EPA-660/3-73-020
January 1974 Ecological Research Series
Effects of Temperature on
Diseases of Salmonid Fishes
^
Office of Research and Development
U.S. Environmental Protection Agency
Washington, D.C. 20460
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and
Monitoring, Environmental Protection Agency, have
been grouped into five series. These five broad
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was consciously planned to foster technology
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2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
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This report has been assigned to the ECOLOGICAL
RESEARCH series. This series describes research
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influences. Investigations include formation,
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c
EPA-660/3-73-020
January 1974
EFFECTS OF TEMPERATURE ON DISEASES
OF SALMONID FISHES
By
J. L. Fryer
K. S. Pilcher
Oregon State University
Corvallis, Oregon 97331
Project 18050 DIJ
Program Element 1BA021
Project Officer
Dr. Gerald R. Bouck
Western Fish Toxicology Laboratory
Environmental Protection Agency
Corvallis, Oregon 97330
Prepared for
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, D.C. 20460
For sale by the Superintendent of Documents, U.S. Government Printing Office, Washington, D.C. 20402 - Price $1.85
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EPA Review Notice
This report has been reviewed by the Environmental
Protection Agency and approved for publication.
Approval does not signify that the contents necessarily
reflect the views and policies of the Environmental
Protection Agency, nor does mention of trade names or
commercial products constitute endorsement or recommenda-
tion for use.
ii
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ABSTRACT
The effect of x/ater temperature on infections of salmonid fish was
investigated. Chondrococcus colunnaris infection was studied in rain-
bow trout, coho and spring chinook salmon; Aeromonas salmonicida in-
fection in coho and spring chinook salmon; and Aeromonas liquefnciens
infection in steelhead trout. In all cases mortality rates were high
ru
at 64 to 69°F; moderate at 54° to 59°F; and low or zero at 39° to 49°F.
Progress of the infections was accelerated at higher temperatures, and
progressively retarded at decreasing temperature levels.
In infection of coho with Ceratomyxa shasta, mortality was high at
69 F, low at 49 to 54 , and zero at 39 to 44 F. This infection in
rainbow trout resulted in high mortality at all temperatures except
39 . In both cases the course of the disease was most rapid at higher
temperatures, and became progressively slower as the temperature de-
creased.
For infection of kokanee salmon fingerlings with sockeye salmon virus,
the temperature range of 54 to 59 F was optimal. In this range mortality
rates were high, and the course of the disease most rapid. At higher
temperatures mortality rates were lower, and at 39 to 44 F, progress
of the disease was retarded, though total mortality was often high.
This report was submitted in fulfillment of Project Number 18050 UIJ,
under the sponsorship of the Office of Research and Monitoring, En-
vironmental Protection Agency.
iii
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CONTENTS
Section
I
II
III
IV
V
Conclusions
Recommendations
Introduction
Equipment Design and Fabrication Phase
Effect of Water Temperature on Infection of
Salmonids bv Aerorionas Salmonicida and Aeromonas
Liquefaciens
Page
1
5
7
9
11
VI Effects of Water Temperature on Infection of
Salmonids by Chondrococcus Columnaris
VII Effects of Water Temperature on Infection of
Salrnonids by the Parasitic Protozoan Ceratomyxa
Shasta
VIII Effect of Water Temperature on Infection by the
Sockeye Salmon Virus (I11N)
IX Acknowledgments
X References
XI Appendices
55
69
89
91
93
v
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FIGURES
PAGE
RELATIONSHIP BETWEEN WATER TEMPERATURE AND LOG OF TIME 20
TO DEATH AFTER INFECTION OF JUVENILE COIIO SALMON WITH
AEROMONAR SALMONICIDA
EFFECT OF TEMPERATURE ON GROWTH RATE OF AEROMONAS 22
SALMONICIDA IN PEPTOHE-BEEF-EXTRACT-GLUCOSE BROTH
RELATIONSHIP BETWEEN WATER TEMPERATURE AND LOG OF TIME 27
TO DEATH AFTER INFECTION OF JUVENILE CHINOOK SALMON
WITH AEROMONAS SALMONICIDA
RELATIONSHIP BETWEEN WATER TEMPERATURE AND LOG OF TIME 45
TO DEATH AFTER EXPOSURE OF JUVENILE RAINBOW TROUT TO
CUONDROCOCCUS COLUMNARIS
RELATIONSHIP BETWEEN WATER TEMPERATURE AND LOG OF TIME 48
TO DEATH AFTER EXPOSURE OF JUVENILE COIIO SALMON TO
CUONDROCOCCUS COLUMNARIS
RELATIONSHIP BETWEEN WATER TEMPERATURE AND LOG OF TIME 51
TO DEATH AFTER EXPOSURE OF JUVENILE CHINOOK SALMON TO
CliONDROCOCCUS COLUMNARIS
RELATIONSHIP BETWEEN WATER TEMPERATURE AND LOG OF TIME 62
TO DEATH AFTER EXPOSURE OF JUVENILE RAINBOW TROUT TO
CERATOMYXA SHASTA
RELATIONSHIP BETWEEN WATER TEMPERATURE AND LOG OF TIME 66
TO DEATH AFTER EXPOSURE OF JUVENILE COHO SALMON TO
CERATOMYXA SHASTA
VI
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TABLES
No. Page
1 Effect of Water Temperature on Aeromonas Salmonicida 17
Infection in Juvenile Coho Salmon
2 Recovery of Aeromonas Salraonicida by Culture of Kidney 18
Tissue of Juvenile Coho Salmon
3 Effect of Water Temperature on Aeromonas Salmonicida 24
Infection in Juvenile Spring Chinook Salmon
4 Recovery of Aeromonas Salmonicida by Culture of Kidney 26
Tissue of Juvenile Spring Chinook Salmon
5 Effect of Water Temperature on Aeromonas Liquefaciens 31
Infection in Juvenile Steelhead Trout
6 Recovery of Aeromonas Liquefaciens by Culture of Kidney 32
Tissue of Juvenile Steelhead Trout
7 Effect of Water Temperature on Chondrococcus Columnaris 44
Infection in Juvenile Rainbow Trout
8 Effect of Water Temperature on Chondrococcus Columnaris 46
Infection in Juvenile Coho Salmon
9 Effect of Water Temperature on Chondrococcus Columnaris 49
Infection in Juvenile Spring Chinook Salmon
10 Incidence of Ceratomyxa Shasta and Mean Time to Death 60
Post-exposure of Juvenile Rainbow Trout Exposed to
Water Containing the Infective Stage of the Organism
and then Placed in Temperature Regulated Disease Free
Water
11 Incidence of Ceratomyxa Shasta and Mean Time to Death 64
Post-exposure of Juvenile Coho Salmon Exposed to
Water Containing the Infective Stage of the Organism
and then Placed in Temperature Regulated Disease Free
Water
12 Mortality Among Fingerling Kokanee Salmon Resulting 74
from Various Concentrations of Sockeye Salmon Virus
in the Aquarium Water at 54 F
13 Mortality Amonti Fingerlinp, Kokanee Salmon Resulting 75
from Various Concentrations of Sockeye Salmon Virus
in the Aquarium Water at 54 F
vii
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No. Pape
14 Effect of Water Temperature on Mortality of 1.1 gm 77
Kokanee Salmon Fingerlings Exposed to Sockeye Salmon
Virus
15 Effect of Water Temperature on Mortality of 2.9 p,m 78
Kokanee Salmon Finp.erlinps Exposed to Sockeye Salmon
Virus
16 Mean Time to Death for 2.9 gm Kokanee Salmon Finper- 80
lings Exposed to Sockeye Salmon Virus
17 Effect of Water Temperature on Mortality of 0.11 gm 81
Kokanee Salmon Fry Exposed to Sockeye Salmon Virus
18 Mean Time to Death for 0.11 p,m Kokanee Salmon Fry 33
Exposed to Sockeye Salmon Virus
19 Effect of Water Temperature Mortality of 0.95 pm 84
Kokanee Salmon Fry Exposed to Sockeye Salmon Virus
20 Mean Tine to Death for 0.95 sm Kokanee Salmon Fry 86
Exposed to Sockeye Salmon Virus
viii
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SECTION I
CONCLUSIONS
1. Water temperatures of 59 F and above produce high mortality rates
in juvenile coho salmon injected with Aeromonas salmonicida. Even at
49 and 54 losses may exceed 40 percent.
2. Mortality rates of coho salmon injected with _A. salmonicida are
very low at temperatures of 39 and 44 F.
3. The mean time to death of coho salmon injected with A. salmonicida
is estimated to be 3.5 days at 69 F, and this increases steadily as
water temperature decreases, to a maximum of 31 days at 39 F.
4. The effect of temperature on the growth rate of A. salmonicida in
vitro appears to be similar to its effect on the rate of progress of the
infection in fish.
5. Among spring chinook salmon injected with ^. salmonicida, the
mean time to death is estimated to be 2.9 days at 74 F, and this in-
creases progressively as water temperature decreases, to a maximum of
18.4 days at 39°F.
6. The percentage of fatal infections among steelhead trout injected
with Aeromonas liquefaciens, is high at temperatures of 64 F and above,
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moderate at 54 and 59 F, and zero at 49 F and below.
7. When coho and spring chinook salmon, and rainbow trout are in-
fected with Chondrococcus columnaris by water contact, the percentage
of fatal infections is high at temperatures of 64 F and above, moderate
at 59 F and approaches zero at 49 F and below.
8. A temperature of 54 F is close to the threshold for development
of fatal infection of salmonids by Chondrococcus coluranaris.
9. The percentage of fatal infections in rainbow trout infected with
Ceratomyxa shasta is high at water temperatures between 74 and 44 F,
10. The mean time to death of rainbow trout infected with £. shasta is
approximately 14 days at 74 F, increasing to approximately 155 days
at 44 F. Fish continually held at 39 F are not believed to develop
fatal infection.
11. The percentage of fatal infections among coho salmon infected
with C_. shasta, is high at 64 F and above, moderate at 54 to 59 , and
approaches zero at 49 and below.
12. The effect of temperature on the progress of infection by C. shasta
appears similar in rainbow trout and coho salmon, as indicated by the
mean time to death. There is a distinct difference in the effect of
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temperature on susceptibility of these two species. Coho are not
equally susceptible at all temperatures, while rainbow trout are.
13. Among kokanee salmon fingerlings exposed to the sockeye salmon
virus mortality rates are high at water temperatures from 39 to 59 F,
and significantly lower at 64 and above.
14. While fatal infections due to the virus may be high at 39 F, the
mean time to death is much longer than at higher temperatures.
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SECTION II
RECOMMENDATIONS
Water temperatures in many rivers of the Pacific Northwest from May
through October are in a range favorable for the progress of the important
infectious diseases of salmonids. During this period threshold temp-
eratures for these diseases are reached and a maximum of 70 F is not
uncommon. Temperatures favorable to the host generally occur from
November through April. It is therefore recommended that no additional
sources of heat should be allowed to enter these rivers. Added heat during
the period from May through October could only serve to further enhance
the severity of these diseases. Increasing water temperatures from
November through April would shorten the period when conditions are most
favorable for the host.
Data collected in this laboratory over the past 5 years as part of
another study indicate that the threshold temperature for initiation of
infection by Ceratomyxa shasta is approximately 50 F. Results in these
studies revealed that once animals are infected with this organism fatal
disease develops over a wide range of temperatures. Therefore it appears
that when temperatures exceed 50 F in waters where this agent occurs,
disease and deaths can be expected.
Water temperature should be considered before trout or salmon are re-
leased into streams or lakes. Releases should not be made when the
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temperature exceeds 50 to 53 F.
Evidence gathered during this investigation indicates that infection with
the sockeye salmon virus occurs over a wide range of temperatures. As a
result no practical recommendation could be made pertaining to changes
of temperature in waters containing this infectious agent. It is con-
ceivable that temperatures above 64 F could be used to control progress
of the disease in fish rearing facilities equipped with water treatment
or reuse systems which could eliminate other pathogens.
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SECTION III
INTRODUCTION
The chief objective of the work described in this report has been to
determine the effect of water temperature upon the course of and
mortality from the more Important infectious diseases of the salmonid
fish native to rivers of the Pacific Northwest.
The diseases which have been studied have included those caused by
Ceratomyxa shasta, Chondrococcus columnaris, Aeromonas salmonicida,
Aeromonas liquefaciens, and the Oregon sockeye salmon virus. Fish
species which have been used in these studies were juvenile coho and
chinook salmon and steelhead trout. Fingerling kokanee salmon were used
in experiments with the virus.
The general experimental plan which has been followed with each disease
agent has been to infect groups of susceptible fish of a given species
by the most appropriate method, and to hold these groups in tanks of
flowing water, controlled at one of several temperature levels. Eight
temperatures, from 39 F to 74 F, with 5 increments, have been provided.
For each experimental temperature, groups of 50 or more infected fish
have been employed, distributed equally between 2 tanks. Parallel groups
of normal uninfected fish have been held under identical conditions.
All experimental fish have been observed daily for appearance of symp-
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toms, lesions, or fatal infections. Dead fish were removed immediately,
and were autopsied and the appropriate organs examined culturally for
the presence of the specific pathogen. Observations were continued until
no further deaths occurred.
The effect of the various water temperatures upon each type of infection
has been judged by the fraction of the group of fish held at each tem-
perature that developed fatal infection caused by the specific pathogen,
and by the mean death time for those that succumbed in each group.
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SECTION IV
EQUIPMENT DESIGN AND FABRICATION PHASE
Before experiments dealing with the effect of water temperature on
infectious diseases of fish could be undertaken, special equipuent for
holding experimental animals at various temperatures had to be designed
and fabricated. This phase of the project was submitted to engineering
firms for bids, and was ultimately carried out by the Corvallis firm of
Cornell, Rowland, Hayes and Merryfield.
The equipment provided for holding fish consists of 64 covered fiber-
glass tanks or aquaria of about 21 gallon capacity. Sixteen of these
were new and 48 were already installed in the fish disease laboratory.
Water is supplied to the laboratory from a well, at a constant tempera-
ture of 54 F. Eight of the 64 tanks are supplied with flowing water at
that temperature. Eight tanks are supplied with water heated to one of
the following temperatures: 59 , 64 , 69 and 74 F; and 8 tanks receive
water chilled to 49 , 44 and 39 F. The rate of flow of these various
streams of heated and chilled water is variable, with a maximum of 1.0
gallon per minute per tank. The temperature of each stream is automat-
ically controlled by a recorder-controller and mixing valve within a
range of ± 0.5 F. An alarm system gives warning of any failure that
might develop in temperature control.
The heated water is supplied by a gas fired boiler capable of supplying
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each of the 4 heated water streams at a rate of 8 gallons per minute.
The refrigerated water is produced by a stainless steel chiller of
special design and custom built, with a capacity adequate to supply
each of the 3 refrigerated streams at 8 gallons per minute. All equip-
ment, piping and valves that come in contact with the water supply
are of stainless steel or polyvinyl chloride, to eliminate possible
toxicity to fish. A small frame building, 16 x 10 ft. was constructed
to house the boiler, chiller and air compressor.
In order to provide protection against possible failure of the well
water supply to the laboratory, and to permit the periodic overhaul of
the pump in the laboratory well, it was necessary to make an alternate
source of water available. Piping was installed to connect the labora-
tory to an existing well located at a distance of about 150 yards. A
new pump with a capacity of 300 gallons per minute was required to
deliver the required volume of water from this second well.
10
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SECTION V
EFFECT OF WATER TEMPERATURE ON INFECTION OF SALMONIDS BY AEROMONAS
SALMONICIDA AND AEROMONAS LIQUEFACIRNS
Materials and Methods
Two strains of Aeromonas salnonicida were used in the work reported
here. Strain 5-G was isolated from the kidney of a coho salmon during
an outbreak of furunculosis at the Siletz Hatchery in Oregon. Stock
cultures were maintained by cultivating the organism in peptone-beef
extract-glucose broth, centrifuging, resuspending the cells in sterile
skim milk, and lyophilizing. The second strain, SS-70 was isolated
from the kidney of a chinook salmon at the South Santiam Hatchery in
Oregon. It was passed through a series of 13 transfers in juvenile coho
salmon by intraperitoneal inoculation of a suspension of kidney tissue
from the fish infected in the preceding transfer. Kidney tissue from
the last fish in the series was then macerated, suspended in skim milk
and lyophilized.
Aeromonas liquefaciens, strain K-l, was isolated from the kidney of
shad during an epizootic in Coos Bay, Oregon. Stock cultures were main-
tained on peptone-beef extract-glucose agar covered with a layer of
neutral mineral oil. This medium contains 10 gm of peptone (Difco) 5 gm
of glucose, 10 gm of beef extract, 5 gm of sodium chloride, and 15 gm
of agar, per liter.
11
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Experimental fish employed in these experiments were juvenile coho, or
spring Chinook salmon, or juvenile steelhead trout. Their average weight
ranged from 10 to 30 grams in different experiments. They were generously
donated for this project in relatively large numbers by the Oregon Game
Commission and the Fish Commission of Oregon.
Experimental infections in fish were produced by the intramuscular or
intraperitoneal injection of 0.05 ml of a 48 hour culture of the organism
grown in brain heart infusion broth (Difco Labs) or peptone-beef extract-
glucose broth (PBG) and resuspended in frog Ringer saline at pH 6.9-7.0.
The composition of the PBG medium with agar is described subsequently
in this report. The broth is prepared in the same manner, but without
the agar. The bacterial concentration was adjusted to represent from
0.5 to 2.0 LDcn doses, based on an earlier titration of the same organ-
ism in the same fish species held at 54 F. It would have been distinctly
preferable to use a more natural method for establishing infection, but
preliminary experiments and previous experience indicated that exposure
of fish to high concentrations of these aeromonas species in their water
supply, or the presence of infected fish in the tank with normal sus-
ceptible ones, could not be relied upon to produce fatal infections in
a large percentage of those exposed. These organisms while pathogenic
for fish, do not always possess highly invasive properties.
The method used to temper fish to the various experimental water
temperatures was as follows: When first received from the hatchery,
12
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the fish were placed in holding tanks supplied with well water at 54 F.
At the beginning of an experiment, the number of fish to be used in one
experimental group were transferred to an 18 gallon tank, also supplied
with 54 F water. Water at the next temperature increment, either 49 or
59 F, from one of the controlled streams, was then introduced at the
rate of about one half gallon per minute. Within 1 to 1% hours, the water
in the tank had reached the 49 or 59 level. The fish were then held
at the new temperature for 48 hours. Water at the next temperature incre-
ment, either 44 or 64 was then introduced into the tank at the same
rate as before, and the new temperature maintained for 48 hours. This
process was repeated until groups of fish had been adjusted to each of
the eight experimental temperature levels covering the range from 39
to 74 F at 5 degree intervals.
For the cultural examination of experimental fish at necropsy small
fragments of kidney tissue were streaked on plates of Furunculosis Agar,
Difco, modified by the addition of 1 gram of skim milk solids per liter
of medium. Plates were incubated at room temperature (about 22 C) for
48 hours. Colonies producing, zones of clearing on this medium were
inoculated on two plates of regular Furunculosis Agar, tubes of Oxidative-
Fermentative Medium, Difco, and Arginine Decarboxylase Medium, Difco.
One of the plates was incubated at 37 C to inhibit jV. salmonicida, the
other at room temperature to permit grov/th. The latter plate was then
used for determining morphology, the Gram reaction, motility, catalase
and cytochrome oxidase reactions. j&. salmonicida is a Gram negative,
13
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non-motile rod that produces clearing on the Furunculosis Agar with
casein, fails to grow at 37 C, and forms catalase and cytochrome
oxidase. /L. liquefaciens differs by growing at 37 C and in being motile.
The experimental design adopted in this work required the use of sixteen
18 gallon aquaria for each experiment. Thus 2 tanks were provided for
each of the 8 water temperatures. Eight tanks, one at each temperature,
were assigned to groups of fish to be infected with the pathogen being
studied, while the remaining eight were assigned to groups of uninfected
control fish that received sham injections. The number of fish per tank
was at least 25, and in some experiments was increased to 35. Two com-
plete and identical experiments were conducted concurrently, each one
consisting of 8 groups of infected fish and 8 control groups. The pur-
pose of this plan was to provide information concerning the degree of
variation to be expected between groups of fish receiving, insofar as
possible, exactly the same treatment.
The terms rainbow trout and steelhead trout are used in the text of this
report. It should be understood that both terms refer to a single species,
i.e. Salmo gairdneri, but steelhead are anadramous while rainbow trout
do not migrate to the ocean.
14
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Experimental Phase
Effect of Temperature on Infection with Aeromonas saliaonicida
The effect of water temperature on experimental infection of juvenile
coho salmon (Oncorhynchus kisutch) with Aeromonas salmonicida, strain
5-G was studied in two experiments. In each of these, 400 fish averaging
28 grams in weight, were distributed at random among 16 tanks, 25 fish
per tank. Each tank contained 18 gallons of well water, flowing at a
rate of 0.5 gallons per minute. Eight tanks contained fish to be in-
fected, and eight contained fish to be used as uninfected controls.
One tank in each group of eight received flowing water at 74 F, another
pair received water at 69 F, a third pair received water at 64 F, and
so on, so that the range of temperatures from 74 F to 39 F was covered,
with groups of fish maintained at each 5 degree increment of temperature.
The two experiments, involving 800 experimental fish, were carried out
concurrently.
After tempering of the fish to the various temperature levels, those
to be infected received an intramuscular injection of 2 LD- of a 48
hour broth culture of _A. salmonicida, strain 5-G diluted in frog Ringer
saline. An LD,-n was the approximate number of bacteria causing death
in 50% of a group of 20 to 30 gram coho salmon injected with the organ-
isms intramuscularly and held at 59 F for 5 days after the last death
occurred. Control fish received a sham injection of 0.05 ml of a sterile
15
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filtrate from a similar culture diluted to the same extent. Dead fish
were collected daily, each was autopsied, and kidney tissue samples
were cultured. This bacterium has been found to be recoverable from
the kidney of about 74% of fish succumbing from this infection. All
experimental groups were observed over a 55 day period.
Results of the two experiments are shown in Table 1. It is apparent
that among the infected groups, the per cent mortality decreased in a
stepwise manner from 100% at 69 F to 12% and 14% at 44 and 39 respec-
tively. In three instances a 5 degree reduction in water temperature
did not significantly influence the per cent mortality; this is evident
at 69 and 64 , at 54 and 49 , and at 44 and 39 . However mortality
was significantly lower at 59 than at 64 , at 54 than at 59 , and
at 44 than at 49 (Appendix, page 95). The data indicate that the
development of fatal disease in juvenile coho due to this organism
was suppressed at water temperatures of 39 to 44 , and was enhanced
progressively at temperatures of 49 , 59 and 64 .
The results of culturing kidney tissue from the infected groups of fish
are recorded in Table 2. Aeromonas salmonicida was recovered from the
majority of the individuals in each temperature group that succumbed to
the infection. These cultural recoveries strengthen the evidence that
death was due to the aeromonas infection. It may be presumed that the
remaining fish in the groups at 59 and below from which the organism
was not recovered also died from the infection, since control fish that
16
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had received a shatn injection and were held under the sane conditions,
all remained healthy. Aerononas salmonicida was not found in the
kidney of any of the infected fish that survived to the end of the
experiment. This may be seen in the data of Table 2. Presumably the
bacteria injected had been disposed of by body defense mechanisms
in these individuals. In order to provide further evidence on this
question, these surviving fish were transferred to tanks supplied with
water at 64 F, a temperature favorable to development of this infection,
and were held at this temperature for a 10 day period. No deaths occurred,
and when these fish were sacrificed and autopsied, ^. salmonicida was not
recovered from the kidney of any of them.
A linear relationship between the log of the number of days to death
and water temperature was observed and confirmed by regression analysis
(Fig. 1). A correlation coefficient of -0.8850 was calculated and found
to be highly significant (Appendix, page 108). This relationship indicates
that progress of the fatal infection was accelerated at the higher tem-
peratures of 69 and 64 , retarded at the intermediate temperatures,
and still further retarded at the low temperatures of 39 and 44 .
This effect of water temperature upon the average time from infection
until death could be an expression of the combined influence of tempera-
ture on growth of the bacterium and upon the defense mechanisms of the
host. In order to shed some light on this question the growth rates of
the above strain of A. salmonicida were determined at each of the tem-
19
-------
lOOr-
kj
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'39 44 49 54 59 64
WA. TER TEMPERA TURE C'F)
74
Fig. 1. Relationship betvee.n v;a!:er tpnoeraturc e.nd log of ti^
to death r.ftcr infection of juvenile echo salmon with
Aeronon&c salmonicida.
-------
peratures used in the fish experiment.
A flask containing 200 ml of peptone-beef extract-glucose broth was
inoculated xjith the organism to give a concentration of 1.9 x 10 cells
per ml. The inoculated medium was then distributed in 2 ml aliquots in
screw cap culture tubes. A group of these tubes was then incubated at
each of the 8 temperatures.
Growth was measured by determining optical density at 650 my at various
intervals during an 80 hour incubation period. The growth rates observed
are shown in Fig. 2.
£. salropnicida grew very slowly at 39 and 44 , and the rate of growth
increased progressively with each 5 degree increase in temperature,
reaching a maximum at 69 . Thus the effect of temperature on the growth
rate of the organism in vitro appears to follow a pattern closely sim-
ilar to its effect on mortality among infected fish. In other words
high temperatures resulting in the most rapid growth of ^. salmonicida
in vitro also resulted in the shortest mean time to death among infected
fish, while low temperatures resulting in very slow growth rates i.n
vitro were associated with the longest mean times to death. No informa-
tion is available concerning the possible influence of temperature on
host defense mechanisms, but in any case the data of Fig. 2 indicate that
the effect of temperature controls the growth of the bacterium which
in turn has a major controlling influence on mortality in infected
21
-------
20 30 40 50 50 70
'INCUBATION PERIOD IN HOURS
60 90
Fig. 2. Effect of temperature on growth iar,e of Aero'.i'.onas
salmorJcida in peptoue-beaf-extracL- gl._,;ose broth.
-------
juvenile coho salmon.
The effect of temperature on infection of juvenile spring chinook salmon
(Oncorhynchus tshawytscha) by Aeromonas salmonicida was studied in two
experiments. As in the work with the coho salmon, 400 fish averaging
10 gn in weight, were used in each experiment. They were distributed
at random among the 16 tanks, 25 fish per tank and tempered to the
various temperatures in the manner previously described. The groups of
fish to be infected received an intraperitoneal injection of 1.4 LD
(about 425 organisms) doses of a 48 hour broth culture of A_. salmonicida
(strain SS-70). Control fish received a sham injection of 0.05 ml of
sterile physiological saline. As in the coho experiments, dead fish
were collected daily, autopsied, and cultures made from kidney tissue.
All experimental groups were observed over a period of 35 days.
The results of these two experiments are presented in Table 3. It is
apparent that there was variation in the percent mortality observed at
the different temperatures, and some of the differences were statisti-
cally significant (Appendix, page 96), but the consistent reduction in
mortality with decreasing temperature which was found in the coho ex-
periments, was not observed with the spring chinook. The results obtained
in 6 of the 8 temperature groups would have been compatible with such
a trend, but the mortality noted at 59°, and at 39° were both higher
than would have been expected, and in each case was significantly
different from the values for the adjacent temperature groups. Reasons
23
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for these irregular results are not apparent, though it appears that
some variable other than temperature has influenced them. The experi-
mental method differed in two details from the coho experiments. ,A.
salmonicida strain SS-70 was used because the stock cultures of strain
5-G, used with the coho, became non-viable for unknown reasons. The
route of inoculation was intraperitoneal instead of intramuscular.
However there seems to be no reason to assume that either of these
differences could account for the irregularities. The experiments will
be repeated when juvenile spring chinook salmon are again available.
The results of culturing kidney tissue from the fatally infected fish
are shown in Table 4. Aeromonas salrnonicida was recovered from the
majority of these fish in each temperature group. Inoculated fish that
survived were not cultured in this case.
The average interval between infection and death was determined for the
infected chinook salmon by combining data from the two experiments. It
was found to be 2.9 days at 74 , and to increase progressively as water
temperature decreased, reaching a maximum of 18.4 days at 39 . When the
log of the interval was plotted against temperature a linear relation-
ship was revealed, exactly as in the coho salmon experiments. Confirma-
tion of this relationship was again obtained by regression analysis
(Fig. 3). A correlation coefficient at -0.8229 was calculated and found
to be highly significant (Appendix, page 109). This figure show that in
the chinook salmon also, the time to death was retarded at the lowest
25
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39 44 49 54 59 64
WATER TEMPERATURE ("FJ
69
74
Fig, 3, Relationship between water Lemperaturp and log of tima
to dent?i after infection of juvenile Chinook salwon
with Aeromon^.s dalinonicida.
27
-------
temperature levels, accelerated at the intermediate temperatures, and
still further accelerated at the highest temperatures. At 39 and 44 ,
the chinook succumbed to the infection nearly twice as rapidly as did
the coho, though at 54 and above, the average time till death was
closely similar for the two species.
28
-------
Effect of Temperature on Infection with Aerononas liquefaciens
Two experiments were carried out to determine the influence of water
temperature on experimental infection of juvenile steelhead trout (Salmo
gairdneri) with Aeromonas liquefaciens, strain K-l. In each of these, 560
fish averaging 25 grams in weight, were distributed at random among 16
tanks, 35 fish per tank. They were tempered to the various water tem-
peratures as described previously. Croups of fish to be infected received
an intramuscular injection of 0.5 LUrn doses (about 2.2 x 10 organisms)
of a 24 hour culture of A_. liquefaciens in peptone-beef extract-glucose
broth. Control fish were injected with 0.05 ml of a sterile filtrate of
the same culture, diluted to the same extent. All groups were held at
their respective temperatures for 27 days. Dead fish were collected
daily, autopsied, and cultures made from kidney tissue. Infected fish
surviving at the end of this period were sacrificed and examined in the
sane manner. Ten control fish from each of the following temperature
groups were also examined by kidney culture at the end of the experi-
ment: 74°, 69°, 64°, 59°, and 54°F.
The mortality data from these experiments are presented in Table 5. The
highest percent mortality occurred among the infected fish at 74 F,
while all of the uninfected controls survived this high temperature.
Mortality was significantly reduced at 69 and was essentially the same
at 64 . A further significant reduction is evident at 59 and 54 , where
38.6 and 40.1 per cent of the fish died, respectively. Progress of the
29
-------
infection was apparently halted at 49 , 44 and 39 , as no deaths occurred
at these temperatures. Thus temperatures from 54 to 74 were favorable
for the development of this infection in steelhead, and increased mor-
tality was correlated with increased temperature in this range.
The mean time to death appeared to be about twice as long at 54 as at
74 , as indicated by the data in Table 5. However the influence of tem-
perature on the mean time to death was less striking than in A^. salmoni-
cida infections, possibly due largely to the fact that no deaths occurred
at 49 or lower.
The results of culturing kidney tissue from the infected groups of
fish are presented in Table 6. It may be noted that among the infected
fish that died, 68 to 93 percent in the various temperature groups,
yielded cultures of A^. liquefaciens. These data provide supporting
evidence that death of these fish was caused by infection with this
organism. It may be presumed that the remaining fish in these groups,
from which the organism was not recovered, also died from the infection,
since of the control fish at the same temperatures that had received a
sham injection, all but one remained healthy. It is also of interest
that among the surviving but infected groups of fish, some still
harboured the organism in the kidney; 23 per cent of the survivors at
74 yielded positive cultures, and recoveries decreased to 17 per cent
at 64 , 7 per cent at 59 and 2 per cent at 54 . None of those at 49
yielded the organism. Because of the small numbers of fish from which
30
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the organism was recovered, the differences in percent positive
cultures between any two temperature levels differing by a 5 increment
are not statistically significant. However, the 28.0% value at 69°
differs significantly from the 7.0 and 2.4% and 0 values at 59°, 54°
and 49 , respectively. Also the 17.4% value at 64 differs significantly
from the 2.4% value at 54 (Appendix, page 107).
These data suggest that within the temperature range studied, the
higher temperatures favor survival of the pathogens in the tissues of
the host, while the lower temperatures enhance the mechanisms that
clear the microorganisms from these tissues.
Presumably the survivors were individuals possessing greater resis-
tance to this bacterium than those that succumbed; however, they might
serve as a reservoir of pathogens for later outbreaks when conditions
are favorable.
Among 50 control fish that were examined by culture, 49 were negative
and A_. liquefaciens was isolated from one.
At the end of the 27 day experimental period, the infected fish that
had been held at 39 , 44 , and 49 , and among which no deaths had
occurred, were transferred to tanks supplied with water at higher tem-
peratures. The 39 groups were transferred to 59 water, the 44° groups
to 64 water, and the 49 groups to 69 water. This was done to deter-
33
-------
mine whether it was possible that small numbers of /L. liquefaciens cells
were surviving in some organ or tissue, and might be activated at the
elevated temperatures and produce a fatal infection. These fish were ob-
served for a period of 5 days at the higher temperatures, but no deaths
occurred and all appeared healthy at the end of this period. This sug-
gested that the organisms originally injected in these fish held at the
3 lower temperature levels had died out and been disposed of by the
defense mechanisms of the animals.
-------
Discussion
The work originally contemplated on the effect of water temperature on
aeromonas infections is incomplete. It was planned to study A^. salmoni-
cida and j\. liquefaciens infection in coho and chinook salmon and in
steelhead trout. Progress has been slower than anticipated for several
reasons. These have included the appearance of natural infections in
populations of experimental fish, difficulties with temperature control
equipment, and the sudden occurrence of high concentrations of dissolved
nitrogen in the well water supply.
However the data reported indicate that fatal infection in coho salmon
due to _A. salmonicida was suppressed at 39 to 44 F, and mortality was
progressively higher at temperatures from 49 to 64 . This was evident
from the mortality rates and from the average intervals from infection
until death, which were longest at the low temperatures, and decreased
progressively with increasing temperature. In the case of juvenile spring
chinook infected with this organism, the effect of temperature on mor-
tality rates was irregular, suggesting the influence of some uncontrolled
variable in the experiment; however it was again observed that the in-
fection progressed slowly at the low temperatures and at progressively
higher rates as the temperature increased. Fatal infection of steelhead
trout with _A. liquefaciens was prevented in the range of 39 to 49 ;
temperatures from 54 to 74 were favorable for development of this
infection, and the mortality rate increased with temperature in this
35
-------
range. Hence the limited data available thus far are consistent with
the view that water temperatures above the range of 44 to 49 may
cause increasing mortality from aeromonas infections in some salmonid
species.
Development of a Differential Plating Medium
for Aeromonas Species in Water Samples
During the course of this work the need arose for a bacterial culture
medium that would permit the counting of aeromonas species in water
specimens in the presence of other common bacterial flora. A medium
was desired that would produce counts comparable to those obtained with
the best plating media, while at the same time inhibiting groxjth of some
other organisms found in water, and exhibiting differential colony
reactions that would permit the recognition of aeromonas colonies. A
relatively large number of formulations were compared with respect to
the above properties and the following one was ultimately selected:
Peptone-Beef Extract-Glycogen (PBG) Agar
Bacto Peptone 10 grams liter
Beef extract 10 " "
Glycogen 4 " "
NaCl 5 " "
Sodium lauryl sulfate 0.1 " "
Brom thymol blue 0.1 " "
Agar 15 "
final pH 6.9-7.1
36
-------
Sodium lauryl sulfate was included in the medium as a selective agent
for inhibition of some Gram positive bacteria. Broin thymol blue serves
as both an indicator of pH change as well as adding to the inhibitory
effect of sodium lauryl sulfate. Glycogen was included as the only car-
bohydrate because the aeromonas species are among the relatively few
bacteria reported to be capable of fermenting this polysaccharide.
For use in plating a water sample, 1 ml of the sample in the desired
dilution is added to a sterile Petri dish and mixed with 15 ml of the
sterile PEG agar at 45-50 C. After the agar has gelled and the surface
has dried, it is overlaid with about 20 ml of 2.0% agar in distilled
water. If Aeromonas salmonicida is to be isolated or counted, plates
must be incubated at 25 C for 3 days. Under these conditions it has
been found that in addition to the above organism, a number of other
bacteria also produce yellow colonies on this nedium. These include
some species of Citrobacter, Arizona, Edwardsiella, Enterobacter, and
Serratia. However, all of these organisms, as well as Aeromonas hydro-
phila (liquefaciens) form large bright yellow colonies, 1 mm or more
in diameter.
Colonies of ^. salmonicida, Pleisomonas shigelloides, Vibrio anguiliarurn,
and Vibrio parahaemolyticus, developing at this temperature, are pin
point in size, and with a little experience can be readily distinguished
from the former group. Organisms from natural sources producing these
very small colonies can be presumptively identified as one of these
37
-------
four species. Pleisomonas shigelloides can be distinguished by failure
to produce gelatinase. The two Vibrio species produce lysine decarbo-
xylase, but not arginine dihydrolase, reactions which would differen-
tiate them from A^ salmonicida and P_. shigelloides. Production of a
brown pigment and lack of motility will serve to differentiate A. sal-
monicida.
In addition, some colonies of this organism will produce small bubbles
of gas in the agar layer, which has not been observed with any of the
other bacteria mentioned above.
If the medium is to be used for isolation or counting of the Aerompnas
hydrophila-liquefaciens complex, plates should be incubated at: 37 C
for 24 hours. Under these conditions colonies of these organises are
0.3 to 0.5 mm in diameter and bright yellow in color. Those of Arizona,
Citrobacter, Cdwardsiella, Enterobacter and Serratia species are smaller,
about 0.1 to 0.2 mm in diameter, and possess more of an orange color.
The aeromonad colonies are often surrounded by a yelloxtf halo in the
green medium, a characteristic not observed with the other genera men-
tioned above. If there are as many as 200 to 300 A. hydrophila colonies
per plate, the whole plate develops a yellow color, while comparable
plates of the other organisms are greenish in color. Furthermore, some
of the subsurface colonies of A. hydrophila produce small bubbles of
gas in the agar layer, which is another differential characteristic.
The Vibrio and Pleisomonas species do not grow at 37 C, and thus do not
38
-------
require differentiation.
Over 75 species of bacteria have been examined on this medium. Many
Grara positive organisms failed to grow, though Bacillus subtilis and
other members of the genus grew sparsely. All Gram negative organisms
grew, but only those genera listed above produced yellow colonies.
Although the medium has obvious limitations, it has been found to be
useful in monitoring the numbers of both A., salmonicida and A_. hydro-
phi la in hatchery water, measuring their growth rates, and recovering
cultures from viscera of experimentally infected fish. Thus far it
appears to be superior to other available media for these purposes,
but further experience will be required to completely define its use-
fulness and reliability.
The medium has been useful not only in counting aeroraonas organisms in
water, but for isolation of these organisms from fish, and measuring
growth rates at various temperatures.
39
-------
-------
SECTION VI
EFFECTS OF WATER TEMPERATURE ON INFECTION OF SALMONIDS
BY CHONDROCOCCUS COLUMNARIS
Materials and Methods
To determine the effect of water temperature on losses caused by the
myxobacterium Chondrococcus columnaris, coho salmon (Oncorhynchus
kisutch), spring Chinook salnons (Oncorhynchus tshawytscha) and rainbow
trout (Salmo gairdneri) were exposed to a virulent isolate of this bac-
terium. Groups of 25 or 35 fish of the species being tested were tem-
pered to water temperatures ranging from 39 to 74 F at 5 F intervals.
Two control and two experimental groups were held at each temperature.
These animals were tempered in a manner previously described in the ex-
periments with the aeromonads.
The C_. columnaris isolate used in this study was obtained from a lesion
on the gill of an adult spring chinook salmon at the Fish Commission of
Oregon, Dexter Dam Holding Pond, Willamette River. This isolate was
passed in coho salmon fry seven times to increase its virulence. After
the seventh passage the culture was lyophilized. Immediately prior to
each experiment the cells were removed from lyophilization, grown in
cytophoga broth and passed once in the salraonid species being tested.
Several isolates from the final fish passage were collected and pooled
for use in the temperature experiment.
41
-------
To prepare the exposure inoculum the cells were grown in tryptone yeast
infusion broth containing 0.4% tryptone and 3.0% yeast infusion. After
approximately 20 hours at 24 C the optical density was adjusted to 0.1
at 525 my with a Bausch and Lomb Spectronic 20 (1). The fish were then
exposed in the experimental tanks to a 1:20 dilution of the adjusted
broth culture for a 10 minute period. Normal water flow through the
tanks was resumed after the exposure period. This dilution was determined
by plate count to represent approximately 3 to 6 x 10 C. columnaris
cells per ml. Dead fish were collected two times each day and bacterio-
logical cultures v/ere made from the gills and or kidney of each fish.
42
-------
Experimental Phase
Table 7 shows the percent mortality and the incidence of C_t columnaris
in rainbow fry (average weight 2.9 g each) exposed to this bacterium at
different water temperatures. Temperatures of 44 and 39 F were not in-
cluded in this experiment. Fish infected with £. columnaris were observed
at temperatures of 74 F down to 54 F with the larger number of losses
occurring at the higher water temperatures. The greatest difference in
loss occurred between 59 and 64 F. Control fish at 74 F had some mortality
which was not due to C. columnaris.
Not only percent mortality but also the time to death was greatly in-
fluenced by water temperature. This is illustrated in Fig. 4, which
shows the regression line relating water temperature and the log of the
number of days from exposure to death. The equation and the data used
in computing it are shown in Appendix, page 110. A correlation coefficient
of -0.8573 was calculated and found to be highly significant. Thus the
linear relationship between these two variables is demonstrated.
The results of the experiment with coho salmon (average weight 33 g each)
are nearly identical to those observed with rainbow trout (Table 8).
Within three days after exposure, all fish held at 69 and 74 F were
dead. After one month at 64 F a loss of 99% had occurred, as compared
to a 51% loss at 59°F. At 54°F only 4% of the test animals had died.
No deaths due to C. columnaris occurred at 49 F or below. Among the
43
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74
Fig. 4, Relationship between water tcn\pf>ratur: and log of time
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control groups, only a few deaths occurred at the higher temperatures,
and £. columnaris was never isolated from these fish.
Again, a linear relationship between water temperature and the log of
the number of days from exposure to death was observed (Fig. 5). A
correlation coefficient of -0.7699 was calculated, and as with the
rainbow trout experiments, was highly significant (Appendix, page 111).
Fifteen days after the last death occurred fish surviving at 59 were
transferred to 69 water and those surviving at 49 and 54 were trans-
ferred to 64 water. Subsequently, losses due to _C. columnaris occurred
among those groups originally held at 59 and 54 , but not among those
held at 49 . Thus, some of the survivors, when moved to higher water
temperatures, developed the disease.
Results of the spring chinook experiment (average weight, 10.2 g each)
were similar to those observed previously with rainbow trout and coho
salmon, although the percent mortality at temperatures of 59 and above
was lower than in the other experiments (Table 9). Chondrococcus
columnaris was isolated in gill or kidney cultures from approximately
88% of the deaths in fish exposed at 59° or higher; from 31% of those
dying at 54 , and was not recovered from any of those held at 49 or
below. Among the control groups only a few deaths occurred, and the
columnaris bacterium was not isolated from these fish.
47
-------
lOOr-
10
£
1
39
49 54 59 64
WA TER TEMPERA TUR£ (°F)
69
Fig. 5. Relationship between water temperature r.nd log of tine
to death after exposure ol juvenile cc',,o salmon to
Chondrococcus columnaris.
-------
60
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49
-------
The linear relationship between water temperature and the log of the
number of days from exposure to death reported in the experiments with
rainbow trout and coho salmon was again observed (Fig. 6). A correlation
coefficient of 0.7192 was calculated and found highly significant
(Appendix, page 112).
At the end of the 30 day observation period fish surviving at 54 were
transferred to 64 water and survivors in the 49 group were transferred
to 59° water. Fifteen of the 40 fish (38%) moved from 54 to 64°F water
and 4 of the 42 fish (10%) moved from 49 to 59 F water became infected
with C. columnaris.
50
-------
100 r-
I
I
k ,ft_
s
8
s
I
WA TER TEMPERA TURE ( T)
Fig. 6. Relationship between water tcipcratur; and log of tine
to death after cxpopui'e of juvenile i/.lr.ook salmon co
Chondrccoccus columnar!;;.
-------
Discussion
Fish infected with £. columnaris were observed at temperatures from 74 F
down to 54 F with the greater number of losses occurring at the higher
water temperatures. Results of the coho salmon and rainbow trout experi-
ments are nearly identical. With spring chinook the percent mortality
at 59 F and above was lower than in the other species. A temperature of
54 F appeared to be the threshold for infection by £. columnaris. At
each temperature increment tested above 54 F exposure to the columnaris
bacterium resulted in a greater number of infections and deaths.
The time to death was also greatly influenced by water temperature. For
example, in the rainbow trout experiment all fish were dead at one and
four days at 74 and 69 F, respectively, after exposure. At each lower
temperature greater numbers of fish survived the exposure.
ChondrocQccus columnaris was isolated from the gills or kidneys of most
experimental animals in each group. The organism was not isolated from
control groups. The symptoms and pathology observed in the test animals
were similar to those described during epizootics of columnaris disease.
In these experiments the fish were exposed to large numbers of the
columnaris bacterium in the water; consequently isolation from the
kidney mav be a more accurate indication of infection.
No deaths caused by C. columnaris were found in fish held at 49 F or below.
52
-------
However, as previously described, exposed fish held at water tempera-
tures unfavorable for the progress of infection often developed fatal
disease when transferred to water at higher temperatures (64 or 69 ).
53
-------
-------
SECTION VII
EFFECTS OF WATER TEMPERATURE ON INFECTION OF SALMONIDS BY
THE PARASITIC PROTOZOAN CERATOMYXA SHASTA
Materials and Methods
Three species of salmonids were examined in this study; rainbow trout
(Salmo gairdneri) were obtained from the 1969 brood at Roaring River
Hatchery (Oregon State Game Commission). Coho salmon (Oncorhynchus
kisutch) were from the 1970 brood at Fall Creek Salmon Hatchery and
spring chinook salmon (Oncorhynchus tshawytscha) were from the 1970
brood at Marion Forks Hatchery (both hatcheries operated by the Fish
Commission of Oregon). Rainbow trout used as a positive control in
the coho-spring chinook experiment were from the 1970 brood at Leaberg
Trout Hatchery (OSGC).
The only practical method of initiating infection of £. shasta in fish
is by exposure to water known to contain the infectious agent (2).
During the summer and fall months, the Willamette River (below Corvallis)
is a very effective location for producing £. shasta infections in
several species of salmonids. The proximity of the Willamette River to
the laboratory, made this site ideal for exposing fish to this protozoan
in these experiments. The object of the exposure was to initiate at
least a 50 percent infection attributable to £. shasta. From previous
experiments conducted by this laboratory (3) it was estimated that a
-------
48 to 72 hour exposure, to water in which the infectious agent was
present, would suffice for our purposes. All experimental groups were
held in a 96 cu ft live-box situated in the Willamette River current
at the Albany site. Replacement time of water in the live-box was
approximately 10 sec.
The rainbow trout were exposed to the infectious agent for 48 hours,
between September 5 and September 7, 1970. The mean water temperature for
this period was 60.7 F (S.D. » 0.45). The coho and spring chinook salmon
were exposed for 72 hours, between September 17 and September 20, 1971.
The mean water temperature was 59.4 F (S.D. = 0.62) for the interval.
The procedure used to temper fish to laboratory water temperatures
after exposure to C_. shasta was necessarily different from that used in
the bacterial experiments. Previous information had suggested that the
rate at which the £. shasta infection proceeds is temperature dependent.
During the exposure period, therefore, the rate of the infectious process
should be dependent on the river water temperature. Following this
reasoning it was deemed necessary to temper the fish after exposure as
rapidly as possible to the experimental temperatures without causing
severe stress. The rates of change which filled these requirements were
determined by preliminary experiments. A rate of 10 F per hour was used
for rainbow trout and a rate of 6.6 F per hour was used for coho and
spring chinook salmon. These rates of change were achieved by manual
control of the temperature regulating equipment. All fish, both experi-
56
-------
mental and control, were placed into their respective tanks, all of
which contained 54 F water. The control equipment was also initially
set at this temperature. This control equipment was then adjusted by
either increasing or decreasing the temperature of the water coming
into each tank to achieve the desired rate of change of temperature.
When the eight experimental temperatures (74, 69, 64, 59, 54, 49, 44 and
39 F) were reached, the regulating instruments were calibrated and set
for the remainder of the experiment.
It was anticipated that with a natural exposure in the Willamette River
the fish would also become infected with bacterial diseases. Aeromonas
liquefaciens and Chondrococcus columnaris were known to be prevalent in
the river. For this reason prophylactic measures were taken to prevent
their interference in the experiments. The antibiotic of choice in control
of these diseases was Terramycin as TM_.. This compound was given to
the fish incorporated in Oregon Moist Pellet (OMP) diet. Terramycin was
chosen also because of its lack of activity against JC. shasta. In each
experiment all fish including controls were fed TM at a level of 25 g
TM /100 Ib of fish/day starting ten days prior to exposure. A level of
20 g TM -/100 Ib of fish/day was fed post exposure for three days in
the rainbow trout experiment. The treatment was discontinued for four
days, resumed for five additional days at which time the level was re-
duced to a dose of 5 g TM--/100 Ib of fish/day. To protect coho and
spring chinook salmon a 30 min bath in water containing 5 yg/ml of
soluble terramycin was given to the exposed fish before placing them
57
-------
in the laboratory tanks. They were fed at the 20 g TM /100 Ib of
fish/day level for the interim of the experiment.
Dead fish were collected at least once daily and either examined while
fresh or were frozen for later autopsy. Examination of dead animals
consisted of microscopic observation (400 x, bright field) of wet mounted
samples of intestinal scrapings. Slides containing two or more of the
spore stage of £. shasta were considered as positive diagnoses.
The experimental design used for the rainbow trout experiment was the
same as that used for the bacterial investigations described in this
report. In order to conserve laboratory facilities during these very
long-term experiments with £. shasta, the design was modified for the
coho-spring chinook experiment. The experimental design was altered
according to recommendations from the project's statistician. This
modified design consists of conducting two concurrent experiments in
each tank using two species of fish distinguished by removal of opposing
pectoral fins. The second modification eliminated the use of two control
groups for two experimental groups at each temperature, and substituted
one control group. In the design used xd.th rainbow trout 25 fish
averaging 11.5 g were placed in each tank. With the modified design 25
coho salmon averaging 14 g and 25 spring chinook salmon averaging 8.7 g
were placed in each tank. Rainbow trout used as an exposure control in
the latter experiment averaged 15 g; 35 of these fish were exposed and
35 used as unexposed controls.
53
-------
Experimental Phase
In these experiments with £. shasta two types of information were ob-
tained. Quantal mortality data, expressed both as percent infection
and quantitative response data expressed as mean time to death in days
have been gathered. From this data some qualitative inferences have
been drawn regarding effects of temperature changes in the host-parasite
relationship.
Table 10 summarizes the mortality data acquired from two concurrent
replications of an experiment utilizing rainbow trout. The data is
arranged to show the effect of temperature on the £. shasta infection.
The number of fish infected with £. shasta from experimental group 2
of both the 74 F and 69 F temperatures are lower than expected. This
is due to a fatal C. columnaris infection in several fish prior to the
time that they would have died of £. shasta. It call be seen, however,
from all other experimental groups in the 74 F to 44 F range that tem-
perature has little or no effect on the percent mortality due to £.
shasta. This data suggests that rainbow trout may have no means to com-
bat this parasite in this temperature range. At 39 F no mortality due
to C_. shasta was observed even after 237 days post exposure. However,
when these same fish were tempered thereafter from 39 F to 64 F over a
two week period and held for an additional four week period at 64 F, six
percent succumbed to C. shasta infections.
59
-------
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-------
Another parameter has been analyzed to determine the effect of tempera-
ture on the infection process. This second parameter is the dependence
of the mean time to death (of dead specimens diagnosed positive for C_.
shasta) on temperature. When the log of the mean time to death in days
is plotted against temperature a straight line should be obtained when
the host shows either no defense against or a logarithmic interaction
with the infectious organism. In this experiment with rainbow trout such
a straight line function was obtained. This further supports the idea
that rainbow trout are not able to overcome a £. shasta infection be-
tween 74 F and 44 F. If this assumption is correct, the temperature
dependence of the mean time to death reflects the effects of temperature
on the growth rate of the parasite.
At 39 F the host parasite relation is markedly altered since no mortality
occurred. _C. shasta may lie dormant in the fish which could explain the
onset of the disease after the fish were elevated in temperature.
A linear relationship between water temperature and the log of the
number of days from exposure to death was confirmed by regression
analysis (Fig. 7). A correlation coefficient of -0.9830 was calculated
and found highly significant (Appendix, page 113).
This experiment was terminated at each temperature when it was reasonable
to assume no additional deaths would occur due to JC. shasta. Fish re-
maining at termination were examined for the presence of C. shasta spores.
61
-------
1000 r-
1
100
£
s
10
39
44
49
54
59
64
69
WATER TEMPERATURE (°F)
Fig, 7. Relationship between water temperature, and log of time
to death after exposure of juvenile rainbow trout to
Ceratpmyxa shasta.
-------
Only two positive fish (at 44 F) were found. These fish appeared to have
been recovering from the infection.
A second pair of experiments with _C. shasta utilized the modified design
explained previously.
Coho salmon and spring chinook salmon were the host animals in this
study. The spring chinook portion of this experiment was terminated after
the third week. When the coho salmon died at 74 F and 69 F (prior to the
death of any spring chinook), the hacterial load, mostly of Aeromonas
liquefaciens overcame the drug treatment. This bacterium killed all ex-
perimental spring chinook at 74 and 69 . The spring chinook were left
in the tanks at the lower temperatures so as not to alter the results
of the remaining coho experiment.
Mortality data from the coho salmon portion of the experiment is reported
in Table 11. The results are noticeably different from that of the ex-
periment with rainbow trout. In this study, in contrast to the rainbow
trout experiment, the percent mortality attributable to C_. shasta does
not remain constant with decreasing temperature. It is instead reduced
at 64 F and below with no mortality below 49 F. The previous experimental
results (with rainbow trout) indicate that _C. shasta can multiply in
infected fish at temperatures down to 44 F when uninhibited. Coho salmon,
therefore, may well be able to somewhat inhibit C_. shasta development at
64 F and below.
63
-------
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Although not as reliable in this experiment as in the rainbow trout
experiment due to lower numbers of deaths, the mean time to death
analysis remains a valid parameter. A plot of the log of the mean time
to death verses temperature again yielded a straight line. Confirmation
of this linear relationship was obtained by regression analysis (Fig. 8).
The correlation coefficient obtained was -0.9574, which was highly
significant (Appendix, page 114).
It appears that at 74 F neither species of fish has the ability to
retard the growth of £. shasta. At 64 F and below the portion of the
coho population able to resist a fatal £. shasta infection increases with
decreasing temperature (Table 11). The portion of the population unable
to resist fatal infection may, however, interact with the parasite to
protract the mean time to death.
Thirty-five rainbow trout were also exposed at the same time as the coho
salmon and were then held at 54 F. These fish were used to determine
whether the Willamette River exposure would give repeatable results from
one year to the next. Sixty-eight percent of these fish were diagnosed
positive for £. shasta and the mean day of death was 67.7 days. These
results were very near those observed with rainbow trout the previous
year (Fig. 7).
65
-------
lOOOr
lOOf
£
to1
44 49 54 59
WATER TEMPERATURE (°F)
64
69
\
74
Fig. 8. Relationshop between water temperature and log of time
to death after exposure of juvenile c&>.o salmon to
Ceratcmyxa shasta.
66
-------
Discussion
It is alxrays the goal of this type of laboratory experiment to be able
to apply the results to natural and management situations. It is felt that
the results obtained in these experiments are applicable in these areas.
The short period of exposure to infection used resulted in fish dying
with identical pathology to that of fish held continuously in the
Willamette River until death. The mortality may be somewhat higher in
a natural situation since the fish are exposed to the infectious agent
for a longer period of time.
Since the water temperature during the exposure was near 60 F nothing
can be said about initiation of jC. shasta infection at colder water ten-
peratures. However, Keith Johnson working in this laboratory, has ini-
tiated an infection of jC. shasta in Cutthroat trout (Salmo clarki) when
the Willamette River had a maximum temperature of 48 F (exposure time,
3 days).
It has been shown in this study that rainbow trout held at water tem-
peratures between 74 F and 44 F have little or no ability to overcome
an infection of C_. shasta once initiated. Coho salmon on the other hand
do seem to be able to interact with the parasite. In this species the per-
cent of the population which is susceptible to the disease diminishes with
decreasing temperature. In both species, the mean time to death, of fish
diagnosed positive for £. shasta, is inversely related to temperature.
67
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SECTION VIII
EFFECT OF WATER TEMPERATURE ON INFECTION BY THE SOCKEYE SALMON VIRUS (IHN)
Materials and Methods
The virus used in the work reported here was isolated in this laboratory
in 1958 from diseased fish collected at the Willamette River Salmon
Hatchery in Oregon during an epizootic associated with a high mortality
rate. Stocks of this agent have been maintained since that time by prop-
agation in cultures of a salmonid cell line. Properties of the virus have
been reported (4). Fish used in these experiments have all been fingerling
kokanee salmon (Oncorhynchus nerka) generously supplied without charge
by the Oregon Game Commission.
Because of the limited number of 21 gallon experimental tanks available
at the main fish disease laboratory, it has not been possible to carry
on experiments with all 5 fish pathogens concurrently in those facil-
ities. Hence, in order to expedite the acquisition of data, work with
the sockeye salmon virus has been done with the aid of one gallon
glass aquaria, held in individual incubators at one of several tempera-
tures. This equipment was practical to use because of the small size
of the experimental fish. The xrater supply for these vessels was derived
from the Corvallis Municipal supply, and had been processed to remove
chlorine and fluoride by the Fisheries and Wildlife Department at Oregon
State University. The water in all of these small aquaria was changed
69
-------
at 48 hour intervals. The fish were fed Rangen's Salmon Mash twice a
day in amounts slightly in excess of their usual consumption.
When fish were first received from a hatchery, they were distributed
among a number of aquaria at a water temperature very close to that of
the hatchery water. In the case of fry averaging less than 1 gm in
weight, 30 fish or less were placed in each 1 gallon aquarium. For
those averaging 1 to 3 gm, the maximum number in each aquarium was 15.
The aquaria were then placed in incubators at one of the desired ex-
perimental temperatures and allowed to gradually adjust to the new
temperature over a 24 hour period. It was realized that from a physio-
logical viewpoint this was not an adequate acclimation procedure, but
in actual practice it did not appear to create any difficulties.
Fish were exposed to the virus infection by adding measured amounts of
standardized virus suspension to the water of their aquaria. Exposure
periods of either 24 or 48 hours were allowed, after which the virus
was removed and replaced by fresh water. Virus assays were carried out
by the plaque method, using monolayer cultures of salmonid cell line
CHSE 214, as described elsewhere (5).
Exposed fish that died 5 days or more after exposure were considered
to be virus infected if they showed symptoms characteristic of the
disease, and if 90% or more of unexposed controls held under the same
conditions remained healthy. Typical symptoms included hemorrhages at
70
-------
the base of pelvic and pectoral fins, extrusion of strings of milky
fecal material, and development of a very dark body color. Control
fish never showed any of these symptoms. The laboratory method for de-
tection of the virus in the organs of dead fish, although used in a few
instances, was considered to be impractical for application to large
numbers of fish.
71
-------
Experimental Phase
Preliminary Experiments
Before attempting to study the effect of water temperatures on infection
with the sockeye salmon virus, it was necessary to determine an appro-
priate concentration of virus for initiating the experimental infection.
In the first experiment bearing on this point, groups of 20 to 22 finger-
ling kokanee salmon, averaging 0.3 gm in weight were exposed to different
concentrations of virus in their water for a 24 hour period. Concentra-
tions varied from 2570 to 11 plaque forming units per ml, (pfu) at 3
fold intervals. All aquaria were held at 54 F and deaths recorded over
a 24 day period. The results are shown in Tahle 12. A second experiment
of the same type was carried out as soon as the first was completed. In
this case the available kokanee fingerlings had increased to an average
weight of 0.94 gm. The results are presented in Table 13.
In both preliminary experiments 90 to 100% of the fish exposed to 11 or
more pfu of virus per ml, succumbed to the infection; hence the cumula-
tive percent mortality did not increase with increasing virus concen-
tration. Deaths were distributed over a much longer time period at the
lower concentrations than was observed with the maximum virus level. This
was probably because some of the fish exposed to the lower virus con-
centrations only became infected when exposed to virus being shed from
others that had been infected during the initial exposure. Differences
72
-------
in the distribution of deaths with time among the experimental groups
are reflected in the values for the mean time to death in Tables 12 and
3. In both cases this value is minimal where the virus concentration
was maximal, and vice versa. In comparing Tables 12 and 13 it may be
noted that in the case of the 0.3 gm fish the mean time to death for
all virus concentrations was consistently shorter than the comparable
figure for the 0.94 gm fish.
The results of these two experiments indicated that any of several virus
concentrations within the range which was studied could be used success-
fully for exposure of fish in temperature experiments. In order to allow
as much latitude as possible for observing effects of water temperature
it appeared desirable to use a lower concentration (e.g. 32 pfu/ml) for
fish in the same weight range, i.e. 0.3 to 1.0 gm.
Effects of Water Temperature on Virus Infection
In the first experiment dealing with the effect of water temperature on
the virus infection, the available kokanee fingcrlinps had an average
weight of 1.1 gm. Incubator facilities for only 4 temperatures were
available and those selected were 74 , 64 , 54 and 39 F. For each tem-
perature level 20 fish were infected and another group of 20 held as
controls. Two complete experiments were carried on concurrently. Dead
fish were collected daily and deaths were presumed to be due to virus
infection, unless they occurred in less than 5 days after exposure, or
73
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cause.
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groups held at 74 F were lost due to failure in the aeration apparatus.
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at 39 , as indicated by the longer average interval between expiosure
and death.
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selected were 72 , 69 , 64 , 59 and 54 F. The number of fish in each
infected and each control group was increased from 20 to 40, arid two
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The results of these two experiments are presented in Table 15, In this
76
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case none of the experimental groups showed 100% mortality. The maximum
average mortality for the two experiments was 61.2%, which occurred at
54 F. The mortality rate decreased at 59 and was still lox^er at 64 and
69 . The 72 temperature was at the threshold of tolerance for these
fingerlings, as shown by a similar mortality rate in controls and in-
fected groups. These experiments seen to indicate that within the tem-
perature range covered, 54 F was the most favorable for the development
of fatal infection in fish of this size, while at 64 and 69 the disease
was partially suppressed and mortality was significantly lower. This
result appears to confirm the findings in the first experiments. No
consistent relationship between temperature and the nean time to death
was observed, as may be seen in Table 16, although this period was
longest among the 69 group.
The next pair of experiments was conducted during the following year
when kokanee salmon fry were available again. At this time the fish
were very small, and the average weight of those used in the experi-
ments was 0.11 gm. Temperatures that were compared in these eperiments
were 69 , 59 , 54 , 49 , and 39 F. For each temperature level in each
experiment, 30 fish were exposed to virus and a similar group of 30
were held as controls. For these small fish, the virus concentration
used to produce infection was 32 plaque forming units/ml of aquarium
water. The exposure period was 24 hours.
Table 17 presents the results of this second pair of experiments. The
79
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percent mortality, as measured by the combined data of the two experi-
ments was very low among the fish held at 39 and 49 F. It approached
100% however in the 54 and 59 groups, and declined again to a signifi-
cant extent in the 69 groups. This was despite the fact that about half
of the control fish at 69 succumbed to effects of this relatively high
temperature. The data thus indicated that the temperature range of 54
to 59 was near optimal for development of fatal infection under these
experimental conditions, and that infection was markedly retarded at
49 or lower, and to a lesser degree at 69 . The mean survival times
for fish that died in each temperature group are shown in Table 18.
The infection apparently progressed most rapidly at 54 and 59 , where
mortality was also greatest. It ran a slower course at both lower and
higher temperatures.
A third pair of experiments was next carried out with a population of
kokanee salmon fry whose average weight was 0.95 gm. The experimental
design was very similar to that in the previous experiments with the
0.1 gm fish, with the exception that only 20 fish were used in each
small aquarium because of their larger size. In addition to the 5 ex-
perimental water temperatures used previously, groups of fish held at
64 F v/ere included. The virus concentration in the water during the ex-
posure period was 32 plaque forming units/ml.
Results of the third pair of experiments appear in Table 19. They are
quite different from those obtained with the 2.9 gm and the 0.11 gm
82
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fish. In this case the final per cent mortality showed little relation-
ship to water temperature. It was high at all temperatures, ranging from
90% to 97.5% at the four lower temperature levels. It declined slightly
to 75% at 64 (a value significantly different from 97.5% (Appendix,
page 106). The distinct protective effective of the 39 and 49 tempera-
tures, observed with the 0.11 gm fish was lacking in these experiments.
There is however an indication of a retarding effect of the 39 tempera-
ture on the course of the disease in Table 20, where the mean time to
death at that temperature is about double the comparable value at the
other temperatures. It is noteworthy that the 0.95 gm fish used in these
experiments were obtained from the same population of kokanee salmon fry
as the 0.11 gm fish. The data seem to indicate that these larger fish had
become more susceptible to the virus infection; this is suggested by the
high mortality at the low temperatures, and the fact that the mean sur-
vival times were distinctly shorter at comparable temperature levels.
Two further experiments with this virus in kokanee salmon populations
were started, but had to be abandoned because of the development of
natural bacterial infections, despite the presence of 2 ppra of oxytetra-
cvclene in the water.
85
-------
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Discussion
It is in one sense unfortunate that all of the experiments reported with
the sockeye salmon virus could not have been carried out with experimental
fish of approximately the same size and from the same population. It
appears that factors related to age or size, and perhaps to environmental
or genetic background may have influenced some of the results obtained.
However it was necessary to use kokanee fry or fingerlings at whatever
time and from whatever source they became available.
One set of two experiments with 2.9 gm fish and one pair of experiments
with 1.1 gm fish indicated that a water temperature of 54 F produced a
higher percent mortality in infected fish than higher temperatures,
up to 69 , and that the course of the disease was slower at the latter
temperature. In another pair of experiments with 0.11 gm fish, tempera-
tures of 54 and 59 both resulted in over 90% mortality, while very
few fatal infections occurred at 39 and 49 . The course of the infec-
tion was also most rapid at 54 and 59 , as measured by the average
time from infection until death. These results are not in conflict with
experiments of Amend, (1970) who reported that mortality among fingerling
sockeye salmon exposed to this virus was reduced to a low level if the
fish were held at 68 F for 4 to 6 days after exposure (6). Work reported
from this laboratory has indicated that this virus replicates abundantly
in sockeye salmon cell cultures in the temperature range from 50 F to
68 F. Replication was retarded and virus yields were lower at 39 F, and
87
-------
no replication occurred at 73.4 F (4). Thus the indications from experi-
ments reported here that the temperature range from 54 to 59 is near
optimal for development of fatal infection due to this virus in fingerling
kokanee, is not out of line with other relevant data.
The experiments reported in Table 19 with fish of 0.95 gm average weight
do not show the marked reduction in percent mortality in 64 water that
was found in the experiments of Table 14 and 15. Presumably some unrec-
ognized variable factor may be responsible for this difference. It is
also of interest to note that only in the case of the very small fry,
averaging 0,11 gm in weight, was the percent mortality reduced to a
very low level (Table 17) by the low temperatures of 39° and 49°. This
might suggest that these tiny fish were more resistant to the virus in-
fection than larger fry from the same population (Table 19).
88
-------
SECTION IX
ACKNOWLEDGMENTS
This project was supported by the Environmental Protection Agency over
a three year period beginning April 1, 1969 and ending March 31, 1972.
The total funds provided by this agency amounted to $182,355. The
assistance provided by Dr. Donald A. Hilden and Dr. Gerald R. Bouck,
who have served as Project Officers, is acknowledged with sincere
thanks.
A major contribution to this project was made by the Fish Commission of
Oregon and the Oregon Game Commission. In fact, without the support of
these agencies none of the work described in this report would have been
possible. They provided the large numbers of juvenile salmon and trout
required for the experiments, without charge. The monetary value of these
fish has been roughly estimated at about $3000 per year for each of the
two years during which experimentation was carried on.
Another important contribution to the project was made by the Department
of Microbiology at Oregon State University. All of the experimentation
with sockeye salmon virus in finperling kokanee salmon was carried on
by Philip McAllister, a graduate research assistant in the above depart-
ment. During 2 years of work on this phase of the project, he was sup-
ported entirely by an NDEA fellowship, so that all of his work was really
donated to the project.
89
-------
-------
SECTION X
REFERENCES
1. Pacha, R. E., and Ordal, E. J., "Histopathology of Experimental
Columnaris Disease in Young Salmon." J. Comp. Path. 77, pp. 419-A23.
1967.
2. Sanders, J. E., Fryer, J. L., and Gould, R. W., "Occurrence of the
Myxosporidan Parasite Ceratomyxa Shasta, in Salmonid Fish from the
Columbia River Basin and Oregon Coastal Streams." A symposium on
Diseases' of Fishes and Shellfishes. Special Publication No. 5.
American Fisheries Society, Washington, D. C. 1970.
3. Sanders, J. E., Fryer, J. L., Leith, D. A., and Moore, K. D.,
"Control of the Infectious Protozoan Ceratomyxa Shasta by Treating
Hatchery Water Supplies." The Progressive Fish-Culturist. Vol 34,
No. 1. 1972.
4. Wingfield, W. H., Fryer, J. L., and Pilcher, K. S., "Properties of
the Sockeye Salmon Virus (Oregon Strain) (33719)." Proc. Soc. Exp.
Biol. Hed. 130. pp. 1055-1059. 1969.
5. McCain, B. B., Fryer, J. L., and Pilcher, K. S., "Antigenic Relation-
ship in a Group of Three Viruses of Salmonid Fish by Cross Neutrali-
zation (35724)." Proc. Soc. Exp. Biol. Med. 137, pp. 1042-1046. 1971.
91
-------
6. Amend, Donald F., "Control of Infectious Hematopoietic Necrosis
Virus Disease by Elevating the Water Temperature." J.Fish Res. Bd.
Canada, 27:265-270. 1970.
92
-------
SECTION XI
APPENDICES
Page No.
A. Analyses of Final Percent Mortality Data in Text Tables.
1. Text Table 1. Aeromonas Salmonicida in Coho Salmon. 95
2. Text Table 3. Aeromonas Salmonicida in Chinook Salmon. 96
3. Text Table 5. Aeromonas Liquefaciens in Steelhead 97
Trout.
4. Text Table 7. Chondrococcus Columnaris in Rainbow 98
Trout.
5. Text Table 8. Chondrococcus Colunnaris in Coho Salmon. 99
6. Text Table 9. ghondrococcus Columnaris in Chinook 100
Salmon.
7. Text Table 10. Ceratomyxa Shasta in Rainbow Trout. 101
8. Text Table 11. Ceratomyxa Shasta in Coho Salmon. 102
9. Text Table 14. Sockeye Salmon Virus in 1.1 Gram 103
Kokanee Salmon.
10. Text Table 15. Sockeye Salmon Virus in 2.9 Gram 104
Kokanee Salmon.
11. Text Table 17. Sockeye Salmon Virus in 0.11 Gram 105
Kokanee Salmon.
12. Text Table 19. Sockeye Salmon Virus in 0.95 Gram 106
Kokanee Salmon.
B. Chi Square Analyses of Percentages of Surviving Fish 107
Yielding Cultures of Aeromonas Liquefaciens. Text Table 6.
C. Regression Analyses. Relation Between Water Temperature
and Log of Number of Days to Death.
1. Text Fig. 1. Aeromonas Salmonicida in Coho Salmon. 108
2. Text Fig. 3. Aeromonas Salmonicida in Chinook Salmon. 109
3. Text Fig. 4. Chondrococcus Columnaris in Rainbow Trout. 110
93
-------
Page No.
4. Text Fig. 5. Chondrococcus Columnaris in Coho Salmon. Ill
5. Text Fig. 6. Chondrococcus Columnaris in Chinook 112
Salmon.
6. Text Fig. 7. Ceratomyxa Shasta in Rainbow Trout. 113
7. Text Fig. 8. Ceratomyxa Shasta in Coho Salmon. 114
94
-------
*ANQVfll2 - ONI/TWO FACTO? AMLY3IS OF VARIANCE.
ORfGON STATE UNIVERSITY COMPUTE? CENTi*
OS-3 VE3.3.5
DATC - iO/i(*/72
EXP 'TYPE
EXP TYPE X TF-1P
TOTftL
PROBLEM 1-0! 2NO TRY
OF SS. MS
___ J. i. 69637aJJ!E_ 0<« JL'JLlil?1?-3£ 0'» 426
7 6.228<«6d75fc. 03 8.99781250E 02 ~>2Cc0939
j!6 _ 7, 0850UOOOE Oj?_>_jL±L83 ?t»CCt_ _2i
31 «».59337186c 0<*
SOURCE MEftNS
EXP TYPt
59
TEMP
Cj)3
21
EXP rYPf; x
( C '/ p
(EXP
t>2
(CON
66
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.75000
( 7<4)
( 5 0 0 3 0
i •>«•)
4 00100
I 7'<)
.00030
t S1*)
Q
(CON )
11. 06250
(
52.
(
23.
(EXP ,
10 r,
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<«&
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<*
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70
-------
- CNc/TK'O FACTOR ANALYSIS OF VARIANCE,
OREGON ST41T UNIVERSITY COMF-UTER
SOURCE-
EX? T_yp£
TEMP
EXP IY°z X TEMP
ERROR
TOTAL
03-3 VER.3.5
DATE - 1C/1W72
1 #•»****
PRO81.El I-Oi 2ND TRY
OF SS MS
1 3_« 7*1-150 0 OF. _ 0<4 3,.731?50C OE J!_'*)
TOO
(CON )
2.25000
(
(
30.
(EXP ,
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60.
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2.
(CON ,
69)
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U9)
C
37
22
(EXP
72
(EXP
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2
(CON
fc
(
P
(
•
f
•
9
•
t
•
•f
•
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<»'*)
OOCOO
00
00
00
00
6«0
003
<*-o
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3
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(EXP
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68.
( C ON
(CON
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2.
00000
39)
00000
59)
•OOCQQ
39)
00000
59)
u
39)
OOCOO
cf Firuil Pcroert tbrtalitj DaU in Toxt Ttbls 5, A. Salregnieida
in SpriBg Cilacos 5i'l»on.
=:2} x= 2,12 tor- ?=0.05; i - 2.92 for P- 0,03: ^75-7.0356
slraU^cant, P\ffax-ease " U.92 pcr^t for P=0,C5
56
-------
*ANOVA12 - ONI/TWO FACTOR ANALYSIS OF VARIANCE, OS-3 V'EP.3.5
OREGON STATE UNIVERSITY COMPUTER CENTER DATE -10/14/72
SOURCE
_EXP JYPE
TEMP
EXP TYPE X TEMP
.ERROR
TOTAL
PROBLEM I-DI 2ND TRY
OF SS MS
1 1.050a8753E C4 i.050857£j£
7 7.7511<«7i9t- 03 1.107J!Q&7ttE 03
7 7.73682219E 03 1.105E«*6C3E 03
16 9.13965000E 02 5.74415625E 01
19.2771
19.246*
31 2.69179397E 0<«
SOURCE
MEANS
EXP TYPE
(EXP )
3G.U2500
TESP
(
40.
(
20.
EXP TYPE X
(EXP ,
81.
(EXP ,
40 .
(CON ,
(CON ,
74)
72500
^4)
00000
TEMP
74)
i»E09Q
54)
00000
74)
0
54)
0
(CON ) " " " " ""
(
32.
(
(EXP ,
64.
(EXP ,
(CON ,
(CON ,
69)
12500
49)
0
69)
25000
49)
0
69)
0
49)
0
(
33.
(
(EXP ,
67.
(EXP ,
(CON ,
. (CON ,
f '<)
57?D3
l»lj)
0
b'v)
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<*^5
B
6^}
J
t><-. J
0
zo
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38
(EXP
(CON
1
(CON
( 59)
.00(300
( 39)
_ _ . G-. ..
, 595
.55000
, 39)
Q
, 59)
.45COC
, 39)
0
£nalyeia of Fin*,l Percent Mortality Data In Text r-^lo 5,
In Stcelb&ad Trout.
=2j t=2.12 for P = 0,05$ t-2.92 for P~ 0.0i>
= 7.5790
Laset elgaif Sctrt differenc* = 16.07 f»rcent for r» ~ 0.05
» » • « » - 22,13 " * " « =0.01
97
-------
r « »»»»«.*(
*ANOVAi2 - ONE/TWO FACTOPx ANALYSIS OF VARIANCE.
OREGON STATE UNIVERSITY COMPUTER CENTER
OS-3 VCR.3.ti
DATE - 12/Oa/72
PROBLEM I-DJ GP3-PT1 __ _
SOURCE
EXP TYPE
TEMP .
EXP TYPE X TEMP
ERROR
TOTAL
SOURCE MEANS
EKP TYPE
(EXP >
56.61106
TEMP
( Si
6 3 o 0 0 3 8 3
( *>)
7.0Q300
EXP TYPE X TE*e
(EXP , 8)
IOO.CQ30Q
(EXP , s)
8» OGOOO
(COK . S»
26.000QG
(CON , <*»
6.003CQ
OF SS MS . _F...
1 1.5366890SE 0<« 1.5368S905E 0«» 26o,3<»35
5 1.33285267E 0<» 2.6657C53^E 03 «*9.1*»5«»^
5 9.35E19Q69E 03 i.871C361
31.00C30
-------
<'ANOVAi2 - ONE/TWO FACTOR AMLYSIS OF VARIANCE.
OREGON STATE UNIVERSITY COMPUTER CENTER
GS-3 YER.3.5
CA IE - 32/05/72
#* **«.«««*** 9
PROBLEM I-DI GP2-PT1
SOURCE OF SS
EXP TYPE 1 l.'«57S2935E 0<» 1-
TEMP 7 1.729627i3E 0<» 2.
EXP TYPE x TEMF 7 i,61261572E 0<» 2.
ERROR 16 1.<»2<«26165E 02 8.
TOTAL 31 <*.81<»51<«eiE 0<»
SOURCC MEANS
CXP TYPE
(F.XP ) (CON )
<»l», 29531 1.C0706
TEMP
( S) (7)
50.7m25 , 50.71«»25 51
< *.) (3)
2.65700 0
EXP TYPE X TE*F
(EXP , 9) (EXP , 7) (EXP
100.00000 100.00000 96
(EXP , <•) (EXP , 3) (EXP
(CON , 3) (CON
1.J.2550 0
KS
^702935E
170S9^E
30
-------
OANOVA12 - ONE/TWO FACTOR ANALYSIS OF VARIANCE. OS-3 VER.3.5
OREGON STATE UNIVERSITY COMPUTER CENTER DATE - 12/05/72
PRODLEH t-Oi GP«*-PTi
SOURCE OF SS ... .MS _F
EXP TYPE i ... 8.j_2S33656E OS 8.2293865SE 03 ?JLlil!lf».i_
TEKP T 7.2227i2'»i£ 03 1.031816C6E 03 25.3201
EXP T¥PE X TEMP 7 6.8596299iE 03 1.26566H«iE 03. 31,0586
ERROR 16 6.520129^E 02 |»_.J3750§J^JE_Qi
TOTAL 3i 2.«»9637^18E. 0*>
S_0 URCZ •
EKP TYPE
(EXP )
( 6)
liG.OOOGC
( <«)
13.COOOO
FXP T V PE X
( 7)
36.00000
( 3>
3.000GO
« 6)
29.00000
( 2}
9,00000
C S)
*>-I:3i75
« 1)
2.COGOO
, o
92.CCOOO
(EXP , »i)
20.0000C
(CON , 8)
, 7»
70.00000
(EXP , 3)
6.00000
(COM » 7)
(CON ,
-------
'*****¥»»****«*«•*»»»»
'ANOVA12 - ONE/TWO FACTOR ANALYSIS OF VARIANCE. OS-3 VER.3,5
OREGON STATE UNIVERSITY COMPUTER CENTER OA~TE - 12/05/72
.EM^I-OjL.^l-FT^
SOURCE^ OF SS MS F_
£KP._J_VP£ 1 2.77G9S276E Ofr 2.7709587;? 0** 323.5rn-?
TEMP 6 5.28712083E 03 8. S118&Sf. •£ 02 10.2892
EXP TYPE X TEHF & 5.28712083E_03 .8.8il66COfe£ 02 _ 1C.2692_
_ERROR t3*OOGOG 37^.50000
I 3) -- f 2j ( tj
«)C,COaOO 37.70825 C
EXP TYPE
(EXP , S)
52.00000
(EXP , 3)
so.ooroo
(COK , e»
p_
(CON , " ?t
C
, 7)
7;;.CGOOO
CEXP , 5J
6 6. C 010 C
~"
(CO-; , ?»
0
(EXP , i»)
75.00000
(CON
Tec":*"
. of Dfttc. Ik 'iia- "iblc 3^5 for Percent of Rr-lnbow Trout lafectod
C.
r=
t =2,H for r --0.05} t — 2. $3 for P-= C.01; /
difference — i9.SO p«roant Tor P = 0.05
« • - 27, 58 ° " e f - O.C1
131
-------
'ANOY&12 - ONE/TWO FACTOR ANALYSIS OF VARIANCE.
OREGON STgTE UNIVERSITY COMPUTER CENTER
CS-3 VER.3.5
OATE - 12/05/7?
PROBLEM
SOURCE OF
EXP TYPE 1 <»
TEMP .79
EXP TYPE X TE«P 7 . . k
ERROR 8 3
TOTAL 23. 1
SOURCE HEANS
EXP TYPE
(COM )
0 , ^ 33
TEMP
( 6)
U. 00*00 96
(
0
END OF *ANOV612 EXECUTION.
of D&t& la Text Table 11 for Percent of Cclio S,.",IBOE Infcctsd
with £. Shasta
= 2} t— 2.31 for P - 0.05; t - 3.36 for P-0.01| J3/2 x 42.54= 7.9802
significant dlfforoaco = 18.Z,? Mrecnt for P — C.05
» " " • r - 26.81 * • " ¥= 0.01
102
-------
«ANGVA12 - CNE/THO FACTOR ANALYSIS OF VARIANCE.
OREGON STATE UNIVERSITY COMPUTER CENTER
OS-3 VER.3.5
DATE - 12/05/72
PROBLEM
SOURCE
EXP TYPE
TEMP
EXP 1YPE X TE^P
ERROR
TOTAL
OF
1
2
2
6
11
1.
1.
1.
1.
2.
I-OI GP^-PTl
ss
7250M50E
797075<»6E
797075«;6E
60561279E
10051272E
0<4
03
03
02
Ok
»s ..
1.7250«fJ.50E
8.98537722E
8.98537732F
2.67602132E
0<»
02
02
01
F
G^<».6292
33.5774
33.577U
SqURCE^_
EXP TYPE
TEMP
SEANS
(EX" )•
75.82967
j 61
2C.83325
EXP TYPE X TEMP
(EXP , 6)
<•!. 66650
-------
'»»*«»*»»«»»»»*•»*»»»
»ANOVA12 - ONE/TWO FftCTOO ANALYSIS OF VARIANCE. OS-3 VER.3,,5
ORF.GON STfiTf UNIVERSITY COMPUTER CENTER 01TE - 12/13/72
*»»
SOURCE
EXP TYPE
TEMP
EXP TY»E X
ERROR
TOTAL
*•*»»«»*»(
PROBLEM I-OI GP2-PT1
OF SS US
1 «*,m««03260fc. 03 <*.li»i«C32fcC£ 03
«» 1.19<«&S6i»7E 03 2.9866M17E 02
i* 2,!79259i«2£ 03 5. 9*»eH»e5fcF 02
10 5.13371758E 02 5.1837176'E 01
19 8.23632026E 03
79.9^33
5.7616
11.1
(EXP )
31.6297Q
J)
SOURCE
EXP TYPE
TEMP
( d)
31.31625
EXP TYPE X T£1F
(EXF , ?)
15.75000
(EXP , <*)
61.38250
(CON , 9)
(CON
MEANS
(CON )
<«. 83970
( 7)
12,72»»25
(tXP , 7)
22.88<»50
(CON , 7)
2.S6<«00
( 6)
(EXP , 6)
16.9*250
(CON , 6)
D
( 5)
23,09200
(EXP ,
(CON
5)
5)
0
Aa&lysls of Final Perteit Mortality Data in Ttr*. Table 15. So«ltey» SaLtoH
Virus iu 2.9 oraa Kok&ne« 3,-.Is.o».
r- 2j t --2.23 for P = 0.05j t ^3.17 for P-0.01; /5l783 -7.1998
I^east
«
diff«rea»e — 16.06 pereoat for P --&.05
" • " 22.82 • " " P— 0.01
104
-------
• »»»»»*»*»«•»»**#»»••»*»**»<
*ANOVfcl2 - ONE/TWO FACTOR ANALYSIS OF VARIANCE. OS-3 VER.3.5
OREGON STATE UNTV£
1.61300
(CON )
11.60530
X" ?) "" 1 " "<.T"
51.66650 <»9. 16675
(EXP ;""5) (EXP', i»Y"
9G. 66650 98.3335C
(CON , 5) (CON , <«)
6.66650 Q
""( 3)
2.50000
(EXP , 3)
3.33350
(CON , 3)
1.66650
is of ?ieal Ptrcest Mortality Data 1m Text ?*l»jf. 17. Socke/e Salwon
Virus in O.H Graa Kokane*
-- 5.0797
r z: 2j t-: 2,23 for P -0.05; t = 3.17 for F = 0.01;
11.33
16,10
for P-- 0.05
• P - C,01
J.05
-------
*»»*»*»*»»#»*»**»»»*»»»»-V»*V»*»****»»*»*»»i
*ANOYA1.2 - ONE/TWO FACTOR ANALYSIS OF VARIANCE.
OREGON STATE IWIVESSITY CO^fUTt'R CENTER
i tffff.
1 * * » »*»*»¥ *
OS-3 VER.3.5
DATE - 12/05/72
SOURCE
EXP TYPE
TEMP
EXP TYPE X
ERROR
TOTAL
PROGLF.1 I-Ot GP3-PT1
OF SS MS F
1 3* 8333120.lt. 0«» 3.883312CJE 0<» <*75
5 2.e718556'*E 03 5.3<»37il26t 02 61.0710
10 8,7i»999991E 01 tt.7i»999961E 00.
21 <*.21808053L 0<«
SOURCE «£ANS
EXP TYPE
(EX° ) (CON )
86.73791 <».7&073
TEMP
( 7)
57.01750
( 3)
••8.75000
EXP TYPE X TE1F
vt )
%7. 50000
(EXP , i»>
95.00000
(CON , U)
0
Analysis of Final Pereent Mortality Data in Text Table is, Sockaye Salaoa
¥ima ia 0.95 Or** KoJotase Saluoa,
r jr 2; t -2.2? for P r 0.05; t - 3.17 for F .- O.C-i; ;2 s 8.749 - 4.1833.
Least *i£»ifio*nt. ciiffaroR«»« 9,33 per*e»t \oi- r _ 0.05
* * e " " 13.?6 " " " P:rO,Oi
106
-------
2
X Analysis of Percentages of Surviving Fish Yielding
Cultures of t±. liquefaciens. Text Table 6.
A. Comparison of Percent Positive at 69°F (28%) and 59°F (71%)
Water
Temp.
69°
59°
Total
2
.X
Number
Positive
7
__3
10
- (17.5 x
Number
Negative
18
40
58
2.5 - 40.5 x 7.5)2 x 68
Total
25
43
68
(Yates Modification) 25 x 43 x 10 x 58
- 7.38
2
From x Table for n = 1 „
p = less than 0.01 for this value of x
Hence difference is highly significant.
B. Comparison of Percent Positive at 64°F (17.4%) and 54°F (2.4%)
Water Number Number
Temp. Positive Negative Total
69° 4 19 23
54° !_ 41 4£
Total 5 60 65
X2 " (18.5 x 0.5 - 4.5 x 41.5)2 x 65
(Yates modification) 23 x 42 x 60 x 5
- 7.09
2
From x Table for n = 1
p = less than 0.01 for this value of x
Hence difference is highly significant.
107
-------
REGRESSION ANALXSLS. AERCilCr.'AS S^LMCNTC.i;V
IN CGHO SALMON. TEXT FIG. 1
.3
,3
4
t
•
•
*
a"
ET
B."
2
2
i
1
i
7
7
7
i
5
5
G
•t
1
2
7
*
i
1
2
3
* <>
* * *
TEMPERATURE —— ——->
.I EX IT
RELATION BKl'WfiEH WATZS TEbPERlTORS AMD 100 I'O. OF I^IS TO
DEATH. A. SAIKGKIC1BA INACTION IN COilO SAL!:. ,
Y( 2)- H.^629315?: C!- . +._-2.7651; 3^£-0<: X(
' Y = LOG EO. DAIS TO D2ATH ( D--'.- l-PEP-Vi'UT,!*.
R SQUARFO = .. 7.331'J516 = %. OF VARIATIOK : ^OUKED 1'OR
VAR S.E. OF REGR. ?GEF»
C 6.00 !ie7282C -02
CORREI.ATICN COEFFICIENT = -C.«8i9775 ~ Lir: . F3IATTOHGH1P
AID JOG KO. Or DAIS
TO DEATti
103
-------
REGRESSION ANALYSIS. ATJICMDIIAS
IN CKIKOOX SALMON. TEXT FIG. 3
l.z
ti
,5
.
.<*
.1
13
^ c
<3« ?
M
H«
/>• 3
H J>
•*.!
Z4
3»
2*
?
•
2
3
2 1
7 3
? ^
i 2
1 2
1
1 t»
' • v 2
i
1
1
. . .1 ,
3
1
5
«
7
5
• - ' - • - •
TVKrnpD l f TTT'T?
«
1
*+ 1
1
1
1
1 2
521
7122
* 8 f \*~1T1 f fl' ** '^l1*'^
' 1 f.., I M J t 1 •«*">" fI K ' I ' f i
/.r: LOG /:o. OF DAYS
TO DEATH
109
-------
REGRESSION ANALYSIS. CHOj'IDRCCOCCJS 001 'imi
IN RAINDOW TROUT. TEXT FIG. 4
i
*
k 5 ' "1
2 9 ._._!
1 * *
*
TE^ERATURS
RELATION E3TVSZN WATSR 'lETERATUPJS A:,T) LOG IT, OF DAYS TO
DSATHc c. COLUI-:JARIS liiECTios IK jaiiizc' lacur.
Y( 2)= 3.28061«*6E 00 + -<*,<»9 .-.3^2E-02 X( i
Yr LOG 110. DAYS TO DZ1VTH ( .) =: TEMPERATURE
R SQUARED = .73^8 935«f = _jj OF VARIATi: 7 .ACCOUNTED FOR,
WITH JJ3.
VAR~""""siE. "OF REGRCOEF. " "" T
C l.'t 216916 fbl-OJ 2. 30? ^'--5. ISt 01
i 2.C96-7151?£«03 -2, l^Sii/iflE 01
CORREtATICN CCEFFIClLMT = -0.8572^93^ 1-".::AR RSL'JIONSHIF
r/ii^SN 'i7.MPLRATJJR3
A.D LOG ::0. OF DAYS
"' "" " "~ " TO DEATH
-------
REGRESSION ANALYSIS. CKCIIDROCQCCUo I SLU1-JNARIS
IK COHQ SALMON. TSXT FIG. 5
4'1 - - - t
A 1
.12
1 - 1
^ _
3
gi ' .
g. 7 7
» 7 * C1
3
* *
.
« 2 ''
_tEXIT_ ._ _ . . .„_ ;
RELATION ESTkSEN WATER KIPERATL'IiS AND LOG 17. OF DAIS TO
. . DEATH.. C. COLUMHAR13 IWPECTTOH IN C£;l 3ALMGN
Y( 2)= 2.62703i»3E 03 + -3.353. 30E-0? X( i)
I-- LOG KO. DAK TO DEATH ( 1) r rEKPERATUHS
R. SQUARED =. .59236395 - % OF VARIATI: : ACCOUNTED FOIi
\'l " LIT®.
"VAR S"VET"~OF~ REGR7" COEF^ ^"^"" ~i -—-— '
0 1.18372935E-01 2. 21?: : ';,3 GE 21
1 1.753«i7-'«05E-03 -1.911 " ?2«*E 01 "•"
CGRRCLATICN CCEFFICIcNT = -0.7699796 ^ J .'.THAH RPILVflONSHIP
- --. .... _ . i n LOG KO. cy u
TO DEATH
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REGKESSJOH ANALYSIS, CIIOIIDRGCOCCUS COI,Ul":i/iRIS
115 CHINOOiC SALMON. TEXT FIG. 6
1 1 i i
ill
1 1
1 • i
.1
£-<
5S»
i
i
2
,2
^2
.1
i
1
1
1
p,
&•
125
6
1
7
*
7
1
*
*
J.fiii'lS'JUUiJ.UWi ---~--«~«——<~———^,»
IEXIT
ITIOM 'E2TW3EH WATER TE13TJIATOKS ?,I3) LOG ' •"." OF DAYS TO
DSATH. C. COUKlilARlS IHEECTIOII IN
Y( 2)= 2.755S2'f3E OC + -3.222 j7 86E-Q 2 X( 1)
1= LOG KO. HAYS TO DEATH ( l) : TEMPERATURE
R SQUARED = .51730671- % OF VARIAT' .s ACCOUNTED FOR
1; 'Til
VAR "StE. OF REGR. COEF "."" "" T .
0 l,76ia8738E-Gl 1.562'« v09E 01
7E-03 -1.220^ :^60E 01
CORRELATION COEFFICIENT ~ -0.7102^0^= - ' "^ RSUTIO;«Sni?
I T^SEK Ti^PERATURS
;•.' ) 1X30 150. OF DAYS
""" "~ "" " TO DEATH
112
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REGRESSION ANALYSIS. CERATOMYXA SJLXSTA IN
RAiliBOW TROUT. TEXT F1GC '/
*:
.
.1
*
.
i
3
851
^J . * 1 ..
B * «* " ~ "
S 1 *
3 " j
ET» LOj i;0. Or DAYS
TO DSATH
-------
A
• 1
REGRESSION V; A LYSIS. C?il/.TC>ffia SI^GTA IK
co;:o PAI^ON. TTIT FIG, 8
CH
«<+
o-
&
i
i
j_
i
TEMPERATURE ~
n J^TVBSL'K I'ATT^l lEli'Etif.iUR-*. AK'.* WO NO, OF I'AYS TO
HSATH. 0. SilASTA 31KCT1GH 111 CCHO S.M: ::;
Y( ?)- ^.O
. Y = IOQ NO,
H 09
TC KATH
(I) :: TQ-i
2 X(
S SQUARCO =
=. % OP
ACCOIT:IT£D FOR
LUS.
VAR S.t. OF ^EGR. COE
0 7.««270H3fc5t---Q2
i " 1.105ZJ8P93E-03
.E 01
:E Qi
CORRELATION. CC£F^ ICI-IN f - -C, 95/4965 = },...;,
KO. OF
TO DSATH
ftU.S GOVERNMEMT PRINTING OFFICE 1974 546-317/313 1-3
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SELECTED WATER
RESOURCES ABSTRACTS
INPUT TRANSACTION FORM
He;- •>' No
w
Effects of Temperature On Diseases Of Salmonid Fishes
J. L. FRYER AND K. S. PILCHER
Oregon State University
Department of Microbiology
Office of Research and Monitoring
Environmental Protection Agency
.-*tD
July, 1972
O/gsr :ttwn
18050 DIJ
13. Type-- Repot ~ntt Final
Period Ctvertii Report
4/1/69 - 3/31/72
Environmental Protection Agency, report number, EPA-660/3-73-020,
January 1974.
In all cases mortality rates
to 59°F; and low or zero at 39° to 49°F.
The effect of water temperature on infections of salmonid fish was investigated.
Chondrococcus columnaris infection was studied in rainbow trout, coho and spring
chinook salmon; Aeromonas salmonicida infection in coho and spring chinook salmon; and
Aeromonas liquefaciens infection in steelhead trout.
were high at 64 to 69 F; moderate at 54
Progress of the infections was accelerated at higher temperatures, and progressively
retarded at decreasing temperature levels. In infection of coho with Ceratomyxa
shasta, mortality was high at 69°F, low at 49° to 54°, and zero at 39 to 44 F. This
infection in rainbow trout resulted in high mortality at all temperatures except 39 .
In both cases the course of the disease was most rapid at higher temperatures, and
became progressively slower as the temperature decreased. For infection of kokanee
salmon fingerlings with sockeye salmon virus, the temperature range of 54 to 59 F was
optimal. In this range mortality rates were high, and the course of the disease was
most rapid. At higher temperatures mortality rates were lower, and at 39 to 44 F,
progress of the disease was retarded, though total mortality was often high.
'-'• • Animal diseases, effluents, fish diseases, heated water, infection,
microorganisms, pathogenic bacteria, pathology, thermal pollution, water pollution,
water quality, water temperature
19. S"-
(si
2t'!. SVciir;-. 7 C'ast.
21. A',, of
Page's
22, Price
Send To:
WATER RESOURCES SCIENTIFIC INFORMATION CENTER
US DEPARTMENT OF THE INTERIOR
WASHINGTON, D C 2O24O
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