Discussion

Fish infected with C. 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 C. 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.

Chondrococcus 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 may be a more accurate indication of infection.

No deaths caused by C. columnaris were found in fish held at 49°F or below.

52


TIME TO OEATH AFTER EXPOSURE fdcys)

100

1 4. L. n 1 L 4
39 44 49 54 59 64 69 74

WATER TEMPERATURE (°F)

Fig. 1. Relationship between water temperatur
to death rer dafection of juvenile coh:
Aeromonas salmonicid

log of time
salmon with



RESEARCH REPORTING SERIES

Research reports of the Office of Research and
Monitoring, Environmental Protection Agency, have
been grouped into five series. These five broad
categories were established to facilitate further
development and application of environmental
technology. Elimination of traditional grouping
was consciously planned to foster technology
transfer and a maximum interface in related
fields. The five series are:

1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research

4. Environmental Monitoring

5. Socioeconomic Environmental Studies

This report has been assigned to the ECOLOGICAL
RESEARCH series. This series describes research
on the effects of pollution on humans, plant and
animal species, and materials. Problems are
assessed for their long- and short-term
influences. Investigations include formaticn,
transport, and pathway studies to determine the
fate of pollutants and their effects. This work
provides the technical basis for setting standards
to minimize undesirable changes in living
organisms in the aquatic, terrestrial and
atmospheric environments.



REGRESSION ANALYSIS. CERATOMYXA SHASTA IN

CH TROUT, TEXT FIG. 7

.
*

a
wk
te
1

of.

i
|
1

Renter ee

to

LOG NO. OF DAYS JO, DEATH, TF

oe eee eee ee eee ee ee ey

_TBMPERATURR

SEXIY
RELATION ESTWEON WATOR TEMPERATURES AND LOG | 3
DEATH, OC, SHASTA INFECTION TN RAIN.

OF DAYS TO
TROUT

YC 2)= 3,63973206 69 # 93.4175 4GEWG2 XE 1)
Y= LOG NO. DAYS TO DEATH (1) °: TEMPERATUSS

“ Q”SQUARED = 496647996 = F OF Vane :

VAR S.&. OF REGR, COEFe y

0 265126879

i 443395505
CORRELAT ICA COEFFICIENT



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 C. 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 C. shasta. Fish re-

maining at termination were examined for the presence of C. shasta spores.

61


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 fingerlings had an average
weight of 1.1 gm. Incubator facilities for only 4 temperatures were

° 54° and 39°F, For each tem-

available and those selected were 74°, 64
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


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


Discussion

It is always 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 C. shasta infection at colder water ten-
peratures. However, Keith Johnson working in this laboratory, has ini-
tiated an infection of C. 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 C, shasta, is inversely related to temperature.

67


TIME TO DEATH AFTER EXPOSURE (days)

i900,

TTT]

T

1 1 —_-L 1 1 1 4 a
39 44 49 54 59 64 69 74

WATER TEMPERATURE (°F)

Fig. 3. Kelationship between water temperature and log of time
to death after infection of juvenile chinook salwon
with Aecomo;



*ANOVAL2 ~ ONE/TWO FACTOR ANALYSIS OF VARIANCE. OS-3 VER.365
OREGON STATE UNIVERSITY COMFUTER CENTER DATE = 10/14/72

PERRET RE REE UTTAR EAE ERE E EMER EERE EERE EEEER EEE ERDE CON EEE EE ERE ERE RE

PROBLEM I-Dt 2ND TRY

SOURCE DF ss. . MS oo. oF
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TEMP 7 7e751147419_ 03 1.40730674E 03 1962774
EXP TYPE x TEMP 7 %.73682219E 03 16405545032 03 19.2464 _
ERROR o1

TOTAL . 34 2,69179I97E 04

MEANS _
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40,0000 | _ 3 8
(CON , 74) (CON y 69) (CON 4 64) (CON, 59?
0 epee wre vee Be MOOD
TOT ECON SG) ECON SD ~UCON 9 8} (CON, 39)
0 0 a 7 9

Analysis of Finel Fercent Mortality Data in Text

na 5, f, ldgusfecicns
in Steeibead Troui.

r=2; ¢=2.12 for P=C,05; t=2.92 for P= 0.03; SEAL = T8790

laget etgnificert difference = 16.07 percent for ° = 0,05
. e id al = = 22.13 ° * “ 256,01

7


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32


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

50
the fish incorporated in Oregon Moist Pellet (OMP) diet. Terramycin was
chosen also because of its lack of activity against C. shasta. In each

experiment all fish including controls were fed TM., at a level of 25 g

50
TM,,/100 1b of fish/day starting ten days prior to exposure. A level of
20g TM,,/100 lb 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 ¢ TM,9/100 lb 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

37


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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60


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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 Aeromonas
hydrophila-liquefaciens complex, plates should he incubated at 37°c
for 24 hours. Under these conditions colonies of these organisms are
0.3 to 0.5 mm in diameter and bright vellow 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 yellow 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 V.

io and Pleisomonas species do not grow at 37°C, and thus do not

38


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81


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 colunnaris 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 columnaris.

9. The percentage of fatal infections in rainbow trout infected with

Ceratonyxa shasta is high at water temperatures between 74° and 44°F,

10. The mean time to death of rainbow trout infected with C. shasta is
approximately 14 days at 74°R, increasing to approximately 155 days
at 44°r, 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


ABSTRACT

The effect of water temperature on infections of salmonid fish was

investigated. Chondrococcus columnaris infection was studied in rain-

bow trout, coho and spring chinook salmon; Aeromonas salmonicida in-
fection in coho and spring chinook salmon; and Aeromonas liquefaciens
infection in steelhead trout. In all cases mortality rates were high

1 3
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
oO . .
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°r, progress

of the disease was retarded, though total mortality was often high.

This report was submitted in fulfillment of Project Number 18050 DIJ,

under the sponsorship of the Office of Research and Monitoring, En-

vironmental Protection Agency.

iii


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 A. 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°r, 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


TIME TO DEATH AFTER EXPOSURE (ccys)

1000

TT

100r-
10 = . n 1 4 1 J
39 44 49 54 59 84 69 74
WATER TEMPERATURE (°F}
Fig. 8. Relationshop between water temperature and log of time

to death after exposure of juverile cono salmon to
Ceratomyxa shasta.

66


unless some other factor, such as failure of aeration, was an obvious

cause.

Results of these two experiments are shown in Table 14, The experimental
groups held at 74°r were lost due to failure in the aeration apparatus.
The percent mortality approached 100% among the fish at 39°, and was
nearly 90% at 54°F, However at 64° it was only 41.5%, a definitely
significant reduction (Appendix, page 103). This result suggested that
the higher temperature had exerted a suppressive effect on the infection
in some of the animals. Progress of the infection was apparently slower
at 39°, as indicated by the longer average interval between exposure

and death.

When a second pair of experiments was started, the available kokanee
fingerlings were considerably larger, averaging 2.9 gm in weight. Because
of their size it seemed advisable to expose them to a higher virus
concentration than was used in the first experiments. Accordingly suf-
ficient stock virus was added to the water to give a final concentration
of 600 pfu/ml, and the exposure period was 24 hours. Equipment to pro-
vide 5 water temperatures was available at this point, and temperatures

° °

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, and two

complete experiments were carried out in parallel, as before.

The results of these two experiments are presented in Table 15, In this

76


EPA-660/3-73-020

January 1974 Ecological Research Series

Effects of Temperature on

Diseases of Salmonid Fishes

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Office of Research and Development

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Washington, D.C. 20460


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ERROR 8 3439642933E 02 4.24553657E 04
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ENO OF *ANOVA12 EXECUTION.

Analysis of Data in Text Tablell for Percent of Coto Sxlzon Infected
with &. Shasts

r= 2; t= 2.31 for P = 0.05; t = 3.36 for P= 0.01; BR X 42.54= 7.9802

Leant significant differoney = 16.42 nercent for P = ¢.05
* . . "= 8 = 26,81 " 8 " P= 9,0)

102


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


BERR ETERS REM ERNE SEEDER ELE TV RARME RSE OEE SERED ERES SEEDS EESEE ERE EERE ED
SANGVAL2 = CNE/THO FACTOR ANALYSIS OF VARIANCE. OS-3 VER3.5
OREGON STATE UNIVERSITY COMPUTER CENTER DATE = 12/05/72

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Virus in 1.1 Gram Kokance Sa

<2; t =2.45 for P= 0.05; t =3.71 for P = 0.01;

7

Larst significcnt difference 12.67 peroent for P=
. . " « . 19.19 " & a

103


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 salmonicida 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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78


SECTION III

INTRODUCTICN

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 liguefaciens, 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-


control groups, only a few deaths occurred at the higher temperatures,

and C. 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




SECTION VIIL

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. lience, 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 water 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


sk

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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 salmons (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 salmonid species being tested.
Several isolates from the final fish passage were collected and pooled

for use in the temperature experiment.

41


ROCCOCUS | NLUMNEAR:
T FIG. 5

REGRESSION AWALYSIS, Ct
IN CORO SALMON.

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RELATION ENTAZEN WATER TOIPERATURS AND LOG “>
DEATH.. C. COLUMNARIS INFECTION IN CC.

OF DAYS 10
ADHOR

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Y= LOG NO, DAYS TO DEATH (.1)= TEM

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we ee ee ee eee . o£ LOG KO, CF bays
TO PRATH

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46


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 mu 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 were made from the gills and or kidney of each fish.

42


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 Table 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


percent mortality, as measured by the combined data of the two experi-
ments was very low among the fish held at 39° and 49°R, 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
experinental 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 were 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 9.11 gm

82


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 growth 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 lo "“ "
Glycogen 4 " "
NaCl 5" "
Sodium lauryl sulfate 01" "
Brom thymol blue _ 01" "
Agar 15 " "

final pl] 6.9-7.1

36


Mad Analysis of Percentages of Surviving Fish Yielding

Cultures of A. liquefaciens. Text Table 6.

A. Comparison of Percent Positive at 69°F (28%) and 59°F (71%)

Water Number Number

Temp. Positive Negative Total
69° 7 18 25
59° 3 40 43

Total 10 58 68
2

= (17.5 x 2.5 - 40.5 x 7.5)2 x 68
yates Modification) 25 x 43 x 10 x 58

= 7,38

From 2 Table for n=1
p = less than 0.01 for this value of 2

Hence difference is highly significant.

B. Comparison of Percent Positive at 64°F (17.4%) and 54°R (2.4%)

Water Number Number

Temp. Positive Negative Total
69° 4 19 23
34° 2 43 a

Total 5 60 65
od

= (18.5 x 0.5 - 4.5 x 41.5)” x 65
‘yates modification) 23 x 42 x 60x 5

= 7,09

From 2 Table for n=1 2
p = less than 0,01 for this value of x

Hence difference is highly significant.

107


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 tho Superintendent of Documents, U.S. Government Printing Office, Washington, D.C. 20402 - Price $1.55


PORES REL STEER TENE SEES EERED DORMER SERED EREEEE EERE DED EEAERE RENEE EERE RS

VANGVAL2 <« ONESTNO FACTOR ANALYSIS CF VARIANSEs
OREGON STATE UNIVERSITY COMPUTER CENTER

DATE

OSs-

3 VERLS.5
~ 12705772

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r=2; t=2,12 for P =~ 0.95; t = 2.92 for F

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ERROR 16 6eoS20L2944E 02 4.97508590F OF _
TOTAL _ BL 2449637418 04 So
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Experimental Phase

In these experiments with C. 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 C. shasta infection.
The number of fish infected with C. 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 C. shasta. It cah be seen, however,
from all other experimental groups in the 74°F to 44°P range that tem-
perature has little or no effect on the percent mortality due to C.
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°R, six

percent succumbed to C. shasta infections.

59


TT

TIME TO DEATH AFTER EXPOSURE (days) -
TT 1

A J. U a —

43 54 59 64 Ped 74
WATER TEMPERATURE (°F)

Ou

Fig. 4, Relatiouship between water temperate
to death after ex eof juvenile
Chondresoccus col is.

and log of time
ow trout to



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ERROR | vn 679085000008 02 EI 2500
TOTAL BL 4459337186 04
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Q 0 t 0

Analysin of Final Percont Mortality Date in Text Table ?. A. Salnoaicida
in Coko Salwon.
P= 2) v= 2.22 for P = 0.05; t = 2.92 for P= 0,01; “EE = 4.6546

Least elgnifieant é1fisrunee 14,11 porcont for P= 0.45
ere) 2% 19.43 7 § © Pagel

95


peratures used in the fish experiment.

A flask containing 200 ml of peptone-beef extract-glucose broth was
inoculated with 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 m at various
intervals during an 80 hour incubation period. The growth rates observed

are shown in Fig. 2.

A. salmonicida 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 A. salmonicida
in vitro also resulted in the shortest mean time to death among infected
fish, while low temperatures resulting in very slow growth rates in
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


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17


SORE E RETESET ERUE REED EEEREUETEREEED EE ER EEEERAEDORRERTURTET OEEEEREEEE

SANOVAL2 = ONE/THO FACTOR ANALYSIS OF VARIANCES os:

OREGON STATE UNIVERST

TY COMFUTER CENTER

GATE

3 VER. .S
- 32705/72

PEER EEER REET EOE D UE ER EEEEED ERED ES EERO SUERTE OD EOE SESE TAGE EERE SR ELS

PROBLEM I-D8 GP2-PTL

SOURCE OF ss 4S F
EXP TYPE 1__Le45762935E O4 1 :4E7H2S35E 04 163767905 —-
TEMP 7 1672962713E 04 2.4708958SE 03 277.5777
EXP TYPE xX TENF 7 1¢61261572E 04 2,30402245E 03 25668314
ERROR __ 16 1.42426165E 02 8.90162529E 00
TOTAL 31 G4. BL451481E 04
__. Source HE ANS é _
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(EXP ) (CON )
44029534 1.€0706 .
yeme _ _ weep ee
C §) t ry t 6) { 2)
56.71425 ‘ 50.71425 $1.46706 27214275
« 4) ¢ 3 (2) (4
2.65700 4 0 071625
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8 (EXP > 7 (EXP 6) CEXP oy 5)
100,00000 £00,60000 98 .64855 51.42859
(EXP y 4) (EXP 5 3) (EXP , 2) (EXP 5 i)
4.28550 Q a 0
COON > 38? (CON 4 7 (CON 4 6: (CON 4 5)
16424950 £42850 4 o28556 _. 2485700 .
{CON 5 a) (CON » x (CON » 2s (CON » 4)
1.42850 0 a 2242850

Rnalysis of Final Pereont Mortality Data im Text Tabls °. C, Coluenaria

ro 2; = 2,12 tor P= 0,05; t= 2.92 for P= 0.01. jE

Least elgnificant di
. * *

fferance
. 8

in Coko Salnca.

€.71 * *

$9

6.33 parcent for P= 0.55

19 = 2.9836


Effect of Temperature on Infection with Aeromonas 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. Groups of fish to be infected received

7
an intramuscular injection of 0.5 LD 0 doses (about 2,2 x 10° organisms)

5
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
same manner. Ten control fish from each of the following temperature
groups were also examined by kidney culture at the end of the experi-

©, 59°, and 54°r.

ment: 74°, 69°, 64
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


REGRESSION ANALYSIS, AUROMONAS SALMOUTCIDA
IN CEINOOX SALMON, TEXT FIG. 3

Ae2 2

2.6

o&

5

oo

a4

ot

E

5 a 3 1

ane i 2 1 1

a 1 ee ee
ed 4 3 * 5 2 4

r.2 4 4 7. 7 2 2.2
Aad i 2 5 * 8 4 1
fx

Os ~. v7

9g * *

2

Ro ee
g ¥ *
: ne Lo. oo

TEMPERATURS ———~—————~

J EXIT, . pee wee eee
RELATION BE i TMPERATUSE AND LOG}, oF DAYS “76

DEAVH. A, SALMONICIDA INFECTION IY C

2 ¥C 205 2,12158165 93 + s 39 1)
Y= LOG NO. DAYS TO DwATH (1) +) TEMPERATURE

167720766 = % OF VARIATION
- WIT:

R SQUARE

COUNTED FOR _
LIS

“WAR ” S.E. OF REG2. COEF.
0 5B. SSSGG44SE-02 3.799
4 9.442294225-04 - 2422

CORRELATICN CCEFFICIENT = ~0.822S5083=

G1, OF DAYS
TO DSATH

16s


TIME TO CEATH AFTER EXPOSURE (days)

100,

TTT)

T

TTT TT

a

4. i _
39 44 49 54 59 es 69 74

WATER TEMPERATURE (°F }

Fig. 6. Relationship tetween water tenperatur: aad log of time

to death after oxposure of juvenile +
Chondrecsccus columna

HOOK galmon cto

w
we


SERED ERE EERE SURO E EA ER EEL BEE ERE EERE NESE EER EE SERED ERE REMEBER EEE RES

*ANOVA12 = ONS/TWO FACTOR ANALYSIS OF VARIANCE.

OREGON STATE UNIVERSITY COMFUTER CENTER

PERE ESE EERE EES EE GEER ER PEERDNSENERDEEES HERES SD EEEBEESL ER SEER EER EER EEE

SOURCE
EXP TYPE
TEMP
EXP TYPE X TENE
ERROR

TOTAL

SOURCE
Exe Type
EX?)
B6.75791

TEMP

PROSLEM I-Dt GP3-PT1

MEANS

Con

S701 756
¢ 3?
48. 75090

EXP TYPE X TEMP
\EEXP gD
€€.6€700

(exe.y oD
97.50960

\(con 72
47636500

(con, =

9

Analysis of Final Persent Mortality Data in Text Tebdie ic,

os-3
OATE

VER365
12/05/72

OF ss MS a
1 3,83331201E 94 3,6832i2G3F 94 4438,0710
5 S.S833010KE 02 1.17666021E 92 13.4675
5 2.€71865564E 03 5.24371126— 02 61.0710
10 8,74999981E 01 8.74999SE1E 09
21 4,21808058E 04
(con)
4.76073
rs Onl’ one C5) “Cay
37.50000 46.25000 47.50009
re)
48.75000
(Exe, 6) (EXP yg 5D (EXP yu)
75,00000 99.00c00 95.00000
(Exe yg 4)
97.50000
(CON y 6) (CON 5 Ss (CON a)
0 2.50000 0
(CON ¢ 4)
9

Sockeye Salmon

Virus in 0,95 Gren Kobanes Salus,

r= 23 € 2,22 for P= 9,08; t = 3.17 for P > G.01; }2 x 6.749 = 4.1833.

Least eignifisant diffaronss 9,3
eo . 8 13)

3 pereent vor ¢ ~ 0.0)

" P=0.01


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


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 gm 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 sroups 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


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


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,


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


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77


re [z
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10

ll

12

13

TABLES

Effect of Water Temperature on Aeromonas Salmonicida
Infection in Juvenile Coho Salmon

Recovery of Aeromonas Salmonicida by Culture of Kidney
Tissue of Juvenile Coho Salmon

Effect of Water Temperature on Aeromonas Salmonicida
Infection in Juvenile Spring Chinook Salmon

Recovery of Aeromonas Salmonicida by Culture of Kidney
Tissue of Juvenile Spring Chinook Salmon

Effect of Water Temperature on Aeromonas Liquefaciens
Infection in Juvenile Steelhead Trout

Recovery of Aeromonas Liquefaciens by Culture of Kidney
Tissue of Juvenile Steelhead Trout

Effect of Water Temperature on Chondrococcus Columnaris

Infection in Juvenile Rainbow Trout

Effect of Water Temperature on Chondrococcus Columnaris

Infection in Juvenile Coho Salmon

Effect of Water Temperature on Chondrococcus Columnaris

Infection in Juvenile Spring Chinook Salmon

Incidence of Ceratomyxa Shasta and Mean Time to Death
Post~exposure of Juvenile Rainbow Trout Exposed to
Water Containing the Infective Stage of the Organism
and then Placed in Temperature Regulated Disease Free
Vater

Incidence of Ceratomyxa Shasta and Mean Time to Death
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

Mortality Among, Fingerling Kokanee Salmon Resulting
from Various Concentrations of Sockeve Salmon Virus
in the Aquarium Water at 54°F

Mortality Among Fingerling Kokanee Salmon Resultine

from Various Concentrations of Sockeve Salmon Virus
in the Aquarium Water at 54°F

vii

Page

17

18

24

26

44

46

64

74

75


SORE ERE EERE EEUU REDE ERE EEE E EERO RE MERE SEER E EERE ER ERE ENDER ERE EERE ES

OS-3 VER.3.5
GATE = 12/05/72

*AMOVA12 ~ ONE/TWO FACTOR AMALYSIS OF VARIANCE.
OREGON STATE UNTVERSITY COMFUTER CENTER

EOE E EE EE EE EO ONE EERE EE EOESS EEEES TENE REE EEEREEER ESE

PROBLEM I-08 GPi-PTL

SOURCE oF ss MS F
EXP TYPE _. A Ge 31122945 03 Ge21L229N5E 03 — 244.5855 _
TEMP 4% 9,72105509F 03 2.43026402E 93 9461825
EXP TYPE Xx TEMF 4 Leh7204664E 04 2,930116E1€ 03 113.5538
ERKOR 10 2.E8037747E 02 2.58037717E OL 8
TOTAL 19 2.50107897E 04
SOURCE MEANS ce cee ee
EXP TYPE

(EXP ) (CON )
G70 33340 11.605350
TEMP
Sok SEE 2 ES SOE 9 SURI SORES SO “ne v
41.20700 51.€6650 49,16675 2.50000
t i
3.30650
EXP TYPE X TENG :
HEXP 5 7) (EXP 5) EK ay “(EXP 4 3)
33.33350 95.66650 98433350 3.33350
(EXP y 4)
5.00000
(CON » 79 (CON, 5) (CON y= 4) (con » = 3)
49.08950 6.66659 t 1666650
(CON 4 4) ~~ 7 .
1661300

Analysis of Final Porcest Mortality Data im Text Tabir

2, Sockeye Salxon
Virus in 0.31 Gram Kokanee S> 1:

ra 2; t= 2,23 for P= 9,05; t = 3.17 for F = 0.01; )35,80 = 5.0797

Lent rigaificant difrercose 11.33 persant fer P=
. . oe 8

1éi0 © 9% "= Ps

405


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75


TRG REECE TORRE CERT EE OTE REDE CREER EET REERES

FANQVAL2 = ONE/THO FACTOR ANALYSIS OF VARTAKCE.  CS-3

Sete errrrerereevre cy cy yt as
73 VER.3.5
OREGON STATE UNIVERSITY COMFUTER CENTER DATE = 12/05/72

DORMER EERE E EO UREE EAGER EER ERE RLT ER URMEU ERD UL REEE RECS PUES EERS RARER

PROBLEM IDs GPLPT2

SOURCE | oF Ss MS

04 323.5599

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cHE 02 10.2892

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ERROR 14 1649696719E 03 $,5€419%24E

TOTAL cow coe wn. OM, Se G4B275E5E OH

__. SOURCE MEANS _
wn. EXP TYPE gg vig ee ean
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_ ~ 62091666
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Anelyets of Date iu ‘ont Tsble 19 for sant of £.
with &. Sooate

14 fur P= 0,05; + = 2.98 for P= 6.62; 65.6 = 9.2543

wow Trout Infected

reajtx

Jeust alerifseens diftsrence ~ 19.50 perount for P = 0.05
e J id “8 5 27,58 9 7 e F=0,01


*satqiedoid

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86


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


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49


the fish were placed in holding tanks supplied with well water at 54°r,
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°) for

48 hours. Colonies producing zones of clearing on this medium were
inoculated on two plates of regular Furunculosis Agar, tubes of Oxidative-
Fernentative Medium, Difco, and Arginine Decarboxylase Medium, Difco.

One of the plates was incubated at 37°C to inhibit A. salmonicida, the
other at room temperature to permit growth. The latter plate was then
used for determining morphology, the Gram reaction, motility, catalase

and cytochrome oxidase reactions. A. salmonicida is a Gram negative,




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74


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.


TIME TO DEATH AFTER EXPOSURE (days)

100

b-
10
C
a
ye n 1 4 n = 1 )
3¢ 44 49 54 59 64 69 74
WATER TEMPERATURE (°F)
Fig. 5. Relationship between water temperature and log of time

to death after exposure of juvenile cei. salmon to
Chondrococeus columnaris.





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 76°F neither species of fish has the ability to
retard the growth of C, shasta. At 64°F and below the portion of the
coho population able to resist a fatal C. 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 C. 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


OPTICAL DENSITY

32

28

o 6 2

T + + -
30 40 50 wo 7 60 30
INCUBATION PERICD IN HOURS

Fig. 2. Effect of temperature on growth
salm 2 in peptone--beef~extract-

5 Aevouonas
se broth.

nN
N


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 bacterial 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 ¢. 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


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°Rr (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 C. 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


Experimental Phase

Effect of Temperature on Infection with Aeromonas salmonicida

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°r, a third pair received water at 64°R, 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

0
hour broth culture of A. salmonicida, strain 5-G diluted in frog Ringer
saline. An LD5o 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




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


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84


Sodium lauryl sulfate was included in the medium as a selective agent
for inhibition of some Gram positive bacteria. Brom 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-
bohvdrate 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 PBG 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 medium, 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 A. salmonicida, Pleisomonas shigelloides, Vibrio anguillarum,

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


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 lower 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 seem 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 mav 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


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 Ilatchery 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 C. 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 C. 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 C. shasta. From previous

experiments conducted by this laboratory (3) it was estimated that a

55


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80


SELECTED WATER "Rep 1 No
RESOURCES ABSTRACTS Ww
INPUT TRANSACTION FORM
SR «rb July, 1972
Effects of Temperature On Diseases Of Salmonid Fishes €

Be lormin Organ ston

Report Non

J. L. FRYER AND K. S, PILCHER

Oregon State University
Department of Microbiology 18050 DIT

i. Type’ Repo sad Final
Period Covered Report

Office of Research and Monitoring
Environmental Protection Agency 4/1/69 - 3/31/72

12) Spon sting © yaa or

Environmental Protection Agency, report number, EPA-660/3-73-020,
January 1974.

The effect of water temperature on infections of salmonid fish was investigated.
Chondrococcus columaris 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. In all cases mortality rages
were high 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 Cerato:

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, Scvurity Cass. J 21. AW. of Send To:
Cseport) Pages

5 : WATER RESOURCES SCIENTIFIC INFORMATI
2 Seeurs.y Class. 22, Price US DEPARTMENT OF THE INTERIOR YON CENTER

(re. WASHINGTON, D C 20240



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26


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 30° 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 30°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 A. 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,


require differentiation,

Over 75 species of bacteria have been examined on this medium. Many
Gran 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-

Phila 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 aeromonas organisms in

water, but for isolation of these organisms from fish, and measuring

growth rates at various temperatures.

39


No.

14

15

16

17

18

19

20

Effect of Water Temperature on Mortality of 1.1 pm
Kokanee Salmon Fingerlings Exposed to Sockeye Salmon
Virus

Effect of Water Temperature on Mortality of 2.9 gm
Kokanee Salmon Fingerlings Exposed to Sockeve Salmon
Virus

Mean Time to Death for 2.9 gm Kokanee Salmon Finger-
lings Exposed to Sockeye Salmon Virus

Effect of Water Temperature on Mortality of 0.11 gm
Kokanee Salmon Fry Exposed to Sockeye Salmon Virus

Mean Time to Death for 0,11 gm Kokanee Salmon Fry
L[xposed to Sockeye Salmon Virus

[Effect of Water Temperature Mortality of 0.95 pm
Kokanee Salmon Fry Exposed to Sockeye Salmon Virus

Mean Time to Death for 0.95 gm Kokanee Salmon Fry
Exposed to Sockeye Salmon Virus

viii

78

80

81

83

84

86


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


REGRESSION 4,
COO SALMO?

Gd

pays, 70
PPR Omi

2
NRE

& cee une eeeee . - -
‘:

is vere eter cere coer ee ee wo ee ee ee eee
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TEMPERATURE wneecesace semrnvacre St
_ EXIT. . _ _ .

RELATION BETWEEN VATTR TEM:

DVATH. ©, SRASTA Vi

sUR 104 NO, OF DAYS v0
PION I) CCHO BAL oT

YC 2)5 40404299 69 +
_ YS 16G NO, DAYS TC DEATH

R SQUARED = 031679956 = 4 OF VAR

ACGOTITED FOR
SH LIS,

CORRELATICN

US GOVERNMENT PRINTING OFFICE 1974 546-317/313 1-8 ik


non-motile rod that produces clearing on the Furunculosis Agar with
casein, fails to grow at 37°C, and forms catalase and cytochrome

oxidase. A. 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


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 equipwent 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, Howland, 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°R; and §& 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


CONTENTS

Section Page
I Conclusions 1
Il Recommendations 5
Ill Introduction 7
Iv Equipment Design and Fabrication Phase 9
Vv Effect of Water Temperature on Infection of 11

Salmonids bv Aerononas Salmonicida and Aeromonas
Liquefaciens

VI Effects of Water Temperature on Infection of 41
Salmonids by Chondrococcus Columnaris

VIL Effects of Water Temperature on Infection of 55
Salmonids by the Parasitic Protozoan Ceratomyxa
Shasta
VIII Effect of Water Temperature on Infection by the 69
Sockeve Salmon Virus (IHN)
IX Acknowledgments 89
xX References 91
XI Appendices 93


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Analysis of Final Pereent Mortality Data in Text Tabls 15,

Virus im 2.9 Gram Kokanes Salon.

r= 2; t =2.23 for P =0,05; t = 3.17 for P=0,01;

Soskeya Saluon

least sigaificant differenes == 16,06 percent for P =-G.0!
“ “ . “of 22.82 9 © " P=4,0


1000,

TTT

TIME TO DEATH AFTER EXPOSURE (doys)

1Oo}--
L
L
10: _L J. 1 LL. J. 4 —
33 44 49 54 59 BS 69 74
WATER TEMPERATURE (°F)
Pig. 7. Relationship between water temperature and log of time

to death after exposure of juvenile
Ceratomyxa shasta,

inbow trout to



had received a sham injection and were held under the same conditions,
all remained healthy. Aeromonas salmonicida was not found in the

kidney of anv 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°r, 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, A. salmonicida was not

recovered from the kidney of any of then.

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 ten-
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


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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24


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


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 ppm of oxytetra-

cyclene in the water.

85


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.


lL

2.

3.

5.

SECTION X

REFERENCES

Pacha, R. E., and Ordal, E. J., "Histopathology of Experimental
Columnaris Disease in Young Salmon." J. Comp. Path. 77, pp. 419-423.

1967.

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.

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,

Wingfield, W. H., Fryer, J. L., and Pilcher, K. S., "Properties of
the Sockeye Salmon Virus (Oregon Strain) (33719)." Proc. Soc. Exp.

Biol. Med. 130, pp. 1055-1059. 1969.

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


BORER EER A DEE EERE EES EERE ERE R ERE HEU EEE GUUREE EER RRER EER ED ERLE TEER EE

FANOVAL2 = CNZ/THO FACTOR ANALYSIS OF VARIANCE. OS-3 VER3.5
OREGON STATE UNIVERSITY COMPUTER CENTER OATE = 10/14/72

PEER EERE OEE ERE R MEAL ERO RE EDR EERE EES ERS EERER ENA EURAR ERE EEREEE

PROBLEM I-06 2ND TRY

source oF ss MS F
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EXP TY? X TEMP 7 24519500008 03 3.599285712 92 7.2713
2 ERROR G2 4e950RHeCE Of
TOTAL o4
EXP TYPE
(EXP ) (CON )
74.00090 2.25000
TEMP . _ a _ a
- aay C69) ¢ C59)
42.0699) 41.00090 37200963 48.00000
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84.09990 $9.00000 72.00009 96,00000
(EXP 4 54) (EXP 49) (EXP y 44D (ExP 39)
70.00990 62.00600 38.00003 66,00006
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0 = ss 2otneo = ONS oe
(CON, 54) (CON 4 49) (CON yu (CON 5 39)
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Analysis cf Final Porcert Mortality Data in Tuxt Tebl: 3, A. Salwonieida

in Spripg Ckincos 3rlmon.

cms

ys 2; % 2,12 for F=0.05; &= 2.92 for P+ 0,08: 49,5 = 7.0356

Least significant, o1fforence 2714.92 poreant for P.
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REGRESSION ANALYSIS, CHONDROSOCCUS COMES ARIS
IN CHINOOK GALION. TEXT FIG. 6

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F 2ATURS
. oe By LOG NO, OF Pays
ft emer mens ~~ TO DEATH

112


in the laboratory tanks. They were fed at the 20 ¢ TM, ,/100 lb 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 C. 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 C. 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 with 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 ¢
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


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SECTION V
EFFECT OF WATER TEMPERATURE ON INFECTION OF SALMONIDS BY AEROMONAS

SALMONICIDA AND AEROMONAS LIQUEFACIENS

Materials and Methods

Two strains of Aeromonas salmonicida 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


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 Columnaris in Coho Salmon. 99
6. Text Table 9. Chondrococcus 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


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 190% 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 organisn

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


Experimental Phase

Table 7 shows the percent mortality and the incidence of C. 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 C. 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


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 vear 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 fingerling 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


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


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 IDs 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




REGRESSION ANATYSIS. AERCIHONAS SALMONTCIn!
‘ DI co70 ON. TEXT PIG, 1

1.

oe

A

Paw rrr
:
:
NANNRB
i
1
:
1
:

ae a4. _- — 3. mye
we ne eee RL
an - cee 3 oe
a: g

- YR 30, QF PATS

DOHC e rH ETO eee eotar erence eeEeoeEoe enc reeuEoree

TEMPERATURS od

2 AEXTT
RELATION BRIVEEN WATZR TEMP
DEATH, A. SAUMOKICIDA INMECTION IN CGi0 SAL,

“32 0 4)

ARATURE

YO 2)=  2.4629715E 02
¥ = LOG BO, DAYS TO WOATH
_R SQUARED = «78315516 = % OF VARTATION 2
: WIT

AYSD FOR

OR.

ed

“VAR” STES OF §
- é 6. OG4872620-02 41915! 736 OL

oe 8,45 0555455-G6 2.9259. 27E 14,

CORRELATICN COSFFLICIE!

$O DEATH

103


REGRESSION ANALYSIS. CHOMDROCOCCUS COL WARIS

v3
we

IN RAI

‘DOW TROUT. TEXT FIG. 4

Se ae vee
ne ce ee _
4 wee eee oe

4 5 i

« 1QG.NQ. OF DAYS TQ DEAT, rst

2. wa 8, wed ne ce oe ee
a a ce
& og : os

eee ee eee eee eee ee eee

TEMPERATURS smmeememeeeeenenet

RELATION SSTVEEN WATER VEMPERATURE AND LOG Yl, OF DAYS TO

ATH, CG, COLUMIARIS IsFOCTION IN RALHEC

¥C 25

3.2806146E 09

Y= LOG NO. DAYS TO DEATH ~

R SQUARED =

nT a
c
i

CORRELATICK

073489354 =

A G2LES1EFE-94
209621512803

CCEFFICIENT

e

1 OF REGR. COEF,

~ 666572593 =

o

ikcuT.

242b-02 XC 1)
TEMPERATURE

4-449.

ACCOUNTED FOR..._

He,

254145145

TO DEATH


4

FIGURES

RELATIONSHIP BETWEEN WATER
TO DEATH AFTER INFECTION 0
AEROMONAS SALMONICIDA

EFFECT OF TEMPERATURE ON G:

SALMONICIDA IN PEPTONE~BEE.

RELATIONSHIP BETWEEN WATER
TO DEATH AFTER INFECTION 0

TEMPERATURE AND LOG OF TIME
F JUVENILE COHO SALMON WITII

ROWTH RATE OF AEROMONAS
F-EXTRACT-GLUCOSE BROTH

TEMPERATURE AND LOG OF TIME
F JUVENILE CHINOOK SALMON

WITH AEROMONAS SALMONICIDA

RELATIONSHIP BETWEEN WATER
TO DEATH AFTER EXPOSURE OF
CHONDROCOCCUS COLUMNARIS

RELATIONSHIP BETWEE
TO DEATH AFTER EXPOSURE OF
CHONDROCOCCUS COLUMNARIS

RELATIONSHIP BETWEEN WATER
TO DEATH AFTER EXPOSURE OF
CHONDROCOCCUS COLUMNARIS

CERATOMY X,

RELATIONSHIP BETWEEN WATER
TO DEATH AFTER EXPOSURE OF

CERATOMYXA SHASTA

RELATIONSHIP BETWEEN WATER
TO DEATH. AFTER EXPOSURE OF
ASTA

TEMPERATURE AND LOG OF TIME
JUVENILE RAINBOW TROUT TO

TEMPERATURE AND LOG OF TIME
JUVENILE COMO SALMON TO

TEMPERATURE AND LOG OF TIME
JUVENILE CHINOOK SALMON TO

TEMPERATURE AND LOG OF TIME
JUVENILE RAINBOW TROUT TO

TEMPERATURE AND LOG OF TINE
JUVENILE COHO SALMON TO

vi

PAGE

20

27

45

48

62

66


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°r 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°r. Replication was retarded and virus yields were lower at 39°F, and

87


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31


4

5.

6.

Text Fig. 5.

Text Fig. 6.
Salmon.

Text Fig. 7.

Text Fig. 8

Chondrococcus Columnaris in Coho Salmon.

Chondrococcus Columnaris in Chinook

Ceratomyxa Shasta

Ceratomyxa Shasta

94

in Rainbow Trout.

in Coho Salmon.

Page No.
111

112

113

114


mine whether it was possible that small numbers of A. 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.

34