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Asdoqne je sainzq—no eatyTsod BurpTerk ysty peqoesur AT[e}ueutiedxa jo uorqrodo1rg

suoulTes yoouTYyo
eTTueanf wory ensst3 Keupf~y Jo vanq{No [eopZoToziejoeq Aq eppofuoMpes Seuomotey jo Azoaovey *Z eTqeL

il


The data presented define the effects of temperature on columnaris
infection without confusion from the activity of other pathogens in the
water, and therefore with somewhat greater precision than reported by
other workers. These results do provide additional guidelines for future
understanding and efforts to control this disease. Considering the
available evidence from all investigators, and the existence of strains
of F. columnaris of high and low virulence, it appears that water
temperatures below 54 F are required for complete suppression of the
disease in salmonid populations that have been exposed to the pathogen.

32


Experimental Phase

The results of the two experiments are presented in Table 9. All of the
exposed fish held at 69 F and 92% of those held at 64 F succumbed within
the 25 day observation period. Mortality dropped significantly to 56% in
the groups held at 59 F, and was only 16% among those held at 54 F. No
deaths occurred at 49 or 39 F, and only one at 44 F. Flexibacter columaris
was isolated from 96 to 100% of exposed fish that died at 54 to 69 F by
culturing gill and kidney tissue.

These experiments clearly demonstrated the effect of temperature on the
incidence of fatal infection in juvenile steelhead trout exposed to this
pathogen. Mortality increased progressively with increasing temperature
in the range from 54 to 69 F. The disease was effectively suppressed at
49 F and below. The 74 F water temperature was not tolerated by these
fish, as all of the unexposed control fish died during the experimental
period. Flexibacter columnaris was not recovered from these animals at
autopsy, and these are presumed to be thermal deaths.

A second effect of water temperature on this infection became apparent

when the mean number of days from exposure to death was plotted on a log
scale against temperature. The relationship between these two variables

was found to be linear, as shown in Fig. 5. A correlation coefficient

of -0.7604 was calcylated and found to be highly significant. The coefficient
of determination (R“) was shown to be 0.5783, indicating that about 58%

of the variation in the log of the time to death was accounted for by

water temperature. The equation and the data used in computing it are

shown in Appendix, page 68. The mean time to death decreased from 7.6

days at 54 F to 1.7 days at 69 F. Thus the progress of fatal infection was
accelerated at the higher temperatures and retarded at the lower temperatures.

Although precise measurements of growth of this strain of F. columnaris
in vitro were difficult because of the strong tendency of the cells to
form clumps in liquid medium, growth rates were determined approximately
at 49 to 74 F by measuring the optical density of static cultures in
tryptone yeast infusion broth. In this temperature range growth was most
rapid at 64 to 74 F. At 59 F there was a lag period of about 47 hours
before growth began, and the growth rate was less than at 64 F. At 54
and 49 F no significant growth was observed, even after 100 hours
incubation. Thus the optimal temperature range for growth in vitro was
correlated with the temperature that produced the highest mortality and
the shortest mean times to death. Furthermore, the 49 and 54 F temperatures,
which were very unfavorable for growth of the organism in vitro were
associated with little or no mortality in the groups of exposed fish.

28


100;-

TIME TO DEATH AFTER EXPOSURE (days)

10 l | _]
39 44 49 54 59 64 69

WATER TEMPERATURE (°F)

Fig. 6. Relationship between water temperature and log of time
to death after infection of juvenile coho salmon with
kidney disease bacteria.

37


SECTION IV
EQUIPMENT DESIGN AND FABRICATION PHASE

The design and fabrication of the equipment used in the project were
described in an earlier report (1).


ANALYSIS OF DAILY MORTALITY DATA USED IN COMPUTING MEAN
VALUES SHOWN IN TEXT FIG. /. A. SALMONICIDS IN CHINOOK
SALMON

*SIPS LOG UNITS 12/16/74 2252 PM
SVAR,3
TREAD, *CHINS2151-2
SSET,3=LN(2)
SNAME,1 TEMP,2 DAYS,3 LNDAYS

SSCATTER) TEMP ALNOAYS
Lower BOUND oF x +90000E o1
UPPER BOUND OF Xt 7220000€ 04
LOWEP 3QUND OF Ys OE 00
UPPER BOUND OF Y8 2.70805E 00
CORRELATION COEFFICIENT = 0. 8606855
i 1 1 1
: 2 3 i
1 i & i 2
rf i i
i i
i 2 4 4
PY 2 6 2 1
5 7 bo.
4
g 4 . 34
° 1 * *
8
Z

To nT

SREGRESS,LNDAYS ON TEMP
LNOAYS = 1.4134E+00

TADD, TEMP
LNOAYS = 5.4010E+¢00 ~6.4E13E-02 TEMP
tS AVTABLE

ANALYSIS OF VARIANCE TABLE
SOURCE OF SUM OF SQUARES MEAN E
TOTAL 108 4.99565192E Of 4 ooseusege ool
REGRESSION 1 3.70067652E 01 3.70067652E O01
RESIOUAL 107 1629497539E 01 1.21025738E-01

R SQUARED = °74077950

65


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02-02 > 08-02 Ov ST 4,67
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07-02 09T-08 SET 09
04%-02 09T-08 SET SY
07-02 OZ@E-09T 697 oe
02-02 > O9T-0¥ LS ST Ag79
O”-0% > 09T-08 €IT 06
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esuel IOITL ia3t} uesy esuel JaITL 4183T} ueSeW sep ut potied eanjeiedusy,
STO1WUOD poyeUpooepA uoTz0efur ysog zeqEM

S193} UOT JEUTINTIZe Jo sTeoordpoey

*j3uean[pe ojeTdwoo s,puneaig uy eTieq0eq aseestTp AeUpTY PETTY JO uoTqoefuT

TesuojTredeazquy eTZurs e ButMoT[oy uowTes oyoo eTTueanf uz sTeaeT ApoqTque BuTzeupan{s3y “ET eTqeL

47


LOG,9 OF RECIPROCAL OF AGGLUTINATING ANTIBODY TITER

! ! it J
15 30 45 60

DAYS POST INJECTION

Fig. 8. Antibody response to an injection of killed Aeromonas
salmonicida cells in juvenile coho salmon held at
various water temperatures.

45


tanks one meter in diameter, supplied with salt water (about 3 gal,

per min.) from the Slough. Vibrio anguillarum is constantly present in
this water, and vibriosis reaches epizootic proportions among resident
salmonids in the warmer months when water temperature rises above 54 F.
The groups of vaccinated fish were held in the salt water at temperatures
favorable for the progress of the disease (51 to 66 F) for 20 days.
During this period fish that died were autopsied and cultures prepared

by inoculating kidney tissue on brain heart infusion agar. Colonies that
developed were examined by Gram stain and those that resembled V.
anguillarum were confirmed by slide agglutination with specific antiserum.

The results of the first experiment are presented in Table 15. Very few
deaths occurred in most of the vaccinated groups and in four of the
seven groups, no deaths were shown to be due to vibriosis.

Among the unvaccinated controls, 44% died and 37% succumbed to infection
with V. anguillarum. The degree of protection estabiished by vaccination
was not influenced by the water temperatures at which the fish were
vaccinated. Those vaccinated at 39 F were protected just as well as the
animals vaccinated at 69 F and in all groups vaccination was highly
effective.

Additional data relating possible effects of water temperature on immuni-
zation was provided by two parallel experiments similar to the one described
above but carried out concurrently. In these experiments the available

coho salmon were smaller than those in the first experiment, having a

mean weight of 3.3 grams. A further modification was made in the temperature
at which the fish were held after vaccination. In the first experiment

the fish were tempered back to 54 F and then heid at that temperature for

a week before being exposed to the infection in Lint Slough. In the
subsequent pair of experiments, after completion of the 15 day vaccination
period, each vaccinated group was held at its respective experimental
temperature for one week before being tempered back to 54 F. The intent

of this modification was to provide a scmewhat longer period during which
the various water temperatures might exert an effect on the immunization
process. An experiment modified in this wey might be more likely to reveal
possible effects of temperature on immunization.

Except for the changes noted, the two parallel experiments were carried
out in the same manner as the first one. The results obtained are shown
in Table 16. Again, immunization was found to be highly effective at all
experimental water temperatures. A small number of deaths from vibriosis
occurred in the 39 and 44 F groups, compared with none in the 69 and

64 F groups, but the numbers are much too small to be significant.

Thus the results confirm those in the first experiment and justify the
conclusion that oral immunization of juvenile coho salmon with this

V. anguillarum vaccine can be carried out effectively at any water
temperatures in the range of 39 to 69 F.

51


SECTION X
ACKNOWLEDGMENTS

This project was supported by the Environmental Protection Agency over
a two and one half year period beginning April 1, 1972 and ending
September 30, 1974. The total funds provided by this agency amounted
to $107,188. The assistance provided by Dr. Gerald R. Bouck, who served
as Project Officer, is acknowledged with sincere thanks.

A major contribution to the project was made by the Fish Commission of
Oregon and the Oregon Wildlife Commission. 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
the fish used during the two and one half year period of the project
has been roughly estimated at about $7000.

55


Experimental Phase
Antibody Response to Killed Aeromonas Salmonicida
at Various Water Temperatures

The levels of agglutinating antibody produced in juvenile coho salmon
held at various water temperatures following a single injection of
killed A. salmonicida cells in Freund's adjuvant are shown in Table 12
and Fig. 8. No significant antibody response was found in fish held
at 39 F during the 60 day observation period. At 49 F a response was
first detected after 30 days, and the level was as high or higher after
60 days. At 54 F the first increase was noted after 30 days, and after
45 days more antibody was present than was found in the fish held at
49 F, Temperatures of 59 and 69 F appeared to be optimal for antibody
production, and the highest concentrations were found in fish held at
these temperatures 45 days after the injection of antigen. In general
these results seem to indicate a progressive enhancement of antibody
production as the temperature increases from 39 to 59 F.

In the original design of this experiment it was planned to obtain some
information on the comparative degrees of protection produced in the
vaccinated fish held at the various experimental temperatures. Twenty

five fish from each temperature group including controls were to be challenged
by the injection of about 2 LD,, of virulent A. salmonicida cells. Before

the challenge, it was necessary to temper all of the experimental fish groups
held below 59 F up to this temperature, so that the effect of water tem-
perature on the percent mortality after challenge would be eliminated.

The tempering process was completed on all groups by the 84th day after

the original antigen injection. Twenty five fish from each group were

then injected with a dose of A. salmonicida cells calculated to represent
about 2 LD,,. This portion of the experiment was unsuccessful however,

as less than 10% of the unvaccinated control fish developed fatal infection.
Hence no information was obtained concerning the effect of temperature

on protective antibody, which is not necessarily identical to agglutinating
antibody.

On the 84th day after the antigen injection, when the tempering process
had been completed, blood specimens were taken from five fish in each
vaccinated and control group and agglutination titers determined. At this
time only two of the vaccinated groups showed higher mean antibody titers
than were found at the 60 day period. The group held initially at 39 F had
a titer of 1:512 compared to 1:45 at 60 days. Apparently the gradual
elevation of temperature to 59 F had enhanced antibody synthesis. In the
groups held at 59 F throughout the entire 84 day period, the titer at

84 days was 1:24,834 compared to 1:2047 at 60 days. No special reason for
this increase is apparent, except that this temperature was in the optimal
range for antibudy formation. In the other temperature groups, the titers
at 84 days were not significantly different from those at 60 days, with
the exception of those originally held at 69 F. In this case the titer
dropped from 1:2,427 at 60 days to 1:323 at 84 days.

43


As in the chinook salmon experiments, a linear relationship between the
log of the time (number of days) to death and water temperature was
observed and confirmed by regression analysis (Fig.,2). The correlation
coefficient was -0.7635, a significant value, and R” was 0.5829 (Appendix,
page 66). The latter figure indicates that about 58% of the variation in
time to death was accounted for by temperature. The mean of times to
death decreased from 20.3 days at 44 F to 2.7 days at 69 F. Thus the
accelerating effect of higher tempertures was again evident.

15


SECTION VII
EFFECT OF WATER TEMPERATURE ON BACTERIAL KIDNEY DISEASE IN SALMONIDS
Materials and Methods

The influence of water temperature on experimental infection with
bacterial kidney disease was studied in juvenile coho salmon and steelhead
trout. Two strains of the bacterium (Corynebacterium sp.) used were
isolated from spring chinook and coho salmon at the Hood River and
Nehalem Hatcheries, respectively. Stock cultures were maintained on
Cysteine blood agar (7) modified by using calf serum in place of whole
blood.

Inoculum for infecting fish was prepared as a suspension of the organism
in physiological saline. In experiments with coho salmon the suspension
contained about 2.5 x 108 bacteria in 0.05 ml, and in steelhead trout
experiments the concentration was about 2.9 x 10° in this volume.

Juvenile coho salmon averaged 6.5 grams in weight, while steelhead trout
averaged 18 grams. Fish in groups to be infected were injected intra-
peritoneally with 0.05 ml of the bacterial suspension, and control fish
received the same volume of sterile physiological saline.

The experimental design was almost the same as previously described in
Section I for A. salmonicida. Fourteen aquaria, each containing 25 fish
were used in each experiment. Seven streams of tempered water, varying

from 39 to 69 F in five degree increments were provided, and two aquaria
were maintained at each temperature. Seven of them, one at each temperature,
were assigned to groups of fish to be infected, while the remaining

seven were used for uninfected controls. The fish were tempered to the
various temperature levels by the method described in Section V. Two
complete and identical experiments were conducted concurrently, each one
consisting of seven infected groups and seven controls.

Dead fish were collected daily during the experimental period, autopsied,
and smears of kidney tissue prepared on glass slides. These were gram
stained and examined microscopically for the presence of the typical
kidney disease bacteria. This criterion was used to establish kidney
disease as the cause of death. Bacterial cultivation of the organism

was considered impractical in this case because of its slow growth rate
and fastidious character.

33


(C/3)*100.
SORDERG Hy 358, (Hy 3d

SANOVA,G,3

SNAME,3 EXPTY9E,& TEMP,6 957 Cy8 PMORT

SVARs10

SNOMINAL 1,3
SREAD, *STEEKLy 1-7
SSET,8

SAWVTABLESS&

prs
Gvco
<
Swuuw
arms
CANT
ren,
rece
a0nNo
wees
Paine

Loo

nt Mortality
. Kidney

/

Disease in Steelhead Trout.

Analysis of Final Pe:
Data in Text Table

SeilN764F OL

14
27

MEAN OF

FREQ
14
14

&#) ERROR
TOTAL
SINTERMEANS 93545 344
EXPTYPE
VALUE
TEMP

iy

for P= 0.05
least significant difference 21.28%

Least significant difference 15.33%
for P=0,01

bdo
geeeaese
ZwWwwwwow
@ OonMsroRnN

EXPTYPE

TEMP

Cd On On SH On OF DO
20 ©6 66 Se SG Fa SO

We Wwe Wu Wd ur Ws
ON CO OF OW OR OO OM

3645263
7.77472

MEAN OF PMCRT
9.80000

FREQ

=a 4+ wa we we we
e © © © &© © oe
wow Ww Ww Ww Ww WwW
wo a So 28 2

° eo
3ez0Sz0 SzaezZOezAezZO0ZO
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2o e eo oe
o

40

A .
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49
Seb
59
6.40
6.90

64

SEND


ABSTRACT

Investigations of the effect of temperature on infections of salmonid
fish were conducted. Aeromonas salmonicida infection was studied in
chinook salmon and steelhead trout; Aeromonas liquefaciens infection in
chinook and coho salmon. In all cases mortality rates were high at 64 to
69 F; usually 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
retarded at lower temperature levels.

Bacterial kidney disease was studied in coho salmon and steelhead trout.
The temperature range of 44 to 54 F was optimal for the development of
fatal infection, as indicated by mortality rates of 78 to 100%. Higher
temperatures had a suppressing effect, which was marked at 69 F.

Temperatures of 59 to 69 F were optimal for the formation of agglutinating
antibody when juvenile coho salmon were injected with a killed suspension
of A. salmonicida. At lower temperatures less antibody was formed, and

no significant amount was produced at 39 F 60 days after injection of
antigen.

Oral immunization of juvenile coho salmon with a vaccine consisting of
formalin killed Vibrio anguillarum cells incorporated in their diet

protected them against fatal infection when the fish were held at temperatures
from 39 to 69 F during immunization.

iii


ANALYSIS OF DAILY MORTALITY DATA USED IN COMPUTING MEAN
VAWWES SHOWN IN TEXT FIG. 3 A. LIQUEFACIENS IN COHO SALMON.
#ST95 LOS UNIT# 91707775 13417 PM

EREAN,*COHIL2 1,19?

BAPPEMO,*CIHIL2 251-2

BSET, T=LNCAd

BSCATTERy

>,Lx0
LOWER 30'N) OF xt &.90000E bt
UPPER 30UI) DE Xt 7.400008 OL
LOWE? t9U'M Je Yt oF 90
UPPER 30UN) SF Y8 2e89027F 30
CORRELATION IFFFICTENT = -0. 7016998
t 1
5
2 1
3 i 1
By t z 3
1
a 2
& 6 : 2
a
5 3 . 4
a
Py . . >
-
a
= . ~ . .
oe TRMPERATURE ST Tc ccs eres ree eee eee aces esse eens

SREGRESS,LNIAYS ON TEMP
LNDAYS = 4e7780E-01

BADD, TMP
ULNDAYS = 346453926400 77.5300E-02 TEMP
PAVTARLE
ANALYSIS OF VARIANCE TA3LE
SOuRtr JF SUM _OF SQUARES MEAN SQUARE
TOTAL 309 1.57508373E 02 5.09737792E-01
REGRESSIIN a 7.755460795E 11 7075546795E 01
RESTOUAL 338 7e995429R4KE O01 2059591878E 01
R SQUARED = ©492382€0

67


ANALYSIS OF DAILY MORTALITY DATA USED IN COMPUTING MEAN

VALUES SHOWN IN TEXT FIG. a. A. SALMONICIDS IN STEELHEAD
TROUT.

*SIPS LOG UNIT# 12/17/74 11843 AP
SVAR,S
SREAD,*STEFS21y1-2
S$SET,3=LN(2)
SNAME,1 TEMP,2 CAYS,3 LNDAYS

LOWER 60 3.90000E 04
UPPER FOUND OF Xt 7.400COE 04
LCWER SOUND OF Y3 6.93147E-04
UPPFR SOUND OF 18 3.3€720F 00
CORRELATION COEFFICIENT = -C. 7635207
4 1 * i
f 2
ls 3 1
= 1
1
a 2 2 1 2
& 1 } 1
z z 3 3
¢
fave € 5 4
oO
. 2 5 * 8
2
i

9g *
_—_________»
oe TRPPATURE TTT os swe cco e ee vee ccnceeccceseceecees
SREGRESS,LNDAYS ON TEMP
LNDAYS = 1.4698E+00

8 AD0, TEMP

LNOAYS = 6.0565E+00 -7.0€9GE-02 TEMF
TAVTABLE

ANALYSIS OF VARIANCE TAALE

SCURCE BF SUM OF SELARES MEAN SCUARE
TOTAL 95 5e27143243€ 01 5 o54887624E-01
REGRESSION 1 3.O7305437E OL JeC7IOS4S7E O1
RESIDUAL 94 2.19837806E 01 2.33870007E-01

R SQUARED = 056296382

66


ANALYSIS OF DAILY MORTA
VAWWES SHOWN IN TEXT FI

DATA USED IN COMPUTING MEAN
« KIDNEY DISEASE IN COHO SALMON.

STP LO UNITS 81407775 17:50 &M
hea eth
LOWER 20U0N) DF 3.90909 91
LBHER 30UN) JF xt AedDUOCE OL
LOWER OUND TF ¥8 4 3L75° 90
UPPZP IOUN)D JE YE & 6MuGF 39
CORPFLATTON SOFFFICTINT 0.7494 363
* 5 1 2
05 * 41
7 * 2 2 1
of 5 F 1
2 3 * 1 ra 1 t
ee
& 5 .
at 2 2 8 7 4
& i i 2
A >
3 6 é t t
i ; :
8 ?
g 1
4 1
cece cece e AEMPRRATURE oO. Pr errr re errr errr rs
BREGPSSa,LVIAY. Ch TEMP
LNIAYS = F,355ZL 4°
tADD, TEMP
LNOAYS = 5.3019F 400 “4Se802GF-02 TEMP
TAVTARLE
ANALYSIS OF VARIANCF TASLC
source dF SUM OCF SQUARES MEAN SCUARE
TOTAL 217 Be oeguatre of 2?.5926N155E -01
REGRESSION 1 fe16152565F 01 3e16152565F 92
REST CUAL 216 PebH4UlO72r i Le 14693505F=04
R SQUARED = 056195456

69


Discussion

The effect of water temperature on infection with A. salmonicida and A.
liquefaciens was studied in coho and spring chinook salmon, and steelhead
trout. Temperature effects on A. salmonicida infections were very similar
in all three salmonid species. Fatal infection was suppressed by tem-
peratures of 39 or 44 F, and was progressively enhanced as temperatures
increased from 49 to 69 F. This was indicated both by the mortality

rates and by the mean times to death, which were longest at the low
temperatures, but decreased progressively as temperatures increased. As
reported previously (1) these effects appear to be related to comparable
effects on growth of the organism in vitro. Such growth was barely
detectable at 39 F, markedly retarded at 44 F, and increased progressively
in rate as the temperature increased from 49 to 69 F. From the mortality
data presented it might be anticipated that natural outbreaks of furuncu-
losis among juvenile salmonids would be most likely when water temperatures
are 50 F or higher.

In the case of the A. liquefaciens infections, the effects of temperature
were similar among the three salmonid species. However, in these experiments
the three lowest temperatures (39, 44 and 49 F) essentially eliminated

any fatal infections in coho salmon and steelhead trout during the
experimental period (1). Thus, suppression was somewhat more complete at
these temperatures than in the A. salmonicida infections. Mortality
increased progressively as water temperature increased from 54 to 72 or

74 F. The effects of temperature on the A. liquefaciens infections also,
appear to be related to the growth rates in vitro at the various experimental
temperatures. Growth was negligible at 39, very slow at 44 F, and increased
in rate with temperature increments of five degrees from 49 to 74 F. The
experimental data on mortality seem to indicate that fatal disease due

to A. liquefaciens probably does not occur in juveniles of the three
salmonid species studied until water temperature rises above 49 F.

26


TIME TO DEATH AFTER EXPOSURE (days)

| ! i 1 ! i J
59 44 49 54 59 64 69 72

WATER TEMPERATURE (°F)

Fig. 1. Relationship between water temperature and the log of
time to death after infection of juvenile chinook salmon
with Aeromonas salmonicida.

12


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14


SECTION VI
EFFECTS OF WATER TEMPERATURE ON INFECTION OF STEELHEAD TROUT BY
FLEXIBACTER COLUMNARIS
Materials and Methods

In an earlier report (1) experiments were described in which the effects
of water temperature on infection by Flexibacter columnaris were inves—
tigated in coho salmon, spring chinook salmon and rainbow trout. These
studies have now been expanded to include juvenile steelhead trout,
which averaged 13 grams in weight. These are the same species as rainbow
trout (Salmo gairdneri), but differ by being anadromous.

The experimental design was essentially the same as previously described
in Section V for experiments with A. salmonicida.

The F. columnaris isolate used in these experiments 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. It 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
a lyophilized specimen was cultivated in Cytophaga broth (2) and passed
once in juvenile steelhead trout. Several isolates from this final fish
passage were collected and pooled to prepare the inoculum for infecting
fish in the temperature experiments. Each isolate was grown in tryptone
yeast infusion broth (3) for about 20 hours at 24 C. The resulting
cultures were then mixed for the preparation of the inoculum. The optical
density of the latter was adjusted to 0.1 at 525 nm. Infection of the
fish in the experimental aquaria was then accomplished by addition of

the mixed culture to the aquarium water. The water supply was cut off for
a 10 minute period and the volume (water plus fish) in the tank reduced
to about 20 liters. A sufficient volume of the cultyre was then added to
give a 1:20 dilution, which represented 3 to 6 x 10° colony forming
units per ml. After the 10 minute exposure period, the flow of water was
resumed.

In control groups of fish which were not exposed to the pathogen, the
volume of water in each tank was reduced to the level in exposed tanks,
and water flow was interrupted and resumed in the same manner. Dead fish
were collected daily during the 25 day period following infection, and
cultures were prepared by streaking gill or kidney tissue on Cytophaga
agar.

27


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ANALYSIS OF DAILY MORTALITY DATA USED IN COMPUTING MEAN
VALUES SHOWN IN TEXT FIG. $~. COLUMNARIS DISEASE IN
STEELHEAD TROUT.

°
ran

wnw

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REGRESS, LOG NO. OF DAYS ON TEMP

LOG NO. OF DAYS = 3.5715E - 01

3 ADD, TEMP
LOG NO. OF DAYS = 3,6361E +00 ~409044E = 02 TEMP
ANALYSIS OF VARIANCE TABLE
SOURCE DF SUM OF SQUARES MEAN SQUARE
TOTAL 181 2.60560919E 01 1.43956309E ~01
REGRESSION 1 1.50676135E O01 1.506761356 -01
RESIDUAL 180 1,09884783E 01 6,10471017E -02

R SQUARED = 57827604
CORRELATION COBFFICIENT = = 0.7604

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53


SECTION I
CONCLUSIONS

1. Water temperatures of 59 F and above produce a high percentage of
fatal infections in juvenile coho and spring chinook salmon, and
steelhead trout injected with Aeromonas salmonicida. Even at 49 and 54 F
from 30 to 70% of the fish may succumb to infection.

2. Mortality rates from infection are very low among juvenile coho
and spring chinook salmon and steelhead trout injected with A. salmonicida
and held at temperatures of 39 and 44 F.

3. The mean time to death after injection with A. salmonicida falls
in the range of 2.3 to 3.5 days for juvenile coho and chinook salmon,
and juvenile steelhead trout held at 69 F. This increases progressively
as water temperature decreases, resulting in a range of 18.4 to 20.3
days among these species at 44 F.

4. The percentage of fatal infections among juvenile coho and chinook
salmon and juvenile steelhead trout injected with Aeromonas liquefaciens
is very high at temperatures of 64 F to 74 F, moderate at 54 and 59 F,
and very low or zero at 39 to 49 F.

5. The mean time to death after injection with A. liquefaciens is

in the range of 1.1 to 1.7 days for juvenile coho and chinook salmon,
and juvenile steelhead trout held at 69 F. At 54 F the infection develops
more slowly, and the average time to death is in the range of 3.1 to

4.7 days.

6. The effect of temperature on the growth rate of A. liquefaciens
in vitro appears to be similar to its effect on the rate of progress
of the infection in fish.

7. When juvenile coho and spring chinook salmon, and steelhead trout
are infected with Flexibacter columnaris by water contact, the percentage
of fatal infections is high at temperatures of 64 to 74 F, moderate

at 59 F, low at 54 F, and approaches zero at 49 F and below.

8. The mean time to death for juvenile coho and chinook salmon and
steelhead trout after exposure to F. columnaris by water contact falls
in the range of 1.7 to 2.5 days at 60 F. At 54 F progress of the disease
is retarded, resulting in a range of 6.7 to 11.0 days.

9. A temperature of 54 F is close to the threshold for development of
fatal infection of salmonids by F. columnaris. All the evidence indicates
that temperatures below 54 F are required for complete suppression of

the disease in salmonid populations that have been exposed to the pathogen.


the eight temperatures used in experiments with fish. Growth was deter-
mined at selected intervals by measuring optical density at 650 nm. The
growth rates observed are shown in Fig. 4. At 39 F growth was barely mea-
surable and was quite slow at 44 F. The rate increased significantly at

49 F and was still greater at 54 F. Temperatures from 59 to 74 F resulted
in further increases in rates, which reached a maximum at 74 F. When these
data are compared with the mortality data in experiments with salmonid
fish it is apparent that the temperatures from 49 to 74 F, which gave

the highest in vitro growth rates, are also those that produced the highest
mortality and the shortest mean times from infection to death in inoculated
fish. The three lowest temperatures, 39, 44 and 49 F, with the lowest
growth rates, were those which consistently suppressed the development

of fatal infections in fish. Thus it seems reasonable to assume from these
in vitro data that the effect of temperature on the growth rate of the
pathogen is in part responsible for the effects of temperature on the
disease process in fish.

24


SNAMES3 EXPTYPE,4 TEMPS Ay 398 PMORT

SSET,B=(A73) "100.

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60


PROBLEM

I-D-COL-1

ANALYSIS OF VARIANCE FOR PMORT
LINE SOURCE OF VARIATION

1)
2)
(3)

C4)

SOURCE

TYPE

TEMP

TYPE

TEMP

EXPTYPE

TEMP*EXPTYPE

ERROR

TOTAL

MEANS

( 1) ( 2)
45275000 14450000
( 1) (2)
100.000 51.000
( 5) 6)
10,000 t)
X TEMP (TEMP
100,000 100.000
16,000 0
100.000 2.000

4.000 0

DF MEAN SQUARE F
7 -5.04392B 03 325442
1 7481250B 03 504.03
7 = 1,68564E 03 108.75

16 = 1.55000E 01

31

( 3) ( 4)
51.000 28,000
(7) ( 8)
1.000 t)

VARIES MOST REPIDLY. )
92,000 56,000
2.000 )
10,000 C)
0 0

Analysis of Final Percent Mortality Data

in Text Table

7 . Columnaris Disease

in Steelhead Trout.

least significant difference 8.21%
for P = 0,05

Least significant difference 11.31%
for P = 0,01

62


SNAME, 7 ZMPT VIC SG TEM, 6 Ay? C8 PMORT

SSET,ASCE7IIEINO.
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PAVTARLE, 5
ANALYSTS GF VAZIANCE FOR PNCRT
LINE Suz? OF VARTATTON .F
(i) Teme 6
© 2) exeryae 1
3) TEMP FE XP TYPE 6
t wD ERI 14
TOTAL 27
BINTERMEANS 9394 TH
EXPTYPE
wate FREQ MEAN CF PMOPT
coh 16 5.96273 3E=01
FXP. 14 6.944024F 41
TEMP
VALUE FREQ MEAN OF PMOOT
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63

MEAN SQUAPE F
1609217E v3 12.35
SI3LA545 Oh 374.92
4.02N00r 03 11.52
BeAGISLE OL

Analysis of Final Percent Mortality
Data in Text Table /0. Kidney
Disease in Coho Salmon.

Least significant difference 20.18%
for P= 0,05.

Least significant difference 28.01%
for P=0,01.


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21


FIGURES

RELATIONSHIP BETWEEN WATER TEMPERATURE AND LOG OF TIME TO
DEATH AFTER INFECTION OF JUVENILE CHINOOK SALMON WITH
AEROMONAS SALMONICIDA

RELATIONSHIP BETWEEN WATER TEMPERATURE AND LOG OF TIME TO
DEATH AFTER INFECTION OF JUVENILE STEELHEAD TROUT WITH
AEROMONAS SALMONICIDA

RELATIONSHIP BETWEEN WATER TEMPERATURE AND LOG OF TIME TO
DEATH AFTER INFECTION OF JUVENILE COHO SALMON WITH

AEROMONAS LIQUEFACIENS

EFFECT OF TEMPERATURE ON GROWTH RATE OF AEROMONAS LIQUEFACIENS

IN PEPTONE BEEF EXTRACT GLUCOSE BROTH

RELATIONSHIP BETWEEN WATER TEMPERATURE AND LOG OF TIME TO
DEATH AFTER EXPOSURE OF JUVENILE STEELHEAD TROUT TO
FLEXIBACTER COLUMNARIS

RELATIONSHIP BETWEEN WATER TEMPERATURE AND LOG OF TIME TO
DEATH AFTER INFECTION OF JUVENILE COHO SALMON WITH KIDNEY
DISEASE BACTERIA

RELATIONSHIP BETWEEN WATER TEMPERATURE AND LOG OF TIME TO
DEATH AFTER INFECTION OF JUVENILE STEELHEAD TROUT WITH
KIDNEY DISEASE BACTERIA

ANTIBODY RESPONSE TO AN INJECTION OF KILLED AEROMONAS

SALMONICIDA CELLS IN JUVENILE COHO SALMON ‘.ELD AT VARIOUS

WATER TEMPERATURES

vi

12

16

23

25

30

37

39

45


CONTENTS

Sections

I Conclusions

II Recommendations

III Introduction

Iv Equipment Design and Fabrication Phase

Vv Effect of Water Temperature on Infection of Salmonids by
Aeromonas salmonicida and Aeromonas liquefaciens

VI Effect of Water Temperature on Infection of Steelhead Trout
by Flexibacter columnaris

VII Effect of Water Temperature on Bacterial Kidney Disease
in Salmonids

VIII Effect of Water Temperature on Antibody Formation in Salmonids

IX Effect of Water Temperature on the Immune Response of Coho
Salmon to Oral Vaccination with a Killed Vibrio anguillarum
Bacterin

x Acknowledgments

XI References

XIL Appendices

27

33

42

49
55
56

57


The results of bacterial cultures of kidney tissue from the infected
groups of fish appear in Table 2. Aeromonas salmonicida was recovered
from 88 to 100% of the mortalities in each experimental group, and
these findings provided confirmatory evidence as to the cause of death.
The organism was not recovered from any control fish examined. It is of
interest to note that the bacterium was also recovered from a considerable
number of the fish that survived at temperatures of 49 F and below. This
was not the case in the coho salmon experiments (1). It seems probable
that the persistence of the bacteria in survivors may be related to the
relatively short duration of the chinook experiments; i.e. 13 days
compared to 55 days in the case of the coho. The longer holding period
might have provided the time necessary for the defense mechanisms of the
surviving fish to clear the invading bacteria from the tissues.

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). The correlation coefficient was -0.8607 and was found to be
highly significant (Appendix, page 65). The R2 value of 0.7407 indicates
that time to death was about 74% dependent on temperature, The mean
values decreased from 12.2 days at 44 F to 2.3 days at 69 F. Thus the
progress of the fatal infection in these fish was accelerated at the
higher temperatures and retarded at the lower temperatures. Values for
the mean time to death were computed on the basis of the fish that died
during the course of an experiment, rather than the total number of

fish in the test group. These values were calculated as the geometric
means of the individual times to death in days.

The effect of temperature on infection of juvenile steelhead trout by A.
salmonicida was studied in duplicate experiments carried out concurrently.
The fish averaged 35 gm, and the infecting dose of the bacterium contained
about 600 colony forming units, or about 2 LDs9- In this case all experi-
mental groups of fish were held at their respective temperatures over a
period of 33 days.

The results obtained are presented in Table 3. The highest water tem-
perature in these experiments was 74 instead of 72 F, which proved to be
too warm for the uninfected control fish, as 96% of them died within the
first few days. At 69 F however only 10% of the controls died, compared
to 96% of the infected groups. Among the latter the percent mortality
decreased with decreasing water temperature from 84% at 64 F to 30% at
54 F, 10% at 44 F, and 2% at 39 F. As in the chinook salmon experiments,
fatal infection was suppressed at 39 and 44 F, and progressively enhanced
as the temperature increased from 49 to 69 F.

Table 4 contains the results of bacterial cultures of kidney tissue from
the infected groups of fish. Aeromonas salmonicida was isolated from 90
to 100% of the fish that died in each temperature group. However, among
fish that survived, it was isolated from only 3 of 200 individuals that
were examined. Apparently natural defense mechanisms had eliminated the
bacterium from the tissues of most of the survivors.

10


15

16

Amount of Diet and Vaccine Consumed in 15

Coho Salmon Averaging 6.5 Grams in Weight,

Water Temperatures

Effect of Water Temperature on the Immune
Salmon to an Orally Administered Bacterin

Effect of Water Temperature on the Immune
Salmon to an Orally Administered Bacterin

viii

Days by Groups of 100
Held at Selected

Response in Coho
of Vibrio anguillarum

Response in Coho
of Vibrio anguillarum

50

52

53


TIME TO DEATH AFTER EXPOSURE (days)

Ww
{e)

Dw)
Oo

fs)

\ \ \ i i }
44 49 54 59 64 69 74

WATER TEMPERATURE (°F)

Fig. 2. Relationship between water temperature and log of time
to death after infection of juvenile steelhead trout
with Aeromonas salmonicida.

16


ENVIRONMENTAL

PROTECTION
AGENCY
EPA-600/3-76-021 _-
Aprit 1876 Ecological Recearth Sane>
LIBRARY

TEMPERATURE, INFECTIOUS DISEASES, AND THE
IMMUNE RESPONSE IN SALMONID FISH

D S7,

SZ
Ky \
cn prove

WN agen’

2
S
3
z,
cA

Environmental Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Duluth, Minnesota 55864


TIME TO DEATH AFTER EXPOSURE (days)

100;-

lo | l |]
39 44 49 54 59 64

WATER TEMPERATURE (°F)

Fig. 7. Relationship between water temperature and log of time
to death after infection of juvenile steelhead trout
with kidney disease bacteria.

39


SECTION II
RECOMMENDATIONS

In an earlier report dealing with the effect of water temperature on
infectious diseases of salmonid fish (1) a major recommendation was advanced
which is restated as follows: 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 temperatures 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 wovld shorten the period when
conditions are most favorable for the host. The experimental evidence
presented in this report confirms and strengthens our original observations
and provides additional validity to this principal recommendation.


10. Water temperatures in the range of 44 to 54 F appear to be optimal
for the development of fatal bacterial kidney disease among juvenile
coho salmon and steelhead trout inoculated with the organism. Percent
mortality is high in this range, moderately high at 59 F. A temperature
of 64 F is less favorable for the disease, as indicated by a significant
reduction in mortality, while at 69 F only 8 to 14% of the inoculated
fish develop fatal disease. It is believed these temperatures retard
progress of this infection.

11. Bacterial kidney disease differs from A. salmonicida and A. liquefaciens

and F. columnaris infections in the fact that it is suppressed by a water
temperature of 69 F. It is a slowly progressing infection in juvenile

coho salmon and steelhead trout. The mean time from inoculation until

death may vary from a range of 21.7 to 33.7 days at 59 F to a range of about
61.0 to 71.8 days at 44 F, At 39 F the process is further retarded
resulting in a mean time to death in the range of 87 to 98 days.

12. Agglutinating antibody against A. salmonicida produced in juvenile
coho salmon in response to a single injection of the killed bacteria in
Freund's adjuvant appears to be influenced by the water temperature at
which the fish are held. Temperatures of 59 to 69 F are optimal for
“antibody production, and the highest concentrations of antibody in the
blood are reached about 45 days after antigen injection. At this time
less antibody is produced at 54 F and still less at 49 F. At 39 F no
significant amount of antibody is formed, even after 60 days.

13. The oral administration of a vaccine consisting of formalin killed
Vibrio anguillarum cells incorporated in the Oregon Moist Pellet diet

to juvenile coho salmon protects these fish against fatal vibriosis when
they are exposed to the infection by residence in estuarine waters
containing this pathogen. Immunization appears to be effective when the
fish are held at any temperature from 30 to 69 F during the 15 day period
of vaccine administration. The oral vaccination does not induce the
formation of circulating antibody against the bacterium.


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35


TIME TO DEATH AFTER EXPOSURE (days)

| | | | a
49 54 59 64 69 74

WATER TEMPERATURE (°F)

Fig. 3. Relationship between water temperature and log of time
to death after infection of juvenile coho salmon with

Aeromonas liquefaciens.

23


Regression analysis of the data in Fig. 7 again confirmed the linear
nature of the relationship between temperature and the mean time to 2
death (Appendix, page 70). The correlation coefficient was -0.7927 and R
was 0.6284, showing that about 63% of the variation in time to death

was accounted for by the regression line.

40


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12

13

TABLES

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 salmonicida
Infection in Juvenile Steelhead Trout

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

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

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

Effect of Water Temperature on Aeromonas liquefaciens
Infection In Juvenile Coho Salmon

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

Effect of Water Temperature on Flexibacter columnaris
Infection in Juvenile Steelhead Trout

Effect of Water Temperature on Bacterial Kidney Disease
in Juvenile Coho Salmon

Effect of Water Temperature on Bacterial Kidney Disease
in Juvenile Steelhead Trout

Agglutinating Antibody Levels in Juvenile Coho Salmon Injected
with Formalin Killed Aeromonas salmonicida in Freund's
Adjuvant and Held at Various Water Temperatures from 39 to 69 F

Agglutinating Antibody Levels in Juvenile Coho Salmon Following

a Single Intraperitoneal Injection of Killed Kidney Disease
Bacteria in Freund's Complete Adjuvant

vii

11

13

14

18

19

21

22

29

35

36

44

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13


Experimental Phase

When the tempering process was completed, oral immunization was begun.

For a 15 day period, all groups of fish were fed Oregon Moist Pellet

diet containing 2 mg of the wet whole cell vaccine per gram of ration.

The diet was fed ad libitum, and because metabolism varies with temperature,
the different groups of fish consumed varying amounts of the vaccine.

The following table shows the amounts consumed by 100 fish averaging 6.5
grams in weight.

Table 14. Amount of diet and vaccine consumed in 15 days by groups of
100 coho salmon averaging 6.5 grams in weight, held at
selected water temperatures.

Temperature at which Total grams of Total milligrams of
fish were held diet consumed vaccine consumed
39 F 157 314
44 F 264 528
49 F 357 714
54 F 366 732
59 F 490 980
64 F 450 900
69 F 473 846

In the experiment with 3.3 gram fish, the amounts of diet and vaccine
consumed were of course considerably smaller, but were influenced by
temperature in a similar manner. During the immunization period a group
of control fish were maintained on the same diet without vaccine at 54 F,

After the 15 day vaccination period all groups of fish were tempered
back to 54 F. Water temperatures were changed by steps of 5 degrees F
and fish were held at the new temperature for 48 hours before the next
change was made. Thus tempering required a week for some groups. In the
first experiment where 6.5 grams fish were used, after all the fish had
been tempered back to 54 F they were held at this temperature for one
week before being challenged by exposure to water containing virulent
V. anguillarum. This was accomplished by transferring the fish to a
salt water rearing impoundment at Lint Slough on the Oregon Coast. At
this facility groups of the vaccinated fish were held in fiberglass

50


SECTION XII

APPENDICES
Page No.
A. Analyses of Final Percent Mortality Data in Text Tables.
1. Text Table 1. Aeromonas Salmonicida in Chinook Salmon. 58
2. Text Table 3. Aeromonas Salmonicida in Steelhead Trout. 59
3. Text Table 5, Aeromonas Liquefaciens in Chinook Salmon. 60
4, Text Table 7. Aeromonas Liquefaciens in Coho Salmon. 61

5. Text Table 9. Flexibacter Columnaris Disease in Steelhead Trout. 62
6. Text Table 10. Bacterial Kidney Disease in Coho Salmon. 63

7. Text Table 11. Bacterial Kidney Disease in Steelhead Trout. 64

B. Regression Analyses. Relation Between Water Temperature and

Log of Number of Days to Death.

1. Text Fig. 1. Aeromonas Salmonicida in Chinook Salmon. 65
2. Text Fig. 2. Aeromonas Salmonicida in Steelhead Trout. 66
3. Text Fig. 3. Aeromonas Liquefaciens in Coho Salmon. 67

4. Text Fig. 5. Flexibacter Columnaris Disease in Steelhead Trout. 68
5. Text Fig. 6. Bacterial Kidney Disease in Coho Salmon. 69

6. Text Fig. 7. Bacterial Kidney Disease in Steelhead Trout. 70

C. Analysis of Replicate Antibody Titers to Determine Least Significant

Difference Between Mean Antibody Titers in Text Table 12. 71

57


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Fig. 5. Relationship between water temperature and log of time
to death after exposure of juvenile steelhead trout to
Flexibacter columnaris.

30


ANALYSIS OF DAILY MORTALITY DATA USED IN COMPUTING :EAN
VALUES SHOWN IN TEXT FIG. 7 . KIDNEY DISEASE IN STEELHEAD

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70


Discussion

The results indicate that the effects of water temperature on experimental
infection of steelhead trout with F. columnaris were very similar to
those in coho and spring chinook salmon (1). In all three salmonid
species, the lowest temperatue at which deaths were observed due to
infection by F. columnaris was 54 F, while mortality increased progressively
with increasing temperature in the range from 54 to 69 F. At 49 F and
below, conditions were unfavorable for progress of the infection. The
effect of temperature on defense mechanisms of the host is not known.
However, the rapid growth of F. columnaris in vitro at temperatures of

64 to 74 F, and the very limited growth at 54 F and below suggest that
the effect of temperature on mortality is due in part to its effect on
growth of the organism in the host's tissues.

The determination of the mean time between exposure to the pathogen and
death of the fish provides a measure of the rate at which fatal infection
develops. In all three salmonid species, this data showed clearly that
progress of the infection was accelerated progressively as the temperature
increased from 54 to 69 or 74 F. At 49 F and below no fatalities
attributable to F. columnaris occurred in any of the three species. It
seems likely that this accelerating effect of increasing temperature may
also be due in some degree to stimulation of growth of the bacterium.

The experiments reported were conducted under controlled conditions in
an experimental fish disease laboratory and the significance of the
results with respect to the effects of temperature on the occurrence

of columnaris disease under natural conditions deserves consideration.

It was important to use water containing no fish pathogens in the experi-
mental aquaria, rather than a natural river water, in order to minimize
the occurrence of disease symptoms and mortality caused by pathogens
other than the one being studied.

Although it cannot be inferred that the results obtained define exactly
the relationship of temperature to mortality from columnaris disease
that could be expected under natural conditions, they are not in conflict
with reported studies of this relationship as it affects the natural
disease. The influence of higher temperatures in streams and hatchery
troughs on incidence of the disease and initiation of the infection has
been reported by other workers (4, 3, 5, 6).

Ordal and Pacha (4) found that under experimental conditions, highly
virulent strains of F. columnaris could initiate infection in fish at
water temperatures as low as 55 F, while strains of low virulence could
do so only when the temperature was increased to 68 F. The strain used
in the experiments described here appears to fall in their category of
intermediate virulence, as it killed 100% of experimental fish exposed
by water contact at 69 F within 96 hours but not within 48 hours.

31


Antibody Response to Killed Kidney Disease Bacteria
At Various Water Temepratures

The results obtained in the experiment in which juvenile coho salmon
received a single injection of killed kidney disease bacteria (Coryne-
bacterium sp.) in Freund's adjuvant are shown in Table 13. Considerable
mortality which was attributed to infection with A. liquefaciens occurred
in the groups of fish held at 64, 69 and 74 F, and it was necessary to
eliminate these groups from the experiment after 30 days. It is apparent
that the results observed in the remaining temperature groups are quite
different from those in the similar experiment with A. salmonicida. In
this case the antibody response was much weaker at all water temperatures.
The antibody titers at 30 days indicated the range from 44 to 54 F had
permitted the formation of more antibody than lower or higher temperatures.
However at 60 and 90 days after injection, no significant differences

are apparent in the antibody levels from fish at 39 to 59 F. The data
obtained in this experiment do not show significant effects of water
temperature on antibody formation in response to a single injection of
the causative agent of bacterial kidney disease. Rather, they indicate
that this bacterium is a considerably weaker antigen than A. salmonicida.

After the final blood samples were collected at the 90 day period, the

25 vaccinated and 25 control fish in each temperature group from 39 to

59 F received a single intraperitoneal injection of about 9 x 108 living
kidney disease bacteria. This challenge was intended to provide information
concerning the degree of protection existing in the vaccinated fish held

at the various temperatures. However this phase of the experiment was
unsuccessful, as only 20 of the 250 fish that were injected with the living
organisms succumbed within an observation period of 120 days. Eight of

the 20 were from vaccinated groups and the remainder were controls.

46


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19


SECTION IX
EFFECT OF WATER TEMPERATURE ON THE IMMUNE RESPONSE OF COHO SALMON
TO ORAL VACCINATION WITH A KILLED VIBRIO ANGUILLARUM BACTERIN
Materials and Methods

The Vibrio anguillarum strain used in these experiments was isolated
from coho salmon which died at Lint Slough, Waldport, Oregon, and was
maintained on Cytophaga Sea water agar (Pacha and Ordal, 1967) at 4 C.
Ten ml of tryptic soy broth were inoculated from a stock culture and
incubated at 25 C for 12 hours. Two ml of this culture were then used to
inoculate each of two one liter volumes of the same medium, which were
also incubated for 12 hours. The resulting two liters of culture then
served as the inoculum for a 30 liter volume of the same broth in a
fermentor. After an incubation period of 10 to 12 hours, 500 ml of a 20%
dextrose solution was added, and incubation continued for an additional
12 hours. Sufficient formalin was then added to give a final concentration
of 0.3% formaldehyde. After one hour the cells were harvested in a
Sharples continuous flow centrifuge, and the wet packed killed whole
cells were frozen and stored at -26 C. For the oral immunization of fish
the wet packed cells, after thawing, were incorporated in the Oregon
Moist Pellet diet (8) in the proper proportion to give a final concen-
tration of 2 mg of vaccine per gm of the ration.

Juvenile coho salmon were used in oral vaccination experiments. In the
first experiment their average weight was 6.5 grams, and in a subsequent
experiment smaller fish averaging 3.3 grams were used. Seven experimental
water temperatures varying from 39 to 69 F at increments of 5 F were
employed in the experiments. One or two groups of 75 to 100 fish were
established at each of the seven temperatures, and the animals were
gradually adjusted or tempered to their respective temperature levels by
the method described in Section V.

49


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58


DISCLAIMER

This report has been reviewed by the Environmental Research Laboratory-
Duluth, U.S. Environmental Protection Agency, and approved for publication.
Approval does not signify that the contents necessarily reflect the views
and policies of the U.S. Environmental Protection Agency, nor does mention
of trade names or commercial products constitute endorsement or recommen—
dation for use.

ii


of a few mutant bacterial cells in the inoculum, which may have had

a greater virulence than the predominant cell population. The mean
number of days from inoculation to death increased to 39 at 54 F, 53 at
49 F and 73 at 44 F. At 39 F it was greater than 83 days.

Two parallel experiments with kidney disease were also carried out in
juvenile steelhead trout. The experimental design and procedures were
the same as in the coho salmon experiments. The influence of water
temperature on mortality from this disease is evident from the data in
Table 11. Again the percent mortality was greatest in the range of 44 to
54 F, varying from about 78 to 98%. It dropped to 49% at 59 F, about 43%
at 64 F, and about 8% at 69 F. At 39 F, about 36% of the inoculated fish
had died from kidney disease when the experiment was terminated. However,
of the 26 survivors, 21 were found by microscopic examination to be
harboring the kidney disease bacterium. Presumably then, as in the coho
salmon experiment, if these fish had been allowed to remain at 39 F for
a longer period, mortality from kidney disease would have approached
100%.

Thus the data from juvenile steelhead also indicated that the range of

44 to 54 F was optimal for the development of fatal infection by this
pathogen, and that higher temperatures had some suppressing effect,

which was greatest at 69 F. A complicating factor in these experiments
was the prevalence of tail rot in the population of steelhead. It is
presumed to be the cause of the deaths among uninoculated control groups
shown in Table 11. However, as mentioned previously, in calculating
percent mortality values due to kidney disease, only those dead fish

were counted which had the specific pathogen. Thus the possible distortion
of the data by non-specific infection should be largely eliminated.

The mean times from inoculation of the bacteria until death of the host
at the various water temperatures are shown in Fig. 7. Although percent
mortality was greatest in the temperature range of 44 to 54 F, the shortest
mean time to death was observed at 59 F. Some animals were apparently
able to overcome the inoculated dose of the pathogen when the body tem-
perature was held at 59 F, but others in the group were not, and in

these animals the disease process progressed more rapidly than in fish
maintained at lower temperatures. As the data show, there was a
progressive increase in the mean time to death as the temperature
decreased from 59 to 39 F. At 64 F the interval was slightly greater

than at 59. However at this temperature the efficiency of detection

of the pathogen in kidney tissue was less than at lower temperatures,
especially in fish that died within two weeks after inoculation. At

69 F the number of deaths was too small to permit a reliable estimation of
the mean time to death.

38


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Analysis of Final Percent Mortality

Data in Text Tabl ° Jigue-
faciens in Sond sd

least significant difference 5.02%
for P = 0.05.

Least significant difference 6.92%
for P= 0,01


SECTION V
EFFECT OF WATER TEMPERATURE ON INFECTION OF SALMONIDS BY

AEROMONAS SALMONICIDA AND AEROMONAS LIQUEFACIENS

Mareriais and Methods

The SS-70 BE-3 scrain of A. saimonicida, which was used in the experiments
reporced nere, was isolated from the kidney of a chinook saimon (Oncorhynchus
tshawycscha) at the South Sanciam Hatchery in Oregon. It was passed

through a series of 13 transfers in juvenile coho salmon (Oncorhynchus
kisucch) 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 sterile skim

milk and lyophilized.

Aeromonas liquefaciens, strain K-1, used in the experimencs with spring
chinook salmon, was isolated from the kidney of a shad (Alosa sapidissima)
during an epizootic in Coos Bay, Oregon. Stock cultures were maintained
on pepcone beef extract giucose agar covered with a layer of neutral
mineral oil. This mediwn contains 10 gm of pepcone (Difco), 5 gm of
glucose, 10 gm of beef extract, 5 gm of sodium chloride, and 15 gm of
agar per lirer. A. liquefaciens scrain JZ-45 was used in the experiments
with coho salmon. It was isolated from a juvenile spring chinook salmon
at the OSU Fish Disease Laboratory where it appeared to be causing fatal
infections in fish held at 64 and 69 F.

The salmonid tisn used 1n tne work reporced here were juvenile coho and
spring chinook salmon, and juvenile steelhead trouc. Their average
weignt ranged from 18 to 35 grams in different experiments. They were
generously donated for this project in relatively large numbers by the
Oregon Wildlife Commission and the Fish Cowmission of Oregon.

Experimencal intectious in fish were produced by the intramuscular or
intraperitoneal injection of 0.05 to 0.1 ml of a culture of the pathogen
in brain heart infusion (BHI) broth or peptone-beef excract-glucose
(PBG) broth suitably diluted to contain a small number of LDsg. Although
it would have been distinctly preferable to use a more natural method
for establishing infection, previous experience with A. saimonicida and
A. liquefaciens indicated that injection was the only method that could
be relied on to produce fatal infection in a large percentage of exposed
fish. These organisms, while pathogenic for fish, do not always possess
highly invasive properties.


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Discussion

The results of these experiments indicating that oral immunization of

coho salmon with V. anguillarum vaccine is as effective at 39 or 44 F

as at 59 to 69 F, on first consideration seem to conflict with the data
reported in Section VIII concerning the effect of water temperature on

the antibody response of coho salmon to an injection of killed cells

of A. salmonicida. In the latter system water temperatures of 59 and 69 F
were found to be optimal for antibody production. At these temperatures
maximum levels of antibody were found after 45 days, while at 39 F very
little antibody was present at this time. However, upon closer scrutiny,
certain differences are evident in the two systems which may help to account
for the apparent conflict in results. Perhaps the most important is the
fact that in the A. salmonicida experiment, agglutinating antibody
circulating in the blood was being measured, while in the oral immunization
experiments with the vibrio vaccine, no agglutinating antibody was found
in the blood. The mechanism by which oral immunity is produced has not

yet been clarified. It could be due to formation of a secretory antibody
localized in the intestinal tract, or possibly to a circulating antibody
that is unable to cause agglutination but may cause lysis of the bacteria.
Finally it might be a cellular type of immunity not associated with any
antibody activity.

Since immunity against vibriosis does not require the presence of agglutina-
ting antibody in the circulation, the effect of water temperature could
well be different from its effect on agglutinating antibody for A.
salmonicida. Another difference between the two systems that could influence
the results lies in the fact that in the A. salmonicida experiment it

was possible to maintain the fish at their respective temperatures
throughout the entire experimental period, while in the V. anguillarum
experiments it was necessary to temper the immunized fish back to 54 F
before they could be transferred to the water of Lint Slough for

challenge. This process required a week for some groups and it is possible
that this brief period of exposure to warmer water could have enhanced

the degree of immunity in the groups held at the lower temperatures.

In the pair of experiments carried out concurrently this difficulty

was minimized but was not eliminated entirely.

The data indicating the non-critical nature of water temperature in the
range of 39 to 69 F in the process of immunization of juvenile salmon
against vibriosis is also of some practical importance. This disease
constitutes a serious threat to the success of marine aquaculture projects.
An oral vaccine of the type described has now been tested rather extensively
under controlled conditions and found to uniformly give a high degree

of protection against the disease (9). The practical application of such

a vaccine will be facilitated by the knowledge that it can be used
effectively over a rather wide range of water temperatures.

54


Experimental Phase
Effect of Temperature on Infection with Aeromonas salmonicida

Experiments designed to determine the effect of water temperature on

experimental infection of juvenile coho and spring chinook salmon with
A. salmonicida have been described in an earlier report (1). However

irregularities in the results obtained with the chinook salmon strongly
indicated that some factors other than temperature were influencing the
mortality data. Accordingly these experiments were repeated and similar
tests carried out for the first time in juvenile steelhead trout (Salmo

gairdneri).

In the experiments with spring chinook salmon, 400 fish averaging 19
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 infected,
and eight contained fish to be used as uninfected controls. One tank in
each group of eight received flowing water at 72 F, another received
water at 69 F, a third received water at 64 F, and so on, so that groups
of fish were maintained at each 5 degree increment of temperature from
39 to 69 F, and also at 72 F. A second identical experiment, requiring
an additional 400 fish, was carried out concurrently.

Fish to be infected received an intraperitoneal injection of 0.1 ml of a
19 hour culture of A. salmonicida strain SS-70 BE-3 in BHI broth diluted
to contain about 5400 colony forming units, or about 18 LDsg. Control
fish received a sham injection of 0.1 ml of sterile phosphate buffered
saline. After injection all groups of fish were held at their respective
temperature for 13 days. The experiments were terminated at that time
due to a disease condition referred to as tail rot in both infected and
control groups at 54 F and below. Dead fish were collected daily, autopsied
and cultures prepared by smearing kidney tissue on plates of BHI agar.
After termination, all surviving fish in infected groups of 5 controls
from each temperature group were autopsied and cultured. Aeromonas
salmonicida colonies were identified by Gram stain, colony morphology,
brown pigment production, and a positive oxidase reaction.

Results of the two experiments are shown in Table 1. Among the infected
groups the mortality exceeded 90% at 69 and 72 F, and in most cases
decreased significantly with each 5 degree reduction in temperature.

Thus it was lower at 64 than at 69 F, at 59 than at 64 F, at 49 than at

54 F, and at 44 than at 49 F. The data show that the development of

fatal infection in juvenile chinook salmon due to A. salmonicida was
suppressed at water temperatures of 39 and 44 F, and enhanced progressively
at temperatures of 49, 54, 64 and 69 F. These results are very similar

to those obtained in comparable experiments with juvenile coho salmon

(1).


EPA-600/3-76-021
April 1976

TEMPERATURE, INFECTIOUS DISEASES, AND THE
IMMUNE RESPONSE IN SALMONID FISH

by

J. L. Fryer
K. S. Pilcher
J. E. Sanders
J. S. Rohovec
J. L. Zinn
W. J. Groberg
R. H. McCoy

Microbiology Department
Oregon State University
Corvallis, Oregon 97331

Grant No. R~800171

Project Officer

Gerald R. Bouck
Western Fish Toxicology Station*
Environmental Research Laboratory—Duluth
Corvallis, Oregon 97330

(4Western Fish Toxicology Station is now attached
to the Corvallis Environmental Research Laboratory
in Corvallis, Oregon 97330)

U.S. ENVIRONMENTAL PROTECTION AGENCY

OFFICE OF RESEARCH AND DEVELOPMENT

ENVIRONMENTAL RESEARCH LABORATORY
DULUTH, MINNESOTA 55804


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29


ANALYSIS OF THE REPLICATE ANTIBODY TITERS IN APPENDIX TABLE A TO DETERMINE
THE LEAST SIGNIFICANT DIFFERENCE BETWEEN MEAN ANTIBODY TITERS IN
TEXT TABLE 12 AND FIG. 8

Twenty nine sets of 5 replicate antibody titers from appendix Table A were
selected for the analysis. Fifteen represented serum samples from vaccinated
fish and 14 were titers from unvaccinated controls. The 15 sets of data from
vaccinated fish included those showing the greatest degrees of variation
among the 5 replicates in each set.

All of the titers were first transformed to logyg values, and the mean of
each set of 5 replicates was determined. Deviations from the mean were
then calculated for each value in each set; each deviation was then squared
and the sum of all these squares determined.
Sum of squares of deviations from mean values = 32.9823
Divisor (sum of degrees of freedom) or (5-1) x 29 = 116

32.9823

Variance = Tie 7 0. 28433

Estimated standard deviation = J0. 28433 0.53322

i Y 2
Standard error of difference 0.53322 ~ 0.53322 -
between means ( 5 af 25 7.23606 * 1.414 = 0.33718

With 116 degrees of freedom and P = 0.05, t = 2.0
Least significant difference = 2.0 x 0.33718 = 0.67436 = 10819 of 4.73

Hence 2 mean titer values in test Table 12 must differ by 0.67436 log units,
or 4.73 fold to be considered significantly different.

71


Effect of Temperature on Infection with Aeromonas liquefaciens

Experiments providing data concerning the influence of water temperature
on experimental infection of juvenile steelhead trout with Aeromonas
liquefaciens have been reported previously (1). Similar studies were
repeated on juvenile coho and spring chinook salmon and the results are
presented below.

Two parallel experiments were conducted concurrently with spring chinook
salmon. Four hundred fish, averaging 18 gm in weight, were used in each
experiment. They were distributed randomly among 16 tanks, 25 fish per
tank. The experimental design was as before except that fish to be
infected received an intraperitoneal injectionjof 0.05 ml of a suspension
of A. liquefaciens in PBS containing 1.22 x 10 organisms, or about 2
LD, . The inoculum was prepared from an 18 hour culture of the bacterium
in Bat broth by washing and resuspending in PBS. Uninfected control fish
received an injection of 0.05 ml of sterile PBS. All groups of fish were
at their experimental temperature levels for 12 days post injection. Dead
fish were collected daily and cultures of kidney tissue were prepared on
BHI agar plates. Identification of A. liquefaciens colonies was made by
agglutination with specific antiserum. At termination of the experiments
all surviving fish in the infected groups, and five uninfected controls
from each temperature group were autopsied and cultures of kidney tissue
prepared on BHI agar.

Results of the two experiments are shown in Table 5. The percent mortality,
which was 78% at 72 F, decreased progressively with decreasing temperatures
to 38% at 54 F, and to less than 15% at 49 F or below. A few deaths
occurred among uninfected controls in all temperature groups. Recovery

of A. liquefaciens by culture of kidney tissue of infected fish and
controls is recorded in Table 6. Ninety four to 100% of fatally infected
fish held at temperatures of 54 to 72 F yielded cultures of the organism,
while no cultures were recovered from any of the infected fish that
survived. Apparently the survivors had been able to eliminate the inoculated
bacteria from the tissues during the 12 day experimental period. The
organism was not recovered from the 20% of the uninfected control fish

that were examined. The mean time to death was very short with this
pathogen and was not significantly affected by temperature in the range
from 59 to 72 F. Apparently fatal infections were suppressed at tem-
peratures of 49 F and below, and were progressively enhanced as the
temperature increased from 54 to 72 F. These results in juvenile chinook
salmon are very much like those previously reported, where the same
pathogen was used to infect steelhead trout (1).

When infection in juvenile coho salmon with A. liquefaciens was studied,
the experimental design was essentially the same as that used with the
chinook but certain details were different. Thirty five coho were used
in each group instead of 25, and their average weight was 25 grams. Fish

17


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Discussion

The experiments described have indicated some definite effects of water
temperature on infection of juvenile coho salmon and steelhead trout
with the bacterium of kidney disease. In general, these effects are very
similar in the two salmonid species. In both cases the temperature
range of 44 to 54 F was found to be optimal for the development of

fatal infections. At higher temperatures, percent mortality declined
progressively, and at 69 F was reduced to values of only 8 to 13%.

The above optimal temperature range seems consistent with the observation
that mortality from the disease in nature is most apparent in the fall
of the year when water temperatures have cooled well below those of
summer.

Experimental kidney disease was observed to be a slowly progressing
infection in both species. The mean interval from inoculation until death
varied from a minimum of about 23 days to more than 89 days, depending
on water temperature. In both coho and steelhead the interval increased
progressively as the temperature decreased from 59 to 39 F.

Kidney disease is the only one of the bacterial infections of salmonids
that have been studied in which temperatures of 64 and 69 F exerted a
suppressive effect on the disease process. In the A. salmonicida, A.
liquefaciens, and F. columaris infections higher temperatures were
associated with the maximum mortality percentages. It seems quite possible
that the much lower optimum range for the kidney disease process may
reflect the optimum temperature range for the growth of the bacterium.
This is not known precisely, but has been reported to be about 59 F (15
c) (7).

4l


OPTICAL DENSITY (650 nm)

*40F

“32

+24

-08

74°F

69°F

T

“16 44°F

39°F
—O
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) 40 80 120 160
INCUBATION TIME (hours)

Fig. 4. Effect of temperature on growth rate of Aeromonas
liquefaciens in peptone beef extract glucose broth.

25


Experimental Phase

The results of duplicate experiments with kidney disease in juvenile
coho salmon are presented in Table 10. The inoculated and control groups
of fish were observed at their respective temperature levels over a
period of 112 days. At this time deaths among experimental fish had
ceased to occur in all groups except those at 39 F. The percent mortality
values in Table 10 were based on dead fish in which the specific pathogen
was demonstrated at autopsy. This was done because the lengthy period
during which these fish were held provided greater opportunity for
occurrence of deaths from causes other than kidney disease. Such deaths
are especially evident among the control groups of steelhead in Table

11.

In Table 10 the combined data from the two experiments with coho show
that mortality approached 100% in the temperature range of 44 to 54 F.

As the temperature increased above 54 F, mortality declined progressively,
and was only 40.8% at 64 F and 13.6% at 69 F. When the experiment was
terminated after 112 days, mortality at 39 F had reached 63.4%, but fish
were still dying from kidney disease. At this time the 15 surviving fish
in the infected groups, and the 46 controls were tempered to 54 F water
by the method described in Section V. Within two weeks all of the fish in
the infected groups had succumbed to kidney disease, whereas none of the
control fish had died. This indicates that the pathogen was still viable
in the inoculated fish held at 39 F, and that progress of the infection
was accelerated by increasing the temperature to 54 F. Presumably if
these fish had been allowed to remain at 39 F for a longer period the
mortality from kidney disease would have approached 100%.

The relationship between water temperature and log of time to death

was linear. This parallels the relationship between these two variables
described previously (Section V and VI) for A. salmonicida and F.
columnaris infections. The regression analysis of the data is shown in
Fig. 6 (and Appendix, page 69. The correlation cgefficient was

-0.7496, a statistically significant value, and R° was 0.5619, indicating
that time to death was about 56% dependent on temperature. The values

for mean time to death decreased from 84.2 days at 39 F to 22.5 days at
69 F. Increasing temperature was associated with shorter survival and
higher mortality.

Although 69 and 64 F were the most unfavorable temperatures for the

production of fatal disease, as shown by the mortality data, fish that

did develop fatal infection at these temperatures exhibited the shortest
survival time between inoculation and death, i.e. 25 and 26 days, respectively.
In other words the disease process progressed more rapidly and completely

in these fish than in others held at 44 to 59 F. The reason for this is

not clear, but it seems possible that the higher temperatures of 69

and 64 F could have exerted a selective influence favoring the growth

34


to be infected received an intramuscular injection of 0.05 ml of an

18 hour culture of A. liquefaciens, strain JZ-45 in BHI broth, diluted

to contain about 2.9 x 10’ cells, or about 1.5 LD59. Uninfected controls
received the same volume of a sterile filtrate of the broth culture,
similarly diluted. All experimental groups were held at their respective
temperatures for 15 days. Dead fish were collected daily and cultures

of kidney tissue prepared on BHI agar plates. Ten control fish from

groups held at 54 F and above were sacrificed and examined by kidney culture
at the end of the experiment.

The data obtained in the coho salmon experiments appears in Table 7.

All of the inoculated fish in the experimental groups held at 64 F and
above succumbed to infection with this pathogen, as did 97% of those held
at 59 F. At 54 F, mortality decreased significantly to 41%, and no deaths
occurred at 49 F or below. All control fish remained healthy during the
experimental period. Results of culturing kidney tissue of fatally
infected fish and control fish are given in Table 8. All of the inoculated
fish that died in groups held at 54 F and above yielded cultures of

A. liquefaciens. This organism was isolated from three specimens among 50
control fish from these five temperature groups that were examined by
culture.

The mortality data from the coho salmon resemble closely the comparable
data from steelhead trout (1) and chinook salmon. Fatal infections did

not develop at water temperatures of 49 F or below, but appeared first

at 54 F, affecting about 41% of the fish. At higher temperatures, mortality
increased rapidly to 100%. It is also evident from the data in Table 7

that the disease process in the infected fish progressed most rapidly at
the higher temperatures. This is indicated by the mean times from infection
to death which increased from about 1 day at 74 and 69 F to 4.7 days at

54 F.

As reported in the A. salmonicida experiments, when the log of the number

of days from inoculation until death was plotted against water temperature,
the relationship was found to be linear in the range from 54 to 69 F (Fig. 3).
Regression analysis revealed a correlation coefficient of -0.7017 (Appendix,
page 67) which was highly significant, and a coefficient of determination

(R°) of 0.4924. The latter indicates that the time to death was about 49%
dependent on temperature.

In view of the fact that percent mortality due to A. liquefaciens

in salmonids increased progressively with increasing water temperature

and that the disease process was accelerated by higher temperatures,

it was of interest to determine the relationship of temperature to the
growth rate of the organism in vitro. Accordingly peptone beef extract
glucose broth was inoculated with the K-1 strain at an initial concentration
of 104 to 105 cells per ml and distributed in 100 x 13 mm screw cap tubes
in three ml aliquots. Groups of these tubes were then incubated at each of

20


SECTION III
INTRODUCTION

The chief objectives of this project were: (a) To determine the effect
of water temperature upon the mortality resulting from the more important
infectious diseases of salmonid fish; and (b) To obtain additional
information concerning the effect of water temperature on antibody
formation and the immune response in salmonid fish.

The diseases which have been studied have included those caused by
Flexibacter columnaris, Aeromonas salmonicida, Aeromonas liquefaciens,
and the organism causing bacterial kidney disease (Corynebacterium sp.).
In an earlier report (1) similar studies of ceratomyxosis and infectious
hematopoietic necrosis were described. Fish species in the experiments
reported here were juvenile coho (Oncorhynchus kisutch) and chinook
salmon (Oncorhynchus tshawytscha) and steelhead trout (Salmo gairdneri).

The general experimental plan consisted of the following: (a) infection
of susceptible fish species by the most appropriate method; (b) subsequent
observation of these fish at one of eight temperatures in flowing pathogen
free water. Eight temperatures from 39 F to 74 F, with 5 increments,
were 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 were observed daily for appearance of symptoms,
lesions, or fatal infections. Dead fish were removed when observed,

and were autopsied and bacteriological cultures prepared from appropriate
organs. Observations were continued until no further deaths occurred.

The effect of the various water temperatures upon each type of infection
was judged by the fraction of the group of fish held at each temperature
that developed fatal infection caused by the specific pathogen, and by
the mean death time for those that succumbed in each group.


SECTION XI
REFERENCES

Fryer, J. L. and Pilcher, K.S. Effects of Temperature on Diseases of
Salmonid Fishes. U.S. Environ. Protection Agency, Office of Res.
and Development. EPA-660/3-73-020. 114 p. 1974.

Anacker, R.L. and Ordal, E.J. Studies on the Myxobacterium
Chondrococcus columnaris. I. Serological typing. J. Bacteriol.
78, pp. 25-32. 1959.

Pacha, R.E. and Ordal, E.J., Myxobacterial Diseases of Salmonids.
In: A Symposium on Diseases of Fishes and Shellfishes. Special
Publication No. 5. Amer. Fish. Soc., S.F. Snieszko, Ed. pp. 243-257. 1970.

Ordal, E.J. and Pacha, R.E. The effects of Temperature on Disease in
Fish. Proc. 12th Pacific Northwest Symp. Water Pollution Res.
pp. 39-56. Nov. 1963.

Colgrove, D.J. and Wood, J.W., Occurrence and Control of Chondrococcus
columnaris as Related to Fraser River Sockeye Salmon. Inter. Pacific
Salmon Fisheries Comm., New Westminster, B.C., Progress Rept. No. 15. 1966.

Fujihara, M.P., Olson, P.A., and Nakatani, R.E. Some Factors in
Susceptibility of Juvenile Rainbow Trout and Chinook Salmon to
Chondrococcus columaris. J. Fish. Res. Bd. Canada 28, pp. 1739-1743. 1971.

Ordal, E.J. and Earp, B.J., Cultivation and Transmission of the
Etiological Agent of Kidney Disease in Salmonid Fishes. Proc. Soc.
Exp. Biol. Med. 92, pp. 85-90. 1956.

Hublou, W.F. Oregon Pellets, The Prog, Fish Culturist. 25:175-180. 1963.
Fryer, J.L., Nelson, J.S. and Garrison, R.L. Vibriosis in Fish.
p. 129-133. In R.W. Moore (ed.) Prog. in Fishery and food science.

Vol. 5. Univ. of Washington Publications in Fisheries New Series.
Seattle. 1972.

56


TECHNICAL REPORT DATA

nope es (Please read Instructions on the reverse before completing)

1. REPORT NO. 2. 3. RECIPIENT’S ACCESSION NO,
EPA~600/3-76-021

4. TITLE AND SUBTITLE 5. REPORT DATE

April 1976 (Issuing Date)
i6. PERFORMING ORGANIZATION CODE

PP RAT

RULTONS DISPASES ANL THE IMM
IN SALMONTIL FISv

7, AUTHOR(S) (8. PERFORMING ORGANIZATION REPORT NO.
ToL. FRYER, ¥.S. PILCELR a
JWS. ROMOWTC, Tot. ZINTOOWT. :

9. PERFORMING ORGANIZATION NAME AND ADDRESS 10. PROGRAM ELEMENT NO,
TIS! EPALTY INIT TBABCS
DEPARTMENT OF MICAORTOLETY 17 CONTRACTIGRANT NO-
ORFGUN STATE MNIVERE ITY

>

LORVALLIS, OREGON 97330 ReRCOL7T

12. SPONSORING AGENCY NAME AND ADDRESS 13. TYPE OF REPORT AND PERIOD COVERED
ENVIRONMENTAL RESEARCH LABORATORY Final: April '73«tarch 1974
OFFICE OF RESEARCH AND DEVELOPMENT 14, SPONSORING AGENCY CODE
U.S. ENVIRONMENTAL PROTECTION AGENCY EPA-ORD

DULUTH, MINNESOTA 55804
75. SUPPLEMENTARY NOTES

76. ABSTRACT

Investigations of the effect of temperature on tafections of salmenid fish
were conducted. Aeromonas saliionicida infcetion was studfed fn chinook salmon and

stcelhead trout; Aeromonas liquefaciens tafcetion was studied tn chinook and coho
saluon. In all cases, mortality rates were high at 64 to 69 Fs; usually moderate at
£4 to 59 F; and low or zero at 39 to 49 F. Progress of the infections was accelerateph
at higher temperatures anc retarded at lower temperatures. bacterial kidney Jiseace|
was studied in choh salmon ard steelhead trout. The tevperature rarge of 44 to 54 wab
optimal for the development of fatal infection as indicated hy mortality rates cf 7%

i 100%. Higher temperatures had a suppressing effcct, which was marked at 69 F.
ewperatures of 53 to 69 F were cptinal for the formation of agglutinating antibody
when juvenile choh salmon were infected with a killcd suspension of A. ca
At lower Cemperatures less aut tho:
at 29 F £9 days after injection of antigen. Cral is
with a vaccine consistedof formalin killed Vibrio
theirs diet , protected them against fate2i infection wh

temperatures fror. 39 to £9 during immunization.

y was formed and ac significant anount was producep

minization of juvenile coho salmon
illarun: ceils incorporated in
en the fish were held at

n7. KEY WORDS AND DOCUMENT ANALYSIS
a. DESCRIPTORS b.1DENTIFIERS/OPEN ENDED TERMS |[c. GOSATI Ficld/Group
SALMON, TACUT, CNVIKONMENTS, PATHOPHYSIOLOGY THERMAL POLLUTION O6/cf/F/M/S/T

WATER PCLLUTION, WASTD VATek, FRESHVATER
DISEASES, ANTIBODIES

18. DISTRIBUTION STATEMENT 19. SECURITY CLASS (This Report) 21. NO. OF PAGES
a
RELEASE TO PUBLIC UNCLASSIFIED li
20. SECURITY CLASS (This page} 22. PRICE
UNCLASSIFIED
EPA Form 2220-1 (9-73) 72

ic US GOVERNMENT PRINTING OFFICE 1976-657-695/5407 Region No. 5-11


SECTION VIII
EFFECT OF WATER TEMPERATURE ON ANTIBODY FORMATION IN SALMONIDS
Materials and Methods

Juvenile coho salmon averaging 34 grams in weight were tempered to experi-
mental temperatures of 39, 44, 49, 54, 59, 64, 69 and 74 F by the method
described in Section V of this report. Each temperature group consisted
of 100 fish, which were divided equally between controls and those to be
injected with a killed suspension of A. salmonicida. Fifty fish at each
temperature received an intraperitoneal injection of 0.1 ml of an
emulsion containing equal parts of a BHI broth culture of the organism,
killed by 0.3% formalin, and Freund's complete adjuvant. The injected
dose represented about 1.5 x 107 cells. Equal numbers of control fish
were injected with the same volume of an emulsion containing equal parts
of 0.85% saline and the adjuvant. At 15 day intervals after the antigen
injection five vaccinated and five control fish at each temperature were
bled and individual antibody titers were determined on each serum sample
by tube agglutination. The experimental groups held at 74 F had to be
eliminated from the experiment because of high mortality rates in both
control and vaccinated groups.

An experiment similar to the above was performed, in which groups of
juvenile coho salmon, held at the same eight water temperatures, received
a single intraperitoneal injection of formalin killed kidney disease
bacteria, emulsified in Freund's complete adjuvant. The organisms were
grown on cysteine serum agar for two to three weeks at 15 C and killed
by the addition of 0.3% formalin. The emulsion was prepared with equal
parts of the killed culture and the adjuvant. The injected dose contained
about 2 x 10? cells. Control fish received a similar injection of an
emulsion containing 0.85% saline with the adjuvant. At intervals of 15
days after injection, five vaccinated fish and five controls from each
temperature group were bled, and the serum specimens titrated for antibody
by tube agglutination.

42


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44


The method used to temper fish to the various experimental water temperatures
was as follows: When received from the hatchery, the fish were maintained
at 54 F. At the beginning of an experiment, the fish to be used were
transferred to an 18 gallon tank, and the water was gradually replaced

with water at the next temperature increment, either 49 to 59 F, over a
period of 1 to 1.5 hours. Fish were held at the new temperature for 48

hours before the cycle was repeated by replacing the water and achieving

the next temperature increment either 44 or 64 F. This process was

repeated until groups of fish had reached the assigned temperature levels
covering the range from 39 to 72 or 74 F at five degree intervals.

Infections were confirmed at necropsy by streaking small fragments of

kidney tissue on plates of BHI agar, which were incubated at room temperature
(about 22 C). Colonies of A. salmonicida were identified by Gram stain,
colony morphology, brown pigment production, and a positive oxidase

reaction. Identification of colonies of A. liquefaciens was made from

the cultures incubated at 37 C by agglutination with specific antiserum

(A. salmonicida does not grow at 37°C).

The experimental design adopted in this work required the use of sixteen
18 gallon aquaria for each experiment. Thus two tanks were provided for
each of the eight 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 essentially
identical experiments were conducted concurrently, each one consisting

of eight groups of infected fish and eight control groups. The purpose

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.