EPA-660/3-73-020
January 1974                           Ecological Research Series
      Effects  of Temperature on
      Diseases of Salmonid Fishes

                                     ^
                                  Office of Research and Development
                                  U.S. Environmental Protection Agency
                                  Washington, D.C. 20460

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            RESEARCH REPORTING SERIES
Research reports of the  Office  of  Research  and
Monitoring,  Environmental Protection Agency, have
been grouped into five series.  These  five  broad
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was  consciously  planned  to  foster   technology
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   1.  Environmental Health Effects Research
   2.  Environmental Protection Technology
   3.  Ecological Research
   4.  Environmental Monitoring
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RESEARCH  series.   This series describes research
on the effects of pollution on humans,  plant  and
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assessed   for   their   long-   and    short-term
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                                                                             c
                                               EPA-660/3-73-020
                                               January 1974
         EFFECTS OF TEMPERATURE ON DISEASES

                 OF SALMONID FISHES
                            By

                     J. L.  Fryer
                    K. S. Pilcher

               Oregon State University
               Corvallis, Oregon 97331
                  Project  18050 DIJ
               Program Element  1BA021
                   Project  Officer

                 Dr. Gerald R.  Bouck
         Western Fish Toxicology Laboratory
           Environmental Protection Agency
               Corvallis, Oregon 97330
                    Prepared  for

         OFFICE OF RESEARCH AND DEVELOPMENT
       U.S.  ENVIRONMENTAL PROTECTION AGENCY
               WASHINGTON, D.C. 20460
For sale by the Superintendent of Documents, U.S. Government Printing Office, Washington, D.C. 20402 - Price $1.85

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                      EPA Review Notice
This report has been reviewed by the Environmental
Protection Agency and approved for publication.
Approval does not signify that the contents necessarily
reflect the views and policies of the Environmental
Protection Agency, nor does mention of trade names or
commercial products constitute endorsement or recommenda-
tion for use.
                             ii

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                               ABSTRACT




The effect of x/ater temperature on infections of  salmonid  fish was

investigated. Chondrococcus colunnaris infection  was  studied  in  rain-

bow trout, coho and spring chinook salmon; Aeromonas  salmonicida in-

fection in coho and spring chinook salmon; and Aeromonas liquefnciens

infection in steelhead trout. In all cases mortality  rates were  high
   ru
at 64  to 69°F; moderate at 54° to 59°F; and low  or zero at 39°  to  49°F.

Progress of the infections was accelerated at higher  temperatures,  and

progressively retarded at decreasing temperature  levels.




In infection of coho with Ceratomyxa shasta, mortality was high  at

69 F, low at 49  to 54 , and zero at 39  to 44 F. This infection in

rainbow trout resulted in high mortality at all temperatures  except

39 . In both cases the course of the disease was  most rapid at higher

temperatures, and became progressively slower as  the  temperature de-

creased.




For infection of kokanee salmon fingerlings with  sockeye salmon  virus,

the temperature range of 54  to 59 F was optimal. In  this  range  mortality

rates were high, and the course of the disease most rapid. At higher

temperatures mortality rates were lower, and at 39  to 44  F,  progress

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




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


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

vironmental Protection Agency.
                                  iii

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                             CONTENTS
Section
I
II
III
IV
V
Conclusions
Recommendations
Introduction
Equipment Design and Fabrication Phase
Effect of Water Temperature on Infection of
Salmonids bv Aerorionas Salmonicida and Aeromonas
Liquefaciens
Page
1
5
7
9
11
VI        Effects of Water Temperature on Infection of
          Salmonids by Chondrococcus Columnaris

VII       Effects of Water Temperature on Infection of
          Salrnonids by the Parasitic Protozoan Ceratomyxa
          Shasta

VIII      Effect of Water Temperature on Infection by the
          Sockeye Salmon Virus  (I11N)

IX        Acknowledgments

X         References

XI        Appendices
55



69


89

91

93
                                 v

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                         FIGURES
                                                           PAGE

RELATIONSHIP BETWEEN WATER TEMPERATURE AND LOG OF TIME      20
TO DEATH AFTER INFECTION OF JUVENILE COIIO SALMON WITH
AEROMONAR SALMONICIDA

EFFECT OF TEMPERATURE ON GROWTH RATE OF AEROMONAS           22
SALMONICIDA IN PEPTOHE-BEEF-EXTRACT-GLUCOSE BROTH

RELATIONSHIP BETWEEN WATER TEMPERATURE AND LOG OF TIME      27
TO DEATH AFTER INFECTION OF JUVENILE CHINOOK SALMON
WITH AEROMONAS SALMONICIDA

RELATIONSHIP BETWEEN WATER TEMPERATURE AND LOG OF TIME      45
TO DEATH AFTER EXPOSURE OF JUVENILE RAINBOW TROUT TO
CUONDROCOCCUS COLUMNARIS

RELATIONSHIP BETWEEN WATER TEMPERATURE AND LOG OF TIME      48
TO DEATH AFTER EXPOSURE OF JUVENILE COIIO SALMON TO
CUONDROCOCCUS COLUMNARIS

RELATIONSHIP BETWEEN WATER TEMPERATURE AND LOG OF TIME      51
TO DEATH AFTER EXPOSURE OF JUVENILE CHINOOK SALMON TO
CliONDROCOCCUS COLUMNARIS

RELATIONSHIP BETWEEN WATER TEMPERATURE AND LOG OF TIME      62
TO DEATH AFTER EXPOSURE OF JUVENILE RAINBOW TROUT TO
CERATOMYXA SHASTA

RELATIONSHIP BETWEEN WATER TEMPERATURE AND LOG OF TIME      66
TO DEATH AFTER EXPOSURE OF JUVENILE COHO SALMON TO
CERATOMYXA SHASTA
                           VI

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                                TABLES
No.                                                              Page

1       Effect of Water Temperature on Aeromonas Salmonicida      17
        Infection in Juvenile Coho Salmon

2       Recovery of Aeromonas Salraonicida by Culture of Kidney    18
        Tissue of Juvenile Coho Salmon

3       Effect of Water Temperature on Aeromonas Salmonicida      24
        Infection in Juvenile Spring Chinook Salmon

4       Recovery of Aeromonas Salmonicida by Culture of Kidney    26
        Tissue of Juvenile Spring Chinook Salmon

5       Effect of Water Temperature on Aeromonas Liquefaciens     31
        Infection in Juvenile Steelhead Trout

6       Recovery of Aeromonas Liquefaciens by Culture of Kidney   32
        Tissue of Juvenile Steelhead Trout

7       Effect of Water Temperature on Chondrococcus Columnaris   44
        Infection in Juvenile Rainbow Trout

8       Effect of Water Temperature on Chondrococcus Columnaris   46
        Infection in Juvenile Coho Salmon

9       Effect of Water Temperature on Chondrococcus Columnaris   49
        Infection in Juvenile Spring Chinook Salmon

10      Incidence of Ceratomyxa Shasta and Mean Time to Death     60
        Post-exposure of Juvenile Rainbow Trout Exposed to
        Water Containing the Infective Stage of the Organism
        and then Placed in Temperature Regulated Disease Free
        Water

11      Incidence of Ceratomyxa Shasta and Mean Time to Death     64
        Post-exposure of Juvenile Coho Salmon Exposed to
        Water Containing the Infective Stage of the Organism
        and then Placed in Temperature Regulated Disease Free
        Water

12      Mortality Among Fingerling Kokanee Salmon Resulting       74
        from Various Concentrations of Sockeye Salmon Virus
        in the Aquarium Water at 54 F

13      Mortality Amonti Fingerlinp, Kokanee Salmon Resulting       75
        from Various Concentrations of Sockeye Salmon Virus
        in the Aquarium Water at 54 F
                                  vii

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No.                                                               Pape
14      Effect of Water Temperature on Mortality  of  1.1  gm         77
        Kokanee Salmon Fingerlings Exposed  to  Sockeye  Salmon
        Virus

15      Effect of Water Temperature on Mortality  of  2.9  p,m         78
        Kokanee Salmon Finp.erlinps Exposed  to  Sockeye  Salmon
        Virus

16      Mean Time to Death for 2.9 gm Kokanee  Salmon Finper-       80
        lings Exposed to Sockeye Salmon Virus

17      Effect of Water Temperature on Mortality  of  0.11 gm        81
        Kokanee Salmon Fry Exposed to Sockeye  Salmon Virus

18      Mean Time to Death for 0.11 p,m Kokanee Salmon  Fry         33
        Exposed to Sockeye Salmon Virus

19      Effect of Water Temperature Mortality  of  0.95  pm          84
        Kokanee Salmon Fry Exposed to Sockeye  Salmon Virus

20      Mean Tine to Death for 0.95 sm Kokanee Salmon  Fry         86
        Exposed to Sockeye Salmon Virus
                                  viii

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                               SECTION I




                              CONCLUSIONS
 1.  Water temperatures of 59 F and above produce high mortality  rates




in juvenile coho salmon injected with Aeromonas  salmonicida.  Even at




49  and 54  losses may exceed 40 percent.
 2.  Mortality rates of coho salmon injected with _A.  salmonicida  are




very low at temperatures of 39  and 44 F.
 3.  The mean time to death of coho salmon injected with A.  salmonicida




is estimated to be 3.5 days at 69 F, and this increases steadily  as




water temperature decreases, to a maximum of 31 days at 39 F.
 4.  The effect of temperature on  the growth rate of A.  salmonicida  in




vitro appears to be similar to its effect on the rate of progress  of the




infection in fish.









 5.  Among spring chinook salmon injected with ^. salmonicida,  the




mean time to death is estimated to be 2.9 days at 74 F,  and  this in-




creases progressively as water temperature decreases, to a maximum of




18.4 days at 39°F.









 6.  The percentage of fatal infections among steelhead  trout  injected




with Aeromonas liquefaciens, is high at temperatures of  64 F and above,

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moderate at 54  and 59 F, and zero at 49 F and below.
 7.  When coho and spring chinook salmon, and rainbow  trout  are  in-




fected with Chondrococcus columnaris by water contact,  the percentage




of fatal infections is high at temperatures of 64 F and above, moderate




at 59 F and approaches zero at 49 F and below.
 8.  A temperature of 54 F is close to the threshold  for  development




of fatal infection of salmonids by Chondrococcus coluranaris.
 9.  The percentage of fatal infections in rainbow  trout  infected  with




Ceratomyxa shasta is high at water temperatures between 74   and  44 F,
10.  The mean time to death of rainbow  trout  infected with £.  shasta is




approximately 14 days at 74 F, increasing  to  approximately 155 days




at 44 F. Fish continually held at  39 F  are not believed  to develop




fatal infection.
11.  The percentage of fatal infections among coho  salmon  infected




with C_. shasta, is high at 64 F and above, moderate at  54   to  59 , and




approaches zero at 49  and below.
12.  The effect of temperature on  the progress of  infection  by  C.  shasta




appears similar in rainbow  trout and coho  salmon,  as  indicated  by  the




mean time to death. There is a distinct difference in the  effect of

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temperature on susceptibility of these  two  species.  Coho  are  not




equally susceptible at all temperatures, while rainbow  trout  are.









13.  Among kokanee salmon fingerlings exposed to  the sockeye  salmon




virus mortality rates are high at water  temperatures from 39   to  59 F,




and significantly lower at 64  and above.
14.  While fatal infections due to  the virus may be high at  39  F,  the




mean time to death is much longer than at higher temperatures.

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                          SECTION II




                        RECOMMENDATIONS









Water temperatures in many rivers of the Pacific Northwest from May




through October are in a range favorable for the progress of the important




infectious diseases of salmonids.  During this period threshold temp-




eratures for these diseases are reached and a maximum of 70 F is not




uncommon.  Temperatures favorable to the host generally occur from




November through April.  It is therefore recommended that no additional




sources of heat should be allowed to enter these rivers.  Added heat during




the period from May through October could only serve to further enhance




the severity of these diseases.  Increasing water temperatures from




November through April would shorten the period when conditions are most




favorable for the host.









Data collected in this laboratory over the past 5 years as part of




another study indicate that the threshold temperature for initiation of




infection by Ceratomyxa shasta is approximately 50 F.  Results in these




studies revealed that once animals are infected with this organism fatal




disease develops over a wide range of temperatures.  Therefore it appears




that when temperatures exceed 50 F in waters where this agent occurs,




disease and deaths can be expected.









Water temperature should be considered before trout or salmon are re-




leased into streams or lakes.  Releases should not be made when the

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temperature exceeds 50 to 53 F.
Evidence gathered during this investigation indicates that infection with




the sockeye salmon virus occurs over a wide range of temperatures.  As a




result no practical recommendation could be made pertaining to changes




of temperature in waters containing this infectious agent.  It is con-




ceivable that temperatures above 64 F could be used to control progress




of the disease in fish rearing facilities equipped with water treatment




or reuse systems which could eliminate other pathogens.

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                          SECTION III




                         INTRODUCTION









The chief objective of the work described in this report has been to




determine the effect of water temperature upon the course of and




mortality from the more Important infectious diseases of the salmonid




fish native to rivers of the Pacific Northwest.









The diseases which have been studied have included those caused by




Ceratomyxa shasta, Chondrococcus columnaris, Aeromonas salmonicida,




Aeromonas liquefaciens, and the Oregon sockeye salmon virus.  Fish




species which have been used in these studies were juvenile coho and




chinook salmon and steelhead trout.  Fingerling kokanee salmon were used




in experiments with the virus.









The general experimental plan which has been followed with each disease




agent has been to infect groups of susceptible fish of a given species




by the most appropriate method, and to hold these groups in tanks of




flowing water, controlled at one of several temperature levels.  Eight




temperatures, from 39 F to 74 F, with 5  increments, have been provided.




For each experimental temperature, groups of 50 or more infected fish




have been employed, distributed equally between 2 tanks.  Parallel groups




of normal uninfected fish have been held under identical conditions.









All experimental fish have been observed daily for appearance of symp-

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toms, lesions, or fatal infections. Dead fish were removed immediately,




and were autopsied and the appropriate organs examined culturally for




the presence of the specific pathogen. Observations were continued until




no further deaths occurred.








The effect of the various water temperatures upon each type of infection




has been judged by the fraction of the group of fish held at each tem-




perature that developed fatal infection caused by the specific pathogen,




and by the mean death time for those that succumbed in each group.

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                              SECTION IV




                EQUIPMENT DESIGN AND FABRICATION PHASE









Before experiments dealing with the effect of water temperature on




infectious diseases of fish could be undertaken, special equipuent for




holding experimental animals at various temperatures had to be designed




and fabricated. This phase of the project was submitted to engineering




firms for bids, and was ultimately carried out by the Corvallis firm of




Cornell, Rowland, Hayes and Merryfield.









The equipment provided for holding fish consists of 64 covered fiber-




glass tanks or aquaria of about 21 gallon capacity. Sixteen of these




were new and 48 were already installed in the fish disease laboratory.




Water is supplied to the laboratory from a well, at a constant tempera-




ture of 54 F. Eight of the 64 tanks are supplied with flowing water at




that temperature. Eight tanks are supplied with water heated to one of




the following temperatures:  59 , 64 , 69  and 74 F; and 8 tanks  receive




water chilled to 49 , 44  and 39 F. The rate of flow of these various




streams of heated and chilled water is variable, with a maximum of 1.0




gallon per minute per tank. The temperature of each stream is automat-




ically controlled by a recorder-controller and mixing valve within a




range of ± 0.5 F. An alarm system gives warning of any failure that




might develop in temperature control.
The heated water is supplied by a gas fired boiler  capable of  supplying

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each of the 4 heated water streams at a rate of 8 gallons per minute.




The refrigerated water is produced by a stainless steel chiller of




special design and custom built, with a capacity adequate to supply




each of the 3 refrigerated streams at 8 gallons per minute. All equip-




ment, piping and valves that come in contact with the water supply




are of stainless steel or polyvinyl chloride, to eliminate possible




toxicity to fish. A small frame building, 16 x 10 ft. was constructed




to house the boiler, chiller and air compressor.









In order to provide protection against possible failure of the well




water supply to the laboratory, and to permit the periodic overhaul of




the pump in the laboratory well, it was necessary to make an alternate




source of water available. Piping was installed to connect the labora-




tory to an existing well located at a distance of about 150 yards. A




new pump with a capacity of 300 gallons per minute was required to




deliver the required volume of water from this second well.
                                  10

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                               SECTION V




   EFFECT OF WATER TEMPERATURE ON INFECTION OF SALMONIDS BY AEROMONAS




                SALMONICIDA AND AEROMONAS LIQUEFACIRNS









                         Materials and Methods









Two strains of Aeromonas salnonicida were used in the work reported




here. Strain 5-G was isolated from the kidney of a coho salmon during




an outbreak of furunculosis at the Siletz Hatchery in Oregon. Stock




cultures were maintained by cultivating the organism in peptone-beef




extract-glucose broth, centrifuging, resuspending the cells in sterile




skim milk, and lyophilizing. The second strain, SS-70 was isolated




from the kidney of a chinook salmon at the South Santiam Hatchery in




Oregon. It was passed through a series of 13 transfers in juvenile coho




salmon by intraperitoneal inoculation of a suspension of kidney  tissue




from the fish infected in the preceding transfer. Kidney tissue  from




the last fish in the series was then macerated, suspended in skim milk




and lyophilized.









Aeromonas liquefaciens, strain K-l, was isolated from the kidney of




shad during an epizootic in Coos Bay, Oregon. Stock cultures were main-




tained on peptone-beef extract-glucose agar covered with a layer of




neutral mineral oil. This medium contains 10 gm of peptone (Difco) 5 gm




of glucose, 10 gm of beef extract, 5 gm of sodium chloride, and  15 gm




of agar, per liter.
                                   11

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Experimental fish employed in these experiments were juvenile coho, or




spring Chinook salmon, or juvenile steelhead trout. Their average weight




ranged from 10 to 30 grams in different experiments. They were generously




donated for this project in relatively large numbers by the Oregon Game




Commission and the Fish Commission of Oregon.









Experimental infections in fish were produced by the intramuscular or




intraperitoneal injection of 0.05 ml of a 48 hour culture of the organism




grown in brain heart infusion broth (Difco Labs) or peptone-beef extract-




glucose broth (PBG) and resuspended in frog Ringer saline at pH 6.9-7.0.




The composition of the PBG medium with agar is described subsequently




in this report. The broth is prepared in the same manner, but without




the agar. The bacterial concentration was adjusted to represent from




0.5 to 2.0 LDcn doses, based on an earlier titration of the same organ-




ism in the same fish species held at 54 F. It would have been distinctly




preferable to use a more natural method for establishing infection, but




preliminary experiments and previous experience indicated that exposure




of fish to high concentrations of these aeromonas species in their water




supply, or the presence of infected fish in the tank with normal sus-




ceptible ones, could not be relied upon to produce fatal infections in




a large percentage of those exposed. These organisms while pathogenic




for fish, do not always possess highly invasive properties.









The method used to temper fish to the various experimental water




temperatures was as follows:  When first received from the hatchery,
                                  12

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the fish were placed in holding tanks supplied with well water at  54  F.




At the beginning of an experiment, the number of fish  to be used in one




experimental group were transferred to an 18 gallon tank, also supplied




with 54 F water. Water at the next temperature increment, either 49   or




59 F, from one of the controlled streams, was then introduced at the




rate of about one half gallon per minute. Within 1 to  1% hours, the water




in the tank had reached the 49  or 59  level. The fish were then held




at the new temperature for 48 hours. Water at the next temperature incre-




ment, either 44  or 64  was then introduced into the tank at the same




rate as before, and the new temperature maintained for 48 hours. This




process was repeated until groups of fish had been adjusted to each of




the eight experimental temperature levels covering the range from  39




to 74 F at 5 degree intervals.
For the cultural examination of  experimental  fish  at necropsy  small




fragments of kidney tissue were  streaked on plates of  Furunculosis Agar,




Difco, modified by the addition  of 1 gram of  skim milk solids  per liter




of medium. Plates were incubated at room temperature  (about  22 C) for




48 hours. Colonies producing, zones of clearing on  this medium  were




inoculated on two plates of regular Furunculosis Agar,  tubes of Oxidative-




Fermentative Medium, Difco, and  Arginine Decarboxylase Medium,  Difco.




One of the plates was incubated  at 37 C to inhibit jV.  salmonicida, the




other at room temperature to permit grov/th. The latter plate was then




used for determining morphology, the Gram reaction, motility,  catalase




and cytochrome oxidase reactions. j&. salmonicida is a  Gram negative,
                                   13

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non-motile rod that produces clearing on the Furunculosis Agar with




casein, fails to grow at 37 C, and forms catalase and cytochrome




oxidase. /L. liquefaciens differs by growing at 37 C and in being motile.
The experimental design adopted in this work required the use of sixteen




18 gallon aquaria for each experiment. Thus 2 tanks were provided  for




each of the 8 water temperatures. Eight tanks, one at each  temperature,




were assigned to groups of fish to be infected with the pathogen being




studied, while the remaining eight were assigned to groups  of uninfected




control fish that received sham injections. The number of fish per tank




was at least 25, and in some experiments was increased to 35. Two  com-




plete and identical experiments were conducted concurrently, each  one




consisting of 8 groups of infected fish and 8 control groups. The  pur-




pose of this plan was to provide information concerning the degree of




variation to be expected between groups of fish receiving,  insofar as




possible, exactly the same treatment.









The terms rainbow trout and steelhead trout are used in the text of this




report. It should be understood that both terms refer to a  single  species,




i.e. Salmo gairdneri, but steelhead are anadramous while rainbow trout




do not migrate to the ocean.
                                   14

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                          Experimental Phase









     Effect of Temperature on Infection with Aeromonas saliaonicida









The effect of water temperature on experimental infection of juvenile




coho salmon (Oncorhynchus kisutch) with Aeromonas salmonicida, strain




5-G was studied in two experiments. In each of these, 400 fish averaging




28 grams in weight, were distributed at random among 16 tanks, 25 fish




per tank. Each tank contained 18 gallons of well water, flowing at a




rate of 0.5 gallons per minute. Eight tanks contained fish to be in-




fected, and eight contained fish to be used as uninfected controls.




One tank in each group of eight received flowing water at 74 F, another




pair received water at 69 F, a third pair received water at 64 F, and




so on, so that the range of temperatures from 74 F to 39 F was covered,




with groups of fish maintained at each 5 degree increment of temperature.




The two experiments, involving 800 experimental fish, were carried out




concurrently.









After tempering of the fish to the various temperature levels, those




to be infected received an intramuscular injection of 2 LD-  of a 48




hour broth culture of _A. salmonicida, strain 5-G diluted in frog Ringer




saline. An LD,-n was the approximate number of bacteria causing death




in 50% of a group of 20 to 30 gram coho salmon injected with the organ-




isms intramuscularly and held at 59 F for 5 days after the last death




occurred. Control fish received a sham injection of 0.05 ml of a sterile
                                  15

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filtrate from a similar culture diluted to the same extent. Dead  fish




were collected daily, each was autopsied, and kidney  tissue samples




were cultured. This bacterium has been found to be recoverable  from




the kidney of about 74% of fish succumbing from this  infection. All




experimental groups were observed over a 55 day period.









Results of the two experiments are shown in Table 1.  It  is apparent




that among the infected groups, the per cent mortality decreased  in  a




stepwise manner from 100% at 69 F to 12% and 14% at 44   and 39  respec-




tively. In three instances a 5 degree reduction in water temperature




did not significantly influence the per cent mortality;  this  is evident




at 69  and 64 , at 54  and 49 , and at 44  and 39 . However mortality




was significantly lower at 59  than at 64 , at 54  than  at 59 , and




at 44  than at 49  (Appendix, page 95). The data indicate that  the




development of fatal disease in juvenile coho due to  this organism




was suppressed at water temperatures of 39  to 44 , and  was enhanced




progressively at temperatures of 49 , 59  and 64 .
The results of culturing kidney tissue from the infected groups  of  fish




are recorded in Table 2. Aeromonas salmonicida was recovered  from the




majority of the individuals in each temperature group  that  succumbed  to




the infection. These cultural recoveries strengthen  the evidence that




death was due to the aeromonas infection. It may be  presumed  that the




remaining fish in the groups at 59  and below from which the  organism




was not recovered also died from the infection, since  control fish  that
                                  16

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had received a shatn injection and were held under the sane conditions,




all remained healthy. Aerononas salmonicida was not found in the




kidney of any of the infected fish that survived to the end of the




experiment. This may be seen in the data of Table 2. Presumably the




bacteria injected had been disposed of by body defense mechanisms




in these individuals. In order to provide further evidence on this




question, these surviving fish were transferred to tanks supplied with




water at 64 F, a temperature favorable to development of this infection,




and were held at this temperature for a 10 day period. No deaths occurred,




and when these fish were sacrificed and autopsied, ^. salmonicida was not




recovered from the kidney of any of them.









A linear relationship between the log of the number of days to death




and water temperature was observed and confirmed by regression analysis




(Fig. 1). A correlation coefficient of -0.8850 was calculated and found




to be highly significant (Appendix, page 108). This relationship indicates




that progress of the fatal infection was accelerated at the higher tem-




peratures of 69  and 64 , retarded at the intermediate temperatures,




and still further retarded at the low temperatures of 39  and 44 .









This effect of water temperature upon the average time from infection




until death could be an expression of the combined influence of tempera-




ture on growth of the bacterium and upon the defense mechanisms of the




host. In order to shed some light on this question the growth rates of




the above strain of A. salmonicida were determined at each of the tem-
                                  19

-------
  lOOr-
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       	(	',	\	I	t
    '39      44       49       54       59       64
                      WA. TER  TEMPERA TURE C'F)
74
      Fig.  1.  Relationship betvee.n v;a!:er  tpnoeraturc e.nd log of  ti^
              to death r.ftcr  infection  of juvenile echo salmon with
              Aeronon&c salmonicida.

-------
peratures used in the fish experiment.
A flask containing 200 ml of peptone-beef extract-glucose broth was




inoculated xjith the organism to give a concentration  of  1.9 x  10   cells




per ml. The inoculated medium was then distributed  in 2  ml aliquots  in




screw cap culture tubes. A group of these tubes was then incubated at




each of the 8 temperatures.
Growth was measured by determining optical density at  650 my at various




intervals during an 80 hour incubation period. The growth rates observed




are shown in Fig. 2.









£. salropnicida grew very slowly at 39  and 44  , and  the rate of growth




increased progressively with each 5 degree increase  in temperature,




reaching a maximum at 69 . Thus the effect of  temperature on the growth




rate of the organism in vitro appears to follow a pattern closely  sim-




ilar to its effect on mortality among infected fish. In other words




high temperatures resulting in the most rapid growth of ^.  salmonicida




in vitro also resulted in the shortest mean  time to  death among infected




fish, while low temperatures resulting in very slow  growth  rates i.n




vitro were associated with the longest mean  times to death. No informa-




tion is available concerning the possible influence  of temperature on




host defense mechanisms, but in any case the data of Fig. 2 indicate that




the effect of temperature controls the growth of the bacterium which




in turn has a major controlling influence on mortality in infected
                                  21

-------
            20    30    40    50     50    70
         'INCUBATION PERIOD IN  HOURS
60     90
Fig. 2.  Effect of temperature on growth iar,e of Aero'.i'.onas
         salmorJcida in peptoue-beaf-extracL- gl._,;ose broth.

-------
juvenile coho salmon.









The effect of temperature on infection of juvenile spring chinook salmon




(Oncorhynchus tshawytscha) by Aeromonas salmonicida was studied in  two




experiments. As in the work with the coho salmon, 400 fish averaging




10 gn in weight, were used in each experiment. They were distributed




at random among the 16 tanks, 25 fish per tank and tempered to the




various temperatures in the manner previously described. The groups of




fish to be infected received an intraperitoneal injection of 1.4 LD




(about 425 organisms) doses of a 48 hour broth culture of A_. salmonicida




(strain SS-70). Control fish received a sham injection of 0.05 ml of




sterile physiological saline. As in the coho experiments, dead fish




were collected daily, autopsied, and cultures made from kidney tissue.




All experimental groups were observed over a period of 35 days.









The results of these two experiments are presented in Table 3. It is




apparent that there was variation in the percent mortality observed at




the different temperatures, and some of the differences were statisti-




cally significant (Appendix, page 96), but the consistent reduction in




mortality with decreasing temperature which was found in the coho ex-




periments, was not observed with the spring chinook. The results obtained




in 6 of the 8 temperature groups would have been compatible with such




a trend, but the mortality noted at 59°, and at 39° were both higher




than would have been expected, and in each case was significantly




different from the values for the adjacent temperature groups. Reasons
                                  23

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for these irregular results are not apparent, though it appears  that




some variable other than temperature has influenced them. The experi-




mental method differed in two details from the coho experiments. ,A.




salmonicida strain SS-70 was used because the stock cultures of  strain




5-G, used with the coho, became non-viable for unknown reasons.  The




route of inoculation was intraperitoneal instead of intramuscular.




However there seems to be no reason to assume that either of these




differences could account for the irregularities. The experiments will




be repeated when juvenile spring chinook salmon are again available.








The results of culturing kidney tissue from the fatally infected fish




are shown in Table 4. Aeromonas salrnonicida was recovered from the




majority of these fish in each temperature group. Inoculated fish that




survived were not cultured in this case.








The average interval between infection and death was determined  for the




infected chinook salmon by combining data from the two experiments. It




was found to be 2.9 days at 74 , and to increase progressively as water




temperature decreased, reaching a maximum of 18.4 days at 39 . When the




log of the interval was plotted against temperature a linear relation-




ship was revealed, exactly as in the coho salmon experiments. Confirma-




tion of this relationship was again obtained by regression analysis




(Fig. 3). A correlation coefficient at -0.8229 was calculated and found




to be highly significant (Appendix, page 109). This figure show  that in




the chinook salmon also, the time to death was retarded at the lowest
                                  25

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39      44       49       54       59       64
                 WATER  TEMPERATURE ("FJ
                                                          69
74
       Fig,  3,   Relationship  between water  Lemperaturp and log of tima
                to dent?i after  infection  of juvenile Chinook salwon
                with Aeromon^.s  dalinonicida.
                                   27

-------
temperature levels, accelerated at the intermediate temperatures, and




still further accelerated at the highest temperatures. At 39  and 44 ,




the chinook succumbed to the infection nearly twice as rapidly as did




the coho, though at 54  and above, the average time till death was




closely similar for the two species.
                                  28

-------
    Effect of Temperature on Infection with Aerononas liquefaciens









Two experiments were carried out to determine the influence of water




temperature on experimental infection of juvenile steelhead trout  (Salmo




gairdneri) with Aeromonas liquefaciens, strain K-l. In each of these, 560




fish averaging 25 grams in weight, were distributed at random among 16




tanks, 35 fish per tank. They were tempered to the various water tem-




peratures as described previously. Croups of fish to be infected received




an intramuscular injection of 0.5 LUrn doses (about 2.2 x 10  organisms)




of a 24 hour culture of A_. liquefaciens in peptone-beef extract-glucose




broth. Control fish were injected with 0.05 ml of a sterile filtrate of




the same culture, diluted to the same extent. All groups were held at




their respective temperatures for 27 days. Dead fish were collected




daily, autopsied, and cultures made from kidney tissue. Infected fish




surviving at the end of this period were sacrificed and examined in the




sane manner. Ten control fish from each of the following temperature




groups were also examined by kidney culture at the end of the experi-




ment:  74°, 69°, 64°, 59°, and 54°F.









The mortality data from these experiments are presented in Table 5. The




highest percent mortality occurred among the infected fish at 74 F,




while all of the uninfected controls survived this high temperature.




Mortality was significantly reduced at 69  and was essentially the same




at 64 . A further significant reduction is evident at 59  and 54 , where




38.6 and 40.1 per cent of the fish died, respectively. Progress of the
                                  29

-------
infection was apparently halted at 49 , 44  and 39 , as no deaths occurred




at these temperatures. Thus temperatures from 54  to 74  were favorable




for the development of this infection in steelhead, and increased mor-




tality was correlated with increased temperature in this range.
The mean time to death appeared to be about twice as long at 54  as at




74 , as indicated by the data in Table 5. However the influence of tem-




perature on the mean time to death was less striking than in A^. salmoni-




cida infections, possibly due largely to the fact that no deaths occurred




at 49  or lower.
The results of culturing kidney tissue from the infected groups of




fish are presented in Table 6. It may be noted that among the infected




fish that died, 68 to 93 percent in the various temperature groups,




yielded cultures of A^. liquefaciens. These data provide supporting




evidence that death of these fish was caused by infection with this




organism. It may be presumed that the remaining fish in these groups,




from which the organism was not recovered, also died from the infection,




since of the control fish at the same temperatures that had received a




sham injection, all but one remained healthy. It is also of interest




that among the surviving but infected groups of fish, some still




harboured the organism in the kidney; 23 per cent of the survivors at




74  yielded positive cultures, and recoveries decreased to 17 per cent




at 64 , 7 per cent at 59  and 2 per cent at 54 . None of those at 49




yielded the organism. Because of the small numbers of fish from which
                                  30

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the organism was recovered, the differences in percent positive




cultures between any two temperature levels differing by a  5   increment




are not statistically significant. However, the 28.0% value at 69°




differs significantly from the 7.0 and 2.4% and 0 values at 59°,  54°




and 49 , respectively. Also the 17.4% value at 64  differs  significantly




from the 2.4% value at 54  (Appendix, page 107).
These data suggest that within the temperature range studied, the




higher temperatures favor survival of the pathogens in the tissues of




the host, while the lower temperatures enhance the mechanisms that




clear the microorganisms from these tissues.








Presumably the survivors were individuals possessing greater resis-




tance to this bacterium than those that succumbed; however, they might




serve as a reservoir of pathogens for later outbreaks when conditions




are favorable.








Among 50 control fish that were examined by culture, 49 were negative




and A_. liquefaciens was isolated from one.








At the end of the 27 day experimental period, the infected fish that




had been held at 39 , 44 , and 49 , and among which no deaths had




occurred, were transferred to tanks supplied with water at higher tem-




peratures. The 39  groups were transferred to 59  water, the 44° groups




to 64  water, and the 49  groups to 69  water. This was done to deter-
                                  33

-------
mine whether it was possible that small numbers of /L. liquefaciens  cells




were surviving in some organ or tissue, and might be activated  at the




elevated temperatures and produce a fatal infection. These  fish were ob-




served for a period of 5 days at the higher temperatures, but no deaths




occurred and all appeared healthy at the end of this period. This sug-




gested that the organisms originally injected in these fish held at the




3 lower temperature levels had died out and been disposed of by the




defense mechanisms of the animals.

-------
                              Discussion









The work originally contemplated on the effect of water  temperature on




aeromonas infections is incomplete. It was planned to  study A^.  salmoni-




cida and j\. liquefaciens infection in coho and chinook salmon and  in




steelhead trout. Progress has been slower than anticipated for  several




reasons. These have included the appearance of natural infections  in




populations of experimental fish, difficulties with  temperature control




equipment, and the sudden occurrence of high concentrations of  dissolved




nitrogen in the well water supply.









However the data reported indicate that fatal infection  in coho salmon




due to _A. salmonicida was suppressed at 39  to 44 F, and mortality was




progressively higher at temperatures from 49  to 64  .  This was  evident




from the mortality rates and from the average intervals  from infection




until death, which were longest at the low temperatures, and decreased




progressively with increasing temperature. In the case of juvenile spring




chinook infected with this organism, the effect of temperature  on  mor-




tality rates was irregular, suggesting the influence of  some uncontrolled




variable in the experiment; however it was again observed that  the in-




fection progressed slowly at the low temperatures and  at progressively




higher rates as the temperature increased. Fatal infection of steelhead




trout with _A. liquefaciens was prevented in the range  of 39  to 49 ;




temperatures from 54  to 74  were favorable for development of  this




infection, and the mortality rate increased with temperature in this
                                  35

-------
range. Hence the limited data available thus far are consistent with




the view that water temperatures above the range of 44  to 49  may




cause increasing mortality from aeromonas infections in some salmonid




species.
             Development of a Differential Plating Medium




                for Aeromonas Species in Water Samples









During the course of this work the need arose for a bacterial culture




medium that would permit the counting of aeromonas species in water




specimens in the presence of other common bacterial flora. A medium




was desired that would produce counts comparable to those obtained with




the best plating media, while at the same time inhibiting groxjth of some




other organisms found in water, and exhibiting differential colony




reactions that would permit the recognition of aeromonas colonies. A




relatively large number of formulations were compared with respect to




the above properties and the following one was ultimately selected:




               Peptone-Beef Extract-Glycogen (PBG) Agar




               Bacto Peptone                10 grams liter




               Beef extract                 10   "     "




               Glycogen                      4   "     "




               NaCl                          5   "     "




               Sodium lauryl sulfate         0.1 "     "




               Brom thymol blue              0.1 "     "




               Agar                         15   "




                 final pH 6.9-7.1
                                  36

-------
Sodium lauryl sulfate was included in the medium as a selective agent




for inhibition of some Gram positive bacteria. Broin thymol blue serves




as both an indicator of pH change as well as adding to the inhibitory




effect of sodium lauryl sulfate. Glycogen was included as the only car-




bohydrate because the aeromonas species are among the relatively  few




bacteria reported to be capable of fermenting this polysaccharide.









For use in plating a water sample, 1 ml of the sample in the desired




dilution is added to a sterile Petri dish and mixed with 15 ml of the




sterile PEG agar at 45-50 C. After the agar has gelled and the surface




has dried, it is overlaid with about 20 ml of 2.0% agar in distilled




water. If Aeromonas salmonicida is to be isolated or counted, plates




must be incubated at 25 C for 3 days. Under these conditions it has




been found that in addition to the above organism, a number of other




bacteria also produce yellow colonies on this nedium. These include




some species of Citrobacter, Arizona, Edwardsiella, Enterobacter, and




Serratia. However, all of these organisms, as well as Aeromonas hydro-




phila (liquefaciens) form large bright yellow colonies, 1 mm or more




in diameter.









Colonies of ^. salmonicida, Pleisomonas shigelloides, Vibrio anguiliarurn,




and Vibrio parahaemolyticus, developing at this temperature, are  pin




point in size, and with a little experience can be readily distinguished




from the former group. Organisms from natural sources producing these




very small colonies can be presumptively identified as one of these
                                  37

-------
four species. Pleisomonas shigelloides can be distinguished by failure




to produce gelatinase. The two Vibrio species produce lysine decarbo-




xylase, but not arginine dihydrolase, reactions which would differen-




tiate them from A^ salmonicida and P_. shigelloides. Production of a




brown pigment and lack of motility will serve to differentiate A. sal-




monicida.









In addition, some colonies of this organism will produce small bubbles




of gas in the agar layer, which has not been observed with any of the




other bacteria mentioned above.









If the medium is to be used for isolation or counting of the Aerompnas




hydrophila-liquefaciens complex, plates should be incubated at: 37 C




for 24 hours. Under these conditions colonies of these organises are




0.3 to 0.5 mm in diameter and bright yellow in color. Those of Arizona,




Citrobacter, Cdwardsiella, Enterobacter and Serratia species are smaller,




about 0.1 to 0.2 mm in diameter, and possess more of an orange color.




The aeromonad colonies are often surrounded by a yelloxtf halo in the




green medium, a characteristic not observed with the other genera men-




tioned above. If there are as many as 200 to 300 A. hydrophila colonies




per plate, the whole plate develops a yellow color, while comparable




plates of the other organisms are greenish in color. Furthermore, some




of the subsurface colonies of A. hydrophila produce small bubbles of




gas in the agar layer, which is another differential characteristic.




The Vibrio and Pleisomonas species do not grow at 37 C, and thus do not
                                  38

-------
require differentiation.









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




Grara positive organisms failed to grow, though Bacillus  subtilis  and




other members of the genus grew sparsely. All Gram negative  organisms




grew, but only those genera listed above produced  yellow colonies.









Although the medium has obvious limitations, it has  been found to be




useful in monitoring the numbers of both A., salmonicida  and  A_. hydro-




phi la in hatchery water, measuring their growth rates, and recovering




cultures from viscera of experimentally infected fish. Thus  far it




appears to be superior to other available media for  these purposes,




but further experience will be required to completely define its  use-




fulness and reliability.









The medium has been useful not only in counting aeroraonas organisms in




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




growth rates at various temperatures.
                                   39

-------

-------
                              SECTION VI




        EFFECTS OF WATER TEMPERATURE ON INFECTION OF SALMONIDS




                      BY CHONDROCOCCUS COLUMNARIS









                         Materials and Methods









To determine the effect of water temperature on losses caused by  the




myxobacterium Chondrococcus columnaris, coho salmon  (Oncorhynchus




kisutch), spring Chinook salnons (Oncorhynchus tshawytscha) and rainbow




trout (Salmo gairdneri) were exposed to a virulent isolate of this bac-




terium. Groups of 25 or 35 fish of the species being tested were  tem-




pered to water temperatures ranging from 39 to 74 F at 5 F intervals.




Two control and two experimental groups were held at each temperature.




These animals were tempered in a manner previously described in the ex-




periments with the aeromonads.









The C_. columnaris isolate used in this study was obtained from a  lesion




on the gill of an adult spring chinook salmon at the Fish Commission of




Oregon, Dexter Dam Holding Pond, Willamette River. This isolate was




passed in coho salmon fry seven times to increase its virulence.  After




the seventh passage the culture was lyophilized. Immediately prior to




each experiment the cells were removed from lyophilization, grown in




cytophoga broth and passed once in the salraonid species being tested.




Several isolates from the final fish passage were collected and pooled




for use in the temperature experiment.
                                  41

-------
To prepare the exposure inoculum the cells were grown in tryptone yeast




infusion broth containing 0.4% tryptone and 3.0% yeast infusion. After




approximately 20 hours at 24 C the optical density was adjusted to 0.1




at 525 my with a Bausch and Lomb Spectronic 20 (1). The fish were then




exposed in the experimental tanks to a 1:20 dilution of the adjusted




broth culture for a 10 minute period. Normal water flow through the




tanks was resumed after the exposure period. This dilution was determined




by plate count to represent approximately 3 to 6 x 10  C. columnaris




cells per ml. Dead fish were collected two times each day and bacterio-




logical cultures v/ere made from the gills and or kidney of each fish.
                                  42

-------
                           Experimental Phase








Table 7 shows the percent mortality and the incidence of C_t columnaris




in rainbow fry (average weight 2.9 g each) exposed to this bacterium at




different water temperatures. Temperatures of 44 and 39 F were not in-




cluded in this experiment. Fish infected with £. columnaris were observed




at temperatures of 74 F down to 54 F with the larger number of losses




occurring at the higher water temperatures. The greatest difference in




loss occurred between 59 and 64 F. Control fish at 74 F had some mortality




which was not due to C. columnaris.
Not only percent mortality but also the time to death was greatly in-




fluenced by water temperature. This is illustrated in Fig. 4, which




shows the regression line relating water temperature and the log of  the




number of days from exposure to death. The equation and the data used




in computing it are shown in Appendix, page 110. A correlation coefficient




of -0.8573 was calculated and found to be highly significant. Thus the




linear relationship between these two variables is demonstrated.









The results of the experiment with coho salmon  (average weight 33 g  each)




are nearly identical to those observed with rainbow trout (Table 8).




Within three days after exposure, all fish held at 69 and 74 F were




dead. After one month at 64 F a loss of 99% had occurred, as compared




to a 51% loss at 59°F. At 54°F only 4% of the test animals had died.




No deaths due to C. columnaris occurred at 49 F or below. Among the
                                  43

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    49
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 54        59         64        i
 WATER  TEMPERATURE (°Fj
74
 Fig. 4,   Relationship between water tcn\pf>ratur: and log of  time
          to  death after exposure of juvenile  r;:.;'.r:bov trout  to
          Chondrococcus colu:yr.aris.

-------



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




and £. columnaris was never isolated from these fish.









Again, a linear relationship between water temperature and the log of




the number of days from exposure to death was observed (Fig. 5). A




correlation coefficient of -0.7699 was calculated, and as with the




rainbow trout experiments, was highly significant (Appendix, page 111).
Fifteen days after the last death occurred fish surviving at 59  were




transferred to 69  water and those surviving at 49  and 54  were trans-




ferred to 64  water. Subsequently, losses due to _C. columnaris occurred




among those groups originally held at 59  and 54 , but not among those




held at 49 . Thus, some of the survivors, when moved to higher water




temperatures, developed the disease.
Results of the spring chinook experiment  (average weight, 10.2 g each)




were similar to those observed previously with rainbow  trout and coho




salmon, although the percent mortality at temperatures  of 59  and above




was lower than in the other experiments  (Table 9). Chondrococcus




columnaris was isolated in gill or kidney cultures from approximately




88% of the deaths in fish exposed at 59° or higher; from 31% of those




dying at 54 , and was not recovered from any of those held at 49  or




below. Among the control groups only a few deaths occurred, and the




columnaris bacterium was not isolated from these fish.
                                  47

-------
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     39
49       54       59        64
WA TER  TEMPERA TUR£ (°F)
69
    Fig. 5.  Relationship between water temperature r.nd  log  of  tine
             to death after exposure ol juvenile cc',,o  salmon to
             Chondrococcus columnaris.

-------








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49

-------
The linear relationship between water temperature and the log of  the




number of days from exposure to death reported in the experiments with




rainbow trout and coho salmon was again observed (Fig. 6). A correlation




coefficient of 0.7192 was calculated and found highly significant




(Appendix, page 112).
At the end of the 30 day observation period fish surviving at  54  were




transferred to 64  water and survivors in the 49  group were transferred




to 59° water. Fifteen of the 40 fish (38%) moved from 54 to 64°F water




and 4 of the 42 fish (10%) moved from 49 to 59 F water became  infected




with C. columnaris.
                                   50

-------
   100 r-
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                       WA TER TEMPERA TURE ( T)
     Fig.  6.  Relationship between water tcipcratur;  and  log  of  tine
              to death after cxpopui'e of juvenile i/.lr.ook salmon co
              Chondrccoccus columnar!;;.

-------
                              Discussion
Fish infected with £. columnaris were observed at temperatures from 74 F




down to 54 F with the greater number of losses occurring at the higher




water temperatures. Results of the coho salmon and rainbow trout experi-




ments are nearly identical. With spring chinook the percent mortality




at 59 F and above was lower than in the other species. A temperature of




54 F appeared to be the threshold for infection by £. columnaris. At




each temperature increment tested above 54 F exposure to the columnaris




bacterium resulted in a greater number of infections and deaths.
The time to death was also greatly influenced by water temperature. For




example, in the rainbow trout experiment all fish were dead at one and




four days at 74 and 69 F, respectively, after exposure. At each lower




temperature greater numbers of fish survived the exposure.
ChondrocQccus columnaris was isolated from the gills or kidneys of most




experimental animals in each group. The organism was not isolated from




control groups. The symptoms and pathology observed in the test animals




were similar to those described during epizootics of columnaris disease.




In these experiments the fish were exposed to large numbers of the




columnaris bacterium in the water; consequently isolation from the




kidney mav be a more accurate indication of infection.
No deaths caused by C. columnaris were found in fish held at 49  F  or  below.
                                  52

-------
However, as previously described, exposed fish held at water tempera-




tures unfavorable for the progress of infection often developed  fatal




disease when transferred to water at higher temperatures  (64  or 69  ).
                                  53

-------

-------
                              SECTION VII




       EFFECTS OF WATER TEMPERATURE ON INFECTION OF SALMONIDS BY




               THE PARASITIC PROTOZOAN CERATOMYXA SHASTA









                         Materials and Methods









Three species of salmonids were examined in this study; rainbow trout




(Salmo gairdneri) were obtained from the 1969 brood at Roaring River




Hatchery (Oregon State Game Commission). Coho salmon  (Oncorhynchus




kisutch) were from the 1970 brood at Fall Creek Salmon Hatchery and




spring chinook salmon (Oncorhynchus tshawytscha) were from the 1970




brood at Marion Forks Hatchery (both hatcheries operated by the Fish




Commission of Oregon). Rainbow trout used as a positive control in




the coho-spring chinook experiment were from the 1970 brood at Leaberg




Trout Hatchery (OSGC).









The only practical method of initiating infection of £. shasta in fish




is by exposure to water known to contain the infectious agent (2).




During the summer and fall months, the Willamette River (below Corvallis)




is a very effective location for producing £. shasta  infections in




several species of salmonids. The proximity of the Willamette River to




the laboratory, made this site ideal for exposing fish to this protozoan




in these experiments. The object of the exposure was  to initiate at




least a 50 percent infection attributable to £. shasta. From previous




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

-------
48 to 72 hour exposure, to water in which the infectious agent was




present, would suffice for our purposes. All experimental groups were




held in a 96 cu ft live-box situated in the Willamette River current




at the Albany site. Replacement time of water in the live-box was




approximately 10 sec.








The rainbow trout were exposed to the infectious agent for 48 hours,




between September 5 and September 7, 1970. The mean water temperature  for




this period was 60.7 F (S.D. » 0.45). The coho and spring chinook salmon




were exposed for 72 hours, between September 17 and September 20, 1971.




The mean water temperature was 59.4 F (S.D. = 0.62) for the interval.








The procedure used to temper fish to laboratory water temperatures




after exposure to C_. shasta was necessarily different from that used in




the bacterial experiments. Previous information had suggested that  the




rate at which the £. shasta infection proceeds is temperature dependent.




During the exposure period, therefore, the rate of the infectious process




should be dependent on the river water temperature. Following this




reasoning it was deemed necessary to temper the fish after exposure as




rapidly as possible to the experimental temperatures without causing




severe stress. The rates of change which filled these requirements  were




determined by preliminary experiments. A rate of 10 F per hour was  used




for rainbow trout and a rate of 6.6 F per hour was used for coho and




spring chinook salmon. These rates of change were achieved by manual




control of the temperature regulating equipment. All fish, both experi-
                                  56

-------
mental and control, were placed into their respective tanks, all of




which contained 54 F water. The control equipment was also  initially




set at this temperature. This control equipment was  then adjusted by




either increasing or decreasing the temperature of the water coming




into each tank to achieve the desired rate of change of temperature.




When the eight experimental temperatures  (74, 69, 64, 59, 54, 49, 44 and




39 F) were reached, the regulating instruments were  calibrated and set




for the remainder of the experiment.
It was anticipated that with a natural exposure in  the Willamette River




the fish would also become infected with bacterial  diseases. Aeromonas




liquefaciens and Chondrococcus columnaris were known  to be prevalent  in




the river. For this reason prophylactic measures were taken  to  prevent




their interference in the experiments. The antibiotic of choice in control




of these diseases was Terramycin as TM_.. This compound was  given to




the fish incorporated in Oregon Moist Pellet  (OMP)  diet. Terramycin was




chosen also because of its lack of activity against JC. shasta.  In each




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




TM  /100 Ib of fish/day starting ten days prior to  exposure. A  level of




20 g TM -/100 Ib of fish/day was fed post exposure  for three days in




the rainbow trout experiment. The treatment was discontinued for four




days, resumed for five additional days at which time  the level  was re-




duced to a dose of 5 g TM--/100 Ib of fish/day. To  protect coho and




spring chinook salmon a 30 min bath in water  containing 5 yg/ml of




soluble terramycin was given to the exposed fish before placing them
                                  57

-------
in the laboratory tanks. They were fed at the 20 g TM  /100 Ib of




fish/day level for the interim of the experiment.
Dead fish were collected at least once daily and either examined while




fresh or were frozen for later autopsy. Examination of dead animals




consisted of microscopic observation  (400 x, bright field) of wet mounted




samples of intestinal scrapings. Slides containing two or more of the




spore stage of £. shasta were considered as positive diagnoses.









The experimental design used for the rainbow trout experiment was the




same as that used for the bacterial investigations described in this




report. In order to conserve laboratory facilities during these very




long-term experiments with £. shasta, the design was modified for the




coho-spring chinook experiment. The experimental design was altered




according to recommendations from the project's statistician. This




modified design consists of conducting two concurrent experiments in




each tank using two species of fish distinguished by removal of opposing




pectoral fins. The second modification eliminated the use of two control




groups for two experimental groups at each temperature, and substituted




one control group. In the design used xd.th rainbow trout 25 fish




averaging 11.5 g were placed in each tank. With the modified design 25




coho salmon averaging 14 g and 25 spring chinook salmon averaging 8.7 g




were placed in each tank. Rainbow trout used as an exposure control in




the latter experiment averaged 15 g; 35 of these fish were exposed and




35 used as unexposed controls.
                                  53

-------
                          Experimental Phase








In these experiments with £. shasta two types of  information were  ob-




tained. Quantal mortality data, expressed both as percent  infection




and quantitative response data expressed as mean  time  to death  in  days




have been gathered. From this data some qualitative  inferences  have




been drawn regarding effects of temperature changes  in the host-parasite




relationship.








Table 10 summarizes the mortality data acquired from two concurrent




replications of an experiment utilizing rainbow trout.  The data is




arranged to show the effect of temperature on the £. shasta infection.




The number of fish infected with £. shasta from experimental group 2




of both the 74 F and 69 F temperatures are lower  than  expected.  This




is due to a fatal C. columnaris infection in several fish  prior to the




time that they would have died of £. shasta. It call  be seen, however,




from all other experimental groups in the 74 F to 44 F range that  tem-




perature has little or no effect on the percent mortality  due to £.




shasta. This data suggests that rainbow trout may have no  means to com-




bat this parasite in this temperature range. At 39 F no mortality  due




to C_. shasta was observed even after 237 days post exposure. However,




when these same fish were tempered thereafter from 39  F to 64 F over a




two week period and held for an additional four week period at  64  F, six




percent succumbed to C. shasta infections.
                                   59

-------









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60

-------
Another parameter has been analyzed to determine the effect of tempera-




ture on the infection process. This second parameter is the dependence




of the mean time to death (of dead specimens diagnosed positive for C_.




shasta) on temperature. When the log of the mean time to death in days




is plotted against temperature a straight line should be obtained when




the host shows either no defense against or a logarithmic interaction




with the infectious organism. In this experiment with rainbow trout such




a straight line function was obtained. This further supports the idea




that rainbow trout are not able to overcome a £. shasta infection be-




tween 74 F and 44 F. If this assumption is correct, the temperature




dependence of the mean time to death reflects the effects of temperature




on the growth rate of the parasite.









At 39 F the host parasite relation is markedly altered since no mortality




occurred. _C. shasta may lie dormant in the fish which could explain the




onset of the disease after the fish were elevated in temperature.









A linear relationship between water temperature and the log of the




number of days from exposure to death was confirmed by regression




analysis (Fig. 7). A correlation coefficient of -0.9830 was calculated




and found highly significant (Appendix, page 113).









This experiment was terminated at each temperature when it was reasonable




to assume no additional deaths would occur due to JC. shasta. Fish re-




maining at termination were examined for the presence of C. shasta spores.
                                  61

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  1000 r-
1
    100
£
s
     10
      39
44
49
                                54
59
                                   64
69
                      WATER  TEMPERATURE (°F)
      Fig, 7.   Relationship between water temperature, and log of time
               to death after  exposure of juvenile rainbow trout to
               Ceratpmyxa  shasta.

-------
Only two positive fish (at 44 F) were found. These  fish appeared  to  have




been recovering from the infection.
A second pair of experiments with _C. shasta utilized  the modified  design




explained previously.









Coho salmon and spring chinook salmon were the host animals  in  this




study. The spring chinook portion of this experiment  was terminated  after




the third week. When the coho salmon died at  74 F and 69 F  (prior  to the




death of any spring chinook), the hacterial load, mostly of  Aeromonas




liquefaciens overcame the drug treatment. This bacterium killed all  ex-




perimental spring chinook at 74  and 69  . The spring  chinook were  left




in the tanks at the lower temperatures so as  not to alter the results




of the remaining coho experiment.









Mortality data from the coho salmon portion of the experiment is reported




in Table 11. The results are noticeably  different from that  of  the ex-




periment with rainbow trout. In  this study, in contrast to  the  rainbow




trout experiment, the percent mortality  attributable  to C_.  shasta  does




not remain constant with decreasing temperature. It is instead  reduced




at 64 F and below with no mortality below 49  F. The previous experimental




results (with rainbow trout) indicate that _C. shasta  can multiply  in




infected fish at temperatures down to 44 F when uninhibited. Coho  salmon,




therefore, may well be able to somewhat  inhibit C_. shasta development at




64 F and below.
                                   63

-------





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Although not as reliable in this experiment as in the rainbow trout




experiment due to lower numbers of deaths, the mean time to death




analysis remains a valid parameter. A plot of the log of the mean time




to death verses temperature again yielded a straight line. Confirmation




of this linear relationship was obtained by regression analysis (Fig. 8).




The correlation coefficient obtained was -0.9574, which was highly




significant (Appendix, page 114).









It appears that at 74 F neither species of fish has the ability to




retard the growth of £. shasta. At 64 F and below the portion of the




coho population able to resist a fatal £. shasta infection increases with




decreasing temperature (Table 11). The portion of the population unable




to resist fatal infection may, however, interact with the parasite to




protract the mean time to death.









Thirty-five rainbow trout were also exposed at the same time as the coho




salmon and were then held at 54 F. These fish were used to determine




whether the Willamette River exposure would give repeatable results from




one year to the next. Sixty-eight percent of these fish were diagnosed




positive for £. shasta and the mean day of death was 67.7 days. These




results were very near those observed with rainbow trout the previous




year (Fig. 7).
                                  65

-------
  lOOOr
    lOOf
£
     to1
              44       49       54       59
                        WATER TEMPERATURE (°F)
64
69
	\
 74
      Fig.  8.   Relationshop between water temperature and log of time
               to death after exposure of juvenile c&>.o salmon to
               Ceratcmyxa shasta.
                                  66

-------
                              Discussion









It is alxrays the goal of this type of laboratory  experiment  to  be able




to apply the results to natural and management situations. It is  felt  that




the results obtained in these experiments are applicable  in  these areas.




The short period of exposure to infection used resulted in fish dying




with identical pathology to that of fish held continuously in the




Willamette River until death. The mortality may be  somewhat  higher in




a natural situation since the fish are  exposed to the  infectious  agent




for a longer period of time.
Since the water temperature during the exposure was near  60  F  nothing




can be said about initiation of jC. shasta  infection at  colder  water  ten-




peratures. However, Keith Johnson working  in  this  laboratory,  has  ini-




tiated an infection of jC. shasta in Cutthroat  trout  (Salmo clarki) when




the Willamette River had a maximum temperature of  48  F  (exposure  time,




3 days).
It has been shown in this study that rainbow  trout  held at water  tem-




peratures between 74 F and 44 F have little or no ability to  overcome




an infection of C_. shasta once initiated. Coho salmon on the  other  hand




do seem to be able to interact with the parasite. In this species the  per-




cent of the population which is susceptible to the  disease diminishes  with




decreasing temperature. In both species, the mean time to death,  of fish




diagnosed positive for £. shasta, is inversely related to temperature.
                                   67

-------
                             SECTION VIII




EFFECT OF WATER TEMPERATURE ON INFECTION BY THE SOCKEYE SALMON VIRUS  (IHN)









                         Materials and Methods









The virus used in the work reported here was isolated in this laboratory




in 1958 from diseased fish collected at the Willamette River Salmon




Hatchery in Oregon during an epizootic associated with a high mortality




rate. Stocks of this agent have been maintained since that time by prop-




agation in cultures of a salmonid cell line. Properties of the virus  have




been reported (4). Fish used in these experiments have all been fingerling




kokanee salmon (Oncorhynchus nerka) generously supplied without charge




by the Oregon Game Commission.









Because of the limited number of 21 gallon experimental tanks available




at the main fish disease laboratory, it has not been possible to carry




on experiments with all 5 fish pathogens concurrently in those facil-




ities. Hence, in order to expedite the acquisition of data, work with




the sockeye salmon virus has been done with the aid of one gallon




glass aquaria, held in individual incubators at one of several tempera-




tures. This equipment was practical to use because of the small size




of the experimental fish. The xrater supply for these vessels was derived




from the Corvallis Municipal supply, and had been processed to remove




chlorine and fluoride by the Fisheries and Wildlife Department at Oregon




State University. The water in all of these small aquaria was changed
                                  69

-------
at 48 hour intervals. The fish were fed Rangen's Salmon Mash  twice  a




day in amounts slightly in excess of their usual consumption.









When fish were first received from a hatchery, they were distributed




among a number of aquaria at a water temperature very close to  that of




the hatchery water. In the case of fry averaging less than 1  gm in




weight, 30 fish or less were placed in each 1 gallon aquarium.  For




those averaging 1 to 3 gm, the maximum number in each aquarium  was  15.




The aquaria were then placed in incubators at one of the desired ex-




perimental temperatures and allowed to gradually adjust to the  new




temperature over a 24 hour period. It was realized that from  a  physio-




logical viewpoint this was not an adequate acclimation procedure, but




in actual practice it did not appear to create any difficulties.









Fish were exposed to the virus infection by adding measured amounts of




standardized virus suspension to the water of their aquaria.  Exposure




periods of either 24 or 48 hours were allowed, after which the  virus




was removed and replaced by fresh water. Virus assays were carried  out




by the plaque method, using monolayer cultures of salmonid cell line




CHSE 214, as described elsewhere (5).









Exposed fish that died 5 days or more after exposure were considered




to be virus infected if they showed symptoms characteristic of  the




disease, and if 90% or more of unexposed controls held under  the same




conditions remained healthy. Typical symptoms included hemorrhages  at
                                  70

-------
the base of pelvic and pectoral fins, extrusion of strings of milky




fecal material, and development of a very dark body color. Control




fish never showed any of these symptoms. The laboratory method  for de-




tection of the virus in the organs of dead fish, although used  in a few




instances, was considered to be impractical for application to  large




numbers of fish.
                                  71

-------
                          Experimental Phase








                        Preliminary Experiments








Before attempting to study the effect of water temperatures on  infection




with the sockeye salmon virus, it was necessary to determine an appro-




priate concentration of virus for initiating the experimental infection.




In the first experiment bearing on this point, groups of 20 to  22  finger-




ling kokanee salmon, averaging 0.3 gm in weight were exposed to different




concentrations of virus in their water for a 24 hour period. Concentra-




tions varied from 2570 to 11 plaque forming units per ml,  (pfu)  at 3




fold intervals. All aquaria were held at 54 F and deaths recorded  over




a 24 day period. The results are shown in Tahle 12. A second experiment




of the same type was carried out as soon as the first was  completed.  In




this case the available kokanee fingerlings had increased  to an average




weight of 0.94 gm. The results are presented in Table 13.








In both preliminary experiments 90 to 100% of the fish exposed  to  11  or




more pfu of virus per ml, succumbed to the infection; hence the cumula-




tive percent mortality did not increase with increasing virus concen-




tration. Deaths were distributed over a much longer time period at the




lower concentrations than was observed with the maximum virus level.  This




was probably because some of the fish exposed to the lower virus con-




centrations only became infected when exposed to virus being shed  from




others that had been infected during the initial exposure. Differences
                                  72

-------
in the distribution of deaths with time among the experimental groups




are reflected in the values for the mean time to death in Tables  12 and




3. In both cases this value is minimal where the virus concentration




was maximal, and vice versa. In comparing Tables 12 and 13  it may be




noted that in the case of the 0.3 gm fish the mean time to  death  for




all virus concentrations was consistently shorter than the  comparable




figure for the 0.94 gm fish.









The results of these two experiments indicated that any of  several virus




concentrations within the range which was studied could be  used success-




fully for exposure of fish in temperature experiments. In order to allow




as much latitude as possible for observing effects of water temperature




it appeared desirable to use a lower concentration (e.g. 32 pfu/ml) for




fish in the same weight range, i.e. 0.3 to 1.0 gm.









            Effects of Water Temperature on Virus Infection









In the first experiment dealing with the effect of water temperature on




the virus infection, the available kokanee fingcrlinps had  an average




weight of 1.1 gm. Incubator facilities for only 4 temperatures were




available and those selected were 74 , 64 , 54  and 39 F. For each tem-




perature level 20 fish were infected and another group of 20 held as




controls. Two complete experiments were carried on concurrently.  Dead




fish were collected daily and deaths were presumed to be due to virus




infection, unless they occurred in less than 5 days after exposure, or
                                   73

-------




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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 F were lost due to failure in the aeration apparatus.




The percent mortality approached 100% among the fish at 39 , arid 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 expiosure




and death.









When a second pair of experiments was started, the available kokanee




fingerlings were considerably larger, averaging 2.9 gra 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, arid two




complete experiments were carried out in parallel, as before.









The results of these two experiments are presented in Table 15, In  this
                                  76

-------




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case none of the experimental groups showed 100% mortality. The maximum




average mortality for the two experiments was 61.2%, which occurred at




54 F. The mortality rate decreased at 59  and was still lox^er at  64  and




69 . The 72  temperature was at the threshold of tolerance for these




fingerlings, as shown by a similar mortality rate in controls and  in-




fected groups. These experiments seen to indicate that within the  tem-




perature range covered, 54 F was the most favorable for the development




of fatal infection in fish of this size, while at 64  and 69  the  disease




was partially suppressed and mortality was significantly lower. This




result appears to confirm the findings in the first experiments. No




consistent relationship between temperature and the nean time to death




was observed, as may be seen in Table 16, although this period was




longest among the 69  group.
The next pair of experiments was conducted during  the following year




when kokanee salmon fry were available again. At this time  the fish




were very small, and the average weight of those used in  the experi-




ments was 0.11 gm. Temperatures that were compared  in these eperiments




were 69 , 59 , 54 , 49 , and 39 F. For each temperature level in  each




experiment, 30 fish were exposed to virus and a similar group of  30




were held as controls. For these small fish, the virus concentration




used to produce infection was 32 plaque forming units/ml  of aquarium




water. The exposure period was 24 hours.









Table 17 presents the results of this second pair  of experiments.  The
                                  79

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percent mortality, as measured by the combined data of the two  experi-




ments was very low among the fish held at 39  and 49 F. It approached




100% however in the 54  and 59  groups, and declined again to a signifi-




cant extent in the 69  groups. This was despite the fact that about half




of the control fish at 69  succumbed to effects of this relatively high




temperature. The data thus indicated that the temperature range of 54




to 59  was near optimal for development of fatal infection under  these




experimental conditions, and that infection was markedly retarded at




49  or lower, and to a lesser degree at 69 . The mean survival  times




for fish that died in each temperature group are shown in Table 18.




The infection apparently progressed most rapidly at 54  and  59  ,  where




mortality was also greatest. It ran a slower course at both  lower and




higher temperatures.
A third pair of experiments was next carried out with a population  of




kokanee salmon fry whose average weight was 0.95 gm. The experimental




design was very similar to that in the previous experiments with  the




0.1 gm fish, with the exception that only 20 fish were used in  each




small aquarium because of their larger size. In addition to the 5 ex-




perimental water temperatures used previously, groups of fish held  at




64 F v/ere included. The virus concentration in the water during the ex-




posure period was 32 plaque forming units/ml.









Results of the third pair of experiments appear in Table 19. They are




quite different from those obtained with the 2.9 gm and the 0.11  gm
                                     82

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fish. In this case the final per cent mortality showed little relation-




ship to water temperature. It was high at all temperatures, ranging from




90% to 97.5% at the four lower temperature levels. It declined slightly




to 75% at 64  (a value significantly different from 97.5%  (Appendix,




page 106). The distinct protective effective of the 39  and 49  tempera-




tures, observed with the 0.11 gm fish was lacking in these experiments.




There is however an indication of a retarding effect of the 39  tempera-




ture on the course of the disease in Table 20, where the mean time to




death at that temperature is about double the comparable value at the




other temperatures. It is noteworthy that the 0.95 gm fish used in these




experiments were obtained from the same population of kokanee salmon fry




as the 0.11 gm fish. The data seem to indicate that these larger fish had




become more susceptible to the virus infection; this is suggested by the




high mortality at the low temperatures, and the fact that the mean sur-




vival times were distinctly shorter at comparable temperature levels.









Two further experiments with this virus in kokanee salmon populations




were started, but had to be abandoned because of the development of




natural bacterial infections, despite the presence of 2 ppra of oxytetra-




cvclene in the water.
                                  85

-------







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-------
                              Discussion









It is in one sense unfortunate that all of the experiments reported with




the sockeye salmon virus could not have been carried out with experimental




fish of approximately the same size and from the same population. It




appears that factors related to age or size, and perhaps to environmental




or genetic background may have influenced some of the results obtained.




However it was necessary to use kokanee fry or fingerlings at whatever




time and from whatever source they became available.









One set of two experiments with 2.9 gm fish and one pair of experiments




with 1.1 gm fish indicated that a water temperature of 54 F produced a




higher percent mortality in infected fish than higher temperatures,




up to 69 , and that the course of the disease was slower at the latter




temperature. In another pair of experiments with 0.11 gm fish, tempera-




tures of 54  and 59  both resulted in over 90% mortality, while very




few fatal infections occurred at 39  and 49 . The course of the infec-




tion was also most rapid at 54  and 59 , as measured by the average




time from infection until death. These results are not in conflict with




experiments of Amend, (1970) who reported that mortality among fingerling




sockeye salmon exposed to this virus was reduced to a low level if the




fish were held at 68 F for 4 to 6 days after exposure (6). Work reported




from this laboratory has indicated that this virus replicates abundantly




in sockeye salmon cell cultures in the temperature range from 50 F to




68 F. Replication was retarded and virus yields were lower at 39 F, and
                                  87

-------
no replication occurred at 73.4 F  (4). Thus the indications  from  experi-




ments reported here that the temperature range from  54   to 59   is  near




optimal for development of fatal infection due to this virus in fingerling




kokanee, is not out of line with other relevant data.
The experiments reported in Table 19 with fish of  0.95 gm average  weight



do not show the marked reduction in percent mortality in 64  water that



was found in the experiments of Table 14 and 15. Presumably  some unrec-



ognized variable factor may be responsible for this difference. It is



also of interest to note that only in the case of  the very small fry,



averaging 0,11 gm in weight, was the percent mortality reduced  to  a



very low level (Table 17) by the low temperatures  of 39° and 49°.  This



might suggest that these tiny fish were more resistant to the virus in-



fection than larger fry from the same population (Table 19).
                                  88

-------
                              SECTION IX




                            ACKNOWLEDGMENTS









This project was supported by the Environmental Protection Agency over




a three year period beginning April 1, 1969 and ending March  31, 1972.




The total funds provided by this agency amounted  to  $182,355. The




assistance provided by Dr. Donald A. Hilden and Dr.  Gerald R. Bouck,




who have served as Project Officers, is acknowledged with sincere




thanks.









A major contribution to this project was made by  the Fish Commission of




Oregon and the Oregon Game Commission. In fact, without  the support of




these agencies none of the work described in this report would have been




possible. They provided the large numbers of juvenile salmon  and trout




required for the experiments, without charge. The monetary value of these




fish has been roughly estimated at about $3000 per year  for each of the




two years during which experimentation was carried on.









Another important contribution to the project was made by the Department




of Microbiology at Oregon State University. All of the experimentation




with sockeye salmon virus in finperling kokanee salmon was carried on




by Philip McAllister, a graduate research assistant  in the above depart-




ment. During 2 years of work on this phase of the project, he was sup-




ported entirely by an NDEA fellowship, so that all of his work was really




donated to the project.
                                  89

-------

-------
                               SECTION X




                              REFERENCES









1.  Pacha, R. E., and Ordal, E. J., "Histopathology of Experimental




    Columnaris Disease in Young Salmon." J. Comp. Path.  77, pp.  419-A23.




    1967.









2.  Sanders, J. E., Fryer, J. L., and Gould, R. W., "Occurrence  of the




    Myxosporidan Parasite Ceratomyxa Shasta, in Salmonid Fish from the




    Columbia River Basin and Oregon Coastal Streams." A  symposium on




    Diseases' of Fishes and Shellfishes. Special Publication No.  5.




    American Fisheries Society, Washington, D. C. 1970.









3.  Sanders, J. E., Fryer, J. L., Leith, D. A., and Moore, K. D.,




    "Control of the Infectious Protozoan Ceratomyxa Shasta by Treating




    Hatchery Water Supplies." The Progressive Fish-Culturist. Vol 34,




    No. 1. 1972.









4.  Wingfield, W. H., Fryer, J. L., and Pilcher, K. S.,  "Properties of




    the Sockeye Salmon Virus (Oregon Strain) (33719)." Proc. Soc. Exp.




    Biol. Hed. 130. pp. 1055-1059. 1969.









5.  McCain, B. B., Fryer, J. L.,  and Pilcher, K. S., "Antigenic  Relation-




    ship in a Group of Three Viruses of Salmonid Fish by Cross Neutrali-




    zation (35724)." Proc. Soc. Exp. Biol. Med. 137, pp. 1042-1046. 1971.
                                  91

-------
6.   Amend, Donald F., "Control of Infectious Hematopoietic Necrosis




    Virus Disease by Elevating the Water Temperature." J.Fish Res. Bd.




    Canada, 27:265-270. 1970.
                                   92

-------
                              SECTION XI

                              APPENDICES

                                                               Page No.

A.  Analyses of Final Percent Mortality Data in Text Tables.

    1. Text Table 1. Aeromonas Salmonicida in Coho Salmon.        95

    2. Text Table 3. Aeromonas Salmonicida in Chinook Salmon.     96

    3. Text Table 5. Aeromonas Liquefaciens in Steelhead          97
       Trout.

    4. Text Table 7. Chondrococcus Columnaris in Rainbow          98
       Trout.

    5. Text Table 8. Chondrococcus Colunnaris in Coho Salmon.     99

    6. Text Table 9. ghondrococcus Columnaris in Chinook         100
       Salmon.

    7. Text Table 10. Ceratomyxa Shasta in Rainbow Trout.        101

    8. Text Table 11. Ceratomyxa Shasta in Coho Salmon.          102

    9. Text Table 14. Sockeye Salmon Virus in 1.1 Gram           103
       Kokanee Salmon.

   10. Text Table 15. Sockeye Salmon Virus in 2.9 Gram           104
       Kokanee Salmon.

   11. Text Table 17. Sockeye Salmon Virus in 0.11 Gram          105
       Kokanee Salmon.

   12. Text Table 19. Sockeye Salmon Virus in 0.95 Gram          106
       Kokanee Salmon.

B.  Chi Square Analyses of Percentages of Surviving Fish         107
    Yielding Cultures of Aeromonas Liquefaciens. Text Table 6.

C.  Regression Analyses. Relation Between Water Temperature
    and Log of Number of Days to Death.

    1. Text Fig. 1. Aeromonas Salmonicida in Coho Salmon.        108

    2. Text Fig. 3. Aeromonas Salmonicida in Chinook Salmon.     109

    3. Text Fig. 4. Chondrococcus Columnaris in Rainbow Trout.   110
                                  93

-------
                                                           Page No.

4. Text Fig. 5. Chondrococcus Columnaris in Coho Salmon.     Ill

5. Text Fig. 6. Chondrococcus Columnaris in Chinook          112
   Salmon.

6. Text Fig. 7. Ceratomyxa Shasta in Rainbow Trout.          113

7. Text Fig. 8. Ceratomyxa Shasta in Coho Salmon.            114
                              94

-------
*ANQVfll2  - ONI/TWO  FACTO?  AMLY3IS OF  VARIANCE.
ORfGON  STATE UNIVERSITY COMPUTE? CENTi*
                                                           OS-3   VE3.3.5
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          - CNc/TK'O FACTOR  ANALYSIS  OF  VARIANCE,
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                                      03-3   VER.3.5
                                    DATE -  1C/1W72
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           slraU^cant, P\ffax-ease " U.92 pcr^t for P=0,C5
                                   56

-------
 *ANOVA12 - ONI/TWO FACTOR  ANALYSIS OF  VARIANCE,       OS-3   V'EP.3.5
 OREGON STATE  UNIVERSITY COMPUTER CENTER              DATE  -10/14/72
   SOURCE

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             PROBLEM  I-DI  2ND TRY

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          »     »    •     «    »   - 22,13  " *    "  « =0.01
                                    97

-------
                            r « »»»»«.*(
*ANOVAi2  - ONE/TWO FACTOPx ANALYSIS  OF VARIANCE.
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                                                 OS-3  VCR.3.ti
                                               DATE - 12/Oa/72
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-------
<'ANOVAi2 - ONE/TWO FACTOR AMLYSIS OF  VARIANCE.
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-------
OANOVA12  -  ONE/TWO FACTOR  ANALYSIS OF VARIANCE.        OS-3  VER.3.5
OREGON STATE  UNIVERSITY COMPUTER CENTER              DATE - 12/05/72


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-------
                                                  '*****¥»»****«*«•*»»»»
 'ANOVA12 - ONE/TWO FACTOR ANALYSIS OF VARIANCE. 	   OS-3  VER.3,5
 OREGON STATE  UNIVERSITY COMPUTER  CENTER              OA~TE - 12/05/72


                          .EM^I-OjL.^l-FT^	

   SOURCE^      	OF	SS	MS	F_	

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 EXP TYPE
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                           «   •    - 27, 58  °  "    e  f - O.C1
                                     131

-------
'ANOY&12 - ONE/TWO FACTOR  ANALYSIS  OF  VARIANCE.
OREGON  STgTE UNIVERSITY COMPUTER CENTER
  CS-3   VER.3.5
OATE  -  12/05/7?
PROBLEM
SOURCE OF
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TOTAL 23. 1
SOURCE HEANS
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U. 00*00 96
( 
0
END  OF *ANOV612 EXECUTION.
              of D&t& la Text Table 11  for Percent of Cclio S,.",IBOE Infcctsd
                                    with £. Shasta
       = 2} t— 2.31 for P - 0.05; t - 3.36 for P-0.01|  J3/2 x 42.54= 7.9802

           significant dlfforoaco = 18.Z,? Mrecnt for P — C.05
        »    "    "      •  r   -  26.81  *  •    "  ¥= 0.01
                                   102

-------
«ANGVA12  - CNE/THO  FACTOR ANALYSIS OF VARIANCE.
OREGON  STATE UNIVERSITY COMPUTER CENTER
                                                          OS-3  VER.3.5
                                                        DATE  - 12/05/72
PROBLEM
SOURCE
EXP TYPE
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ERROR
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797075<»6E
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         75.82967
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-------
'»»*«»*»»«»»»»*•»*»»»
»ANOVA12  -  ONE/TWO FftCTOO ANALYSIS OF  VARIANCE.        OS-3  VER.3,,5
ORF.GON  STfiTf  UNIVERSITY  COMPUTER CENTER               01TE  - 12/13/72
                                                                    *»»
  SOURCE

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TOTAL
                        *•*»»«»*»(

                     PROBLEM  I-OI  GP2-PT1

                       OF           SS              US

                        1   «*,m««03260fc.  03   <*.li»i«C32fcC£  03

                        «»   1.19<«&S6i»7E  03   2.9866M17E  02

                        i*   2,!79259i«2£  03   5. 9*»eH»e5fcF  02

                       10   5.13371758E  02   5.1837176'E  01

                       19   8.23632026E  03
                                                                79.9^33

                                                                 5.7616

                                                                11.1
           (EXP )
         31.6297Q
               J)
SOURCE

EXP TYPE



TEMP
           (    d)
         31.31625

EXP TYPE  X T£1F
      (EXF  ,    ?)
         15.75000
      (EXP  ,    <*)
         61.38250
      (CON  ,    9)
      (CON
                 MEANS
                             (CON )
                            <«. 83970
                             (    7)
                           12,72»»25
(tXP ,   7)
   22.88<»50
                        (CON ,   7)
                            2.S6<«00
                                               (   6)
(EXP ,   6)
   16.9*250
                  (CON ,    6)
                             D
                                          (    5)
                                       23,09200
                                                            (EXP ,
                                                            (CON
                                                                     5)
                           5)
                             0
      Aa&lysls of Final Perteit Mortality Data in Ttr*. Table  15.  So«ltey» SaLtoH
                        Virus iu 2.9 oraa Kok&ne« 3,-.Is.o».

      r- 2j t --2.23 for P = 0.05j t ^3.17 for P-0.01; /5l783 -7.1998
      I^east
        «
                      diff«rea»e  — 16.06 pereoat  for P --&.05
                  "     •   "      22.82  •  "    "  P— 0.01
                                   104

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                     • »»»»»*»*»«•»»**#»»••»*»**»<
*ANOVfcl2  -  ONE/TWO FACTOR ANALYSIS  OF VARIANCE.        OS-3  VER.3.5
OREGON  STATE UNTV£
1.61300
(CON )
11.60530

	 X" ?) "" 1 " "<.T"
51.66650 <»9. 16675



(EXP ;""5) (EXP', i»Y"
9G. 66650 98.3335C


(CON , 5) (CON , <«)
6.66650 Q





""( 3)
2.50000



(EXP , 3)
3.33350


(CON , 3)
1.66650


           is of ?ieal Ptrcest Mortality Data 1m Text ?*l»jf. 17. Socke/e Salwon
                          Virus in O.H Graa Kokane*
                                      -- 5.0797
      r z: 2j t-: 2,23 for P -0.05; t = 3.17 for F = 0.01;
             11.33
             16,10
                                               for P-- 0.05
                                                •  P - C,01
                                 J.05

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*»»*»*»*»»#»*»**»»»*»»»»-V»*V»*»****»»*»*»»i
*ANOYA1.2  - ONE/TWO  FACTOR ANALYSIS OF  VARIANCE.
OREGON  STATE IWIVESSITY CO^fUTt'R CENTER
                         i tffff.
                                                              1 * * » »*»*»¥ *
                                                           OS-3   VER.3.5
                                                         DATE - 12/05/72
  SOURCE

EXP  TYPE

TEMP

EXP  TYPE X

ERROR

TOTAL
                      PROGLF.1  I-Ot  GP3-PT1

                        OF           SS              MS             F

                         1  3* 8333120.lt.  0«»   3.883312CJE  0<»  <*75

                         5  2.e718556'*E  03   5.3<»37il26t  02    61.0710

                        10  8,7i»999991E  01   tt.7i»999961E  00.

                        21  <*.21808053L  0<«
SOURCE «£ANS 	 	
EXP TYPE
(EX° ) (CON )
86.73791 <».7&073
TEMP
( 7)
57.01750
( 3)
••8.75000
EXP TYPE X TE1F
vt )
%7. 50000



(EXP , i»>
95.00000


(CON , U)
0


     Analysis of Final Pereent Mortality Data in Text Table is,   Sockaye Salaoa
                         ¥ima ia 0.95 Or** KoJotase Saluoa,
     r jr 2;  t -2.2? for P r 0.05;  t  - 3.17 for F .- O.C-i; ;2 s 8.749 - 4.1833.

     Least *i£»ifio*nt. ciiffaroR«»«  9,33 per*e»t \oi- r _ 0.05
       *    *     e     "    "     13.?6  "  "    "  P:rO,Oi
                                 106

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          2
         X  Analysis of Percentages of Surviving  Fish Yielding


               Cultures of t±. liquefaciens. Text  Table  6.
A.  Comparison of Percent Positive at  69°F  (28%)  and  59°F  (71%)
Water
Temp.
69°
59°
Total
2
.X
Number
Positive
7
__3
10
- (17.5 x
Number
Negative
18
40
58
2.5 - 40.5 x 7.5)2 x 68
Total
25
43
68

(Yates Modification)  25 x 43 x 10 x  58


                                   -  7.38

      2
From x  Table for n = 1                                  „

                  p = less than 0.01  for this value  of  x


Hence difference is highly significant.




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


Water              Number               Number

Temp.             Positive             Negative              Total


 69°                  4                   19                   23


 54°                  !_                   41                   4£


Total                 5                   60                   65


X2                 " (18.5 x 0.5 - 4.5 x 41.5)2 x  65
(Yates modification)  23 x 42 x 60 x  5


                                   -  7.09

      2
From x  Table for n = 1

                  p = less than 0.01  for this value  of  x


Hence difference is highly significant.
                                  107

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          REGRESSION ANALXSLS. AERCilCr.'AS S^LMCNTC.i;V
                IN CGHO SALMON.  TEXT FIG.  1
   .3
   ,3
   4

   t

   •

   •

   *
  a"
  ET
  B."
2
2
i



1
i
7
7
7



i
5
5
G
•t

1

2
7
*
i


1
2
3
* <>
* * *
                   TEMPERATURE ——	——->
  .I EX IT	  			
     RELATION BKl'WfiEH WATZS TEbPERlTORS AMD 100 I'O. OF I^IS TO
     DEATH.  A.  SAIKGKIC1BA INACTION IN COilO SAL!:. ,

      Y(  2)-   H.^629315?:  C!-     .    +._-2.7651; 3^£-0<: X(
    '  Y = LOG EO. DAIS TO D2ATH              ( D--'.- l-PEP-Vi'UT,!*.


    R  SQUARFO =   .. 7.331'J516 =  %. OF VARIATIOK : ^OUKED 1'OR
        VAR    S.E.  OF REGR.  ?GEF»
          C       6.00 !ie7282C -02
CORREI.ATICN  COEFFICIENT  =  -C.«8i9775  ~ Lir: . F3IATTOHGH1P
                                             AID JOG KO. Or DAIS
                                                  TO DEATti
                                  103

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           REGRESSION  ANALYSIS. ATJICMDIIAS
                 IN CKIKOOX SALMON. TEXT FIG.  3
l.z
ti
,5
.
.<*
.1
13
^ c
<3« ?
M
H«
/>• 3
H J>
•*.!
Z4
3»
2*
?
•

2

3
2 1
7 3
? ^
i 2
1 2
1
1 t»
' • v 2





i
1

1
. . .1 ,
3
1
5
«
7
5
• - ' - • - •


TVKrnpD l f TTT'T?
«

1

*+ 1
1
1
1
1 2
521
7122
* 8  f \*~1T1 f fl' ** '^l1*'^
                                              ' 1 f.., I M J t 1 •«*">" fI K ' I ' f i

                                           /.r: LOG /:o. OF DAYS
                                                 TO DEATH
                               109

-------
          REGRESSION  ANALYSIS. CHOj'IDRCCOCCJS 001  'imi
                 IN RAINDOW TROUT. TEXT FIG. 4
                 i
 *
                k              5           ' "1
                2 	  9  		._._!

                1              *             *
                                             *
                TE^ERATURS
    RELATION E3TVSZN WATSR 'lETERATUPJS A:,T) LOG IT,  OF DAYS  TO
       DSATHc c. COLUI-:JARIS liiECTios IK jaiiizc'  lacur.
     Y(  2)=  3.28061«*6E 00         +  -<*,<»9  .-.3^2E-02 X(  i
      Yr LOG 110. DAYS TO DZ1VTH              (  .) =: TEMPERATURE
    R SQUARED  =    .73^8 935«f = _jj OF VARIATi: 7 .ACCOUNTED FOR,
                                          WITH  JJ3.
    VAR~""""siE. "OF  REGRCOEF. " ""       T
       C       l.'t 216916 fbl-OJ       2. 30? ^'--5. ISt  01
       i       2.C96-7151?£«03      -2, l^Sii/iflE  01
CORREtATICN  CCEFFIClLMT  = -0.8572^93^  1-".::AR RSL'JIONSHIF
                                              r/ii^SN 'i7.MPLRATJJR3
                                              A.D LOG ::0. OF DAYS
"'  ""	             	"  "~    "                 TO DEATH

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            REGRESSION ANALYSIS. CKCIIDROCQCCUo I SLU1-JNARIS
                  IK COHQ SALMON.  TSXT FIG. 5
4'1      - -  -   t        	
 A              1
 .12
                1            -  1
 ^              _

                3
gi   '          .

g.              7              7

 »              7              *             C1

3
                               *             *


 .

 «                             2             ''


_tEXIT_			._	_    .        . .„_	;		
    RELATION ESTkSEN WATER KIPERATL'IiS AND  LOG  17. OF DAIS  TO
    .  .  DEATH..  C. COLUMHAR13 IWPECTTOH IN C£;l 3ALMGN


    Y(  2)=  2.62703i»3E  03         +  -3.353.  30E-0? X(  i)
       I-- LOG KO. DAK TO DEATH         ( 1) r  rEKPERATUHS


    R. SQUARED  =.   .59236395  - % OF VARIATI: : ACCOUNTED FOIi
                                           \'l " LIT®.

    "VAR    S"VET"~OF~ REGR7" COEF^   ^"^""    ~i	-—-— '
        0        1.18372935E-01        2. 21?: : ';,3 GE 21
        1        1.753«i7-'«05E-03       -1.911 " ?2«*E 01 "•"


CGRRCLATICN  CCEFFICIcNT =  -0.7699796 ^  J .'.THAH RPILVflONSHIP

 	-		--.	....  _			 .  i n LOG KO.  cy u
                                                  TO DEATH

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         REGKESSJOH ANALYSIS,  CIIOIIDRGCOCCUS COI,Ul":i/iRIS
                115 CHINOOiC SALMON. TEXT FIG. 6
           1       1       i       i
                   ill
                           1       1
                           1  •     i
  .1
£-<
5S»
i


i


2
,2



^2
.1

i
1
1
1
p,
&•
125
6
1
7
*
7
1
*
*
                  J.fiii'lS'JUUiJ.UWi ---~--«~«——<~———^,»
  IEXIT
        ITIOM 'E2TW3EH WATER  TE13TJIATOKS ?,I3) LOG ' •"." OF DAYS  TO
         DSATH.  C.  COUKlilARlS  IHEECTIOII IN
     Y( 2)=   2.755S2'f3E  OC        +  -3.222 j7 86E-Q 2 X(  1)
      1= LOG KO. HAYS TO DEATH            ( l) :   TEMPERATURE


     R  SQUARED  =    .51730671-  % OF VARIAT'  .s ACCOUNTED FOR
                                              1; 'Til
      VAR  "StE.  OF REGR.  COEF ".""    ""   T    .
        0        l,76ia8738E-Gl        1.562'«  v09E  01
                           7E-03       -1.220^  :^60E  01
CORRELATION  COEFFICIENT  ~  -0.7102^0^=  - ' "^ RSUTIO;«Sni?
                                              I  T^SEK Ti^PERATURS
                                              ;•.' ) 1X30 150. OF DAYS
"""  	  "~	              ""	"       TO DEATH
                                112

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            REGRESSION ANALYSIS.  CERATOMYXA SJLXSTA IN
                    RAiliBOW TROUT. TEXT F1GC '/
*:
 .
 .1
 *
 .
i
3
           851
^J                 .  *          1       ..
B                   *  	   «*            " ~	"
S                   1          *

3        	"	      	      j
ET»                              LOj i;0. Or DAYS
                                                   TO DSATH

-------
        A
          • 1
                        REGRESSION V; A LYSIS. C?il/.TC>ffia SI^GTA IK
                                co;:o PAI^ON. TTIT FIG,  8
         CH
         «<+
         o-
         &
                                 i
                                 i
                j_
                i
                          TEMPERATURE ~
                   n  J^TVBSL'K  I'ATT^l lEli'Etif.iUR-*. AK'.* WO NO, OF I'AYS TO
                  HSATH.  0.  SilASTA 31KCT1GH 111 CCHO S.M: ::;
Y(  ?)-   ^.O
.  Y = IOQ NO,
                                    H  09
                                  TC KATH
          (I) :: TQ-i
                       2  X(
                S  SQUARCO =
=.  % OP
                                                ACCOIT:IT£D FOR
                                                 LUS.
                 VAR    S.t.  OF  ^EGR.  COE
                   0        7.««270H3fc5t---Q2
                   i   "     1.105ZJ8P93E-03
                                                 .E 01
                                                 :E Qi
          CORRELATION. CC£F^ ICI-IN f  -  -C, 95/4965 =  },...;,
                                                                  KO. OF
                                                                TO DSATH
ftU.S GOVERNMEMT PRINTING OFFICE 1974 546-317/313  1-3

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SELECTED WATER
RESOURCES ABSTRACTS
INPUT TRANSACTION FORM
                                              He;-  •>' No
                                                                w
   Effects of Temperature On Diseases Of Salmonid Fishes
J. L. FRYER AND K. S. PILCHER
Oregon State University
Department of Microbiology
                      Office of Research and Monitoring
                      Environmental Protection Agency
                                                                     .-*tD
                                                                            July, 1972
                                                                           O/gsr :ttwn
                                                                   18050 DIJ
                                                               13.  Type--  Repot  ~ntt Final
                                                                   Period Ctvertii   Report
                                                                   4/1/69 - 3/31/72
Environmental  Protection Agency,  report number, EPA-660/3-73-020,
January  1974.
                                                       In all cases mortality rates
                                           to 59°F;  and low or zero at 39° to 49°F.
The effect of water temperature on infections  of  salmonid fish was investigated.
Chondrococcus columnaris infection was  studied in rainbow trout,  coho and spring
chinook salmon; Aeromonas salmonicida infection in coho  and spring chinook salmon; and
Aeromonas liquefaciens infection in  steelhead  trout.
were high at 64  to 69 F; moderate at 54
Progress of the infections was accelerated  at  higher  temperatures, and progressively
retarded at decreasing temperature levels.   In infection of coho  with Ceratomyxa
shasta, mortality was high at 69°F,  low at  49° to 54°, and zero at 39  to 44 F.   This
infection in rainbow trout resulted  in  high mortality at all temperatures except 39 .
In both cases the course of the disease was most  rapid at higher  temperatures, and
became progressively slower as the temperature decreased.  For infection of kokanee
salmon fingerlings with sockeye salmon  virus,  the temperature range of 54  to 59 F was
optimal.  In this range mortality rates were high, and  the course of the disease was
most rapid.  At higher temperatures  mortality  rates were lower, and at 39  to 44 F,
progress of the disease was retarded, though total mortality was  often high.
 '-'•       •    Animal diseases,  effluents,  fish diseases,  heated water, infection,
microorganisms, pathogenic bacteria,  pathology,  thermal  pollution, water pollution,
water quality, water temperature
                       19.  S"-
                           (si

                       2t'!.  SVciir;-. 7 C'ast.
                                        21.  A',, of
                                            Page's

                                        22,  Price
Send To:
WATER RESOURCES SCIENTIFIC INFORMATION CENTER
US DEPARTMENT OF THE INTERIOR
WASHINGTON, D  C 2O24O

-------