 3. A biologist analyzes water quality on-site at a farm pond (Photograph by M. Stuckey).
Fig. 4. Biologists electroshock fish in a hatchery water source. Fish suspected of being disease carriers will be taken
back to the laboratory for further examination (Photograph by M. Stuckey).
Fig. 5. A rainbow trout shows the white necrotic areas in the kidney that indicate a bacterial kidney disease (BKD)
infection (Photograph by C. Camenisch).
using the proper media to ensure that samples ar-
rive at the laboratory in good condition. As much
information as possible about the circumstances
and appearance of the disease condition is recorded
on site (19). Moribund fish may show typical disease
signs (Figs. 5 and 6). This information combined
with laboratory identification of the infectious agent
gives the investigator the most complete picture of
the problem. While in the field, every effort is made
to explore even the most unlikely possible causes.
In cases where mortalities are caused by external
parasites and bacteria, examining material on site
is the best way to identify the problem. External
protozoans may not transport well because the
holding environment becomes disadvantageous to
the parasites. Likewise, pathogenic bacteria in fish
are often overgrown by other faster-growing, com-
mensal and saprophytic bacteria during transport.
Sample analysis
Protocols. Analysis of the samples follows pro-
tocols of the individual laboratory (2421). If an in-
fectious agent is the cause of mortality, it is
generally easy to isolate a sufficient number of the
organisms for identification. If the fishes are ex-
hibiting signs typical of a particular disease and the
agent is isolated, diagnosis can be accomplished
relatively quickly. Further information on the role
Fig. 6. An initial examination of a large striped bass starts with external examination. The biologists inspect the fishs
gills for abnormal morphology and color and check for parasites (Photograph by M. Stuckey).
of the disease agent in those particular mortalities
can be obtained from diagnostic samples, includ-
ing such information as the titer of the virus in the
materials collected or the relative virulence of a
bacterial pathogen. A generalized approach is the
best for first attempts to diagnose a fish health
problem (Fig. 7). In some cases, a disease agent
may be isolated and identified but the quantitative
numbers of the pathogen are not sufficient to be
considered responsible for the mortalities. Water
quality and environmental parameters, such as ad-
dressed in Proceedings of the Bio-Engineering
Symposium for Fish Culture (22), Fish Hatchery
Management (23), and Water Quality in Channel
Catfish Ponds (24), need to be assessed to deter-
mine their contribution to mortalities. In addition,
the fish pathologist may consult further with re-
search facilities or toxicology laboratories to assist
in the diagnosis of the problem. Nutritional defi-
ciencies and even genetic aberrations also need to
be considered.
Fish parasities. Parasitic infestations are treated
with chemicals applied to the water, rather than
fed to the fish. When parasitic loads on fishes be-
come very high, treatments may not be effective
because some of the microorganisms escape the
full dose of treatment compound. Also, in some
cases so much damage and weakening of the fish
has taken place from the heavy parasitic load that
the fish will not survive the application of the
chemical treatment.
Diagnosticians are often called on by commer-
cial and sport fishermen to investigate feral fish
kills (19,25). Mortalities in wild fish can be more
diverse in cause, and generally include more un-
knowns and variables compared to mortalities oc-
curring in fish held in the more controlled hatchery
environments. Mitigation of fish health problems
in feral systems is also more complicated. Treat-
ment of fish held in large impoundments or
streams may not be cost effective. Likewise, when
poor environments or toxicants are responsible for
mortalities, the logistics of correcting the problem
may be impossible. Nevertheless, it is often impor-
tant to identify the causes of fish mortalities in wa-
ters under agency regulation, Since the public is
often involved in reporting fish mortalities of sport
fish, it becomes important to assure those using the
natural waters that the investigation of the cause is
ongoing, especially if there is a potential human
health hazard.
Microscopic examination. Expanded micros-
copy technologies are used in fish pathology. While
more advanced techniques may be out of reach for
smaller laboratories, most biologists are aware of
federal or state laboratories that perform routine
histology on properly preserved samples (26-30).
Histopathologic examination of fish can be very
useful when the role of infectious agents is unclear
or when a nutritional or toxicological basis for a
mortality is suspected (Fig. 8). Scanning electron
microscopy (SEM) is beginning to be used for
Obsewational
Indices
Inked
brood Stock
NZ
NH3 Clinical
Chemisti
Stress Tests
Fig. 7. Fish health monitoring involves using skills from various disciplines.
Fig. 8. A tissue sample is taken from a rainbow trout thymus for histological examination. The tissue can be placed
in a fixative, such as Bouins solution, and later studied for histopathological changes (Photograph by M. St uckey).
pathogen identification and an increasing amount
of information is being accumulated using trans-
mission electron microscopy (TEM) particularly
to elucidate life stages of known pathogens (31)
and explore disease conditions and their causes
(32,33).
Blood samples. Hematological assays are fre-
quently used for monitoring fish health. The wide
statistical variation in results from hematocrit, leu-
kocrit, serum protein, and other samples is well
recognized and often makes interpretation diffi-
cult. Repeated sampling of an individual fish can
cause changes in blood parameters and osmotic
changes may interfere when plasma is diluted (34-
36). Nevertheless, the ease of taking blood samples
and the large amount of available information
from other veterinary studies makes hematological
assays important tools (37-39). Hematological pro-
files that add to the clinical picture of fish health
problems are relatively easy and inexpensive. Tech-
niques are being developed for measuring the fluc-
tuations of neutrophils and macrophages resulting
from the immunosuppressive effects of bacterial
infections (40,41). Studies have followed the pan-
leukocytokemia induced by infection of Aeromonas
salmonicida (42). Infection with Renibacterium sal-
moninarum causes reductions in circulating erythro-
cytes and leukocytes (43). Reduction in blood sugar
and leukocytes can also indicate the effect of pol-
lutants (44,45). Automated blood analyzers are
used widely in hospitals, but their expense has
hindered use in fish health monitoring. Assays
monitoring the activity of blood cells, such as pro-
liferation of subpopulations of lymphocytes, have
been widely used in fish immunology research
(46,47). Their cost and the necessity of specially
trained technicians presently limits adaptation to
practical application.
Stress effects on disease
The role of stress in fish disease outbreaks is
complex (48-5 1). Fish health practitioners need to
understand the possible effects of cumulative
hatchery stressors on fishes (52). In the natural en-
vironment, stress can adversely affect fish, causing
outbreaks of opportunistic pathogens or unex-
plained spring and spawning mortalities. If a fish
pathogen gains a foothold in a population because
of stressful conditions, then treatments may be
necessary. When no infectious agent is involved,
environmental parameters and hatchery practices
can be manipulated to try and address the mortal-
ities. Decreasing feed, reducing rearing unit densi-
ties, or bringing in another water supply may lower
the numbers of mortalities or improve the condition
of the fish. Both the field biologist in charge and
the laboratory diagnostician should work closely
together in developing strategies to reduce the
number of possible stressors. If recurring seasonal
stressors are present at a facility, the pathologist
can assist in planning cultural or environmental
changes to lessen the effects of the stressors during
these predictable times. Some diagnostic centers
may have wet lab facilities to hold fish for stress
tests that may include subjecting the fish to tem-
perature changes or giving injections of a cortico-
steroid. By these methods, asymptomatic carrier
fish may be stressed to the point of developing dis-
ease signs (Fig. 9).
TREATMENT OF FISH DISEASES
Drugs, antibiotics, disinfectants
Treatment by drugs and antibiotics is the most
common method for combating bacterial diseases
of fish. Currently, two antibiotics and their ana-
logues are registered for drug use to food fish: sul-
famerizine and oxytetracycline. Formalin can also
be used for treatment of parasites. Various disin-
fectants can be used to clean facilities and herbi-
cides are commonly used to kill algae and aquatic
plants.
In most European countries, veterinarians pro-
vide diagnostic services to fish farmers and pre-
scribe treatments for disease conditions. In the
United States, fish health practitioners, biologists,
and pathologists regularly advise on the treatments
for infectious diseases. The U.S. Food and Drug
Administration oversees the approval of drugs and
chemicals in fisheries, and the current list of ap-
proved compounds is in Schnick (53). Compounds
not specifically listed for food fish use cannot be
used on fishes in hatcheries because these fish are
destined for human consumption by direct market-
ing or stocking for anglers. Whenever therapeutic
agents are administered, specific requirements for
Fig. 9. Some fish disease
COUl rse of a disease. Also,
atur e changes or drug exl
stuc :key).
laboratories may have wet labs to hold fish for longer periods of time for studying the
fish in controlled laboratory environments can be placed under s #tress induced by tern] 3er-
yosures to help confirm the presence of disease agents in carrier fish (Photograph by M.
Fig. 10. Immunization by intraperitoneal injection, as demonstrated here with a goldfish, can be economically, justi-
fied when individual fish are valuable. A multi-injector system is often used to increase the ease of handling large
numbers of fish (Photograph by M. Stuckey).
application and withdrawal times must be met
(5435).
Other strategies for control of recurring infec-
tious problems include culling of infected parental
stock, managing environmental and cultural stress
conditions at the hatchery, administering specific
treatments, improving water quality, and disinfect-
ing ponds. Each hatchery requires its own specific
programs (1, 56-58).
Immunization of fish against disease
Immunization is another method of control and
prevention of fish diseases (59-66). In the United
States, bacterins and vaccines are licensed by the
U.S. Department of Agriculture, and fish health
diagnosticians must be aware of licensed immuno-
gens and understand protocols and limitations be-
fore recommending their use. Currently licensed
bacterins in the United States include those for the
prevention of enteric redmouth disease, diseases
caused by Vibrio anguiflarum and K ordalii, and
Aeromonas salmonicida infections (Figs. 10 and
11). The commercial preparation of these bacterins
is relatively simple; most use formalin to kill the
bacteria, then further purify the antigen to amplify
the host immune response. Future vaccine and
Fig. Il. In a production facility, immunization is usually by bath, shower, or flush treatment. The photograph shows
rainbow trout being immunized through an apparatus that showers the bacterin, Yersinia ruckeri, on the fish (Pho-
tograph courtesy of T. Goodrich).
bacterin preparations will include some genetically
engineered by recombinant DNA methods. Prep-
arations made by isolating specific genes and viru-
lence factors may have enhanced potency and
efficacy (67-69). Present research also focuses on
conjugate bacterins (70) and the addition of stim-
ulants for boosting nonspecific defense mecha-
nisms as well as specific immune responses (71).
EQUIPMENTS, SUPPLIES, AND MATERIALS OF
A FISH DlAGNOSTIC LABORATORY
Equipment
Standard laboratory equipment such as cen-
trifuges, water baths, pH meters, balances, vortex
mixers, and spectrophotometers are required for a
basic fish disease diagnostic laboratory (Table 2).
Laboratories that are more research-oriented might
have sonicators, high-speed centrifuges, freeze
dryers (lyophilizers), enzyme linked immunosorbant
assay (EL&A) readers, fluorescent microscopes,
and fermentors. Sterile work areas, glassware, plas-
tic tubing, disposable plasticware, and pipettes are
necessary for assays of samples suspected of con-
taining viral infections.
Expendable materials
Expendable laboratory equipment for a fish
health laboratory is more tenuous to recommend
because each laboratory has its own specialities. A
list divided by the major disciplines in studying fish
disease pathogens is given in Table 3. The basic
laboratory biologics, reagents, and supplies for
proper diagnosis are often difficult to keep up-to-
date and in stock. If held under reasonable temper-
atures, powdered bacteriological media has a long
shelf-life. Liquid media for the growth and main-
tenance of cell cultures for viral diagnostics have a
shorter shelf life. Cell cultures themselves require
constant attention. Antisera for serodiagnostics,
unless lyophilized, may degrade within a few
months. These biologics become important re-
agents in immunological assays, such as fluorescent
antibody tests (FAT), enzyme immunosorbent as-
says (ELISA), and simpler assays such as aggluti-
nation and precipitation.
Antisera for fish disease diagnosis
The U.S. Fish and Wildlife Service supplies
antisera made through the research section at the
National Fish Health Research Laboratory, Kear-
neysville, West Virginia (72) to its own diagnostic
laboratories. For the past 15 years we have pro-
duced and distributed to qualified pathologists and
researchers polyclonal antisera made in rabbits and
goats against major fish pathogens. The develop-
Table 2. Facility and equipment needs
of the fish health laboratory
Item Purpose
Facility:
At least 300 square feet
of laboratory space
Fish tanks with flowing
water
Distilled and/or deion-
ized water supply
Oversized sink and
cleanup area
Autoclave
Equipment:
Refrigerators, freezers
and temperature-
controlled incubators
Low and high-speed
centrifuges
Hematocrit centrifuge
Water baths
Bunsen burner, gas
source
Stove with oven
Microwave oven
Microscope, general
and fluorescent
Sterile hood or room
Spectrophotometer
More specialized
equipment:
Voltage meters
Lyophilizer
ELISA reader
Fermentor
Cryostat
Automatic tissue
processor
Ultra-low temperature
freezer and/or liquid
nitrogen tank
Automatic dishwashers
Vacuum source
Air pressure source
Safety and first aid
supplies
Depends upon size of
operation
Hold live fish for short
and long-term
observations
High quality water for me-
dia preparation
General use
Sterilization of media and
contaminated refuse
Temperature control for
preservation and con-
trolled growth of
pathogens
Sample processing
Blood hematocrits and
leukocrits
Temperature control during
processing of samples
Heating and sterilization of
tools
Kitchen model for general
use
Rapid heating of media
and samples
Sample observation
Cell culture preparation,
media preparation,
pathogen transfer
Density and colormetric
readings
Electrophoresis, gel and
liquid
Freeze drying of specimens
Immunoenzyme assays
Preparation of large bacte-
ria batches
Processing frozen samples
for histological
observations
Rapid histological
preparations
Long term storage of
samples
Cleaning laboratory
glassware
Aspiration of samples
General laboratory use
Safety
Table 3. Expendable supplies for
a fish health laboratory
Virology
Cell cultures
Culture media, media components including fetal
bovine serum
Physiological saline
Antibiotics
Buffers
Culture flasks, glass or plastic
Culture plates, 96, 24, 12 or single wells
Tubes, caps, pipettes, measuring and media mixing
glassware
Virus cultures for controls
Serum neutralization/ELISA reagents for immuno-
logical assays
Bacteriology
Bacteriological media for isolation and growth
Biochemical test reagents or test systems for identi-
fication of organisms
Bacteriological stains
Petri plates, tubes, pipettes, bacteriology loops for
culture transfers, slide coverslips
Glass slides and coverslips
FAT/ELISA reagents for immunological assays
Immunology
Materials for immunological assays:
ELISA, electrophoresis, immunoblot, gel diffu-
sion reagents, test tubes, microtiter plates, nee-
dles, syringes, centrifuge tubes, pipettes.
Hematology supplies:
Hematocrit tubes
Refractometer
Parasitology
Slides, coverslips, formalin for direct examination
and sample preservation
Materials for plankton centrifuge or trypsin diges-
tion of cartilage
FAT/serological reagents for confirmation of
parasites
Histology
Tissue processing supplies, imbedding media
Histological stains
Slides, coverslips
ing techniques for the production of more specific
monoclonal antibodies permits more definitive and
rapid identifiction of fish pathogens. Antisera
against many of the fish disease pathogens are now
offered on the commercial North American and
European markets by several companies.
The ideal diagnostics laboratory would have
complete facilities for handling, growing, and pro-
cessing the parasitic, bacterial, and viral disease
agents of fish. Budgets limit media stock, the avail-
ability of continuous cell lines, and fish-holding fa-
cilities for in vivo virulence studies, so biologists
must determine priorities for their geographical
area. Warm water fish and cold water fish have
different kinds of disease problems. For instance,
some salmonid hatcheries are often in areas where
furunculosis disease, caused by Aeromonas salmon-
icida, is endemic and causes mortalities if environ-
mental parameters such as water temperatures are
not controlled. If it were truly known that this was
the only etiological agent in a fish population, the
biologist could direct his laboratory toward rapidly
controlling that problem. However, if a biologist
is conducting tests at several hatcheries without
previous indications as to which pathogens might
be endemic, then a wider base of laboratory meth-
odologies need to be established. This problem was
evident in the recent isolation of viral hemorrhagic
septicemia virus (VHSV) in chinook (Oncorhyn-
thus tshawytscha) and coho salmon (Oncorhyn-
thus kisutch) in the northwestern United States.
Because the viral agent had not been previously
reported as occurring on the North American con-
tinent, the biologists did not have on hand the se-
rological tools to give a rapid identification of
VHSV. Samples from two locations revealed that
an unknown virus was present. Later, specific anti-
sera was obtained by the fish pathologists in the
United States from European colleagues for viral
neutralization studies. Positive identification was
confirmed by the immunoblot assay, using a highly
specific monoclonal antibody to the VHSV agent.
Indeed, this example helps illustrate that well-
equipped fish diagnostic laboratories with highly
trained staff can face unexpected disease problems
and the pathologists have to be creative to adapt or
acquire new methodologies.
FISH HEALTH INSPECTION PROGRAMS
Inspection and importation
For the laboratory to perform inspection work
in response to the requirements of fish health im-
portation regulations, the facility must be equipped
to sample healthy looking animals for specific dis-
ease agents. Because these fish may be asymptom-
atic carrier animals, it is a more time-consuming
process than that of examining fish for the cause
of mortalities during an active epizootic (73,74).
Inspection programs are based on the identifi-
cation of particular fish pathogens in both feral
and hatchery populations. It is strictly a plus or mi-
nus situation (present or not present) as to whether
the pathogen is present, and makes no assessment
on whether that pathogen or any other pathogen is
the cause of a disease situation at that location.
Many potential pathogens can be present in a pop-
ulation without causing disease. For instance, if
cultural or environmental conditions are favorable,
Yersinia ruckeri will not cause enteric redmouth
disease in a hatchery even if the pathogen is iden-
tified during a routine inspection. Likewise, there
may not be an outbreak of infectious pancreatic
necrosis (IPN) even if the pathogen is harbored
within the population.
Inspections and surveys are an important part
of the cooperation needed between the hatchery bi-
ologists and the diagnostic laboratory. Biologists
can set up sampling equipment pondside for tak-
ing sex product samples from brood fish. Speci-
mens are taken back to the laboratory for further
processing and analysis.
List of fish pathogens in inspection programs
Inspections vary by state and individual Federal
agency policies. Common pathogens include the
following:
.
.
.
.
.
.
.
.
.
.
Infectious Pancreatic Necrosis Virus (IPNV)
Infectious Hematopoietic Necrosis Virus (IHNV)
Viral Hemorrhagic Septicemia Virus (Egtved Vi-
rus) (VHSV)
Channel Catfish Virus (CCV)
Yersinia ruckeri (causative agent of Enteric Red-
mouth disease)
Aeromonas salmonicida (causative agent of
Furunculosis)
Renibacterium salmoninarum (causative agent of
Bacterial Kidney Disease)
Ceratomyxa Shasta (causative agent of Cerato-
myxosis)
Myxobolus cerebralis (causative agent of Whirl-
ing Disease)
PKX (causative agent of Proliferative Kidney
Disease).
Other pathogens where protocols are still in de-
velopment or used only in certain regions include
the following:
l Erythrocytic Inclusion Body Syndrome Virus
(EIBSV)
l Viral Erythrocytic Necrosis Virus (VENV)
l Epitheliocystis organism
l Epizootic Epithiliotropic Disease Virus (EEDV)
l Sturgeon Virus
l Mad River Virus.
Techniques for inspection
Examples of application. Improved technologies
increase the capabilities of detecting very low lev-
els of organisms. With Renibacterium salmonina-
rum for instance, the fluorescent antibody technique
is a method of identification (20). This technique
is in common use in inspection laboratories, and
has been an effective tool for identifying stocks of
fish carrying the R. salmoninarum organism. Re-
cent comparisons (75-77) demonstrated that the
enzyme linked immunosorbent assay may be a
more sensitive technique for identification of R.
salmoninarum. The ELISA technology is imple-
mented in many locations, but unavailability of re-
agents and equipment has delayed its use as a
common tool. Other serological tests are becoming
available to fish health practitioners (2,70,78-83).
Epizootic Epitheliotropic Disease Virus (EEDV) is
a disease agent that is not readily manipulated or
isolated in the laboratory even during epizootics,
and, therefore, isolation from asymptomatic car-
riers is difficult (8485). The current technique for
identifying EEDV involves standardized heat and
stress testing to induce disease in potential carriers
and electron microscopic identification of the ap-
propriate viral particle. This technology is not
commonly available to disease diagnosticians.
For the majority of program pathogens, ade-
quate laboratory techniques have been standard-
ized to ensure isolation from asymptomatic carriers
(20,86). Many of these techniques are time con-
suming, with strict attention to sample integrity
and preservation being required. The main diffi-
culty with inspection work is still that the fishes are
generally not dying from pathogens at the time of
inspection. The fishes may be infected, but there
are limits of the sampling and examination tech-
niques (87).
Bacteriology. Several generalized schemes for
the identification of fish bacterial pathogens have
been published (88-90). General bacteriology ref-
erences can be helpful in composing identification
schemes for the less common bacterial pathogens
(91-94). Tube and plate tests used in other micro-
biology disciplines may be used to test the bio-
chemical reactions of the culturable pathogens.
Commercial identification systems have also been
used for identification of fish pathogens. The Fish
Disease Control Center in La Crosse has been
using the Minitek system (Baltimore Biological
Laboratory, Cockeysville, MD) for identifying the
culturable program pathogens since 1981. Minitek
uses a wide range of enzyme substrates with small
amounts of culture inoculum and is read by the
color changes in the substrate tabs. This system,
and others like it, relies on the characteristic bio-
chemical reactions of organisms to generate a code,
which then identifies that organism within a per-
centage probability. The La Crosse laboratory has
worked out the codes for the most common fish
pathogens, as well as other commonly isolated
aquatic environmental bacteria. The API ZYM
system (Analytab Products, New York, NY) and
Quantum II (Abbott Laboratories, Chicago, IL)
have also been tested with pathogens isolated from
fish (95, 96). Whether classical or automated sys-
tems are used, presumptive identification is often
confirmed with serological testing to definitively
identify an organism as a bacteriological pathogen.
Virology. Even as work is proceeding with the
development of immunoblot and other serological
assays for the identification of fish viruses (6,97,
98), the most common method of virus identifica-
tion still involves its isolation in cell culture. This
involves the use of quality, virus-specific, suscep-
tible cell lines, and proper sample processing to en-
sure virus viability (99-101). Once characteristic
cytopathic effect (CPE) is seen in the cell sheet,
then the putative virus can be identified by infec-
tivity neutralization or other serological assays.
References for new techniques. The material in
Table 4 provides information on protocols used
recently to detect fish pathogens. Several of the
techniques described in these papers are meant for
use in the research laboratory, or for use when
working with one specific pathogen. For inspection
purposes and when screening for pathogens in a
population where the disease history is not known,
it is always best to use the broadest possible tech-
niques to assure finding all possible pathogens in
that population. Customizing techniques to con-
centrate on one particular disease agent may be ad-
vantageous in some situations, where management
of that disease is the highest priority, but some
other pathogen might go undetected.
Inspection certification. Fish health officials, re-
lying on results from a diagnostic laboratory, are
often thought to certify fish populations as being
free of disease or disease agents. Such is not the
case and is a misunderstanding of the inspection
sampling process. Fish health inspection sampling
is based on a statistical table (Table 5) that lists
suggested sample sizes based on percentages of
prevalence of the particular agent in the population
(20). Most fish health regulations require a cycle of
two years of freedom from identification of spe-
cific pathogens. This is because one inspection is
limited by the assumed prevalence and the confi-
dence level of the statistical table and the sampling
procedures. When a fish health inspector signs a
report of inspection results, they are indicating
concurrence with the results of that particular in-
spection, not the fish health status of those fish at
a time other than the date the inspection sampling
takes place. The inspector is not certifying that the
facility is specific pathogen free in lots other than
those listed in the inspection form (for instance, the
inspection results would not apply to a batch of
fish that arrived on the station the day after the
samples were taken). The inspector is not certify-
ing the fishes are disease or pathogen free, merely
that the pathogens inspected for were not found
during that particular inspection visit. The identi-
fication of a pathogen does not necessarily mean
that the fish are suffering from disease caused by
that agent, only that the organism is present at that
facility.
The inspection process is strengthened by his-
torical data from a facility. While a single inspec-
tion may confirm the presence of a pathogen, the
limits described in the statistical process mean that
the negative case is not adequately assured by a sin-
gle inspection. Repeated sampling at the statisti-
cally based levels gives a better indication of the
true status of the facility, with a much more accu-
rate record developing over time showing what
pathogens are actually there. This is especially true
in feral populations, where the animals are more
dispersed and sampling adequate numbers for sta-
tistical validation is a problem.
One of the most difficult decisions faced by the
diagnostic pathologist is the determination as to
whether an infected hatchery should be disinfected
and the fish destroyed. This problem comes up
when a disease pathogen is discovered in a previ-
ously clean hatchery. Decisions may be particu-
larly difficult when only a few individual pathogens
are isolated from fish not exhibiting any signs of
the disease or a single isolation of a program
pathogen in a hatchery of six million fish. This is
occurring more frequently as our biochemical and
immunological assays become more sensitive and
we can detect the sequestered pathogens at lower
infection levels. The destruction of valuable fish,
especially broodstock or endangered species, may
be difficult to justify. Laboratory pathologists oc-
casionally have to participate in decisions that are
controversial. Their decisions are often made
public.
MONITORING FISH HEALTH
An increasingly common practice of monitoring
fish health involves using the Utah Fish Health-
Condition Profile developed by Ron Goede and
the Utah Department of Natural Resources (102,
103). It is a field technique of systematic examina-
tion of blood parameters and internal and external
appearances. Over time, data on individual facili-
ties and wild populations are developed, which al-
low the biologists to assess changes in the condition
Table 4. References giving examples of biochemical and serological techniques used for recent diagnostic cases
and in research studies for the identification and further characterization of some major fish disease agents
Viral pathogens:
Channel Catfish Virus (CCV)
117 (NEW, Dia), 118 (NEU, Dia), 119 (BIOCH-G, Dia), 120 (NAP, Tech Dev), 121 (BIOCH-G, Dia)
Infectious Hematopoietic Necrosis Virus (IHNV)
122 (BIOCH-G, Dia), 123 (BIOCH-G), 124 (BIOCH-G, Res), 125 (IMB, Res), 126 (BIOCH-G, Res), 127
(BIOCH-G, Tech Dev), 128 (IMB, Dia), 98 (FAT, Dia), 129 (ELISA, Dia)
Infectious Pancreatic Necrosis Virus (ZPNV)
130 (ELISA, Dia), 97 (CA, Dia), 131 (NEU, Res), 132 (BIOCH-G, Dia), 133 (BIOCH-G, Dia), 134 (ELISA,
PPN, Tech Dev)
Viral Hemorrhagic Septicemia Virus (VHSV)
135 (BIOCH-G, Dia), 136 (ELISA, Tech Dev), 137 (IMB, Res)
Bacterial pathogens:
Aeromonas hydrophila
138 (BIOCH-G, Dia), 139 (BIOCH-G, Dia), 140 (BIOCH-G Dia), 141 (BIOCH-G, Res)
Aeromonas salmonicida
142 (IOCH-G, Dia), 143 (BIOCH-G, Dia), 144 (ELISA, Dia), 145 (BIOCH-G, Dia) 146 (ELISA, Dia), 147 (CA,
Tech Dev), 148 (BIOCH-G, Dia), 149 (Bioch-G, Res), 150 (BIOCH-G, Dia)
Edwardsiella sp.
151 1981 (BIOCH-G, Res), 152 (FAT, Dia), 153 (BIOCH-G, Res), 154 (BIOCH-G, Dia), 155 (ELISA, Dia)
Ftexibacter columnaris
156 (BIOCH-G, Dia), 157 (BIOCH-G, Dia), 158 (BIOCH, Res)
Renibacterium salmoninarum
159 (PPN, Dia), 160 (BIOCH-G, Dia), 161 (FAT, Dia), 162 (FAT, Dia), 163 (CA, Dia), 164 (BIOCHEM-G,
Dia), 165 (BIOCH-G, Dia), 166 (FAT, Dia), 167 (ELISA, Dia), 168 (ELISA, Res), 169 (FAT, Dia), 170 (IMB,
Res), 171 (BIOCHEM-G, Dia), 172 (BIOCH-G, Dia), 173 (BIOCH-G, Dia), 77 (ELISA, FAT, CA, AGG)
Vibrio sp.
174 (BIOCH-G, Dia), 5 (IMB, Dia), 175 (ELISA, Dia), 176 (BIOCH-G, Dia), 158 (BIOCH-G, Dia), 177
(BIOCH-G, Dia)
Yersinia ruckeri
178 (BIOCH-G, Dia), 179 (BIOCH-G, Dia), 136 (Dia), 146 (ELISA, Dia), 180 (Bioch-G, Dia), 181 (BIOCH-G,
Tech Dev), 182 (BIOCH-G, Dia)
Protozoan pathogens:
Ceratomyxa Shasta
183 (EX, Dia)
Myxosoma (Myxobolus) cerebralis
135 (EX, Dia), 184 (EX, Res), 185 (EX, Dia), 186 (EX, Res), 187 (EX, Res), 188 (EX, Tech Dev), 189 (EX, Tech
Dev), 190 (FAT, Tech Dev), 191 (FAT, Res)
*Assays (AGG, agglutination; BIOCH-G, biochemical and growth characteristics; CA, coagglutination; ELISA, en-
zyme immunosorbent assay; FAT, fluorescent antibody technique; NAP, nucleic acid probe; EX, gross examination;
IMB, immunoblot; NEU, viral or serum neutralization; PPN, precipitin or gel diffusion) and the purposes (Dia, di-
agnostics; Res, research; Tech Dev, technique development) are given.
of the populations. This technique is applied by
field and hatchery personnel (4,104). If a labora-
tory program is combined with this systematic ap-
proach of monitoring fish health changes, the
diagnostician is better equipped to make recom-
mendations to the biologists from this more com-
plete picture than from individual examinations at
irregular intervals.
Other ways of monitoring fish health have been
developed. Some rely on particular physiological
changes in fish tissues. Wolke et al. (105) reported
that following the characteristics of pigmented
macrophages in the fish kidney was useful as an in-
dicator of fish health. Orme and Lemm (106) pre-
sented a procedure for classifying lens cataracts
that arose as a result of inadequate diets. Many of
these different monitoring techniques have features
that can be used in given situations. Most biolo-
gists recommend using several parameters for con-
firmation of changes in fish health.
Table 5. Sample size required for lots of fish for deter-
mining assumed pathogen prevalence (from Ref. 20).
The table is generated at the 95% confidence level.
At 2% At 5% At 10%
Prevalence Prevalence Prevalence
Size of Size of Size of
Lot Size Sample Sample Sample
50 50 35 20
100 15 45 23
250 110 50 25
500 130 55 26
1,000 140 55 27
1,500 140 55 27
2,000
145 60 27
4,000
145 60 2-l
10,000 145 60 27
100,000 or more 150 60 30
CERTIFYING AND REGULATING AGENCIES
The signature of a qualified pathologist or in-
spector is required as testimony that certain disease
agents are not present, before transportation of
fish across state or international boundaries is al-
lowed. Many foreign countries are justifiably cau-
tious about the introduction of diseases into new
geographical areas, so strict procedures are
followed.
The American Fisheries Society/Fish Health
Section (AFS/FHS) periodically revises and pub-
lishes a manual entitled The Fish Health Blue
Book-Procedures for the Detection and Identifica-
tion of Certain Fish Pathogens (20). In Canada,
The Department of Fisheries and Oceans publishes
Fish Health Protection Regulations: Manual of
Compliance (86) that sets forth guidelines to con-
trol the spread of fish disease agents. The U.S.
Fish and Wildlife Service relies on its Fish Health
Policy and Implementation Guidelines for sam-
pling and laboratory protocols.
Inspection of salmonids and salmonid fish
products imported by the United States is governed
by legislation enacted by the United States Con-
gress in 1969 and appears in the Code of Federal
Regulations Part 50 16.13-33 (107). Title 50 gives
the U.S. Fish and Wildlife Service the authority to
certify certain individuals as qualified to inspect
and clear fishes for transportation into the United
States. Inquiry about necessary qualifications be-
fore moving fish must be made to the U.S. Fish
and Wildlife Service offices in Washington, D.C.
Fish imported into the U.S. must meet Title 50 re-
quirements as well as those of the state into which
the fish are due to be transported. The Canadian
government certifies individuals with adequate lab-
oratory facilities through its regulations to per-
form inspections in keeping with the Manual of
Compliance.
The question arises: How is the fish pathologist
known to be qualified to give this judgment? Sev-
eral programs have been set up by various agencies
to give credibility to qualified fish disease patholo-
gists. The AFS/FHS has two classifications of fish
health specialists: the Fish Health Inspector must
have a basic academic education in biological sci-
ences and work experience in fish health laboratory
procedures; the Fish Pathologist requires specialized
training including specific course work in addition
to a Bachelors degree. The Fish Pathologist is also
required to take a comprehensive examination by
the AFS/FHS Certification Committee.
The Office of International Epizootics, in Paris,
France, works closely with European and other
countries in matters pertaining to the transportation
of domestic and wild animals, including fish. Most
European countries recognize a veterinarians signa-
ture for certification of fish inspection. The Euro-
pean Association of Fish Pathologists holds meetings
every two years to discuss fish health problems and
publishes procedures, regulations, and protocols
(108), but is not involved with certification.
In Japan, importation and certification of spe-
cific pathogen-free fish is usually done by a
veterinarian. The Asian Fisheries Society, a newly
formed organization, is looking forward to estab-
lishing regulations for international transportation
of fish and fish products in southeast Asia.
KEEPING CURRENT IN FISH HEALTH
Importance of communication
Diagnostic techniques evolve and change in re-
sponse to new challenges. The individual fish
pathologist faces the problems of how to keep cur-
rent with the times and his or her peers. Patholo-
gists are responsible for reporting findings that are
important to other laboratories, especially if new,
virulent pathogens are causing contagious diseases.
Professional meetings offer opportunities to dis-
cuss common problems and disseminate new infor-
mation. Local and regional meetings are often
informal and promote a good interaction among
biologists; national and international meetings usu-
ally offer more direct scientific reporting. Diag-
nosticians should be encouraged to attend.
Journals and periodicals on fish health. Current
journals and books are important sources of infor-
mation on the latest techniques and diseases. The
diagnostician should have several of these at hand.
Notable journals to be considered are Journal of
Aquatic Animal Health, Diseases of Aquatic Or-
ganisms, Fish and Shellfish Immunology, Journal
of Fish Biology, Journal of Fish Diseases, Trans-
actions of the American Fisheries Society, Cana-
dian Journal of Fisheries and Aquatic Sciences,
Journal of Wildlife Diseases, Aquaculture, Pro-
gressive Fish Culturist, and Annual Review of Fish
Diseases. Several newsletters on fish health, such
as the AFS/Fish Health Section Newsletter, play
important roles in the more rapid dissemination of
important trends and events. The Fish Disease
Leaflet series is published through the National
Fish Health Research Laboratory and presents ex-
cellent reviews of current selected diseases.
Textbooks on fish health. There are several
excellent books on fish diseases (109,110) and fish
pathogens (1490,111). There are also several excel-
lent reviews detailing more about hatchery man-
agement and fish health (23,112). Refresher courses
are offered by governmental agencies and private
consultants. The Fish Health Management and
Disease Diagnosis course offered by the U.S. Fish
and Wildlife Service at Leetown, West Virginia,
offers 5-6 months of intensive training directed to
the pathologists of the U.S. Fish and Wildlife Ser-
vice. Excellent fish disease courses are given at sev-
eral universities in North America. Introductory
material and workshops for beginning hatchery
personnel or field biologists are also available
(113-116).
FUTURE TRENDS FOR FISH DISEASE
DIAGNOSTIC LABORATORIES
Although future trends in fish disease diagno-
sis and control are difficult to predict, they most
probably follow the pathways of modern medical
and veterinary research. Fish health laboratories
will become more centralized and larger, justifying
purchases of expensive analyzing equipment and
the employment of highly trained personnel. Au-
tomated blood analyzers for precise determinations
of serum and plasma constituents and flow cytom-
eters for counting numbers of a lymphocyte pop-
ulation may accurately reveal the influence of
subclinical infections. The biochemical and immu-
nological assays will become more sensitive and
easier to perform, upgraded by greater antibody
specificity and antigen purification. Even now, kits
for isolation and identification of fish pathogens
are being refined and marketed. ELISA, immuno-
blot, and other sensitive conjugated assays will be
simplified to give presumptive evidence and facil-
itate pondside diagnosis. Quality antisera is becom-
ing available, greatly increasing the sensitivity and
reliability of the serological assays (Fig. 12). In ad-
IMMUNODIAGNOSTIC ASSAYS
Fig. 12. The production of antisera for the serological identification of fish disease agents can be done by isolating
the disease pathogen and obtaining a culture in sufficient quantities so rabbits can be injected for the production of
polyclonal antibody. An alternative method is to inject mice with the pathogen and establish monocional, antibody-
producing cell lines by the fusion of the individual antibody-producing cells with myeloma cell lines.
dition, contemporary biotechnology is progressing
rapidly toward genomic sequencing of fish patho-
gens. Matching specific DNA and RNA segments
and amplification by the polymerase chain reaction
(PCR) assay will further increase credibility of fish
disease diagnosis. Given these developments and
the tremendous expansion of diagnostic research,
fish pathologists someday may define our fishery
environments biochemically. Fish and water sources
will be described in terms of antigens and whole
pathogens present, and we will monitor their de-
structive potential in relation to nonspecific defense
mechanisms and specific immune responses of the
fish.
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
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