Ecological Research Series
SURVIVAL AND IMMUNE RESPONSE OF COHO
SALMON EXPOSED TO COPPER
Environmental Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Corvallis, Oregon 97330
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development, U.S. Environmental
Protection Agency, have been grouped into five series. These five broad
categories were established to facilitate further development and application of
environmental technology. Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in related fields.
The five series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5. Socioeconomic Environmental Studies
This report has been assigned to the ECOLOGICAL RESEARCH series. This series
describes research on the effects of pollution on humans, plant and animal
species, and materials. Problems are assessed for their long- and short-term
influences. Investigations include formation, transport, and pathway studies to
determine the fate of pollutants and their effects. This work provides the technical
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This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia 22161.
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EPA-600/3-77-031
March 1977
SURVIVAL AND IMMUNE RESPONSE OF
COHO SALMON EXPOSED TO COPPER
by
Donald G. Stevens
Western Fish Toxicology Station
Corvallis Environmental Research Laboratory
Corvallis, Oregon 97330
CORVALLIS ENVIRONMENTAL RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U. S. ENVIRONMENTAL PROTECTION AGENCY
CORVALLIS, OREGON 97330
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DISCLAIMER
This report has been reviewed by the Corvallis Environmental Research
Laboratory, U. S. Environmental Protection Agency, and approved for publica-
tion. Mention of trade names or commercial products does not constitute
endorsement or recommendation for use.
11
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FOREWORD
Effective regulatory and enforcement actions by the Environmental Protection
Agency would be virtually impossible without sound scientific data on
pollutants and their impact on environmental stability and human health.
Responsibility for building this data base has been assigned to EPA's Office
of Research and Development and its 15 major field installations, one of which
is the Corvallis Environmental Research Laboratory, (CERL).
The primary mission of the Corvallis Laboratory is research on the effects of
environmental pollutants on terrestrial, freshwater, and marine ecosystems;
the behavior, effects and control of pollutants in lake systems; and the
development of predictive models on the movement of pollutants in the
biosphere.
This report describes part of the research on copper pollution being conducted
at WFTS. Other reports are being prepared which describe additional research
with copper, cadmium, and zinc.
A. F. Bartsch
Director, CERL
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ABSTRACT
Vaccination with Vibrio anguillarum by oral administration during copper
exposure and intraperitoneal injection prior to copper exposure was employed
to investigate the effects of copper upon survival and the immune response of
juvenile coho salmon (Oncorhynchus kisutch). Following copper exposure the
survivors were challenged under natural conditions to V_. anguillarum, the
causative agent of vibrios is in fish.
Copper concentrations of 18.1 yg/liter and higher caused significant
mortality among coho fry during 30 days of exposure. The exposure of copper
bioassay survivors to a natural challenge against _V. anguillarum in seawater
caused significant mortality among those fish from concentrations of copper at
13.9 yg/liter and higher. The reduced number of dead fish positive for V_.
anguillarum from the challenge suggests that sublethal copper stress and
difficulty with seawater adaptation may have caused several deaths.
Significant mortality occurred among coho finger!ings exposed to 24.6
yg/liter copper and higher for 31 days. Most of the survivors of these con-
centrations were unable to adapt to seawater and died within the first three
days of challenge. Significant mortality also occurred during adaptation of
survivors from 18.2 yg/liter copper where the mean mortality resulting from 31
days exposure was only 2%. The antibody level against \l_. anguillarum,
measured by agglutinin titer, was significantly reduced in fish exposed to
this concentration of copper when compared to that developed in control
animals.
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CONTENTS
Foreword i i i
Abstract iv
List of Figures vi
List of Tables vii
Acknowledgements viii
1. Introduction 1
2. Conclusions 2
3. Recommendations 3
4. Literature Review 4
Toxicity of Copper to Fish 4
Heavy Metals and the Immune Response 4
Stress and Disease 5
Vibrios is 6
5. Methods and Materials 7
Preliminary Bioassays 7
Copper Bioassay and Oral Vaccination 8
Copper Bioassay and Vaccination by Injection. . 9
Determination of Agglutination Titers.. , . . . 11
Statistical Analysis 12
6. Results 13
Preliminary Bioassays for the Oral Vaccination
Experiment 13
Copper Bioassay and Oral Vaccination 13
"Preliminary Bioassay for the Vaccination by
Injection Experiment 19
Copper Bioassay and Vaccination by Injection. . 26
Antibody Titers Developed Following Injection,
Copper Exposure, and Seawater Challenge. ... 26
7. Discussion 30
References 33
vn
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FIGURES
Number Page
1. Mortality of coho fry exposed to copper during the
preliminary bioassays conducted prior to the oral
vaccination test with indicated variations in
alkalinity and hardness 14
2. Mean mortality of coho salmon fry exposed to copper
during the oral vaccination bioassay 15
3. Mean mortality of coho salmon fry during natural
• challenge to V_. anguillarum following 30 days exposure
to copper 21
4. Mortality of coho fingerlings exposed to copper during
the preliminary bioassay for the vaccination by in-
jection test with variations of alkalinity and
hardness 23
vm
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TABLES
Number Page
1. Precision of Microtiter Agglutination Test Using a
Pooled Serum Sample 11
2. Mortality of Coho Salmon Fry During Simultaneous Oral
Vaccination and Copper Exposure in Freshwater 16
3. Chemical Characteristics of the Water in the Aquaria During
the Oral Vaccination Bioassay 17
4. Measured Total Copper Concentrations Taken During the Oral
Vaccination Bioassay 18
5. Chemical Characteristics of the Water in the Lint Slough
Holding Tanks During the Challenge Following the Oral
Vaccination Bioassay 20
6. Mortality of Coho Salmon Fry During the Natural Seawater
Challenge to V_. anguillarum Following Simultaneous Oral
Vaccination and Copper Exposure 22
7. Measured Total Copper Concentrations From Experimental Tanks
During the Injected Bacterin Bioassay 24
8. Chemical Characteristics of the Water in the Experimental
Tanks During the Injected Bacterin Bioassay 25
9. Mortality of Finger!ing Coho Salmon Vaccinated by Injection,
Exposed to Copper and Transferred to Seawater for a Natural
Challenge to V_. anguillarum 27
10. Mean Agglutinating Antibody Titers Developed in Fingerling
Coho Salmon from a Single IP Injection with V_. anguillarum
Bacterin Prior to Copper Exposure and Natural Challenge. . . 28
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ACKNOWLEDGEMENTS
My appreciation is expressed to the staff at the Western Fish Toxicology
Station Corvallis who provided assistance during the course of this study.
Dr. Gerald Bouck is thanked for providing the administrative support and
guidance so vitally needed. Jan Wold and Don Samuelson performed the routine
water chemistry analysis and provided bioassay assistance. Dr. Gary Chapman
and Dr. Alan Nebeker are thanked for their review commentaries. Dr. Ronald
Garton is acknowledged for his administrative support. The manuscript was
typed by Gwen White and Deidra M. Boczkiewicz.
The Department of Microbiology at Oregon State University provided
significant assistance during this study. The guidance by Dr. John Fryer is
gratefully acknowledged. Sincere thanks are extended to Dr. John Rohovec and
Jerry Zinn for their assistance throughout the study.
Another important contribution to the study was made by the Oregon
Department of Fish and Wildlife which provided the fish and allowed the use
of the Lint Slough facility. The assistance of Robert Garrison and Ernie
Keiski at that facility is gratefully acknowledged.
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SECTION 1
INTRODUCTION
The establishment of water quality standards to protect aquatic life is
based largely upon criteria developed under laboratory conditions. Acute and
chronic bioassays have been used to establish copper toxicity. Chronic tests
are used to select a "no effect level" which is defined as the highest con-
centration that has no adverse effect on survival, growth, and reproduction.
During these studies, environmental conditions are usually ideal when compared
to those found in nature. Stress from environmental conditions can be an
important factor in allowing an outbreak of infectious disease to occur among
fish populations. Wild fish populations contain many fish which are resistant
to disease but are carriers of pathogens capable of infecting nonresistant
fish. These pathogens are released continuously into the aquatic environment
and may survive long enough to infect other fish.
For many years marine aquaculture operations have suffered tremendous
losses among cultured fish species from Vibrio anguillarum, the etiological
agent of vibriosis. Renewed interest in mariculture of salmonid fish has
prompted considerable research toward the control of this bacterial disease.
Injected vaccines provide good protection and may be practical for fish
farming operations but are impractical for hatchery-type production. Oral
vaccine incorporated into the diet has been shown to be effective and may be
an economical means of protecting juvenile fish against _V. anguillarum. Both
types of vaccine were employed in this study.
The purpose of this work was to determine the mortality from copper
exposure and the effect of copper exposure on the immune response in juvenile
coho salmon (Oncorhynchus kisutch). Continuous-flow diluters were used to
expose the fish to constant concentrations of copper for approximately one
month. The copper levels were designed to cover the range of concentrations
from a no-effect level to that level causing nearly 100% mortality. A
natural challenge to _V. anguillarum was to be used to determine if the immune
response was affected~by the copper exposure. Antibody titers were measured
in the injected fish to determine the magnitude of the immune response in
fish exposed to the different copper concentrations and subsequent challenge.
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SECTION 2
CONCLUSIONS
1. A sub-lethal concentration (18.2 yg/liter) of copper significantly
reduced the level of antibody produced in finger!ing coho salmon vaccinated
by intraperitoneal injection.
2. Fingerling coho salmon, vaccinated or unvaccinated, surviving con-
centrations of 32.9, 24.6, and 18.2 yg/liter copper suffered significant
mortality adapting to seawater.
3. Coho salmon fry, orally vaccinated during exposure to copper concen-
trations of 26.6, 18.1, and 13.9 yg/liter suffered significant mortality when
placed in seawater.
4. The low percentage of coho salmon fry positive for V_. anguillarum
suggests that sub-lethal copper stress and poor sea water adaptation may have
caused a number of deaths during the seawater challenge.
5. Copper concentrations of 18.1 yg/liter and higher caused significant
mortality among coho salmon fry during 30 days of exposure to copper.
6. Fingerling coho salmon suffered significant mortality when exposed
31 days to copper concentrations of 24.6 yg/liter and higher.
7. Increased alkalinity and hardness decreased the toxicity of copper
to coho salmon fry.
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SECTION 3
RECOMMENDATIONS
Data from this study indicate that sublethal levels of copper interfered
with both the immune response and the mechanism for seawater adaptation.
Therefore, it is recommended that toxicity bioassays using smolt size
anadromous fish be designed to test such latent physiological effects.
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SECTION 4
LITERATURE REVIEW
TOXICITY OF COPPER TO FISH
Copper, in the form of soluble salts, naturally occurs in lakes and
streams only in trace amounts up to about 20 yg/liter. Pollution from
industrial uses, mine drainage, corrosion of copper and brass tubing, and
aquatic herbicides probably accounts for the majority of the sources. Fish
and other aquatic life are very sensitive to copper and considerable research
has been conducted in this area. Doudoroff and Katz (1953), and McKee and
Wolf (1963) have summarized earlier research.
Long-term or chronic exposure of fish to copper can produce sublethal
effects which do not affect survival or appearance of adult fish but may
affect spawning activity (Mount 1968; Mount and Stephan, 1969). McKim et_
al., (1970) studied the chronic effect of copper on several blood character-
istics and were able to show that during the first 21 days of exposure to
various copper concentrations the fish appeared to be under considerable
stress as indicated by changes in the blood parameters. Most of the changes
were not detectable after about 11 months exposure indicating a possible
accommodation or adaptation.
HEAVY METALS AND THE IMMUNE RESPONSE
Heavy metals have been shown to cause other physiological and morpho-
logical changes in fish which could be used to detect heavy metal poisoning.
Jackim et_ aj_., (1970) reported changes in activities of five liver enzymes
after exposing fish and fish liver homogenates to salts of various metals.
Using killifish (Fundulus heteroclitus) exposed to 96 hour TL (50% mortality
after 96 hours exposure) concentrations of copper, they demonstrated a
reduced activity with most of the enzymes tested. Winter flounder
(Pseudopleuronectes americanus) exposed to high and medium concentrations of
copper exhibited morphological changes in gill, kidney, and liver tissue
(Baker, 1969). The necrosis of kidney hemopoetic tissues suggested that
copper induces hemolytic anemia as an initial lesion.
Suppression of some immune mechanisms has been demonstrated with several
heavy metals. Nontoxic levels of lead increased the susceptibility of mice
to Salmonella typhimurium (Hemphill et_ aj_., 1971). Mice were injected with
different amounts of lead nitrate over a 30 day period. The percent mortality
was directly related to lead intake when challenged with an LD50 (dose lethal
to 50% of the animals) of a culture of S_. typhimurium. The specific mechan-
ism involved in this study was not identified.
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There is a great amount of research today directed toward finding anti-
tumor drugs which do not suppress the immune response. Suppression of anti-
body plaque-forming cells with cis-Platinum (II) Diamminodichloride was
demonstrated in mice by Khan and Hill (1971). It was thought that the anti-
tumor effect of the platinum was due to stimulation of immunity, however, the
study did not substantiate that idea. There was in fact a suppression of
normal antibody forming cells. In a similar study Jones ejt al_. , (1971)
investigated the effects of cadmium on the synthesis of specific antibody.
Rats receiving cadmium one week and two weeks prior to antigen showed
opposite antibody responses. Rats receiving cadmium two weeks prior to
antigen had an enhanced antibody response. However, those injected one week
prior to antigen had the response delayed and suppressed. The exact mechan-
ism affected by cadmium was not identified.
The effects of copper on the immune response of vertebrates has received
some attention through nutritional studies in small animals. Excess copper
in a ration fed to mice reduced the phagocytic activity of leukocytes, the
lysozyme titer and the bactericidal action of blood serum (Kolomiitseva ejt^
a!., 1969). In most nutritional studies there appears to be an optimum level
of copper which allows the maximum response of the mechanism involved.
Investigators working with drugs used to treat trypanosome infections
found that colloidal copper interfered with the action of the drugs (Jancsb
and Jancsb, 1934; Ormerod, 1961). It was determined that the copper blocked
phagocytosis by the reticuloendothelial system which potentiated the action
of the drugs.
STRESS AND DISEASE
Fish in natural water are continuously exposed to pathogens which are
capable of causing disease. However, disease only occurs when the proper
relationship between the fish, the pathogen, and the environment is estab-
lished (Snieszko, 1974). Natural waters are not stable and can vary both
physically and chemically depending upon natural factors or the activities of
man. Many instances of pollution-caused fish kills lack specific data about
the effects of certain chemicals upon the fish and how these chemicals may or
may not allow invasion of the fish by disease organisms. A convincing
observation on the combined effect of pollution, high water temperature, and
low flow on fish deaths attributed to Aeromonas hydrophlia was described by
Pippy and Hare (1969). Migrating Atlantic salmon (Salmo salar) and some
resident fish were affected by the outbreak. Toxic concentrations of copper
and zinc coupled with low water level and high water temperature preceded the
outbreak in the Miramichi River in New Brunswick, Canada. Isolates of A.
hydrophlia capable of killing test fish were isolated from moribund fish.
These kinds of environmental changes place fish in a state of stress
which can be an important factor in the outbreak of infectious disease
(Wedemeyer, 1970). When the stress requires an adjustment beyond the fish's
capabilities, it is lethal, and while physiological disorders or disease
result from sublethal stress, fish are not without some capacity to combat
disease organisms. It is well established that they are capable of an immune
response which can afford protection against certain pathogens (Ridgway ejt
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al_., 1966; Snieszko, 1970). A recent review by Corbel (1975) summarizes the
current available information on the immune response in fish.
VIBRIOSIS
One particular disease commonly found in fish from marine environments
is vibriosis. Vibrio-associated diseases have been described for many years
in wild and captive populations of marine fish (summarized by Anderson and
Conroy, 1970). The first report of vibriosis in Pacific salmon, specifically
caused by V_. anguillarum, was described by Cisar and Fryer (1969). An epi-
zootic, which killed more than half a million juvenile fall chinook salmon,
occurred in the Lint Slough salt water rearing impoundment near Waldport,
Oregon. Pure cultures of V_. anguillarum, capable of causing infection and
death in inoculated test fish, were isolated from 78% of the dead fish
examined. Research in the use of oral and injected vaccines for control of
vibrio disease has shown encouraging results (Hayashi et^ aj_., 1964; Nelson,
1972; Fletcher and White, 1973; Rohovec et_ al_., 1975). These investigators
showed that both oral vaccination and vaccination by injection can provide
good protection against vibriosis. High serum antibody titers were obtained
by injection, resulting in fish more resistant to vibrio attack. Fryer et_
al_., (1972) reviewed the etiology, pathology, and distribution of vibriosis.
Control of the disease with drugs and results of early studies with oral
vaccines are described in detail. Further research into the methods of
vibriosis control, especially through oral immunization, were reported by
Rohovec (1974). The investigator developed several bacterins which could be
used both orally and parenterally. He determined that both wet-packed whole
cells and formalin-killed lyophilized whole cells of the organism were
effective immunogens. Intraperitoneal injection of formalin-killed whole
cells mixed with Freund complete adjuvant provided protection against a
natural challenge to V_. anguillarum. Other parameters such as length of
vaccination period, concentration of vaccine, and optimum water temperatures
are described.
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SECTION 5
METHODS AND MATERIALS
PRELIMINARY BIOASSAYS
Prior to beginning each immunization experiment, one or more preliminary
bioassays were conducted to determine the range of copper concentrations to
be used. Ten fish were used in the two highest concentrations in each bio-
assay. Each test was continued for at least 72 hours.
The apparatus used to expose coho fry to copper for both the preliminary
bioassays and the oral immunization test was a proportional diluter similar
to that described by Mount and Brungs (1967). Five concentrations and a
background control were randomly assigned to duplicate sets of six aquaria.
The aquaria measured 27 x 21 x 52 cm and each held approximately 27 liters of
water. The diluter was designed to deliver one liter to each aquarium during
every cycle (97 seconds/cycle) with a continuous flow to the aquaria. The
copper stock solution was fed to the diluter from a marriotte bottle.
The apparatus used for copper exposure during the injected bacterin test
as well as its preliminary bioassay was a continuous-flow serial diluter
described by Garton (1976). The diluter was designed to deliver two liters
per minute to each of 12 aquaria with continuous flow. Each aquarium
measured 25 x 39 x 90 cm and contained approximately 74 liters of water. The
stock solution of copper was introduced to the dilution apparatus by a
metering pump. The stock solutions for all tests were prepared with copper
chloride (CuCl2:2H20 reagent grade) and acidified with H2S04 (0.1 ml/liter)
to prevent precipitation. The stock solutions were prepared using well
water.
Each aquarium was covered with 1/4-inch mesh plastic screen and the area
around the sides of the aquaria was shrouded with black plastic curtains to
prevent disturbance from room activities. -Gentle aeration was provided to
each aquarium to insure adequate oxygen levels. The photoperiod was con-
trolled to simulate natural photoperiod during the spring months when use of
the laboratory would cause unnatural lighting.
Water samples (500 ml) for copper analysis were collected twice a week
from each concentration. Each sample was acidified with 12.5 ml of HN03 and
evaporated to dryness. The samples were brought up to a volume of 25 ml with
a 2.5% HN03 solution to give a 20 fold concentration. These samples were
analyzed by flame atomic absorption spectrophotometry. Water samples for
copper analysis taken during the injection bioassay were analyzed by flame-
less atomic absorption spectrophotometry. The water used for all bioassays
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was obtained from wells 30 meters from the Willamette River at Western Fish
Toxicology Station (WFTS), Corvallis, Oregon.
Dissolved oxygen, total hardness, alkalinity, and pH determinations were
conducted daily. Measurements were conducted according to the American
Public Health Association (1971). Temperatures were controlled and continu-
ally recorded during the copper bioassays.
COPPER BIOASSAY AND ORAL VACCINATION
Coho salmon fry, 45 to 50 mm in fork length with a mean weight of one
gram, were obtained from the Fall Creek Hatchery, Oregon Department of Fish
and Wildlife. They were trucked to the Western Fish Toxicology Station
(WFTS) and held in a 200-gallon circular tank. The fish were fed Oregon
Moist Pellet (OMP) diet for three days and then switched to Oregon Test Diet
(OTD). The formulation of the OTD used was a modification ±-' of that given
by Lee et_ al_., (1967). The diet was prepared in one-liter batches. Follow-
ing overnight refrigeration, the gelled diet was sliced and forced through
screens with 1/16-inch mesh. The cut pieces were weighed out in daily
rations for each aquarium and frozen in plastic bags.
The OTD used to vaccinate the coho fry was prepared as above except for
the addition of bacterin during the preparation. Formalin killed lyophilized
whole cells of V_. anguillarum from the Department of Microbiology at Oregon
State University were used in preparation of the bacterin. The cells had
been grown in Tryptic Soy Broth, formalin killed, washed in buffered saline,
lyophilized, and stored under vacuum until used in the bacterin. ^
One hundred fish were placed in each of 12 aquaria by stratified random
assignment of groups of five fish. They were acclimated to the apparatus and
fed OTD (at 6% of body weight) for six days. Beginning on the seventh day,
the OTD with bacterin was fed and the copper exposure was initiated.
The copper solution was introduced into the diluter and the aquaria were
allowed to come up to concentration. The 95% replacement time in the aquaria
was approximately two hours. The diluter was designed to provide duplicate
copper concentrations (40, 28, 19.6, 13.7, 9.6 yg/liter) and a background
control. The aquaria were checked for dead fish before and after feeding
each day. The OTD with bacterin was fed once a day slowly enough to allow
total consumption of the diet. The amount of diet fed each aquarium*ber day
was adjusted weekly according to the mortality incurred. This would provide
equivalent amounts of bacterin per aquaria according to the number of fish
present at various concentrations. Copper exposure was terminated following
a 30-day exposure. The fish were then held under control conditions for a
10-day rest period and fed OMP. During this period the aquarium which
received the highest concentration of copper, and where all fish had died,
was rinsed thoroughly and supplied with freshwater without copper. One
hundred stock fish were placed in this aquarium to serve as an unvaccinated
control during the seawater challenge.
Modification by J. H. Wales, Food Science and Technology, O.S.U.
2Bacterin provided by John Rohovec, O.S.U. Department of Microbiology.
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After the 10-day rest period the fish were trucked to Lint Slough for a
challenge to V_. anguillarum. The Lint Slough facility at Waldport, Oregon is
a marine rearing impoundment operated by the Oregon Department of Fish and
Wildlife in which epizootics of vibriosis in juvenile chinook salmon
(Oncorhynchus tshawytscha) have been reported, with mortality as high as 90%
of the fish (Cisar and Fryer, 1969). The coho were placed in fiberglass
tanks containing 68 liters of a 1:2 mixture of seawater and freshwater.
After 20 minutes the mixture was brought up to 1:1. Following 30 minutes at
1:1, seawater was pumped directly into the tanks from the slough at a rate of
four liters per minute. The challenge was continued for 25 days. The fish
were fed OMP (all they would eat once a day), and dead fish were noted and
removed twice a day.
Dead fish were placed in plastic bags, labeled, and stored on ice when
examined the same day as removal. When examination was delayed, the fish
were frozen until the day of necropsy. Examination of the dead fish included
visual observation for gross pathological symptoms, aseptic dissection and
bacteriological culture of kidney and liver tissue. Furunculosis and Brain
Heart Infusion agar plates were innoculated and examined for typical colonies
of V_. anguillarum after 24 hours incubation at 30 C. Presumptive tests on
typical colonies included microscopic examination of the gram reaction, and
observation for morphology and motility using phase contrast microscopy.
Suspected isolates were confirmed with V_. anguillarum antiserum in rapid
slide agglutination tests using the suspected isolates as antigen (Kolmer e_t
al., 1951). Death was attributed to V_. anguillarum when all these tests were
positive.
COPPER BIOASSAY AND VACCINATION BY INJECTION
Coho salmon (mean weight of seven grams) raised at WFTS were selected
for this study. These fish were obtained as eggs from the Fall Creek
Hatchery, Oregon Department of Fish and Wildlife. One hundred fish were
assigned to each of 12 aquaria as in the previous test. They were fed OMP
(at 2% of body weight per day) throughout the acclimation period and bio-
assay.
On the sixth day, the fish were not fed and the next day the fish in
tanks 7 to 12 were removed, anesthetized with MS-222 (Tricane Methane-
sulfonate)^, and injected intraperitoneally with _V. anguillarum bacterin.
The V_. anguillarum isolates were obtained from stock cultures in the Depart-
ment of Microbiology at Oregon State University. Isolate L.S. 71 was cul-
tured from spring chinook at Lint Slough in 1971. Stock cultures of this
isolate were maintained in Cytophaga Seawater Agar deeps at 4 C (Pacha and
Ordal, 1967).
Routine antigen preparation for mass cell culture, immunization, and
agglutination were produced as follows. Typical V_. anguillarum colonies were
selected from Furunculosis Agar flats, examined for motility, and checked by
gram stain and rapid slide agglutination.
3Kent Chemicals Ltd.
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Confirmed colonies were used to inoculate Tryptic Soy Broth in 15ml test
tubes. Following 24 hours growth, 2 ml were inoculated into 32-ounce bottle
slants prepared with 100 ml of Furunculosis Agar.
After 48 hours at room temperature the cells were harvested with 10 ml
of sterile phosphate buffered saline (PBS) (Williams and Chase, 1968) using
glass beads to free the colonies. Before dilution to 50 ml with PBS the
culture was checked for purity. Treatment beyond this step varied depending
upon intended use.
For agglutination the cells were washed three times in PBS, centrifuged
at 2500 rpm for 15 minutes, and resuspended in PBS to an optical density (OD)
of 0.85 at 525 nm (approximately 5 x 109 cells/ml) as determined with a
Bausch and Lomb Spectronic 20. Cells for agglutination were prepared and
used on the same day.
The cell suspension used for injection was killed with 0.3% formalin and
stored for 24 hours at 4 C before sterility was checked by inoculating two
tubes of TS broth and two FA plates. If sterile, the cells were washed three
times with PBS, resuspended to an.OD of 0.85 at 525 nm and mixed 1:1 with
Freund complete adjuvant (PCS). -^ The cell suspensions and FCA were blended
for seven minutes using a microhomogenizer. The blended vaccine was loaded
into a 10-ml syringe which was used to fill 70 one-mi tuberculin syringes.
Fish were injected just dorsal of the pelvin fin by the intraperitoneal (IP)
route with 0.1 ml of the bacterin-FCA mixture. Anesthesia in an aqueous
solution of MS-222 was necessary to facilitate handling without injury.
After the injections were completed, the last fish injected was allowed to
recover for 30 minutes before the copper solution was introduced. Nominal
(expected) concentrations of copper were 35, 26.3, 19.7, 14.8, 11.1 yg/liter
and a control. The duplicate tanks (1 to 6) were used as an unvaccinated
control bioassay. These fish were not anesthetized or injected and were
intended to demonstrate the effects of copper alone.
Copper exposure was continued for 31 days followed by a two-day rest
period before transportation of the survivors to the Lint Slough facility for
challenge. During the copper exposure at WFTS and challenge at Lint Slough
the fish were fed OMP and the dead fish removed daily after feeding.
Transportation to Lint Slough was accomplished in 5 gallon carboys and
required approximately 1% hours time. Oxygen was bubbled into the Containers
during transport. Upon arrival at Lint Slough the fish were placed in the
tanks and the seawater allowed to replace the freshwater as in the previous
experiment. Salinity levels during this challenge period ranged from 24.1 to
26.4 parts per thousand with a mean of 25.3.
Vibrio-caused deaths normally occur in Lint Slough during the summer
months when the temperature is above 12 C. An effective challenge
^Difco Laboratories
10
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would normally produce heavy mortality between the fourth and tenth days
after the fish are introduced to seawater. The water temperature at the
beginning of this challenge was slightly above 12 C and dropped continuously
until it reached 10 C by the tenth day of the challenge. Consequently the
challenge was not very effective and the fish were sacrificed to determine if
antibody titers had developed against V_. anguillarum. Blood samples were
drawn from sacrificed fish on the 16th and 34th day of exposure to seawater.
On the second sampling day the remaining vaccinated fish and the unvaccinated
fish held at 18.2 yg/liter copper were returned to freshwater. These fish
were transported to the Fish Disease Laboratory of the Department of Micro-
biology and held in tanks similar to those used at Lint Slough. The remain-
ing unvaccinated fish were counted and destroyed.
DETERMINATION OF AGGLUTINATION TITERS
The fish to be sacrificed were anesthetized and blood was collected by
severing the caudal peduncle just posterior to the adipose fin. Blood was
collected in 4 mm OD glass tubes, plugged with clay, and stored at room
temperature for one hour prior to overnight refrigeration at 4 C. Following
overnight storage the tubes were centrifuged at 1800 rpm for 10 minutes.
After centrifugation the tubes were etched and broken at the serum clot
interface and the serum drawn into 50 yl capillary tubes. These tubes were
plugged with clay at the uncalibrated end and frozen for future use. During
storage the capillary tubes were kept in test tubes sealed with Parafilm $-'
to prevent sublimation.
Agglutinating antibody titers were determined by the microtiter method
(Witlin, 1967; Benenson et.aj_., 1968). Several styles of microtiter plates
were tested for clarity of end points. A disposable V-bottom plate was
selected since it had a highly transparent bottom. The determination of
agglutination titers by the microtiter method provides a high degree of
accuracy in titer determination from small serum samples. The precision of
the microtiter method used is shown in Table 1.
TABLE 1. PRECISION OF MICROTITER AGGLUTINATION TEST
USING A POOLED SERUM SAMPLE
Standard 95% confidence
Serum Replications Mean error interval
(titer'1) (Log10 value) (titer"1)
Pooled 16 4277 .01881 3900-4690
sample
5American Can Company
11
-------
On the day of antibody titration, the capillary tubes containing serum
samples were removed from the freezer and spread out in a horizontal position
to prevent leakage during thawing. While these were thawing, 50 yl of 0.85
saline diluent were added to all the wells in one or two plates. When the
serum had thawed, the plugged end of the 50 yl capillary tube was broken off
and the tube drained to the index line. Often it was necessary to carefully
expel bubbles which formed inside the tube during thawing.
The 50 yl serum sample was added to well number one and mixed by drawing
the mixture into the capillary tubes several times. A small rubber bulb was
used to facilitate accurate dispensing of the tube contents. The 50 yl
microtiter diluters were heated to near incandescence, air cooled, wet in
saline, and blotted before each dilution. Initially, each day, a blotter
with calibrated circles would be used to check for proper delivery by each
diluter. Following blotting, the diluter was gently placed in the first row
of wells and allowed to take up the mixture. After gently mixing by four
reciprocal rotations the diluters were placed in the second well and mixed as
before. This procedure, repeated until the end of the plate, provided the
two-fold dilutions. Following the titration the diluters were blotted,
rinsed twice in saline and once in distilled water, then heated to near
incandescence.
The V^. anguillarum antigen, adjusted to 0.85 OD at 525 nm, was mixed and
added to each well using a 50 yl pipetter. Three additional wells received
only saline and antigen in order to serve as an auto-agglutination control.
The plate was gently shaken in all directions and covered with an adhesive
mylar sheet. These plates were incubated for two hours at room temperature
and then stored for sixteen hours at 4 C prior to reading.
A uniform spread of agglutinated bacterial cells over the entire bottom
of the well constituted a positive agglutination reaction. A button of cells
at the very bottom of the V-shaped well constituted a negative reaction. The
microtiter plates were read over a dark surface and checked with a broad
field microscope for accuracy. During later titrations it was necessary to
extend the dilution into a second plate in order to reach the higher end
points.
STATISTICAL ANALYSIS
The copper concentrations and fish were assigned to aquaria by grat-
ified random methods. Antibody titers determined by agglutination are
reported as the reciprocal of the maximum dilution showing a positive re-
action. Reciprocal titer values were converted to the Log10 for statistical
analysis. Means calculated on titer determinations after conversion to Log10
were geometric and unpaired statistics were used for comparison of means.
Statistical analysis of most of the data was completed using the CDC 3300
computer at Oregon State University.
12
-------
SECTION 6
RESULTS
PRELIMINARY BIOASSAYS FOR THE ORAL VACCINATION EXPERIMENT
During the spring and fall, chemical parameters of the water supply at
the WFTS vary considerably, depending on the amount of rainfall and the
Willamette River stage. The alkalinity during the two preliminary bioassays
ranged from 20 to 53 mg/1 as CaC03 while the hardness ranged from 20 to 83
mg/1 as CaC03 (Figure 1). Increased alkalinity and hardness decrease the
toxicity of copper to fish (Stiff, 1971).
The alkalinity and hardness were decreasing during the early part of the
first preliminary bioassay when nominal copper concentrations of 40 and 28
yg/liter were tested. The mortality during this bioassay (Figure la) indi-
cated that the copper concentrations were too high to obtain the desired
survival. The day the second preliminary bioassay was started, the alkalin-
ity and hardness began to increase. During the next 72 hours both the
alkalinity and hardness continued to increase and consequently the mortality
at the nominal 28 yg/liter concentration during the second bioassay (Figure
Ib) was considerably less than that observed during the first bioassay.
COPPER BIOASSAY AND ORAL VACCINATION
In this experiment coho salmon fry were orally vaccinated during a 30-
day exposure to various concentrations of copper. Deaths were recorded daily
through 30 days of exposure and the 10-day rest period. There were no
survivors at a copper concentration of 34.4 yg/liter and only 7% survived at
26.6 yg/liter (Figure 2). Mean mortality at 18.1 yg/liter reached 41.5% and
greater at higher copper concentrations, all of which were significant (P =
0.05) when compared to the control (Table 2). At the two lowest copper
concentrations of 13.9 and 11.6 yg/liter the copper bioassay mortality was
not significantly different (P = 0.05) from the controls.
The oral vaccination experiment was begun when the hardness and alkalin-
ity were relatively high (79.5 and 53 mg/liter, respectively). These began
to decrease and after six days were about 50% lower than at the beginning of
the copper exposure. Mortality at the two highest copper concentrations was
delayed, when compared to the preliminary bioassay data (Figure 1), and was
probably due to hardness and alkalinity dynamics. The hardness and alkalin-
ity remained low for the duration of the experiment as indicated by the means
and ranges in Table 3. The measured copper concentrations are shown in
Table 4.
13
-------
=40.4 yg/l
=28.5jjg/l
10
30 40 50
Time (Hours)
70
B
100
80
^ 60
*-
c
80
0)
60 o
X
40 o
20 -
o
10 20 30 40 50 60 70
Time (Hours)
Figure 1. Mortality of coho fry exposed to copper during the preliminary
bioassays conducted prior to the oral vaccination test with indicated
variations in alkalinity and hardness.
14
-------
lOOr
90
80
70
.-t 60
50
40
30
20
o
-------
TABLE 2. MORTALITY OF COHO SALMON FRY DURING SIMULTANEOUS ORAL VACCINATION AND
COPPER EXPOSURE IN FRESH WATER
Mean Copper
Concentration
(yg/ liter)
34.4
26.6
18.1
13.9
11.6
Vaccinated
control
(3.2 pg/liter)
Percent Mortal i
in Test Aquari
A
100 1
96
48
8
3
3
ty
a
B
00
90
35
5
7
1
Mean Percent
Mortality*
100**
93**
41 .5**
6.5
5
2
* Thirty days copper exposure plus ten day rest period
** Significantly different from the control at P = 0.05
-------
TABLE 3. CHEMICAL CHARACTERISTICS OF THE WATER IN THE AQUARIA DURING
THE ORAL VACCINATION BIOASSAY
Characteristic
Dissolved oxygen (nig/liter)
PH
Alkalinity (mg/liter as CaC03)
Hardness (mg/liter as CaCO-J
Temperature C
Number of
Samples
41
29
29
31
33
Mean of
Determinations
9.6
7.3*
24.8
31.9
12.9
Standard
Deviation
±0.5
±0.1
±6.3
±13.9
±0.37
Range
8.7 -
7.1 -
19.0 -
20.0 -
12.1 -
10.8
7.5
53.0
79.5
13.8
Median
-------
TABLE 4. MEASURED TOTAL COPPER CONCENTRATIONS TAKEN DURING THE ORAL VACCINATION BIOASSAY
co
Nominal Copper
Concentrations
(yg/liter)
40
28
19.6
13.7
9.6
Control
Mean Measured
Number of
samples
11
12
8
6
9
13
Copper Concentration
(yg/liter)
34.
26.
18.
13.
11.
3.
4
6
1
9
6
2
Standard
Deviation
±3.1
±3.7
±2.0
±1.7
±1.7
±1.4
Range
(yg/liter)
27.2 -
18.7 -
13.5 -
11.4 -
8.6 -
1.9 -
38.1
31.2
19.7
15.5
13.8
6.0
-------
One of the first indications of stress by copper was anorexia (loss of
appetite). By the end of the first 24-hour period, fish in the 34.4 and 26.6
yg/liter tanks were eating an estimated 75% of their ration. After 48 hours,
these fish were eating less than 50% of their food and fish at all levels of
copper appeared to be in stress as indicated by the uneaten food in the
aquaria. Control fish were eating 100% of the food being fed. The appetites
of all groups of fish improved considerably during the rest period when they
were fed OMP.
Following the 10-day rest period the fish were transferred to Lint
Slough where fish began to die on the second day of exposure to seawater.
Temperature and salinity varied daily, the magnitude of each being determined
by the height of the tide and the amount of fresh water entering the slough
(Table 5). The number of deaths continued to increase in all groups of fish
exposed to copper during the seawater exposure.
The highest percent mortality among the fish challenged occurred in the
unvaccinated control group (Figure 3). All groups of fish receiving bacterin
were protected to some degree when compared to the unvaccinated control-
Fish exposed to 13.9 yg/liter copper and above suffered significant mortality
(P = 0.05) when compared to the control, while those exposed to 11.6 yg/liter
had similar mortality to the control, indicating that the lowest copper level
had little, if any, effect on the immune response and subsequent survival.
Fish surviving copper exposure at higher levels may have had difficulty
adapting to seawater, however it is difficult to attribute death to this one
factor. Many of those fish which died during challenge from concentrations
of 13.9 yg/liter copper and above were very thin and apparently never went
back to feeding and may have died from starvation. The percentage of thin
fish ranged from 18 to 47% among the dead fish from 11.6, 13.9, and 18.1
yg/liter copper.
The results of the examination of dead fish indicate that, with the
exception of the fish held at 26.6 yg/liter, 50 to 63% of the vaccinated fish
that died were positive for V^. anguillarum (Table 6). Many of the fish that
were negative for V_. anguillarum were the debilitated fish that never began
to eat again after copper exposure. These fish, though very debilitated,
could have been protected through the small amounts of vaccine they inoested
during copper exposure. It is assumed that the remainder of the fish nega-
tive for V_. anguillarum were not infected and died from the stress of copper
exposure ?nd seawater adaptation. The percentage of recovery of _V.
anguillarum from fish tissues was very high in the unvaccinated control group
which received no vaccine and were not exposed to copper.
PRELIMINARY BIOASSAY FOR THE VACCINATION BY INJECTION EXPERIMENT
The preliminary bioassay for this experiment was conducted for seven
days. Ten fingerling coho were placed in each aquaria and a copper stock
solution prepared to deliver concentrations of 50, 35, 24.5, 17.1, and 12.0
yg/liter copper. Within 72 hours, 100% of the fish were dead at 50 yg/liter
and at the end of the 7-day test, 40% of the fish were dead at 35 y/liter
(Figure 4). The latter concentration was chosen as the highest concentration
in the injection experiment.
19
-------
PO
o
TABLE 5. CHEMICAL CHARACTERISTICS OF WATER IN THE LINT SLOUGH HOLDING TANKS DURING THE
CHALLENGE FOLLOWING THE ORAL VACCINATION BIOASSAY
Characteristic Units
Dissolved Oxygen trig/liter
pH
Alkalinity ing/1 iter as CaCCU
Hardness ing/liter as CaCCL
Temperature C
Salinity parts per 1000
Number of
Analysis
2
1
1
1
21
5
Determination
9.2*
7.9
40
900
16.7*
19.3*
Range
9.0 - 9.3
11.7 - 24.4
10.1 - 23.4
* Mean Value
-------
100
90
80
£> 70
"6
o 60
o
4_
-------
TABLE 6. MORTALITY OF COHO SALMON FRY DURING THE NATURAL SEAWATER CHALLENGE TO
V. ANGUILLARUM FOLLOWING SIMULTANEOUS ORAL VACCINATION AND COPPER EXPOSURE
ro
Mean
Copper
Concentration
(ng/D
26.6
18.1
13.9
11.6
Vaccinated
Control
Unvaccinated
Control
Test
Tank
A
B
A
B
A
B
A
B
A
B
Number of
Dead Fish
3(4)**
8(10)
37(47)
48(63)
55(81)
39(77)
31(84)
22(68)
16(80)
36(76)
92(96)
Number of
Fish Examined*
3
8
22
44
47
32
30
17
15
36
42
Percent Number
Mortality From Positive For
Challenge^ V. anguillarum
Mean ± S D
77.5 ± 3.5t 1
2
77.3 ± 1.7t 14
26
59.2 ± 12. 2t 30
16
34.5 ± 3.3 16
10
33.6 ± 19.3 9
18
96 38
Percent
Positive For
V. anguillarum
33
25
63
59
63
50
53
58
60
50
90
* The number of fish examined is smaller than the number of dead fish because of a lab accident and
because partially decomposed fish were not cultured.
** Number of fish per tank at the beginning of challenge.
t Significantly different from the control at P = 0.05.
-------
100 r
ro
CO
80
60
c
o 40
^
0)
Q_
20
= 35pg/liter
100
80
60
t/>
T3
O
40
20 c
o
Time (Days)
Figure 4. Mortality of coho finger!ings exposed to copper during the preliminary bioassay for the
vaccination by injection test with variations of alkalinity and hardness.
-------
TABLE 7. MEASURED TOTAL COPPER CONCENTRATIONS FROM EXPERIMENTAL TANKS DURING
THE INJECTED BACTERIN BIOASSAY
ro
-p.
Nominal Copper
Concentration
(yg/liter)
Number of
Samples
Measured Copper
Concentration
(yg/liter)
Mean ±
35
26.3
19.7
14.8
11.1
24
24
24
24
24
32.
24.
18.
13.
10.
9 ±
6 ±
2 ±
6 ±
1 ±
S D
4.0
3.1
2.2
1.7
1.4
Range
(yg/liter)
21.8 -
16.3 -
12.2 -
9.2 -
6.9 -
38.8
29.0
21.6
15.7
11.8
-------
IX)
en
TABLE 8. CHEMICAL CHARACTERISTICS OF THE WATER IN THE EXPERIMENTAL TANKS DURING THE INJECTED BACTERIN
BIOASSAY
Characteristic
Dissolved Oxygen
PH
Alkalinity
Hardness
Temperature
Units
mg/ liter
mg/liter as CaCO^
ing/liter as CaCO,
C
Number of
Analysis*
223
30
30
30
38
Determinations
Mean ±
9.1 ±
**
7.2 ±
21.1 ±
21.7 ±
12.2 ±
S D
0.5
0.1
2.0
1.1
0.3
Range
7.4
6.7
13.0
20.0
11.5
- 10.6
- 7.4
- 26.0
- 25.0
- 12.8
* Analysis conducted at least weekly in each concentration
** Median
-------
COPPER BIOASSAY AND VACCINATION BY INJECTION
In this bioassay the coho fingerlings, after being injected with the V_.
anguJllarum bacterin, were exposed for 31 days to copper concentrations
ranging from 32.9 to 10.1 yg/liter (Table 7). This experiment was conducted
in early fall resulting in relatively stable alkalinity and hardness values
(Table 8). Significant mortality occurred only at the two highest copper
concentrations and no significant differences in mortality were observed
between vaccinated and unvaccinated fish held at any copper concentration
(Table 9). A significant proportion of the mortality occurred between the
fifth and fifteenth day of the bioassay.
During the copper bioassay the appetites of the fish at the higher
concentrations diminished, but the fish remained in good condition, showing
no signs of disease at the end of the exposure period. Fish were held for 48
hours following termination of the copper exposure before being transported
to Lint Slough for the seawater challenge. By the end of the 48 hour rest
period all fish appeared to be feeding normally and only one fish died during
that period.
During the first two days of seawater exposure heavy mortality occurred
among the survivors of the copper bioassay, especially in the groups which
were exposed to the highest copper concentrations (32.9 and 24.6 yg/liter)
(Table 9). All of the fish that died during the first three days were nega-
tive for V_. anguillarum as were most of the fish that died during the first
seven days of seawater exposure. The lack of Vibrio ascribed deaths in the
unvaccinated fish during the period from the fourth to seventh day indicated
the challenge was insufficient. The greatest number of dead fish positive
for V_. anguillarum occurred among the fish exposed to 18.2 yg/liter copper.
Temperatures by the eleventh day of the challenge had dropped from 12.2
to 10.0 C and only occasional deaths were appearing throughout all groups.
The number of deaths, regardless of cause, reflected the concentration of
copper in both the vaccinated and unvaccinated groups.
On the 16th day of the challenge, between 25 and 30 fish were sacrificed
from the vaccinated controls and the group of fish exposed to 18.2 yg/liter
copper. The blood was withdrawn and prepared for antibody titration.
Analysis indicated that antibody titers had developed sufficiently so that
they could be measured and therefore it was decided that all the fisN»would
be removed from the seawater for antibody analysis. Similar samples to those
taken above were removed from the groups exposed to 13.2 and 10.1 yg/liter
copper just prior to transporting the fish back to the laboratory on the 34th
day of exposure to seawater.
ANTIBODY TITERS DEVELOPED FOLLOWING INJECTION, COPPER EXPOSURE, AND SEAWATER
CHALLENGE
Blood samples for antibody titer determination were collected at two
different times. The first samplings, described above, were taken during the
seventh and ninth weeks after injection. The remainder of the blood samples
were collected 16% weeks after injection. A significant increase in antibody
26
-------
TABLE 9. MORTALITY OF FINGERLING COHO SALMON VACCINATED BY INJECTION, EXPOSED TO COPPER, AND
TRANSFERRED TO SEAWATER FOR A NATURAL CHALLENGE TO V. ANGUILLARUM
Mean Copper
Concentration
(yg/liter) Treatment*
32.9
24.6
18.2
13.6
10.1
Control
U
V
U
V
U
V
U
V
U
V
U
V
Percent Mortality
During Bioassay
(31 days)
68
69
17
18
4
0
0
1
0
0
0
0
Percent Mortality
Not Attributed
to Vibriosis**
100
100
94
90
16
20
6
8
3
0
2
4
Percent Mortality
Positive for
Vibriosist
--
--
10
3
7
1
6
0
4
0
* U and V represent
** Deaths in seawater
t Deaths in seawater
unvaccinated
, of bioassay
, of bioassay
and vaccinated groups of
survivors, negative for
survivors, positive for
fish.
V. anguillarum.
V. anguillarum.
-------
no
CO
TABLE 10. MEAN AGGLUTINATING ANTIBODY TITERS DEVELOPED IN FINGERLING COHO SALMON FROM A
SINGLE IP INJECTION WITH V. ANGUILLARUM BACTERIN PRIOR TO COPPER EXPOSURE AND NATURAL CHALLENGE*
Mean Copper
Concentration
(ug/liter)
Control
10.1
13.6
18.2
Mean liters 50 Days
After Injection
(titer'1)
3,068 (24)**
1,087 (23)t
Mean liters 68 Days
After Injection
(titer"1)
3,492 (21)
6,502 (21)
Mean liters 115 Days
After Injection
(titer'1)
29,569 (54)
15,578 (55)
29,077 (58)
8,192 (31)t
* Vaccinated fish with titers of 32 and below were not included in the statistical analysis.
** Number of individual serum samples titered in parenthesis.
t Significantly different from the control at P - 0.05.
-------
titers occurred between the first and last sampling times (Table 10). At
both sampling times the antibody titers at 18.2 yg/liter copper were signifi-
cantly lower than the controls. At the last sampling the mean titer for
those fish from 10.1 yg/liter copper was considerably lower than the controls
while those at 13.6 yg/liter copper were equal to the controls, indicating
that the copper may have enhanced antibody production.
A sample of 20 unvaccinated fish was taken during the second sampling
from the group of fish held at 18.2 yg/liter copper to be used to establish
the background titers present in the fish. The mean reciprocal titer from
these fish was 19.9 dilutions with a range of 8 to 32 dilutions. Less than
4% of the titers from vaccinated fish fell within this range and were con-
sidered to be a result of poor immune response or more likely an improper
injection. The day after injection several aquaria had small amounts of
adjuvent floating on the surface indicating that some injections were made
into the intestinal tract instead of into the peritoneal cavity.
29
-------
SECTION 7
DISCUSSION
Because the water supply at the Western Fish Toxicology Station does not
have a constant level of alkalinity and hardness during the rainy season and
these factors affect copper toxicity, preliminary bioassays were necessary to
establish a satisfactory level of copper for each test. Mortality observed
in the preliminary bioassays with coho fry indicated what effect alkalinity
and hardness would have on the toxicity of copper to fish. Lloyd and Herbert
(1962) found a linear relationship between hardness and the lethal threshold
concentration for rainbow trout. Stiff (1971) suggested that the difference
in the toxicity of copper in hard and soft water may be due to a copper
carbonate complex which is less toxic than the free cupric ion and whose
formation is favored at higher alkalinities. The decreasing alkalinity and
hardness during the first five days of the oral immunization bioassay in-
creased the toxic effect of the copper to the fry and resulted in higher
mortality at both 34.4 and 26.6 yg/liter copper.
The first test conducted was designed to determine what effect copper
exposure would have upon the protection provided to coho salmon fry by oral
immunization with a V_. anguillarum bacterin. The fish were fed bacterin
daily while being exposed to various copper concentrations. McKim and Benoit
(1971) found that 17.5 yg/liter copper had a severe effect on survival of
juvenile trout where slightly less than three months exposure resulted in 15%
mortality in water with an alkalinity and hardness between 40 and 45 mg/liter
The data from the coho fry bioassay indicate that 18.1 yg/liter has a
definite effect (mean--42% mortality) on survival of coho fry exposed to
copper for 30 days. Mortality continued during the ten day rest period, but
appeared to be reaching an asymptote by that time.
Looking at the effects of copper upon challenge survival, the level of
significant effect begins at 13.9 yg/liter. The stress of copper to* these
fish, and the additional stress of seawater and possibly vibriosis, produced
further mortality. They could also have died from starvation whether or not
they were protected. The development of salinity tolerance in coho salmon
has been shown to occur in the fry stage and is related to fish size (Conte
et_ a_]_., 1966). Coho fry 3 to 4 cm in length had a median survival time
greater than 30 days when held at a salinity of 26 parts per thousand.
Therefore, these fish under normal conditions should have been able to
survive the transfer to seawater.
In the study using coho finger!ings vaccinated by injection, mortality
at the two highest copper concentrations (32.9 and 24.6 yg/liter) was
30
-------
significantly greater than the controls (P = 0.05). The few deaths occurring
at 18.2 lag/liter copper and below suggest that these concentrations are safe
(non-lethal) for fingerling coho salmon under similar water conditions.
When the survivors from the two highest concentrations were placed in
seawater nearly all the fish died within three days. This time period is
generally not long enough for V_. anguillarum infection to cause death. This
was supported by the fact that all the dead fish were negative when examined
for V_. anguillarum. The survivors at 18.2 yg/liter copper also suffered
significant losses but fish at the lower copper concentrations and control
fish suffered little mortality from stress of placement into seawater. The
data indicate that concentrations of copper down to 13.6 yg/liter which
appear to be non-lethal levels for coho fingerlings can inhibit their ability
to adapt to seawater. Recent studies conducted by Lorz and McPherson (1976)
produced similar results. Yearling coho, exposed to 20 yg/liter copper for
144 hours without any mortality, suffered nearly 40% mortality when placed in
seawater. Longer exposure to copper at 20 yg/liter caused only slight
mortality in freshwater but increased the mortality significantly in seawater,
The efficacy of the challenge diminished considerably as the water
temperature dropped to near 10 C. The number of deaths following acclimation
failed to increase and there was little difference in the number of deaths
between vaccinated and unvaccinated fish. Since the fish were injected with
a bacterin, a humoral antibody response had been initiated and could be
measured by titration. The remaining fish were sacrificed and the blood
collected for antibody measurement.
Antibody titrations conducted on unvaccinated fish indicated the
presence of natural antibody titers which were at 32 dilutions or below.
There were also a few vaccinated fish with titers at that level. These, in
part, would result when the inoculum was injected into the intestine by
mistake, initiating little, if any, measurable immune response. There is
also a certain portion of any population that is immunologically incompetent.
It is also quite possible that in some fish the stress involved in handling
as well as the copper exposure completely blocked the immune response.
The means of the agglutinin titers of serum samples taken 50 days after
vaccination from fish exposed to 18.2 yg/liter copper indicated a significant
(P = 0.05) reduction in antibody concentration. This reduction prevailed
throughout the entire vaccination period (115 days) in the group of fish held
at 18.2 yg/liter of copper. However, at 13.6 yg/liter copper exposure, there
is an enhanced antibody response compared to that of the fish held at 10.1
yg/liter copper.
The exact mechanism involved in the reduction of the antibody titer is
not known. Further research would be needed to determine if copper directly
inhibits antibody production or whether chronic stress caused by the copper
is directly involved with the reduced antibody production. The metabolic
changes which take place during stress are directed toward increasing the
possibility of survival; however, these changes often make the organism more
susceptible to infection (Wedemeyer, 1970). Studies conducted on fish in
stress indicate that the metabolic changes which take place are similar to
31
-------
those of higher vertebrates (Hoar, 1957). In studies conducted with sockeye
salmon, Fagerlund (1967) demonstrated that stress from fright and handling
increased the level of corticosteroids present in the fish. Little work,
however, has been done with fish to relate levels of corticosteroids with
various aspects of the immune response such as antibody formation.
As pollution control methods improve there will undoubtedly be fewer catas-
trophic fish kills by heavy metals such as copper. However, as these studies
indicated, sublethal levels of copper or other pollutants may have a signifi-
cant effect upon physiological responses such as acclimation to seawater and
may interfere with a successful immune response to disease organisms.
32
-------
REFERENCES
American Public Health Associates. 1971.
examination of water and wastewater.
874 pp.
Standard methods for the
13th Ed. A.P.H.A., New York.
Anderson, J. I. W. and D. A. Conroy. 1970. Vibrio disease in marine
fishes. In: A symposium on diseases of fishes and shellfishes,
ed. by S. F. Snieszko. Washington, D. C. American Fisheries
Society. Special Publication No. 5, pp. 266-272.
Baker, J. T. P. 1969. Histological and electron microscopical observa-
tions on copper poisoning in the winter flounder (Pseudopleuronectes
americanus). Journal of Fisheries Research Board of Canada.
26:2785-2793.
Benenson, A. S., A. Saad, and W. H. Mosley. 1968. Serological studies
in cholera. 2. The vibriocidal antibody response-of cholera
patients determined by a microtechnique. Bulletin of the World
Health Organization. 38:277-285.
Cisar, J. 0. and J. L. Fryer.
Chinook salmon. Bulletin
5:73-76.
1969. An epizootic
of Wildlife Disease
of vibriosis in
Association.
Conte, F. P., H. H. Wagner, J. Fessler, and C. Gnose. 1966. Development
of osmotic and ionic regulation in juvenile coho salmon
Oncorhynchus kisutch. Comparative Biochemistry and Physiology.
18:1-15.
Corbel, M. J. 1975. The immune response in fish:
of Fish Biology. 7:539-563.
a review. Journal
Doudoroff, P. and M. Katz. 1953. Critical review of literature on
the toxicity of industrial wastes and their components to fish.
II. The metals as salts. Journal of Sewage and Industrial Wastes.
25:802-839.
Fagerlund, U. H. M. 1967. Plasma cortisol concentration in relation to
stress in adult sockeye salmon during the freshwater stage of
their life cycle. General and Comparative Endocrinology. 8:197-
207.
33
-------
Fletcher, T. C. and A. White. 1973. Antibody production in the plaice
(Pleuronectes platessa L.) after oral and parenteral immunization
with Vibrio anguillarum antigens. Aquaculture. 1:417-428.
Fryer, J. L., J. S. Nelson, and R. L. Garrison. 1972. Vibriosis in
fish. Progress in Fishery and Food Science. 5:129-133.
Garton, R. 1976. A simple continuous-flow toxicant delivery system.
Western Fish Toxicology Station, Corvallis, Oregon (in manuscript).
Hayashi, K., S. Kobayashi, T. Kamata, and H. Ozaki. 1964. Studies
on the Vibrio-disease of rainbow trout (Salmo gairdneri irideas).
II. Prophylactic vaccination against the Vibrio-disease. Journal
of Faculty of Fisheries, Prefectural Univerisity of Mie. 6:181-
191.
Hemphill, F. E., M. L. Kaeberle, and W. B. Buck. 1971. Lead suppression
of mouse resistance to Salmonella typhimurium. Science. 172:1031-
1032.
Hoar, W. S. 1957- Endocrine organs.
Academic Press, New York. Vol. 1
In: The physiology of fishes.
447 pp.
Jackim, E., J. M. Ham!in, and S. Sonis. 1970. Effects of metal
poisoning on five liver enzymes in the killifish (Fundulus
heteroclitus). Journal of Fisheries Research Board of Canada.
27:383-390.
Jancs6, N. von and H. von Jancso. 1934. Mikrobiologische grundlagen
der chemotherapeutischen wirkung. I. Mitteilung: Wirkungsmechan-
ismus des germanins (Bayer 205) bei -trypanosomen. Zentralblatt
fur Bakteriologie, Parasitenkunde, und Infektionskrankheiten.
132(5/6):257-292.
Jones, R. H., R. L. Williams, and A. M. Jones. 1971. Effects of heavy
metal on the immune response. Preliminary findings for cadmium
in rats. Proceedings of the Society for Experimental Biology
and Medicine. 137:1231-1236.
Khan, A. and J. M. Hill. 1971. Immunosuppression with cis-platinum
(II) diamminodichloride: Effect on antibody plaque-forming spleen
cells. Infection and Immunity 4(3):320-321.
Kolmer, J. A., E. H. Spaulding, and H. W. Robison. 1951. Approved
laboratory technique. 5th Ed. New York, Appleton-Century Crafts.
1180 p.
Kolomiitseva, M. G., F. M. Voznesenskaya, and E. A. Isaeva. 1969.
Immunobiological reactivity of the animal organism in association
with different copper content in the rations. Journal of Micro-
biology, Epidemiology, and Immunobiology. 46(8):99-104.
34
-------
Lee, D. J., J. N. Roehm, T. C. Yu, and R. 0. Sinnhuber. 1967. Effect
of u3 fatty acids on .the growth rate of rainbow trout, Sal mo
gairdnerii. Journal of Nutrition. 92:93-97.
Lloyd, R. and D. W. M. Herbert. 1962. The effect of the environment
on the toxicity of poisons to fish. Journal of Institution of
Public Health Engineers. 61:132-145.
Lorz, H. W. and B. P. McPherson. 1976. Effects of copper or zinc in
freshwater on the adaptation to seawater and APTase activity, and
the effects of copper on migratory disposition of coho salmon
(Oncorhynchus kisutch). Journal Fisheries Research Board of
Canada. 33:2023-2030.
McKee, J. E. and H. W. Wolf, eds. 1963. Water Quality Criteria. The
Resources Agency of California, State Water Quality Control Board.
Sacramento, Calif. Publication No. 3-A.
McKim, J. M., G. M. Christensen, and E. P. Hunt. 1970. Changes in the
blood of brook trout (Salvelinus fontinalis) after short-term and
long-term exposure to copper. Journal Fisheries Research Board of
Canada. 27:1883-1889.
McKim, J. M. and D. A. Benoit. 1971. Effects of long-term exposures
to copper on survival, growth, and reproduction of brook trout
(Salvelinus fontinalis). Journal Fisheries Research Board of
Canada. 28(5):655-662.
Mount, D. I. 1968. Chronic toxicity of copper to fathead minnows
(Pimephales promelas, Rafinesque). Water Research. 2:215-223.
Mount, D. I. and W. A. Brungs. 1967. A simplified dosing apparatus
for fish toxicology studies. Water Research. 1:21-29.
Mount, D. I. and C. E. Stephan. 1969. Chronic toxicity of copper to
the fathead minnow (Pimephales promelas) in soft water. Journal
Fisheries Research Board of Canada. 26:2449-2457.
Nelson, J. S. 1972. Oral immunization for the control of vibrios is
in Pacific salmon. Master's thesis. Corvallis. Oregon State
University. 48 numb, leaves.
Ormerod, W. E. 1961. The study of volutin granules in trypanosomes.
Transactions of Royal Society of Tropical Medicine and Hygiene.
55:313-327.
Pacha, R. E. and E. J. Ordal. 1967. Histopathology of experimental
columnaris disease in young salmon. Journal of Comparative Pathology
77:419-423.
35
-------
Pippy, J. H. and G. M. Hare. 1969. Relationship of river pollution to
bacterial infection in salmon (Salmo salar) and suckers (Catostomus
commersoni). Transactions of the American Fisheries Society.
98(4):685-690.
Ridgway, G. J., H. 0. Hodgins, and G. W. Klontz. 1966. The immune
response in teleosts. In: Phylogeny of Immunity (Eds. R. T.
Smith, et_a]_.). p. 199-208. Univeristy of Florida Press,
Gainesville.
Rohovec, J. S. 1974. Oral and parenteral immunization for the control
of Vibrio anguillarum, the etiological agent of vibriosis in
salmonid fish. Doctoral Thesis. Con/all is. Oregon State University
82 numb, leaves.
Rohovec, J. S., R. L. Garrison, and J. L. Fryer. 1975. Immunization of
fish for the control of Vibriosis. Proceedings of the Third U.S.-
Japan Meeting on Aquaculture at Tokyo, Japan, October 15-16, 1974:
Special Publication of Fishery Agency, Japanese Government and
Japan Sea Regional Fisheries Research Laboratory. Niigata, Japan.
Snieszko, S. F. 1970. Immunization of fishes: a review. Journal of
Wildlife Diseases. 6:24-30.
Snieszko, S. F. 1974. The effects of environmental stress on outbreaks
of infectious diseases of fishes. Journal of Fish Biology.
6:197-208.
Stiff, M. J. 1971. Copper/bicarbonate equilibria in solutions of
bicarbonate ion at concentrations similar to those found in natural
water. Water Research. 5:171-176.
Wedemeyer, G. 1970. The role of stress in the disease resistance of
fishes. In: A symposium on diseases of fishes and shellfishes,
ed. by S. F. Snieszko. Washington, D. C. American Fisheries
Society. Speical Publication No. 5, 30-35.
Williams, C. A. and M. W. Chase. 1968. Methods in immunology and
immunochemistry. New York Academic Press. Vol. II, p. 459.
Witlin, B. 1967. Detection of antibodies by microtitrator techniques.
Mycopathologia. 33:241-257.
36
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO.
EP EPA-600/3-77-031
3. RECIPIENT'S ACCESSI Or* NO.
4. TITLE AND SUBTITLE
5. REPORT.DAT
Survival and Immune Response of Coho Salmon Exposed
to Copper
"March 1977
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
Donald G. Stevens
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Western Fish Toxicology Station
Corvallis Environmental Research Laboratory
1350 S. E. Goodnight Road
Corvallis, Oregon 97330
10. PROGRAM ELEMENT NO.
1BA608
11. CONTRACT/GRANT NO.
12. SPONSORING AGENCY NAME AND ADDRESS
U. S. Environmental Protection Agency -Corvallis, OR
Corvallis Environmental Research Laboratory
200 SW 35th St.
Corvallis, Oregon 97330
13. TYPE OF REPORT AND PERIOD COVERED
Final - in house
14. SPONSORING AGENCY CODE
EPA/600/02
15. SUPPLEMENTARY NOTES
is.ABSTRACT Vaccination with Vibrio anguillarum by oral administration during cupper
exposure and intraperitoneal injection prior to copper exposure was employed to invest!
gate the effects of copper upon survival and the immune response of juvenile coho
salmon (Oncorhynchus kisutch). Following copper exposure the survivors were challengec
under natural conditions to _V. anguillarum, the causative agent of vibriosis in fish.
Copper concentrations of 18.1 ug/liter and higher caused significant mortality
among coho fry during 30 days of exposure. The exposure of copper bioassay survivors
to a natural challenge against V. anguillarum in seawater caused significant mortality
among those fish from concentrations of copper at 13.9 ug/liter and higher. The
reduced number of dead fish positive for V. anguillarum from the challenge suggests
that sublethal copper stress and difficulty with seawater adaption may have caused
several deaths.
Significant mortality occurred among coho finger!ings exposed to 24.6 ug/liter
and higher for 31 days. Most of the survivors of these concentrations
copper
were
unable to adapt to seawater and died within the first three days of challenge. Sig-
nificant mortality also occurred during adaptation of survivors from 18.2 ug/liter
copper where the mean mortality resulting from 31 days exposure was only 2%. The
antibody level against V_. anguillarum, measured by agglutinin titer, was significantly
reduced in fish exposed to this concentration of copper when compared to that
dpyplnned in control animals.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.IDENTIFIERS/OPEN ENDED TERMS
COSATI Field/Group
Salmon
Freshwater fishes
Coho salmon
Effects of copper
06/F,S,T
B. DISTRIBUTION STATEMENT
RELEASE TO PUBLIC
19. SECURITY CLASS (This Report)
UNCLASSIFIED
21. NO. OF PAGES
44
20. SECURITY CLASS (This page)
UNCLASSIFIED
22. PRICE
EPA Form 2220-1 (9-73)
37
U S GOVERNMENT PRINTING OFFICE: 1977—797-541/106 REGION 10
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