AN ABSTRACT OF THE THESIS OF
Matthew G. Mesa for the degree of Doctor of Philosophy in Fisheries Science presented
on January 28. 1999. Title: Ecological Influence of Bacterial Kidney Disease on
Juvenile Spring Chinook Salmon: Effects on Predator Avoidance Ability.,
Smoltification. and Physiological Responses to Stress.
Abstract approved:
Redacted for Privacy
Alec G. Mau le
Abstract approved:
Redacted for Privacy
Carl B. Schreck
Juvenile chinook salmon (Oncorhynchus tshawytscha) were experimentally
infected with Renibacterium salmoninarum (Rs), the causative agent of bacterial kidney
disease (BKD), to investigate the effects of BKD on three aspects of juvenile salmonid
performance: (1) predator avoidance ability; (2) smoltification; and (3) physiological
responses to stress. For these experiments, fish with different Rs-infection profiles
(created by using an immersion challenge method) were sampled to assess physiological
change and subjected to various performance tests during disease progression.
When equal numbers of Rs-challenged and unchallenged fish were subjected to
predation by northern pikeminnow (Ptychocheilus oregonensis) or smallmouth bass
(Micropterus dolomieui), Rs-challenged fish were eaten in significantly greater numbers
than controls by nearly two to one.
A progressively worsening infection with Rs did not alter the normal changes in
gill ATPase and condition factor associated with smoltification in juvenile chinook
salmon. A dramatic proliferation of BKD was associated with maximal responses of
indicators of smoltification, suggesting that the process of smoltification itself can
trigger outbreaks of disease.
When Rs-infected fish were subjected to three 60-s bouts of severe handling that
were separated by 48-72 h, this experience did not lead to higher infection levels or
increased mortality when compared to diseased fish that did not receive the stressors.
Furthermore, the kinetics of plasma cortisol, glucose, and lactate over 24-h following
each stressor were similar between fish with moderate to high BKD and those that had
low or no detectable infection. Fish with moderate to high Rs infections had higher
titers of cortisol and lactate prior to each application of the stressor and were also unable
to consistently elicit a significant hyperglycemia in response to the stressors when
compared to fish with low infection levels.
During all experiments, fish consistently developed decreased hematocrits and
blood glucose levels and increased levels of cortisol and lactate as the disease worsened,
indicating that BKD is stressful, particularly during the later stages.
Collectively, these results illustrate the impact of BKD on juvenile salmonids
and have also ascribed some ecological significance to this disease beyond that of direct
pathogen-related mortality.
Ecological Influence of Bacterial Kidney Disease on Juvenile Spring Chinook Salmon:
Effects on Predator Avoidance Ability, Smoltification, and Physiological Responses to
Stress
by
Matthew G. Mesa
A THESIS
submitted to
Oregon State University
in partial fulfillment of
the requirements
for the degree of
Doctor of Philosophy
Presented January 28, 1999
Commencement June 1999
Doctor of Philosophy thesis of Matthew G. Mesa presented on January 28, 1999
APPROVED:
Redacted for Privacy
Co-Major Professor, representing Fisheries Science
Redacted for Privacy
Co-Major Professor, representing Fisheries Science
Redacted for Privacy
Head of Department of Fisheries an
ildlife
Redacted for Privacy
Dean of Gradu e School
I understand that my thesis will become part of the permanent collection of Oregon
State University libraries. My signature below authorizes release of my thesis to any
reader upon request.
Redacted for Privacy
Matthew G. Mesa, Author
Acknowledgments
During my research and writing of this thesis, I somehow managed to have a
couple of children (with my wife, of course), build a house, and hold down a full-time
job. Friends and family would occasionally ask me "man, how did you do it ?", but
really the answer was quite evident: this endeavor was simply not possible without the
willing, generous, and untiring efforts of many people. This last bit of writing is saved
to recognize those persons that contributed--in a variety of ways--to the completion of
this thesis.
From the outset, my wife, Mary, was a school teacher, mother, homemaker, and
a friend. She sacrificed as much as I did, and therefore deserves equal credit for this
thesis. She knows what this meant to me, and I love her. My two daughters, Taylor and
Lauren, brought joy to my life, the sense of wonder that accompanies childhood, a
constant source of inspiration to maintain my focus, and an endless reservoir of respite
from the daily routine. A special thank-you goes to: my parents, Gilbert and Hazel
Mesa, for believing in me and teaching me to keep my eye on the ball and eschew
excuses; my brothers and sisters, Brian, Maree, and Ted, who never fully understood
what I was doing but always thought it was "great"; and my "in-law" family: Bob and
Doris Sapp, Bob and Anne Chudek, and Katie and Steve Mammen, who simply always
seemed to care.
Numerous individuals are on my "wonderful persons" list for their physical and
intellectual efforts, advice and friendship, and guitar playing and beer drinking: Craig
Barfoot, Jen Bayer, Brian Beckman, Pat Connolly, Tim Counihan, Speros Doulos,
Diane Elliott, Bill Gale, Dena Gadomski, Susan Gutenberger, Karen Hans, Glen
Holmberg, Jack Hotchkiss, Pete Kofoot, Al Kolok, Jerry McCann, Connie McKibben,
Bill and Susan Muir, Todd Olson, Ron Pascho, Jim Petersen, Jim Rockowski, Sally
Sauter, Robin Schrock, Cam and Terri Sharpe, Rip Shively, Stan Smith, Matt and Mary
Swihart, Marne Thomasen, Scott VanderKooi, Matt Vijayan, Joe Warren, Lisa Weiland,
and Margo Whipple. This thesis was not possible without you, and I thank you.
Sincere gratitude goes to my co-major professors, Alec Mau le and Carl Schreck,
for their advice and assistance, and for shaping my development as a scientist. I respect
these gentlemen immensely because they epitomize professionalism in every way I can
think of, and I feel extremely fortunate to have had the pleasure of knowing and
working with them. I thank my graduate committee, Ken Funk, Gordie Reeves, Paul
Reno, and Steve Schroder, for starting this whole thing off in the beginning and making
sure it got done right in the end. Finally, a heartfelt thanks goes out to Tom Poe, Bill
Nelson, Jim Seelye, Al Fox, and Frank Shipley, my supervisors and lab directors, for
their professional and financial support and for providing me with this opportunity.
You guys held up your end of the bargain, I hope I made good on mine.
Contributions of Authors
Dr. Alec G. Mau le, Thomas P. Poe, and Dr. Carl B. Schreck were advisors and editors
on all manuscripts included in this work. In addition, Alec and Tom were invaluable in
helping to secure funding for completion of this work.
TABLE OF CONTENTS
Page
CHAPTER 1. Introduction
1
Objectives
8
References
9
CHAPTER 2. Vulnerability to Predation and Physiological Stress Responses in
Juvenile Chinook Salmon Experimentally Infected with Renibacterium
salmoninarum
16
Abstract
17
Introduction
18
Methods
19
Results
25
Discussion
37
Acknowledgments
43
References
44
CHAPTER 3. Influence of Bacterial Kidney Disease on Smoltification in Juvenile
Salmonids: is it a Case of Double Jeopardy ?
48
Abstract
49
Introduction
50
Methods
52
Results
57
Discussion
73
Acknowledgments
81
References
82
TABLE OF CONTENTS (continued)
Page
CHAPTER 4. Interaction of Bacterial Kidney Disease and Physical Stress in
Juvenile Chinook Salmon: Physiological Responses, Disease Pathogenesis,
and Mortality
89
Abstract
90
Introduction
91
Methods
94
Results
98
Discussion
118
Acknowledgments
126
References
126
SUMMARY AND CONCLUSIONS
132
BIBLIOGRAPHY
138
LIST OF FIGURES
Page
Figure
2.1.
Mean (and SE) ELISA optical densities at selected time intervals
after spring chinook salmon had received a waterborne challenge with
Renibacterium salmoninarum and for unchallenged control fish during
1992 and 1994
26
2.2.
Cumulative mortality of spring chinook salmon after receiving a waterborne
challenge with Renibacterium salmoninarum and for unchallenged control
fish during 1992 and 1994
29
2.3.
Mean (+ SE) fork lengths (mm) and weights (g) for spring chinook salmon
that had received a waterborne challenge with Renibacterium salmoninarum
and for unchallenged control fish during 1992 and 1994
29
2.4.
Mean (and SE) concentrations of plasma cortisol, lactate, and glucose for
spring chinook salmon that had received a waterborne challenge with
Renibacterium salmoninarum and for unchallenged control fish during
3.1.
3.2.
1994
29
Mean (+ SE) fork lengths (mm) and weights (g) for spring chinook salmon
that had received a waterborne challenge with Renibacterium salmoninarum
and for unchallenged control fish during_1993 and 1994
58
Mean (+ SE) condition factor for spring chinook salmon that had received
a waterborne challenge with Renibacterium salmoninarum and for
unchallenged control fish during 1993 and 1994
58
3.3.
Mean (and SE) ELISA optical densities at selected time intervals after spring
chinook salmon had received a waterborne challenge with Renibacterium
salmoninarum and for unchallenged control fish during 1993 and 1994 . . . . 62
3.4.
Cumulative mortality of spring chinook salmon after receiving a waterborne
challenge with Renibacterium salmoninarum and for unchallenged control
fish during 1993 and 1994
65
3.5.
Mean (and SE) gill Na, + K+-ATPase activity, and concentrations of plasma
cortisol, glucose, and lactate for spring chinook salmon that had received a
waterborne challenge with Renibacterium salmoninarum and for
unchallenged control fish during 1993
68
LIST OF FIGURES (continued)
Page
Figure
3.6.
4.1.
4.2.
Mean (and SE) gill Na+, IC-ATPase activity, and concentrations of plasma
cortisol, glucose, and lactate for spring chinook salmon that had received a
waterborne challenge with Renibacterium salmoninarum and for
unchallenged control fish during 1994
71
Mean (and SE) fork lengths and weights for spring chinook salmon during
the 16-week holding and monitoring period of our BKD/stress response
experiments
99
Mean (and SE) ELISA absorbances at selected time intervals after spring
chinook salmon had received a waterborne challenge with Renibacterium
salmoninarum and for unchallenged control fish during our BKD/stress
response experiments
99
4.3.
Mean (and SE) hematocrit (A), and concentrations of plasma cortisol (B),
glucose (C), and lactate (D) for spring chinook salmon that had received a
waterborne challenge with Renibacterium salmoninarum and for unchallenged
control fish during the 16-week holding and monitoring period of our
103
experiment
4.4.
Cumulative mortality of spring chinook salmon after receiving a waterborne
challenge with Renibacterium salmoninarum during the 16-week holding
and monitoring period of our experiment
107
4.5.
Mean (and SE) plasma cortisol concentrations of Rs-challenged and
unchallenged juvenile spring chinook salmon subjected to three 60-s periods
of handling and mild agitation and for fish not exposed to the stressors . . . 110
4.6.
Mean (and SE) plasma glucose concentrations of Rs-challenged and
unchallenged juvenile spring chinook salmon subjected to three 60-s periods
of handling and mild agitation and for fish not exposed to the stressors . . . 114
4.7.
Mean (and SE) plasma lactate concentrations of Rs-challenged and
unchallenged juvenile spring chinook salmon subjected to three 60-s periods
of handling and mild agitation and for fish not exposed to the stressors . . . 114
LIST OF TABLES
Page
Table
2.1.
Results of experiments for juvenile chinook salmon experimentally infected
with Renibacterium salmoninarum and subjected to predation by northern
pikeminnow
34
2.2.
Results of experiments for juvenile chinook salmon experimentally infected
with Renibacterium salmoninarum and subjected to predation by smallmouth
36
bass
4.1.
Mean (and SD) number of days to death and total number of fish that died
during the 16 week holding and monitoring period of our BKD /stress
response experiment
109
Ecological Influence of Bacterial Kidney Disease on Juvenile Spring Chinook
Salmon: Effects on Predator Avoidance Ability, Smoltification, and Physiological
Responses to Stress
CHAPTER 1
Introduction
Diseases, broadly defined to include those due to viruses, bacteria, protozoans,
fungi, and helminths, can have profound effects on plant and animal communities and
individuals. Diseases can alter community structure when an epizootic occurs, as
evidenced by pathogens that have destroyed much of the endemic Hawaiian Island bird
fauna (Warner 1968), altered the distribution of the North American moose Alces alces
(Anderson 1981), and almost completely eliminated the English elm Ulmus campestris
from England (Begon et al. 1986). Besides causing outright mortality, diseases can
have more subtle, sub-lethal effects on individual organisms. Diseases may adversely
affect the performance capacity of an organism, which I define as all of the life
functions an individual must do in order to persist. Organisms performing at a suboptimal level because of poor health may be less competitive, have reduced genetic
fitness, and ultimately succumb to other sources of mortality besides the disease itself.
My research, involving the pathogen-host relation between the causative agent of
bacterial kidney disease (BKD) Renibacterium salmoninarum (Rs) and juvenile spring
chinook salmon Oncorhynchus tshawytscha, investigated the effects of this disease on
selected aspects of juvenile salmonid performance. The performance characteristics I
2
assessed--predator avoidance ability, smoltification, and physiological responses to
stress--elucidated some of the sub-lethal effects of BKD and helped define how it
affects survival of fish in the wild.
Bacterial kidney disease has long been a significant contributor to mortality of
juvenile salmonids in hatcheries in the Columbia River basin and in virtually all parts of
the world where salmonids occur (Fryer and Sanders 1981; Elliott et al. 1989). The
disease is not only considered to be a major obstacle to the successful culture of
salmonids, but is prevalent in and presumably impacts wild populations (Ellis et al.
1978; Mitchum et al. 1979; Paterson et al. 1979; Elliott and Pascho 1992; Sanders et al.
1992). During hatchery residency of juvenile salmonids BKD causes chronic mortality,
which may be exacerbated when fish enter salt water (Ellis et al. 1978; Paterson et al.
1979; Fryer and Sanders 1981; Banner et al. 1983). The pathogen can be transmitted
horizontally and is the only bacterial disease known in fish to be transmitted vertically
(Bullock et al. 1978; Evelyn et al. 1989). Control of BKD is difficult to achieve, and,
because large gaps persist in our knowledge of the infection (Elliott et al. 1989), this
disease will continue to receive a considerable amount of research interest.
Most research on BKD has, understandably, focused on the etiology,
epidemiology, pathogenicity, and control of the disease. Thus, little is known about the
effects BKD may have on fish performance capacity (sensu Schreck 1981) that may
affect survival. The recent development of more sensitive and quantitative detection
methods for Rs, such as the enzyme-linked immunosorbent assay (ELISA; Pascho and
Mulcahy 1987; Turaga et al. 1987) has allowed more accurate determination of the
3
prevalence and levels of Rs infections in salmonid populations (Pascho et al. 1987).
Consequently, fish have been classified as having low, moderate, and high infection
levels based on ELISA results (Pascho et al. 1991). This implies that differing infection
levels may have some adaptive significance (e.g., fish with low infection levels may
have higher survival than fish with moderate levels), but thus far such levels appear to
be of clinical significance only. Because there are few data on causes of fish mortality
outside of hatcheries, the impact of BKD on migrating hatchery and wild fish has been
difficult to evaluate (Sanders et al. 1992).
Aside from direct mortality, the effects of BKD on salmonids include a variety
of sublethal physiological and hematological changes (Wedemeyer and Ross 1973;
Bruno 1986; Bruno and Munro 1986; Iwama et al. 1986). Similar changes have been
reported in fish with viral (MacMillan et al. 1980; Haney et al. 1992) or parasitic
infections (Laidley et al. 1988; Rand and Cone 1990). It appears, however, that effects
of sub-optimal fish health on performance characteristics have been mainly limited to
studies of fish with parasitic infections. For example, parasite infections have been
shown to reduce the maximum swimming speed of European smelt Osmerus eperlanus
and eel Anguilla anguilla (Sprengel and Luchtenberg 1991), reduce the cardiac output
of rainbow trout 0. mykiss (Tort et al. 1987), increase the susceptibility of rainbow trout
to hypoxia (Woo and Wehnert 1986), and increase the oxygen consumption of
sticklebacks Gasterosteus aculeatus (Lester 1971). Parasitic infections may not,
however, significantly increase the vulnerability of fish to predation (Vaughn and Coble
1975; Milinksi 1985). Fish diseases appear to have been an important force in
4
structuring fish communities in the Pacific Northwest (Li et al. 1987); it is clear from
the lack of data on the ecological impacts of disease that possible mechanisms behind
this structuring force will remain elusive.
Despite uncertainty in mortality estimates, predation is now considered to be a
significant contributor to the previously unexplained mortality of out-migrating juvenile
salmonids in the Columbia River (Rieman et al. 1991). Predator avoidance ability is
one aspect of performance capacity that has obvious ecological significance, but the role
that Rs infection may play in predator-prey interactions is unknown. The effects of suboptimal fish health on predator avoidance ability has focused mainly on stress induced
effects. For example, several studies have shown increased vulnerability to predation of
prey exposed to a variety of stressors, including temperature shock (Coutant 1973;
Yocum and Edsall 1974), contaminants (Hatfield and Anderson 1972; Kania and O'Hara
1974), and handling or agitation (011a and Davis 1989; 011a et al. 1992; Mesa 1994). If
similar effects occur in fish infected with sub-clinical levels of Rs, it would elucidate at
least one proximate cause of mortality in migrating juvenile salmon.
As mentioned above, mortality of juvenile salmon infected with BKD may
accelerate when fish enter salt water, perhaps due to the stress associated with the
transition from fresh to salt water (Fryer and Sanders 1981; Banner et al. 1983).
Surprisingly, the effects of BKD on processes that may be associated with this
mortality, such as the parr-smolt transformation and enhancement of hypoosmoregulatory ability, have received little attention.
5
In juvenile anadromous salmonids, the parr-smolt transformation is a series of
physiological, morphological, and behavioral changes associated with the transition
from fresh water to salt water (Hoar 1976; Folmar and Dickhoff 1980). One of the most
characteristic features of smoltification is enhanced sea water adaptability of fish.
Smoltification involves many complex processes and, if infection with Rs disrupts some
or all of these, it may explain the apparent high mortality of infected fish in sea water.
Smoltification is partly under neuroendocrine control, and a variety of hormones serve
diverse functions during the parr-smolt transformation (Barton et al. 1985; Young 1986;
Prunet et al. 1989; Madsen 1990a, 1990b). For example, cortisol functions in
mobilizing stored energy (Specker 1982; Franklin et al. 1992) and enhancing hypoosmoregulatory ability by stimulating gill Nat, K+-ATPase and chloride cell activity
(Richman and Zaugg 1987; McCormick and Bern 1989; Avella et al. 1990; Madsen
1990a; Bisbal and Specker 1991). Because cortisol is produced in the interrenal tissue,
the affinity of Rs for the kidney and its subsequent pathogenesis could conceivably
impair interrenal function. Indeed, chronically elevated cortisol has been implicated in
the failure of salmon to adapt to sea water, perhaps resulting from the inability of fish to
achieve ionic and osmotic homeostasis (Avella et al. 1990; Franklin et al. 1992). The
possibility also exists that a systemic infection with Rs could impair the release or
functions of other hormones important to smoltification and sea water adaptability,
including prolactin and growth hormone (Prunet et al. 1989; Madsen 1990b). An
understanding of how BKD affects the physiological processes involved with
6
smoltification could help in explaining mechanisms behind mortality of juvenile salmon
not caused directly by predation.
Although it is recognized that stress can predispose fish to disease (see Ellis
1981 for a review), less is known about how disease per se acts as a stressor or how
disease affects the ability of fish to cope with secondary, exogenous stresses. Various
pathogens are known to elicit characteristic stress responses in fish (Robertson et al.
1987; Laidley et al. 1988; Haney et al. 1992; Olson et al. 1992). Bacterial kidney
disease elicits several significant physiological changes before evidence of clinical
disease, including decreases in hematocrit, hemoglobin, total plasma protein, and
plasma glucose (Suzumoto et al. 1977; Bruno 1986; Bruno and Munro 1986; Iwama et
al. 1986; Turaga et al. 1987). The response of plasma cortisol, a well documented
indicator of stress in fish, to infection with Rs is less clear. Donaldson (1981) quoted
unpublished observations that cortisol levels increase whereas Suzumoto et al. (1977)
suggested cortisol levels may decrease with the progression of BKD. In general, the
literature on cortisol elevation due to disease is sparse and equivocal (Olson et al. 1992).
Because much of the work examining the physiological responses of salmonids to BKD
has been done using intraperitoneal injection as the method of infection, the results may
not reflect responses of fish under more natural conditions. In this regard, the use of a
waterborne, immersion challenge (which I used for all my experiments) to infect fish
may be more appropriate. The advantages of immersion challenges have been discussed
by Murray et al. (1992) and include a reduced likelihood to overwhelm the innate BKD
resistance of fish, a closer simulation of the natural routes of infection, and no additional
7
stress due to handling. Thus, the immersion challenge provides an opportunity for
studying Rs infections under more natural conditions.
There is a paucity of information concerning how diseased fish might respond
physiologically to secondary, exogenous stressors, even though this may be critical to
their survival. During their downstream migration, juvenile salmon are commonly
subjected to multiple stressors. For example, fish passing through dams are subjected to
traveling screens, gatewells, fish sorters, turbines and spill, all of which can have
cumulative stress effects on the fish (Matthews et al. 1986; Maule et al. 1988). Multiple
disturbances can elicit severe physiological and behavioral responses in salmonids
(Barton et al. 1986; Pickering and Pottinger 1987; Maule et al. 1988; Peters et al. 1988;
Mesa and Schreck 1989; Mesa 1994), yet whether BKD affects compensatory stress
responses or worsens after exposure to stress is not known. Given the
immunosuppressive effects of stress and elevated cortisol levels in salmonids (Maule et
al. 1987; Tripp et al. 1987; Maule et al. 1989), the potential for accelerated pathogenesis
of BKD and maladaptive stress responses seems high. Barton et al. (1986) showed that
juvenile chinook salmon with chronic fin rot and infected with the coldwater disease
bacterium Cytophaga psychrophila were unable to achieve physiological compensation
after exposure to repeated, acute handling stresses. In their fish, plasma cortisol and
glucose remained chronically elevated and mortality was high after the stresses. Similar
findings in juvenile salmonids with sublethal Rs infection profiles could partly explain
any mortality during the freshwater phase of their migration.
8
Objectives
The overall goal of my research was to provide insight into whether sub-lethal,
infectious disease significantly compromises selected aspects of animal performance.
Such reduced performance might adversely affect the survival of organisms, thereby
leading to an ultimate reduction in fitness. To accomplish this goal, I examined the
pathogen-host system of Renibacterium salmoninarum and juvenile salmonids to
determine whether different infection levels affect the ability of fish to avoid predators,
undergo smoltification in freshwater, and show compensatory, physiological responses
to exogenous stressors. Specifically, I determined:
1. The vulnerability to predation of juvenile spring chinook salmon with
differing levels of Rs infection.
2. The effects of a chronic, progressive infection with Rs on selected aspects of
smoltification in yearling juvenile chinook salmon.
3. The physiological responses of Rs-infected and uninfected juvenile
salmonids after they had been exposed to a series of multiple, acute stressors and
assessed if exposure to such stressors worsened established Rs infections and led to
increased mortality.
4. Some stress-related physiological changes in juvenile spring chinook salmon
during the long-term, chronic progression of the disease.
This research examined physiological and behavioral responses of fish to
different levels of Rs-infection and concomitant stress. Collectively, it provides a much
9
needed understanding of the functional, ecological significance of BKD and the role it
plays in affecting the survival of fish in the wild.
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cortisol concentrations during salinity challenges of coho salmon (Oncorhynchus
kisutch) at smolt and post-smolt stages. Aquaculture 91:359-372.
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10
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11
Haney, D.C., D.A. Hursh, M.C. Mix, and J.R. Winton. 1992. Physiological and
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12
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13
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14
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15
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16
CHAPTER 2
Vulnerability to Predation and Physiological Stress Responses in Juvenile Chinook
Salmon Experimentally Infected with Renibacterium salmoninarum
Matthew G. Mesa14, Thomas P. Poe', Alec G. Maule', and Carl B. Schreck3
'U.S. Geological Survey, Biological Resources Division
Western Fisheries Research Center, Columbia River Research Laboratory
5501A Cook-Underwood Road, Cook, Washington 98605, USA
2Oregon Cooperative Fishery Research Unit, Department of Fish and Wildlife
Oregon State University, Corvallis, OR 97331-3803
3Oregon Cooperative Fish and Wildlife Research Unita
Oregon State University, Corvallis, Oregon 97331-3803, USA
aCooperators are Oregon State University, the Oregon Department of Fish and Wildlife,
and the U.S. Geological Survey.
Published in Can. J. Fish. Aquat. Sci. 55:1599-1606.
17
Abstract
We experimentally infected juvenile chinook salmon (Oncorhynchus
tshawytscha) with Renibacterium salmoninarum (Rs), the causative agent of bacterial
kidney disease (BKD), to examine the vulnerability to predation of fish with differing
levels of Rs infection and assess physiological change during progression of the disease.
Immersion challenges conducted during 1992 and 1994 produced fish with either a low
to moderate (1992) or high (1994) infection level of Rs during the 14-week postchallenge rearing period. When equal numbers of treatment and unchallenged control
fish were subjected to predation by either northern pikeminnow (Ptychocheilus
oregonensis, formerly the northern squawfish) or smallmouth bass (Micropterus
dolomieui), Rs-challenged fish were eaten in significantly greater numbers than controls
by nearly two to one. In 1994, we also sampled fish every two weeks after the
challenge to determine some stressful effects of Rs infection. During disease
progression in fish, plasma cortisol and lactate increased significantly whereas glucose
decreased significantly. Our results indicate the role BKD may play in predator-prey
interactions, thus ascribing some ecological significance to this disease beyond that of
direct pathogen-related mortality. In addition, the physiological changes observed in
our fish during the chronic progression of BKD indicate that this disease is stressful,
particularly during the later stages.
18
Introduction
Bacterial kidney disease (BKD), caused by Renibacterium salmoninarum (Rs),
is a significant contributor to mortality of juvenile salmonids in hatcheries in the
Columbia River basin. The disease is not only considered to be a major obstacle to the
successful culture of salmonids, but is prevalent in and presumably has some impact on
wild populations (e.g., Sanders et al. 1992). The disease causes chronic mortality
during hatchery residency of juvenile salmonids and mortality may be exacerbated when
fish enter salt water (Fryer and Sanders 1981; Banner et al. 1983). Control of BKD has
been difficult to achieve and, because large gaps persist in our knowledge of the
infection (Elliott et al. 1989), this disease will continue to receive a considerable amount
of research interest.
Little is known about the effects BKD may have on selected aspects of fish
performance capacity, such as predator avoidance ability. In addition, there is scant- and equivocal--information on whether infection with Rs per se is stressful and thus
activates the hypothalamic-pituitary-interrenal (HPI) axis and elicits physiological stress
responses (Suzumoto et al. 1977; Donaldson 1981). Because the impact of BKD on
migrating hatchery and wild fish has been difficult to evaluate (Sanders et al. 1992),
examining the effects of BKD on ecologically relevant performance characteristics
should at least partially elucidate the functional significance of BKD and the role it
plays in the survival of fish in the wild.
19
The recent development of more sensitive and quantitative detection methods for
Rs antigen, such as the enzyme-linked immunosorbent assay (ELISA; Pascho and
Mulcahy 1987) has allowed researchers and pathologists to more accurately determine
the prevalence and level of severity of Rs infections in salmonid populations (Pascho et
al. 1987). Consequently, fish have been qualitatively classified as having low,
moderate, and high levels of Rs antigen--corresponding to infection severity--based on
ELISA results (Pascho et al. 1991; Elliott et al. 1997). This implies that differing
infection levels may have some adaptive significance, but thus far such levels appear to
be of clinical significance only. We attempt to ascribe some ecological significance to
infections of Rs by examining the vulnerability to predation of juvenile spring chinook
salmon (Oncorhynchus tshawytscha) with differing Rs infection levels and also
assessing stress-related physiological changes in fish during the long-term, chronic
progression of the disease.
Methods
We conducted two series of experiments to evaluate the predator avoidance
ability of Rs-infected prey, one series in 1992 using northern pikeminnow
(Ptychocheilus oregonensis, formerly the northern squawfish) as predators and another
in 1994 using smallmouth bass (Micropterus dolomieui) as predators. We also sampled
Rs-infected prey for physiological assessment during disease progression, but only in
1994. Several aspects of our methods differed between years as indicated subsequently.
20
Test fish.--Juvenile subyearling spring chinook salmon were used for all
experiments. All fish were offspring from adults with a negative or low Rs infection
level as determined by ELISA segregation (Pascho et al. 1991). In both years, we
obtained fish (1992: mean length ± SE = 84.6 + 1.4 mm; mean weight = 6.7 + 0.34 g, N
= 50; 1994: mean length = 101.8 + 0.95 mm; mean weight = 12.9 + 0.38 g, N = 40)
from Entiat National Fish Hatchery (Washington) and transported them to our
laboratory using a truck with a large aluminum tank and aerated water of about 10°C.
In 1992, upon arrival, about 750 fish were placed indoors in each of four 1.2-m-
diameter circular tanks receiving 10 + 1°C well water at a rate of about 4 L/min. Fish
in two adjacent tanks were classified as controls and not challenged with Rs whereas
fish in the other two tanks were quarantined and classified as Rs-challenged (treatment)
groups. In 1994, we placed 250 fish into each of six 1.5-m-diameter tanks receiving
12+1°C water, three tanks holding control fish and three tanks holding treatment fish.
Volumes in all tanks were adjusted to yield low rearing densities that ranged from 7.5 to
9.5 g/L. Fish were fed twice daily with moist pellets (at a ration of about 3% body
weight per day) and kept under an ambient photoperiod simulated with lights and timers
that produced a gradual intensity dusk and dawn. In 1992, all control fish were marked
by clipping their adipose fin several weeks prior to the start of experiments. In 1994,
we marked all treatment fish.
In 1992, we used six northern pikeminnow, collected from the Columbia River
by electrofishing, as predators. They averaged about 400 mm in fork length and were
held indoors in a 7.6-m-long, 1.2-m-wide, 1.2-m-deep flowing water raceway
21
maintained at 17 + 1°C. In 1994, we used 14 smallmouth bass as predators, also
collected from the Columbia River by electrofishing. They averaged 295 mm in length
and seven fish were placed in each of two 3.75-m-diameter circular tanks receiving 15 ±
1°C well water. These tanks were 1-m-deep and lined with gravel and cobble substrates
with several pieces of 0.25-m-long, 15-cm-diameter PVC pipe randomly scattered along
the bottom to serve as cover for the predators. All tanks were surrounded with curtains
to minimize outside disturbance and fish were held under a simulated ambient
photoperiod. All predators were fed a maintenance diet of juvenile chinook salmon and
predation tests, which occurred in the same tanks, did not commence until predators
were consistently feeding.
Bacterial immersion challenges.--Treatment fish were experimentally infected
with Rs using an immersion challenge procedure similar to those described by Murray
et al. (1992) and Elliott and Pascho (1995). The bacterium used for the challenges was
Rs, isolate DWK 90. A 1 mL aliquot of the isolate was grown in 1 L of KDM-2 broth
media with serum at 15°C for 9 d. Log phase cells were then harvested by
centrifugation and re-suspended in cold, sterile peptone-saline (0.1% w/v peptone +
0.85% NaCl). The concentration of Rs cells in the final suspension was determined by
a membrane filtration-fluorescent antibody test, spectrophotometry, and culture on
KDM-2 agar media.
The immersion challenge procedure differed slightly between years. In 1992,
the water level in all four tanks was first lowered to 500 L by inserting new standpipes.
Inflow water was shut off and oxygen from tanks and air from aquarium pumps was
22
added to each tank via airstones. The two treatment tanks each received 1 L of broth
culture containing about 1.9 x 10 9 bacteria/mL, thus yielding a challenge dose of about
3.8 x 106 bacteria/mL. Control tanks each received 1 L of sterile peptone-saline. The
liquid was poured throughout the tank and the challenge continued for 24 h, after which
time water was partially drained from each tank, the original standpipes replaced, and
flow re-established. In 1994, fish in each tank were divided into two 108 L sterilized
plastic buckets filled with 76 L of water from the tanks. The water level in the tanks
was then lowered by inserting new standpipes and the buckets were then placed back
into the tanks, which then served as water baths to help maintain temperatures during
the challenge. We then poured 323 mL of broth containing 3 x 109 bacteria/mL into
each bucket containing treatment fish, thus yielding a challenge dose of 1.27 x 10'
bacteria/mL. Buckets containing control fish each received a similar aliquot of sterile
peptone-saline. Lids were then placed on the buckets and airstones connected to
aquarium pumps were inserted through the lids and into the water. Again, challenges
continued for 24 h before re-inserting the original standpipes and liberating fish back
into the tanks. The challenge in 1992 was conducted on 30 April, and in 1994 on 1
June.
Holding and sampling.--After the 24-h challenges had ended, fish were
maintained as described above (at 12-13°C) for a period of at least 100 d. To monitor
progression of the disease during the holding period and to estimate the time when
predation trials should commence, in 1992 we necropsied 50 control and 50 treatment
fish for analysis of whole kidneys by ELISA at 2, 6, 10, 12, 13, 14 and 15 weeks after
23
the challenge ended. Twenty five fish from each tank were rapidly removed by dip
netting and placed in a lethal dose (200 mg/L) of tricaine methanesulfonate. After
recording the length and weight of each fish, the kidney was removed aseptically and
frozen at -80°C for future analysis by ELISA as described by Pascho and Mulcahy
(1987) and modified by Elliott and Pascho (1991). In 1994, we sampled 20 control and
20 treatment fish for kidneys and blood, just before the challenge and at 2, 4, 6, 8, 10,
12, and 14 weeks after the challenge. Fish were processed as previously described,
except that blood samples were collected before removing kidneys by bleeding fish into
ammonium-heparinized capillary tubes after severance of the caudal peduncle. Plasma
was obtained by centrifugation and stored at -80°C for future analysis. Plasma cortisol
was determined by enzyme immunoassay modified from procedures described by
Munro and Stabenfeldt (1984). Plasma glucose and lactate were measured with a
biochemistry analyzer (Yellow Springs Instruments, model 2700D).
Predation experiments.--Predation experiments were composed of several
replicate trials, either conducted in the raceway using northern pikeminnow or in the
3.75-m circular tanks using smallmouth bass. Trials in 1992 were conducted on
consecutive days from 3 August to 20 August which was during weeks 13, 14, and 15
after the Rs challenge, whereas in 1994 we conducted trials from 17 August to 12
September (weeks 11 through 14). Trials in 1994 were not done on consecutive days
because of the relatively low consumption and, presumably, digestion rates by
smallmouth bass. To account for this, we allowed from 2-4 d between predation trials
using smallmouth bass.
24
To begin a trial in 1992, 12 or 13 fish from each prey holding tank (i.e., 25
control and 25 treatment fish) were rapidly transferred at 0800 h into 19 L buckets filled
with water and poured into the upstream end of the raceway. In 1994, only 10 control
and 10 treatment fish were released to predators. In all cases, predation was allowed to
continue for 24 h, at which time all survivors were netted, identified as control or
treatment fish, and measured. In 1992, we also excised the kidneys of survivors for
analysis by ELISA to compare their severity of infection with that of our stock of
control and treatment fish. We were able to observe the tests during daylight hours and
made general behavioral observations, while concealed, from overhead.
Statistical analysis.Because there were no differences in average level of Rs
infection as determined by ELISA absorbances between fish in replicate tanks on each
sampling day (t-tests or ANOVA, P > 0.05), data were pooled for analysis. On each
sampling day, we then compared the mean ELISA absorbances between control and
treatment fish using a two-sample t-test and plotted the frequency distribution of ELISA
values. The positive-negative threshold absorbance values for the ELISA were
calculated as described by Pascho et al. (1987). Fish with a positive Rs infection with
absorbance between the positive-negative threshold (mean absorbance + SD = 0.066 +
0.004; N = 18) and 0.199 were considered to have low infection levels, fish with
absorbance between 0.200 and 0.999 were considered to have moderate infections, and
those with absorbance > 1.000 were considered to have high infection levels (Pascho et
al. 1991; Pascho et al. 1993). Mortalities during the holding period were tallied, plotted
cumulatively, and sometimes necropsied to assess general physical condition. We
25
compared the mean lengths of fish in the treatment and control groups on each sampling
day using two sample t-tests. For the physiological data on each sampling day, we also
found no significant differences (P > 0.05) in average concentrations of cortisol,
glucose, or lactate from fish in each tank and therefore pooled the data for analysis. For
each sample day, we calculated overall mean concentrations for treatment and control
fish and compared them using two-sample t-tests for use with equal or unequal
variances. For all tests, the level of significance was 0.05.
Predation data were analyzed and statistical power considerations assessed in a
manner identical to that of Mesa (1994). A heterogeneity chi-square analysis first
determined that individual tests were homogeneous (P > 0.250; Sokal and Rohlf 1981).
Chi-square goodness-of-fit tests were then used on pooled data to determine if predation
was random (i.e., 50:50) on treated versus control fish.
Results
The immersion challenges were successful, in both years, of producing fish with
established infections of Rs in treatment fish (Figure 2.1), although rate of progression
and average level of infection severity differed. In 1992, mean ELISA absorbance rose
gradually to about 0.6 during the first 10 weeks, and maintained values of about 0.5 to
0.7 thereafter (Figure 2.1). Mean ELISA absorbances of treatment fish were
significantly (P < 0.001) higher than control fish for all sampling days. Control fish
26
Figure 2.1. Mean (and SE) ELISA optical densities at selected time intervals
after spring chinook salmon had received a waterborne challenge with Renibacterium
salmoninarum and for unchallenged control fish during 1992 (upper panel) and 1994
(lower panel). Sample size was 50 fish per treatment in 1992 and 20 fish per treatment
in 1994. Asterisks denote mean values within a time period that differ significantly (P <
0.05).
27
1.0
0
(/)
a
ca
a)
2
0.9- 1992
0.8Controls
BKD
0.70.60.50.40.3n,) _
0.1_
0.0
2.8_
0
2.4_
111
2
a)
0.8-
2
*
0
14
13
12
*
*
*
2
4
0.4_
0.0
11
1994
1.6_
1.2_
*
10
6
2.0_
a
*
11
6
*
8
10
12
Week after BKD challenge
Figure 2.1
14
28
showed either very low or negative infection levels for the duration of the experiment.
Most (87%) fish receiving the challenge had ELISA values corresponding to a low or
moderate infection level. In 1994, mean ELISA absorbance increased rapidly in
treatment fish to values greater than 1.0 for all sample days except the last (Figure 2.1).
Also, control fish showed a significant, but unexpected, increase in average ELISA
values during the last two sample days. Mean ELISA values were significantly (P <
0.0001) higher in treatment fish than in controls for all sample days except the first and
last. Most (86%) treatment fish had ELISA values corresponding to a moderate or high
infection level, whereas most (84%) control fish had a negative or low infection level.
In 1992, mortalities were low in both control tanks, except during weeks 4
through 6 when some minor unexpected (and unexplained) mortality occurred in one
tank (Figure 2.2). In the treatment tanks, fish mortalities were low until week 10 when
they started to increase considerably. In 1994, mortalities were essentially non-existent
in control fish except for a few fish that died during the last two weeks of the
experiment (Figure 2.2). The timing of mortality observed for treatment fish was
similar between the two years of study, but the fish in 1994 experienced 38% more total
mortality than fish in 1992. In both years, treatment fish that died usually showed
clinical signs of BKD, including swollen abdomens, ascites fluid, kidney lesions, and
exopthalmia.
In both years, all fish surviving throughout the experiment fed well, grew, and
were active. In 1992, treatment fish were significantly larger than control fish at all
sample periods except the first (Figure 2.3). In 1994, both groups of fish grew at similar
29
Figure 2.2. Cumulative mortality of spring chinook salmon after receiving a
waterborne challenge with Renibacterium salmoninarum and for unchallenged control
fish during 1992 (upper panel) and 1994 (lower panel). Data represent only fish that
died in rearing tanks during the period of disease progression.
Figure 2.3. Mean (± SE) fork lengths (mm) and weights (g) for spring chinook
salmon that had received a waterborne challenge with Renibacterium salmoninarum and
for unchallenged control fish during 1992 (upper panels) and 1994 (lower panels).
Asterisks denote mean values within a time period that differ significantly (P < 0.05).
Figure 2.4. Mean (and SE) concentrations of plasma cortisol (upper panel),
lactate (middle panel), and glucose (lower panel) for spring chinook salmon that had
received a waterborne challenge with Renibacterium salmoninarum and for
unchallenged control fish during 1994. Asterisks denote mean values within a time
period that differ significantly (P < 0.05).
30
300
250
,8
C
1992
controls
BKD
" 200
=> Z 150
a)
ca
100
E
50
0
500
450
>+
ed
400
350
CO
la
E
w
=
0=
I
I
Ci
1994
I
.4.---*---
/
/
250_
200
150
100_
/f#
,,/
50
#11
0 --,--,--,--,--4
0
I
1I0 1'1 1I2 1I3 14 15 16 17
- 300
Z
7>.. --co
234567
0
1
2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
Week after BKD challenge
Figure 2.2
100
98 -
96_
94_
92 _
90 88 -
86-
84 _
82 80
125
120_
1
*
994
1994
115 -
110_
................
i-------
105_
*
100 - --------
9590
0
2
4
6
6
10
12
Week after BKD challenge
14
16
0
2
4
6
6
10
12
Week after BKD challenge
14
10
16
32
180
160-
,- 140-J
E 120 CD
°
100-
co
80-
o
60-
7)
E
C)
40200
60
55-
J 5012
a' 45E
O 40B 35-
i
ou
302520
110
100-
908070-
6050-
4030
6
2
4
6
6
10
Week after BKD challenge
Figure 2.4
12
14
33
rates for the first 8 weeks, at which time control fish grew significantly faster than
treatment fish (Figure 2.3).
All three indicators of physiological stress showed significant changes in
treatment fish as the disease progressed in 1994 (Figure 2.4). Plasma cortisol was low
and similar between both groups of fish during the first 4 weeks, started to increase at 6
weeks, and then increased significantly in treatment fish during weeks 8 through 12
before declining to control levels by week 14. Although plasma lactate showed an
unexplained increase in both groups at week 4, the trend in plasma lactate after 6 weeks
essentially mirrored that of cortisol except for not showing such a dramatic decrease at
week 14. Plasma glucose was somewhat erratic in both groups, being similar between
groups during the first 4 weeks before decreasing significantly in treatment fish during
weeks 6 through 8. Concentrations of glucose in treatment fish then rose gradually to
levels that were significantly higher than controls at week 14.
Treatment fish were significantly more vulnerable to predation than control fish
in both years of our study (Tables 2.1 and 2.2). In 1992, we conducted a total of 18
replicate trials and released a total of 450 fish to northern pikeminnow. Predators
consumed a total of 280 fish, and on average ( ±SE) consumed 31% ( ±2.0 %) of the fish
released during a trial. Of the 280 fish eaten, 181 (65%) were of the Rs-challenged
group, which differed significantly from random (chi-square = 24.01; P < 0.001). In
1994, we conducted a total of 12 replicate trials and released a total of 240 fish to
smallmouth bass. Predators ate a total of 115 fish, and on average ate 48% ( ±2.8 %) of
the fish released during a trial. Of the 115 fish eaten, 76 (66%) were Rs-challenged,
34
Table 2.1. Results of experiments for juvenile chinook salmon experimentally
infected with Renibacterium salmoninarum and subjected to predation by northern
pikeminnow. P values < 0.100 denote predation rates that differ significantly from
random (50:50).
Number eaten
Replicate
Infected
Control
Test
df
X2
P
1
13
9
1
0.727
0.601
2
9
7
1
0.250
0.623
3
11
4
1
3.267
0.067
4
10
5
1
1.667
0.193
5
9
3
1
3.000
0.079
6
10
11
1
0.048
0.822
7
15
5
1
5.000
0.024
8
15
8
1
2.130
0.140
9
9
7
1
0.250
0.623
10
11
0
1
11.000
0.001
11
11
6
1
1.471
0.223
12
8
5
1
0.692
0.589
13
6
5
1
0.091
0.761
14
8
5
1
0.692
0.589
15
11
9
1
0.200
0.659
35
Table 2.1, continued
Number eaten
Replicate
Infected
Control
Test
df
X2
P
16
13
3
1
6.250
0.012
17
6
5
1
0.091
0.761
18
6
2
1
2.000
0.153
Total
E
181
99
Pooled
Heterogeneity
18
38.826 0.002
1
24.014 0.000
17
14.812 >0.500
36
Table 2.2. Results of experiments for juvenile chinook salmon experimentally
infected with Renibacterium salmoninarum and subjected to predation by smallmouth
bass. P values < 0.100 denote predation rates that differ significantly from random
(50:50).
Number eaten
Replicate
Infected
Control
Test
df
X2
P
1
7
4
1
0.818
0.631
2
6
0
1
6.000
0.014
3
6
6
1
0.000
1.000
4
7
1
1
4.500
0.032
5
6
2
1
2.000
0.153
6
6
5
1
0.091
0.761
7
9
4
1
1.923
0.162
8
7
2
1
2.778
0.092
9
6
4
1
0.400
0.535
10
6
3
1
1.000
0.681
11
5
5
1
0.000
1.000
12
5
3
1
0.500
0.513
Total
E,
76
39
Pooled
Heterogeneity
12
1
11
20.010 0.046
11.904
0.000
8.106 >0.250
37
which also differed significantly from random (chi-square = 11.904; P < 0.001). In
1992, the Rs infection levels of fish that had survived a predation trial were similar to
those observed in the general populations of treatment and control fish during the trials
(data not shown).
Discussion
Our results indicate that fish infected with either moderate or high infection
levels of Rs were significantly more vulnerable to predation by two species of fish with
distinctly different predatory tactics, thus providing some evidence of the functional,
ecological significance of BKD and the role it plays in affecting the survival of fish in
the wild. Also, our physiological data indicate that the HPI axis responds as the Rs
infection progresses, thus providing new insight into the nature and potential effects of
BKD as a chronic stressor. Collectively, our results indicate that the importance of
BKD as a mortality factor may be significantly greater in toto than simply mortality
directly attributed to the pathogen, which is prevalent in populations of salmon in the
wild (Sanders et al. 1992).
The effects of disease on vulnerability to predation has received considerable
attention in a variety of taxa, however such studies have focused almost entirely on the
effects of macro-parasites. Most evidence indicates that parasites are often associated
with altered host behaviors that lead to an increased susceptibility to predation (e.g.,
Hefting and Witt 1967; Moore 1983; Hudson et al. 1992; Lafferty and Morris 1996). In
38
contrast, Coble (1970) and Vaughan and Coble (1975) found no evidence that
infestation with various ectoparasites increased the vulnerability of fathead minnows
(Pimephales promelas), brook trout (Salvelinus fontinalis), or yellow perch (Perca
flavescens) to predation. To our knowledge, our study and that of Hefting and Witt
(1967)--who found that bluegills (Lepomis macrochirus) infected with Flexibacter
columnaris showed an increased vulnerability to predation by bowfin (Amia calva)--are
the only studies that have dealt with the predator avoidance effects of piscine bacterial
pathogens. In many cases, predation is a key aspect of the life cycle of parasites
because it allows the parasite to be transmitted from one host to another. However,
such is not the case with Rs infections since the disease is limited to the Salmonidae
and, as far as we know, transmission occurs only horizontally or vertically and not by
predation. Therefore, unlike many parasitic infestations, predation represents a
substantial adaptive cost to the host and the bacteria.
The most often cited mechanism underlying increases in vulnerability to
predation is alterations in behavior of the diseased organisms. Although changes in
behavior could have played a role in the decreased predator avoidance ability of Rs
infected fish, we did not observe any overt differences in behavior between treatment
and control fish. The decreased ability to avoid predators in our fish was likely due to
the combined effects of behavioral and physiological performance changes as the
disease progresses. For example, Giles (1983) and Sprengel and Liichtenberg (1991)
speculated that an increased vulnerability to predation was a probable consequence of
the altered fright responses and swimming performance they observed in their studies of
39
parasitized fish. From a theoretical perspective, a potential--if not probable--reason for
the decreased predator avoidance ability of Rs-infected fish is that such fish have a
reduced metabolic scope for activity (sensu Fry 1947). We hypothesize that infection
with Rs places considerable energetic demands on fish during the long, chronic
progression of the disease and therefore leaves less energy available to perform other
activities--such as predator avoidance. Evaluating the bioenergetic cost of disease may
help elicit ultimate, rather than proximate, explanations for reduced performance in
diseased organisms.
There were substantial differences in average ELISA values of treatment fish
between years during the time when predation trials were done. Most fish in 1992 were
classified as having a moderate infection level, whereas most fish in 1994 had high
infection levels. Low to moderate infection levels are commonly observed for various
salmonids in the Columbia River (Mau le et al. 1996), whereas fish with high infection
levels are rarely seen, suggesting our 1994 results may have little relevance to fish in the
wild. Although it may not be too surprising that fish with high infection levels were
more vulnerable to predation than were controls, our 1994 results may actually provide
at least a partial explanation for why so few highly infected fish are found in the wild.
Collectively, we believe our results are ecologically relevant because the treatment
effect was evident over a range of infection levels and two different types of predators
produced similar results.
Although considerable evidence shows that various forms of environmental
stress can predispose fish to disease (e.g., Snieszko 1974), less is known about whether
40
the natural disease process per se constitutes a stressor in fish. Various pathogens have
been shown to elicit a variety of general physiological responses in fish, including
hyperglycemia and decreases in hematocrit, plasma osmolality, and plasma protein
(e.g., Byrne et al. 1991; Haney et al. 1992; Grimnes and Jakobsen 1996). Although
such physiological changes could be construed as being part of the stress response to
disease, most studies of this type did not have the explicit objective of evaluating the
stress responses to disease nor were they necessarily designed to do so. Studies that
have addressed the potential stress responses to disease have at least partially focused on
the question of whether disease activates the HPI axis with the resultant secretion of
cortisol, but results have been equivocal. For example, significant increases in plasma
cortisol were not observed in studies dealing with rainbow trout infected with the blood
parasite C. salmositica (Laidley et al. 1988), in rainbow trout infected with
Ichthyophonus hoferi (Rand and Cone 1990), or in chum salmon (0. keta) infected with
erythrocytic necrosis virus (ENV; Haney et al. 1992). These findings led Laidley et al.
(1988) and Rand and Cone (1990) to conclude that activation of the HPI axis and
cortisol secretion may not be a normal component of the stress response to disease. In
contrast, elevated plasma cortisol levels were observed in brown trout (Salmo trutta)
infected with Saprolegnia (Pickering and Christie 1981), in red drum (Sciaenops
ocellatus) infected with a bacterial infection (Robertson et al. 1987), and in Atlantic
salmon (S. salar) having infectious salmon anaemia (ISA; Olsen et al. 1992). Our
results (and those from other as yet unpublished experiments in our laboratory) indicate
that cortisol becomes substantially elevated only during the later stages of 131(13--a time
41
when ELISA values are high, moribund fish are common, and mortalities are rapidly
increasing. To our knowledge, ours is only the third study reporting changes in plasma
cortisol in salmonids infected with Rs, and the only study evaluating physiological
changes in fish subjected to an immersion challenge with Rs. Donaldson (1981)
discussed unpublished observations indicating that cortisol levels in salmonids were
elevated due to BKD and that such levels may increase with an increasing severity of Rs
infection. However, the opposite result was obtained by Suzumoto et al. (1977), who
found that plasma cortisol may have decreased with the progressive development of
BKD. Our results generally support those discussed by Donaldson (1981), except that
we would describe cortisol elevations due to Rs infection as threshold, rather than
progressive, responses. There are several possible reasons for the increase in plasma
cortisol during the later stages of BKD, including stress associated with the apparent
hypoxemia that exists in fish during this time, the secretion of interleukin-lor other
cytokines by activated macrophages, stress associated with the general necrosis and
breakdown of various organ systems, and a reduction in metabolic clearance of cortisol.
The trend in plasma lactic acid after 6 weeks in Rs-infected fish mirrored that of
cortisol and suggests that fish were hypoxic. Lactate is an end product of glycolysis
formed under anaerobic conditions and is a well established indicator of stress due to
fright or severe exertion (Wedemeyer et al. 1990). Just like cortisol, however, increases
in lactate do not always occur as part of the stress response to disease. For example,
Byrne et al. (1991) observed no increases in plasma lactate during the pathogenesis of
bacterial gill disease in brook trout. Also, Haney et al. (1992) noted that lactate actually
42
decreased in chum salmon infected with ENV. Given the rather severe hematological
changes associated with BKD, we believe the probable cause for elevated lactate is the
same as that discussed by Olsen et al. (1992) for their fish, namely a hypoxemia
resulting in poor oxygen transport. Furthermore, duration of elevated lactate in our fish
was much longer than that typically observed in fish subjected to acute stressors. Longlasting elevations in lactate were also reported by Olsen et al. (1992), and we concur
with their observation that such responses must put serious demands on acid-base
balance in fish.
Hyperglycemia is a classic response of organisms to a variety of types of
stressors, yet the response of plasma glucose to disease is highly variable. Our results
revealed a general decrease in plasma glucose through 8 weeks followed by a gradual
increase during weeks 10 through 14. Depressed levels of plasma glucose in fish have
been reported by others assessing the physiological effects of BKD (Wedemeyer and
Ross 1973; Iwama et al. 1986) and cryptobiosis (Laidley et al. 1988). In contrast,
Robertson et al. (1987) noted only mild elevations of glucose in red drum with a
bacterial infection and Rand and Cone (1990) and Haney et al. (1992) reported little
changes in glucose in fish infected with I. hoferi or ENV. Hypoglycemia in fish is
thought to occur primarily because of inanition (Wedemeyer et al. 1990), but reduced
food intake was probably not a major cause of low glucose levels in our fish since they
fed well throughout the experiment. We hypothesize that the reason for low plasma
glucose in our Rs-infected fish was an excessive use of this energy substrate to help
combat the infection. We hope our studies in progress on the metabolic cost of BKD
43
will provide more insight into the dynamics of energy sources used by fish to help
combat chronic, long-lasting infections.
Fish diseases are an important force in structuring fish communities in the
Pacific Northwest (Li et al. 1987), but a lack of data on the ecological impacts of
disease has limited our understanding of the possible mechanisms behind this
structuring force. Our results indicate the role BKD may play in predator-prey
interactions, thus ascribing some ecological significance to this disease beyond that of
direct pathogen-related mortality only. In addition, the physiological changes observed
in our fish during the chronic progression of BKD indicates that this disease is stressful,
particularly during the later stages. More information is needed on the effects of
exogenous stress and physiological changes in Rs-infected fish to further our
understanding of the role BKD may play in affecting the survival of fish in the wild.
Acknowledgments
We thank Ron Pascho, Diane Elliott, Connie McKibben, Marne Thomasen, and
Margo Whipple for their training and assistance in culturing and quantifying Rs; Todd
Olsen, Joe Warren, Jack Hotchkiss, Robin Schrock, Sally Sauter, Stan Smith, Karen
Hans, Scott Vanderkooi, and Lisa Weiland for laboratory and editorial assistance; and
the U.S. Fish and Wildlife Service and personnel at the Entiat National Fish Hatchery
for kindly providing fish for this study. This work was funded by Bonneville Power
44
Administration and is Oregon Agricultural Experimental Station Technical Report
number 11286.
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47
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48
CHAPTER 3
Influence of Bacterial Kidney Disease on Smoltification in Juvenile Salmonids: is it
a Case of Double Jeopardy ?
Matthew G. Mesa''' , Alec G. Maulel, Thomas P. Poe', and Carl B. Schreck3
'U.S. Geological Survey, Biological Resources Division
Western Fisheries Research Center, Columbia River Research Laboratory
5501A Cook-Underwood Road, Cook, Washington 98605, USA
'Oregon Cooperative Fishery Research Unit, Department of Fish and Wildlife
Oregon State University, Corvallis, OR 97331-3803
a, 3Oregon Cooperative Fish and Wildlife Research Unit
Oregon State University, Corvallis, Oregon 97331-3803, USA
aCooperators are Oregon State University, the Oregon Department of Fish and Wildlife,
and the U.S. Geological Survey
"Reprinted from Aquaculture, Volume 174, Matthew G. Mesa, Alec G. Maule, Thomas
P. Poe, and Carl B. Schreck, Influence of Bacterial Kidney Disease on Smoltification in
Juvenile Salmonids: is it a Case of Double Jeopardy?, Pages xxx-xxx, Copyright (1999),
with permission from Elsevier Science"
49
Abstract
We investigated the effects of a chronic, progressive infection with
Renibacterium salmoninarum (Rs), the causative agent of bacterial kidney disease
(BKD), on selected aspects of smoltification in yearling juvenile spring chinook salmon
(Oncorhynchus tshawytscha). After experimentally infecting fish with Rs using an
immersion challenge, we sampled them every two weeks to monitor changes in gill Na, +
KtATPase (ATPase), cortisol, infection level, mortality, growth, and other stressrelated physiological factors during the normal time of parr-smolt transformation in
fresh water (i.e., from winter to spring). A progressively worsening infection with Rs
did not alter the normal changes in gill ATPase and condition factor associated with
smoltification in juvenile chinook salmon. The infection did, however, lead to elevated
levels of plasma cortisol and lactate and depressed levels of plasma glucose, indicating
that the disease is stressful during the later stages. A dramatic proliferation of BKD was
associated with maximal responses of indicators of smoltification, suggesting that the
process of smoltification itself can trigger outbreaks of disease. Our results suggest
mechanisms that probably influence the reported inability of Rs-infected fish to
successfully adapt to sea water.
50
Introduction
Juvenile anadromous salmonids typically undergo smoltification just prior to or
during their seaward migration. This period of development involves a series of
profound changes in physiology, morphology, and behavior that prepare freshwateradapted fish for survival and growth in the marine environment (see reviews by Hoar
1976; Folmar and Dickhoff 1980; Wedemeyer et al. 1980). Smoltification has long
been studied by scientists attempting to understand its underlying biochemical and
behavioral aspects and has recently received attention from a more applied sense as an
important factor in the quality of hatchery-reared salmonids. Survival and growth in the
marine environment depends on successful smoltification (Sheridan et al. 1985), and
aquaculturists are recognizing the importance of understanding factors influencing the
quality of smolts (e.g., Iwama 1992). Although several environmental and biological
factors are known to affect smoltification and subsequent marine survival, such as water
temperature, photoperiod, and fish size (Wedemeyer et al. 1980; Zaugg and Beckman
1990; McCormick et al. 1991; Shrimpton 1996), little is known about the effects of
disease on smoltification in salmonids.
Bacterial kidney disease (BKD) is one of the most serious and prevalent health
problems affecting hatchery-reared and wild salmonids today (Fryer and Sanders 1981;
Sanders et al. 1992). The disease is caused by Renibacterium salmoninarum (Rs), a
slow-growing Gram-positive diplobacillus that produces a chronic, systemic infection
that is often lethal. Efforts to control BKD have had little success (Elliott et al. 1989)
51
and the disease is considered a major obstacle to the successful culture of salmonids
(Fryer and Sanders 1981; Fryer and Lannan 1993).
Although substantial information exists on both smoltification and infection
with Rs in juvenile anadromous salmonids, little is known about the potential effects
BKD may have on the parr-smolt transformation. This is surprising considering the
ubiquity of BKD, the high probability that out-migrating juvenile salmonids undergoing
smoltification are infected with Rs, and that mortality of juvenile salmon infected with
BKD accelerates when fish enter salt water (Fryer and Sanders 1981; Banner et al.
1983; Moles 1997). Although the accelerated mortality observed in Rs-infected fish
when they enter the marine environment may be due to the stress associated with the
transition from fresh water to seawater (Fryer and Sanders 1981; Banner et al. 1983),
other explanations are possible or at least contributory. For example, the relative
success of smoltification in juvenile salmonids during their fresh water migratory phase-which would affect their hypoosmoregulatory ability--would be important to their
subsequent marine survival. However, infection with Rs may disrupt some or all of the
many complex processes associated with smoltification and perhaps hinder the ability of
fish to adapt to seawater.
For the present research, we investigated the effects of a chronic, progressive
infection with Rs on selected aspects of smoltification in yearling juvenile spring
chinook salmon (Oncorhynchus tshawytscha). After experimentally infecting fish with
Rs using an immersion challenge, we monitored changes in gill Na, + IC-ATPase
(ATPase), cortisol, infection level, mortality, growth, and other stress-related
52
physiological factors during the normal time of parr-smolt transformation in fresh water
(i.e., from winter through spring).
Methods
We conducted two experiments to assess the effects of BKD on smoltification in
yearling spring chinook salmon, one in 1993 and another in 1994. Our methods differed
somewhat between years as indicated subsequently.
Test fish.--On 12 January 1993, we obtained about 2000 fish (mean length + SE
= 123.8 + 2.3 mm; mean weight = 21.0 + 1.2 g) from Leavenworth National Fish
Hatchery (Washington), whereas on 19 January 1994 we obtained the same number of
fish (mean length = 121.5 + 2.4 mm; mean weight = 21.7 + 1.5 g) from Little White
Salmon National Fish Hatchery (Washington). All fish were offspring from adults with
a negative or low Rs infection level as determined by enzyme-linked immunosorbent
assay (ELISA; Pascho et al. 1991). Fish were transported to our laboratory using a
truck with a large aluminum tank and aerated water of ambient temperature. In 1993,
upon arrival, we placed 167 fish in each of twelve 0.76-m-diameter circular tanks
receiving 4-5°C well water at a rate of about 4 L/min. In 1994, we placed 333 fish in
each of six 1.5-m-diameter circular tanks under similar water conditions. Loading
densities in all tanks ranged from 11-13 g/L. Fish were fed twice daily with moist
pellets (at a ration of about 2% body weight per day) and kept under an ambient
53
photoperiod simulated with lights and timers that produced a gradual intensity dusk and
dawn.
Bacterial immersion challenges.--The experimental design involved creating
two groups of fish, one group (treatment fish) that received a 24-h waterborne challenge
with Rs and another group (control fish) that was not challenged. Treatment fish were
experimentally infected with Rs using an immersion challenge procedure similar to
those described by Murray et al. (1992) and Elliott and Pascho (1995). The bacterium
used for the challenges was Rs, isolate DWK 90. A 1-2 mL aliquot of the isolate was
grown in 1-2 L of KDM-2 broth media with serum at 15°C for 7-11 d. Log phase cells
were then harvested by centrifugation and re-suspended in cold, sterile peptone-saline
(0.1% w/v peptone + 0.85% NaCl). The concentration of Rs cells in the final
suspension was determined by a membrane filtration-fluorescent antibody test,
spectrophotometry, and culture on KDM-2 agar media.
The immersion challenge procedure differed slightly between years. In 1993,
fish in six adjacent tanks were designated as treatment fish whereas fish in the other six
tanks were designated as controls. First, water level in all tanks was lowered to 167 L
by inserting new standpipes. Inflow water was shut off and air from aquarium pumps
was added to each tank via airstones. The six treatment tanks each received 333 mL of
broth culture containing about 3.9 x 108 bacteria/mL, thus yielding a challenge dose of
about 7.8 x 105 bacteria/mL. Control tanks each received 333 mL of sterile peptone-
saline. The liquid was poured throughout the tank and the challenge continued for 24 h,
after which time water was partially drained from each tank, the original standpipes
54
replaced, and flow re-established. In 1994, fish in each tank were divided into two 108
L sterilized plastic buckets filled with 76 L of water from the tanks (i.e., 167 fish per
bucket). The water level in the tanks was then lowered by inserting new standpipes and
the buckets were then placed back into the tanks, which then served as water baths to
help maintain temperatures during the challenge. We then poured 170 mL of broth
containing about 2 x 108 bacteria/mL into each bucket containing treatment fish, thus
yielding a challenge dose of 4.5 x 105 bacteria/mL. Buckets containing control fish
each received a similar aliquot of sterile peptone-saline. Lids were then placed on the
buckets and airstones connected to aquarium pumps were inserted through the lids and
into the water. Again, challenges continued for 24 h before re-inserting the original
standpipes and liberating fish back into the tanks. The challenge in 1993, which proved
to be successful based on subsequent culture of Rs from the broth on agar media, was
conducted on 2 February. In 1994, we originally challenged fish on 3 February, but we
deemed this procedure unsuccessful since we were unable to obtain any growth of Rs on
agar media from our broth culture. We therefore conducted a second, and successful,
challenge on 1 March 1994.
Holding and sampling offish.- -After the 24-h challenges, fish were maintained
as described for a period of up to 20 weeks. During this time, photoperiod and water
temperature were adjusted periodically to mimic ambient environmental and Columbia
River conditions. Just prior to and every two weeks after the Rs challenge, we sampled
fish to monitor changes in growth, mortality, and various smoltification and stress-
related physiological factors. In 1993, we sampled 24 fish per treatment on each sample
55
day by initially removing 4 fish per tank. However, because of a leaking standpipe
during the challenge and clogged water lines from storm runoff during week 8, we lost
one tank of treatment fish and three tanks of control fish. Because fish were originally
stocked in excess to account for mortality, we were able to adjust the number of fish we
sampled per tank to maintain a consistent sample of 24 fish on each sample day
throughout the experiment. In 1994, we encountered no technical difficulties and
sampled 7 fish per tank for a total of 21 fish per treatment on each sample day.
For all sampling, fish from each tank were rapidly removed by dip netting and
placed in a lethal dose (200 mg/L) of buffered tricaine methanesulfonate. After
recording the length and weight of each fish, we collected blood samples by bleeding
fish into ammonium-heparinized capillary tubes after severance of the caudal peduncle.
Plasma was obtained by centrifugation and stored at -80°C for future analysis. Plasma
cortisol was determined by an ELISA modified from procedures described by Munro
and Stabenfeldt (1984). Plasma glucose and lactate were measured with a biochemistry
analyzer (Yellow Springs Instruments, model 2700D). Next, whole kidneys were
removed aseptically and frozen at -80°C for future analysis by ELISA as described by
Pascho and Mulcahy (1987) and modified by Elliott and Pascho (1991). Finally, we
excised a small piece of gill filament and placed it in 0.5-mL of buffer composed of
sucrose, EDTA, and imidazole for analysis of ATPase activity using the microassay
method described by Schrock et al. (1994).
Statistical analysis.--For all of our data, we found few or no differences between
mean values from fish in replicate tanks so data were pooled for all analyses (ANOVA,
56
P > 0.05). We plotted mean lengths and weights of fish over time and calculated
condition factor for each fish using the formula K = weight/length3 x 100. For each
sampling day, we calculated overall mean ELISA absorbances, mean activity level of
ATPase, and mean concentrations of cortisol, glucose, and lactate and plotted them over
time. The positive-negative threshold absorbance values for the ELISA were calculated
as described by Pascho et al. (1987). Fish with a positive Rs infection with absorbance
between the positive-negative threshold (mean absorbance + SD = 0.066 + 0.004; N =
14) and 0.199 were considered to have low infection levels, fish with absorbance
between 0.200 and 0.999 were considered to have moderate infections, and those with
absorbance > 1.000 were considered to have high infection levels (Pascho et al. 1991;
Pascho et al. 1993). Mortalities during the holding period were tallied and plotted
cumulatively. All data were compared in two ways. We compared seasonal changes in
our data using one-way ANOVA followed by the Student-Newman-Keuls multiple
range test. We also compared data from fish in the treatment and control groups on
each sampling day using two sample t-tests. Before analysis, any data displaying
heterogeneity of variance were log-transformed. For all tests, the level of significance
was 0.05.
57
Results
Body length, weight and condition factor
1993 experiments. During the study, mean length and weight of both groups of
fish increased significantly (Fig. 3.1A and 3.1B; P < 0.0009). There were no significant
differences in mean length or weight between treatment and control fish at any sample
period except for weight at week 18 (P < 0.05). Condition factor dropped markedly
after week 8 in both groups (Fig. 3.2A; P = 0.0001) and tended to decrease thereafter
but not significantly so. There were no differences in mean condition factor between
treatment and control fish at any sample period.
1994 experiments. Mean length of both groups of fish increased significantly
over the course of the experiment (Fig. 3.1C; P = 0.0001 ). Although both groups
displayed a general increase in weight over time (Fig. 3.1D), only treatment fish showed
a significant (relative to earlier samples) increase in weight, which occurred after week
12 (P = 0.0001). As in 1993, there were no significant differences in average length or
weight between treatment and control fish at any sample period except for weight at
week 16. Condition factor dropped significantly (relative to earlier samples) in both
groups of fish by 8-10 weeks (Fig. 3.2B; P = 0.0001) before increasing (relative to the
sample at week 8) in treatment fish during weeks 14 and 16 (P < 0.05) and remaining
low but stable in control fish. Mean condition factor did not differ between treatment
and control fish for the first 8 weeks but was significantly higher (P < 0.02) in treatment
fish during weeks 10-16.
58
Figure 3.1. Mean (± SE) fork lengths (mm) and weights (g) for spring chinook
salmon that had received a waterborne challenge with Renibacterium salmoninarum and
for unchallenged control fish during 1993 (A and B) and 1994 (C and D). Asterisks
denote mean values within a time period that differ significantly (P < 0.05).
Figure 3.2. Mean (± SE) condition factor for spring chinook salmon that had
received a waterborne challenge with Renibacterium salmoninarum and for
unchallenged control fish during 1993 (A) and 1994 (B). Asterisks denote mean values
within a time period that differ significantly (P < 0.05).
145
30
140
28
26
135
24
130
22
125
20
120
18 -
115
16
0
2
4
8
12 14 16 18 20
2
4
8
4
6
12 14 16 18 20
34
145
32
140
135
0
30 -
28
130
26
24
125
22
20
0
2
4
18
8
10 12 14 16
0
2
Week after Rs challenge
8
10 12 14 16
60
1.14
1.12 1.10 1.08
1.06 1.04 -
1.02 1.00 -
0.98 -
0.96 0.94
0
2
4
8
12
14
16
18
20
Week after Rs challenge
0
2
4
6
8
10
12
Week after Rs challenge
Figure 3.2
14
16
61
Rs-infection levels
1993 experiments. The immersion challenge was successful in producing fish
with an established infection of Rs (Fig. 3.3A). Over the course of the study, average
ELISA absorbance increased slightly during the first 12 weeks in treatment fish before
increasing significantly (P = 0.0001) at week 14 and remaining stable thereafter.
Although control fish displayed relatively stable mean ELISA absorbance values, they
did actually differ significantly over time (P = 0.0001), but this was probably due to the
exceptionally low variance in the samples. Treatment fish had significantly (P < 0.05)
higher infection levels than control fish at every sample period except the first two.
Treatment fish during the first 12 weeks were classified as having a low to moderate Rs
infection level, whereas fish from the remaining sample periods had mostly high
infection levels. Control fish were either negative or classified as having a low level of
Rs infection throughout the experiment.
1994 experiments. The second immersion challenge was successful in
producing an established Rs infection in treatment fish, but the infection accelerated
much more rapidly than expected (Fig. 3.3B). After 4 weeks, mean ELISA values in
treatment fish were significantly higher than the initial sample (P = 0.0001) and
continued to increase to a peak at 12 weeks before decreasing slightly during weeks 1416. Control fish showed a steady, but slight, increase in average infection level during
the first 12 weeks before infection level rose dramatically (P = 0.0001) and
unexpectedly during weeks 14-16. Average ELISA absorbance did not differ between
62
Figure 3.3. Mean (and SE) ELISA optical densities at selected time intervals
after spring chinook salmon had received a waterborne challenge with Renibacterium
salmoninarum and for unchallenged control fish during 1993 (A) and 1994 (B).
Asterisks denote mean values within a time period that differ significantly (P < 0.05).
63
1.8
1.6
1.4 -
A
1993
mi. BKD
1
Control
1
1.2 -
1.0 -
0.8 0.6 -
r
0.4 -
0.2 0.0
,
0
(2/1)
2.2
2.0
In
1r*
2
4
(2/18) (3/2)
11*-1
8
I
II
I
I
I
16
18
12
14
(3/31) (4/22) (5/11) (5/25) (6/8)
B
1994
*
*
1.8
20
(6/22)
1.6
1.4
1.2
*
1.0
i
0.8
0.6
0.4
0.2
I*
0.0
0
2
4
6
8
10
12
14
16
(2/24) (3/17) (3/31) (4/14) (4/28) (5/11) (5/25) (6/9) (6/22)
Week (date) after Rs challenge
Figure 3.3
64
groups at week 0, was significantly higher in treatment fish from weeks 2-14, and rose
to levels in control fish that were substantially higher than treatment fish at week 16 (P
< 0.05). Treatment fish had low to moderate infection levels during the first 6 weeks
and high infection levels thereafter. Control fish had low to moderate infection levels
for the first 14 weeks before establishing a high infection level at week 16.
Mortality
In 1993, there were no mortalities in treatment fish during the first 14 weeks but
they increased considerably thereafter; there were no mortalities among control fish
(Fig. 3.4). In 1994, mortalities in treatment fish increased dramatically after 10 weeks
and a few control fish died during weeks 14-16 (Fig. 3.4). Treatment fish in 1994
experienced 38% more total mortality than fish in 1993. In both years, treatment fish
that died showed clinical signs of BKD, including swollen abdomens, ascites fluid,
kidney lesions, and exopthalmia.
Physiological data
1993 experiments. Over the course of the experiment, gill ATPase activity
increased significantly in both groups of fish (Fig. 3.5A; P = 0.0001). Both groups
displayed a similar trend with peak ATPase activities occurring during weeks 12-14 that
were significantly higher than earlier samples. Gill ATPase activity did not differ
between treatment and control fish during any sample period except for the last two
where activity was slightly higher in control fish (Fig. 3.5A).
65
Figure 3.4. Cumulative mortality of spring chinook salmon after receiving a
waterborne challenge with Renibacterium salmoninarum and for unchallenged control
fish during 1993 and 1994. Data represent only fish that died in rearing tanks during the
period of disease progression.
400
360
320
co
-ft
280
°E
6,1
240
w 200
160
mft..
120
E 80
=
40
0
I
I
0
2
I
I
I
4
6
8
I
I
I
I
I
10 12 14 16 18 20 22
Week after Rs challenge
67
Concentrations of plasma cortisol changed significantly over time in both
groups, but the pattern of change was different (Fig. 3.5B). Cortisol titers in treatment
fish were relatively stable during the first 14 weeks before increasing to higher levels
during weeks 16-20; only the sample at week 18 had cortisol titers that were
significantly different than the other samples. The variability within samples also
increased during weeks 16-20 in treatment fish. Control fish showed a distinct trend in
plasma cortisol over time, with concentrations of cortisol being low and stable during
the first 4 weeks before increasing significantly (P = 0.0001) to a peak at week 8 and
decreasing steadily thereafter. The peak in cortisol titer occurred about 6 weeks prior to
the peak in ATPase. Cortisol concentrations were significantly (P < 0.05) higher in
control than in treatment fish at week 8, but then the reverse was true during weeks 1620.
Plasma glucose concentrations were significantly elevated (relative to all other
samples) at the first sample period in both groups (Fig. 3.5C; P = 0.0001). Thereafter,
glucose titers in treatment fish were stable during weeks 2-16 and then declined
significantly (relative to previous samples) during weeks 18-20. Mean glucose
concentrations among control fish never differed in any subsequent sample periods.
Glucose concentrations differed significantly between treatment and control fish at
weeks 14, 18, and 20.
Plasma lactate levels changed significantly over time in both groups of fish (Fig.
3.5D; P = 0.0001). In treatment fish, lactate concentrations were significantly elevated,
relative to all other samples, at weeks 0 and 18. Lactate concentrations in control fish
68
Figure 3.5. Mean (and SE) gill Nat, KtATPase activity (A), and concentrations
of plasma cortisol (B), glucose (C), and lactate (D) for spring chinook salmon that had
received a waterborne challenge with Renibacterium salmoninarum and for
unchallenged control fish during 1993. Asterisks denote mean values within a time
period that differ significantly (P < 0.05).
69
70
60
50
40
30
20
10
0
J'C
g
a)
ta
O
0
=
CD
100
90
80
70 60
50 40
30
40
35 30
25 20
1510
20
16
18
12
14
4
8
2
0
(2/1) (2/18) (3/2) (3/31) (4/27) (5/11) (5/25) (6/8) (6/22)
Week (date) after challenge
Figure 3.5
70
were significantly higher in the first sample when compared to all other samples.
Lactate levels in control fish then remained low and stable during weeks 2-12 before
showing a significant increase at week 14 and decreasing mildly thereafter. The
increase in lactate concentration at week 14 corresponded with the peak in ATPase
activity. Average concentrations of plasma lactate never differed between treatment and
control fish except at week 8 (Fig. 3.5D; P < 0.05).
1994 experiments. Gill sodium, potassium-activated ATPase changed
significantly in both groups over the course of the experiment (Fig. 3.6A; P = 0.0001).
Both groups displayed a similar trend in ATPase with a peak occurring during weeks 68 followed by a rapid decline to initial levels from week 10 to the end of the experiment.
There were no differences in mean ATPase activity between treatment and control fish
during any sample period.
Plasma cortisol concentrations changed significantly over time in both groups of
fish (Fig. 3.6B; P = 0.0001). In treatment fish, cortisol concentrations were low and
stable during the first 4 weeks before increasing (not significantly) at week 6. Cortisol
concentrations were significantly higher at weeks 8 and 12 than samples taken during
the first four weeks and at week 16. In control fish, cortisol titers rose gradually to a
peak at 12 weeks, declined at 14 weeks, and then were elevated again at 16 weeks.
Samples taken at 8, 10, 12, and 16 weeks had cortisol levels that were significantly
higher than the first three samples. The only differences in mean cortisol concentration
between treatment and control fish were at weeks 6 and 16.
71
Figure 3.6. Mean (and SE) gill Na+, K+-ATPase activity (A), and concentrations
of plasma cortisol (B), glucose (C), and lactate (D) for spring chinook salmon that had
received a waterborne challenge with Renibacterium salmoninarum and for
unchallenged control fish during 1994. Asterisks denote mean values within a time
period that differ significantly (P < 0.05).
72
:4"
2.
02
c-
1,0)
ai
o_
1
'c
+ca
it
"E.. 1
zz
E
E
12
10
8
64
2
100
80
60
40 20 0
110
100
90 80 70
60
50 40
40
35
30
25 20 15
10
12
14
16
4
6
8
2
0
(2/24) (3/17) (3/31) (4/14) (4/28) (5/11) (5/25) (6/9) (6/22)
Week (date) after challenge
Figure 3.6
73
In both groups of fish, plasma glucose levels changed considerably over the
course of the experiment (Fig 3.6C; P = 0.0001). In treatment fish, glucose levels
increased slightly during the first 8 weeks and declined significantly (relative to the
sample taken at week 8) during all samples thereafter. In control fish, glucose levels
were slightly erratic during the first 6 weeks before increasing significantly to a peak at
8 weeks. This peak in glucose levels coincided with the maximal activity in ATPase.
Glucose concentrations then declined significantly relative to this peak during weeks
10-14 before displaying a drop that was substantially lower than all other samples.
Mean glucose concentrations were substantially lower (P < 0.05) in treatment than in
control fish at weeks 4, 8, 10, 12, and 14.
Plasma lactate levels also changed significantly over the course of the
experiment in both groups of fish (Fig. 3.6D; P = 0.0001). In treatment fish, lactate
levels increased steadily to a peak at 12 weeks and was erratic thereafter. Lactate
concentrations in control fish were stable during the first 6 weeks, rose significantly
(relative to the first 6 weeks) to elevated levels that persisted during weeks 8-12, and
declined thereafter. As in 1993, peak lactate levels in control fish coincided with the
peak in ATPase activity. Mean concentrations of plasma lactate differed (P < 0.05)
between treatment and control fish at weeks 12 and 16.
Discussion
This research investigated a common, yet essentially unstudied, scenario among
anadromous juvenile salmonids in freshwater, namely fish undergoing the process of
74
smoltification while they are infected with Rs. Our results indicate that a progressive
infection of Rs did not alter normal changes in ATPase and condition factor associated
with smoltification, but did affect trends in concentrations of plasma cortisol, glucose
and lactic acid. Our data from 1993 indicate that the "stress" associated with
smoltification may have triggered a substantial increase in severity of Rs infection level.
Collectively, our results provide some insight into the nature of BKD as a chronic
stressor and can help answer questions regarding the reported inability of Rs-infected
fish to successfully adapt to sea water.
Two of our criteria of smoltification, ATPase and condition factor, were not
affected by a worsening infection with Rs. Gill ATPase activity, which is important in
providing energy for ion transport across gill membranes, has commonly been used as a
quantitative biochemical indicator of the progression of smoltification in migrating
juvenile salmon (Folmar and Dickhoff 1981; McCormick and Saunders 1987; Zaugg
and Beckman 1990). The enzyme displays a characteristic profile during the seaward
migration of salmonids although absolute levels vary among species. Furthermore,
increases in ATPase activity during the seaward migration of many salmonid species
occur in freshwater prior to seawater entry and are thought to be preparatory
physiological adaptations to enhance their hypoosmoregulatory ability (Folmar and
Dickhoff 1981; McCormick and Saunders 1987). The mortality attributed solely to
BKD upon exposure of Rs-infected fish to seawater has been highly variable (Banner et
al. 1983; Sanders et al. 1992; Elliott et al. 1995; Moles 1997) and indicates that Rsinfected fish may be dying of other proximate causes besides disease during seawater
challenges. Indeed, Moles (1997) noted that the osmoregulatory ability of Rs-infected
75
coho salmon 0. kisutch was altered and that diseased fish benefitted from a gradual
acclimation to seawater, thus indicating that reduced osmocompetence of Rs-infected
fish could be a cause of death when such fish are exposed to seawater. Because BKD
did not affect the dynamics of ATPase in our fish, we presume the role ATPase serves
in preparing fish for seawater entry would remain intact and that reduced ATPase
activity would not be a contributing factor to any possible reduced osmocompetence in
Rs-infected fish. However, we recognize that enhanced hypoosmoregulatory ability of
successful smolts is controlled not only by ATPase activity, but also by endocrine
products such as cortisol, growth hormone, and thyroid hormones (e.g., Richman et al.
1987; Prunet et al. 1989; Young et al. 1989). Clearly, further study on the effects of
BKD on other aspects of smoltification will help clarify reasons for the accelerated
mortality of Rs-infected fish upon entry into seawater.
Condition factor is a classic variable used to assess the progression of the parr-
smolt transformation (Hoar 1976; Folmar and Dickhoff 1980). Our data on condition
factor, along with ATPase, confirm that the fish underwent this transformation, at least
to some extent. Decreased condition factor during smoltification reflects a phase of
enhanced catabolism, particularly of body lipid and glycogen reserves (Sheridan et al.
1985; Sheridan 1986). All of our fish fed well and grew during these experiments,
indicating that fish with relatively high Rs infection levels feed and can channel some
energy into somatic growth. The relatively fast increase in condition factor seen in Rsinfected fish in 1994 after week 8 probably reflects an increase in weight due to
accumulation of ascites fluid as the disease progressed. That Rs-infected fish feed and
grow during progression of the disease is important because growth rate and size has
76
also been shown to be a critical factor in successful smoltification (Clarke and
Shelbourne 1985; McCormick and Saunders 1987; Saunders et al. 1994; Shrimpton
1996).
Several studies have reported a rise in plasma cortisol concentrations during
smoltification (e.g., Specker and Schreck 1982; Young et al. 1989; Franklin et al. 1992;
Olsen et al. 1993) that usually occurs prior to or coincident with the rise in ATPase.
The role of cortisol in smoltification has been studied both in vitro and in vivo with
mixed results; some reports have shown cortisol to increase ATPase activity and cause
cellular differentiation and proliferation of chloride cells (e.g., Richman and Zaugg
1987; McCormick et al. 1987; Madsen 1990; Shrimpton et al. 1994), while others have
reported little or no effect (Langdon et al. 1984; Langhorne and Simpson 1986;
Richman et al. 1987; McCormick et al. 1987). Such contrasting results in attempting to
precisely define the role of cortisol during smoltification probably arise from differences
in developmental stage, season, species, and unknown interactions with other endocrine
or physiological mechanisms. In our study, only control fish in 1993 displayed what
might be considered a typical cortisol response related to smoltification. For all of our
other fish, BKD apparently altered the normal dynamics of cortisol during
smoltification. The tendency for cortisol levels to become and remain elevated in Rsinfected fish is consistent with other experiments at our laboratory and probably reflects
a generalized stress response to disease (for a discussion on stress responses to disease
and possible reasons for elevated cortisol in Rs-infected fish, see Mesa et al. 1998). The
implications of such chronic elevations in cortisol titers for fish undergoing
smoltification might include reduced osmoregulatory ability and immunocompetence.
77
Continuous chronic stress, repeated acute stress, and elevated cortisol levels are known
to modify the concentration and affinity of glucocorticoid receptors in gills of coho
salmon (Mau le and Schreck 1991; Shrimpton and Randall 1994) which in turn reduce
the sensitivity and tissue responsiveness of the gills to cortisol. Furthermore, chronic,
but not acute, stress has been shown to increase the clearance rate of cortisol, thus
reducing the residence time of cortisol in the plasma (Patifto et al. 1985). Taken
together, the effects of chronic stress and persistent elevations of cortisol may prevent
this hormone from achieving a maximal response with respect to its presumed role in
smoltification. For example, persistent elevations in cortisol concentrations due to
disease may prevent the surge in ATPase activity that generally develops 1-2 weeks
after SW exposure (e.g., Bjornsson et al. 1989). Chronic stress and elevated cortisol are
also known to reduce functioning of the salmonid immune system (e.g., Maule et al.
1987; Maule et al. 1989; Mazur and Iwama 1993; Pickering 1993; Espelid et al. 1996),
which could allow established infections to proliferate and lead to increased mortality
when fish enter seawater.
Circulating levels of plasma glucose are commonly used as an indicator of stress
and have been examined to some extent during the parr-smolt transformation in
salmonids. Evidence of a change in blood glucose concentration during smoltification
is mixed, with some reports of an increase (Wendt and Saunders 1973; Woo et al. 1978;
Plisetskaya et al. 1988) and other reports of a decrease (Sweeting et al. 1985; Soengas et
al. 1992; Ji et al. 1996). Our results regarding changes in glucose levels during
smoltification were equivocal; our data from control fish in 1994, where the peak in
glucose concentrations coincided with the peak in ATPase activity, perhaps supports the
78
notion of a hyperglycemia during smoltification. The declines in glucose concentrations
we observed in Rs-infected fish (i.e., both treatment groups and the 1994 control fish
after the period of smoltification) were probably due in large part to the effects of
disease. These declines in glucose concentrations in fish infected with Rs occurred
during times of high ELISA values and relatively severe infection levels. Depressed
levels of plasma glucose in fish have been reported by others assessing the
physiological effects of BKD (Wedemeyer and Ross 1973; Iwama et al. 1986) and are
probably due to excessive use of this energy substrate to help combat the infection
(Mesa et al. 1998).
Although our primary purpose in evaluating seasonal changes in plasma lactate
concentrations were related more to disease and stress rather than smoltification, we did
note some interesting patterns in lactate levels during both years of our study that could
be related to smoltification. In control fish, lactate levels rose to a peak coincident with
the peak in ATPase and declined slowly thereafter. In treatment fish BKD apparently
altered any smoltification-related changes in lactate concentrations. Although increases
in lactate concentration in our treatment fish and 1994 control fish at week 16 are
probably due to the apparent hypoxemia elicited by BKD (Mesa et al. 1998), we are
unaware of any studies that have assessed changes in plasma lactate during
smoltification. Lactate is an end product of glycolysis formed under anaerobic
conditions and is a well established indicator of stress due to fright or severe exercise
(Wedemeyer et al. 1990). The reasons why plasma lactate might increase during
smoltification are speculative but could be related to the increased restlessness and
79
migratory behavior of smolting fish or to the ontogenetic changes in complexity of the
hemoglobin system that reflects preadaptations to changes in oxygen transfer, CO2
transfer, and pH regulation (Giles and Vanstone 1976; Hoar 1988). Increased levels of
circulating lactate could be used by smolting fish to: (1) replenish or maintain blood
glucose and glycogen stores via liver gluconeogenesis; (2) serve as a glucose precursor
in the Cori cycle, whereby glycogen-derived muscle lactate is converted to liver glucose
and this glucose is converted to muscle glycogen; or (3) serve as a preferred oxidative
substrate for liver, erythrocytes, heart, and red muscle (e.g., Suarez and Mommsen
1987; Milligan and Girard 1993; Eros and Milligan 1996). However, there are
conflicting reports on changes in liver gluconeogenesis during smoltification, with some
reports indicating an increase (Guillaume et al. 1984; Soengas et al. 1992) and another
showing a decrease (Ji et al. 1996). Also, the importance of the Cori cycle to lactate
metabolism in fish undergoing smoltification is, to the best of our knowledge, unknown.
Thus, it appears that lactate metabolism during smoltification and its potential influence
on this transformation are topics in need of further study.
Our results offer the intriguing possibility that the "stress" associated with
smoltification (Simpson 1985; Franklin et al. 1992) may have triggered a proliferation
of BKD in some of our test fish. In 1993, mean ELISA absorbance values in treatment
fish ranged from about 0.1 to 0.4 through late April (week 12), indicating fish had a low
to moderate level of infection. At week 14, mean ELISA absorbance increased
dramatically to values above 1.0, indicating fish had progressed to a severe level of
infection in just two weeks. This increase in infection level was coincident with peak
ATPase activity and a significant decrease in condition factor. Several lines of
80
reasoning have lead us to conclude that the association of smoltification and BKD
progression is more than just mere coincidence. First, we are unable to attribute the
substantial increase in infection level at week 14 to other exogenous stressors, such as
density or water temperature. Densities at this time were actually relatively low (due to
sampling) and water temperature varied only by about 1°C over the period from week
12 to week 14. Second, Mau le et al. (1987) reported that the salmonid immune system
is depressed during smoltification due to increased cortisol titers or perhaps other
hormones present during smolting, which could allow an established but latent infection
to proliferate. Third, several reports indicate that Rs and its major surface antigen, p57,
are immunosuppressive (Turaga et al. 1987; Sakai et al. 1991; Fredricksen et al. 1997;
Seigel and Congleton 1997) which could exacerbate the already depressed immunity
observed during smoltification. Finally, from an energetics standpoint, the metabolic
cost of smoltification--which we presume would be relatively high given all the
morphological and physiological changes ongoing--may leave less energy available for
disease resistance and thus create conditions amenable for disease proliferation. Why,
then, did we not observe similar changes in infection level in our other groups of fish
that underwent smoltification? There could, for example, be threshold levels of antigen
(or numbers of bacteria) necessary before BKD will proliferate in response to other
stressors or smoltification. This would help explain why our control fish in 1993 never
displayed an increase in severity of infection--perhaps the numbers of bacteria present
and associated antigen level were simply insufficient to contribute to a proliferation of
disease. There could also be stock differences in the relative resistance to BKD, as
indicated by several studies evaluating genetic variation in resistance of salmonids to
81
various diseases (Suzumoto et al. 1977; McIntyre and Amend 1978; Winter et al. 1979;
With ler and Evelyn 1990; Beacham and Evelyn 1992). This could help explain the
difference in infection profiles between control fish in the two years of our study and
also partially explain why control fish in 1994 contracted a severe case of BKD after the
period of smoltification. Considerable evidence shows that various forms of
environmental stress can be an important factor in outbreaks of infectious disease in
fishes (Snieszko 1974; Pickering 1993). We believe our results substantiate this notion
by implicating smoltification as a possible influence on disease outbreaks.
Acknowledgments
We thank Ron Pascho, Diane Elliott, Connie McKibben, Mame Thomasen, and
Margo Whipple for their training and assistance in culturing and quantifying Rs; Karen
Hans, Jack Hotchkiss, Sally Sauter, Robin Schrock, Stan Smith, Scott Vanderkooi, and
Joe Warren for laboratory and editorial assistance; and the U.S. Fish and Wildlife
Service and personnel at the Leavenworth and Little White Salmon National Fish
Hatcheries for kindly providing fish for this study. This work was funded by
Bonneville Power Administration and is Oregon Agricultural Experimental Station
Technical Report number 11365.
82
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89
CHAPTER 4
Interaction of Bacterial Kidney Disease and Physical Stress in Juvenile Chinook
Salmon: Physiological Responses, Disease Pathogenesis, and Mortality
Matthew G. Mesa', Alec G. Maule', and Carl B. Schreck2
'U.S. Geological Survey, Biological Resources Division
Western Fisheries Research Center, Columbia River Research Laboratory
5501A Cook-Underwood Rd.
Cook, WA 98605, USA
2U.S. Geological Survey, Biological Resources Division
Oregon Cooperative Fish and Wildlife Research Unita
Oregon State University
Corvallis, OR 97331-3803, USA
aCooperators are Oregon State University, Oregon Department of Fish and Wildlife, and
the U.S. Geological Survey
Accepted for publication in Transactions of the American Fisheries Society
90
Abstract
We experimentally infected juvenile spring chinook salmon (Oncorhynchus
tshawytscha) with Renibacterium salmoninarum (Rs), the causative agent of bacterial
kidney disease (BKD), to compare the physiological responses of Rs-infected and noninfected fish to a series of multiple, acute stressors and determine if exposure to these
stressors worsens the disease and leads to increased mortality. After subjecting groups
of fish to a waterborne challenge of Rs, we sampled them bi-weekly to monitor
infection levels, mortality, and some stress-related physiological changes. As the
disease worsened, fish developed decreased hematocrits and blood glucose levels and
increased levels of cortisol and lactate, indicating that BKD is stressful, particularly
during the later stages. Eight weeks after the challenge, when fish had moderate to high
infection levels, we subjected them, along with un-challenged control fish, to three 60-s
bouts of hypoxia, struggling, and mild agitation that were separated by 48-72 h. Our
results indicate that the imposition of these stressors on Rs-infected fish did not lead to
higher infection levels or increased mortality when compared to diseased fish that did
not receive the stressors. Furthermore, the kinetics of plasma cortisol, glucose, and
lactate over a 24-h period following each application of the stressor were similar
between fish with moderate to high BKD and those that had low or no detectable
infection. Some differences in the stress responses of these two groups did exist,
however, most notably fish with moderate to high Rs infections had higher titers of
cortisol and lactate prior to each application of the stressor and also were unable to
91
consistently elicit a significant hyperglycemia in response to the stressors. Collectively,
our results should be important to understanding the impact BKD has on the survival of
downstream migrants but we caution that our results represent the combined effects of
one type of stressor and one disease only and probably cannot be applied to other
scenarios.
Introduction
Bacterial kidney disease (BKD) is one of the most serious diseases among
populations of wild and hatchery-reared salmonids worldwide (Fryer and Sanders
1981).
It is caused by the gram-positive bacterium Renibacterium salmoninarum (Rs)
and is a chronic and often lethal disease. The disease has been especially problematic in
the Columbia River basin, where it has had a severe impact at hatcheries and is highly
prevalent in several species of Oncorhynchus during their downriver migration. For
example, Elliott et al.
(1997)
sampled various groups of hatchery and wild spring-
summer chinook salmon 0. tshawytscha at several dams on the Snake and Columbia
rivers during their outmigration and reported that 86-100% of fish tested positive for Rs
antigen by enzyme-linked immunosorbent assay (ELISA). Also, Sanders et al. (1992)
detected Rs in over 20% of the salmonids collected at the terminus of their freshwater
migration in the Columbia River using the relatively insensitive fluorescent antibody
technique (FAT) and suggested prevalence would have been even higher if ELISA was
used. As a corollary to prevalence, severity of Rs infections based on ELISA
92
absorbance values have been variable in populations of fish examined during the early
part of their seaward migration. Although most fish have been classified as having low
infection levels, there have been several instances where a majority of fish have shown
medium to high infection levels (Mau le et al. 1996; Elliott et al. 1997).
Despite our increasing knowledge of the prevalence and severity of BKD in the
Columbia River basin, we still know little about the impact of BKD on the survival of
emigrating fish. Several studies indicate that BKD can affect survival of fish when they
enter and reside in seawater (Banner et al. 1983; Sanders et al. 1992; Elliott et al. 1995;
Moles 1997). Other research suggests that the disease may contribute to mortality of
salmonids during their emigration (Pascho et al. 1993) and fish with moderate to high
Rs infection levels are particularly vulnerable to predation (Mesa et al. 1998). In
contrast, other evidence indicates that some fish with relatively high Rs infections levels
may survive and actually show some degree of recovery (Pascho et al. 1991). Clearly,
further research is necessary to assess the impact of BKD on survival of fish outside of
hatcheries and improve our ability to predict the outcome of Rs infections.
One factor that might affect the prevalence and severity of Rs infections in
juvenile salmonids during their outmigration is exposure to secondary, exogenous
stressors. During their downstream migration in the Columbia River, juvenile salmon
commonly experience an array of stressful events or conditions that occur serially and
can have cumulative effects. For example, migrating juveniles at dams are stressed by
passage through traveling screens, gatewells, fish sorters, turbines, and spillways
(Matthews et al. 1986; Maule et al. 1988). Multiple disturbances can elicit severe
93
physiological and behavioral responses in salmonids (Barton et al. 1986; Mau le et al.
1988; Mesa 1994), yet whether BKD affects compensatory stress responses or whether
the infection worsens after exposure to physical stress is unknown. Given the
immunosuppressive effects of stress (Mau le et al. 1987; Tripp et al. 1987; Mau le et al.
1989) and the role of stress in predisposing fish to disease (Wedemeyer 1970; Pickering
1993), the potential for maladaptive stress responses and accelerated pathogenesis of
BKD after exposure to stress seems high. Several studies have described altered
physiological responses, increased infection rates, and increased mortality due to
disease after exposure of fish to stressors such as multiple handlings (Barton et al.
1986), crowding (Mazur et al. 1993; Wise et al. 1993), and social interaction (Peters et
al. 1988). However, this information cannot be used to predict the responses of Rsinfected fish to other types of stressors.
The objectives of this study were twofold: (1) document and compare the
physiological responses of Rs-infected and non-infected juvenile spring chinook salmon
to a series of multiple, acute stressors; and (2) determine if exposure to exogenous stress
worsens established Rs-infections and leads to increased mortality. This study is part of
a larger research effort designed to ascribe some ecological significance to BKD by
examining the effects of different infection levels on selected aspects of juvenile
salmonid performance. Ultimately, we hope to increase our understanding of the impact
BKD may have on survival of fish in the wild.
94
Methods
Test fish.--Age-0 spring chinook salmon (mean fork length + SE = 97 + 0.9 mm;
mean weight = 11 + 0.3 g) from the Little White Salmon National Fish Hatchery
(Washington) were used in this experiment. All fish were offspring from adults with a
negative or low Rs infection level as determined by ELISA. On 10 July, 1997, we
stocked 205 fish into each of eight 0.86-m-diameter, flow-through tanks receiving 7°C
well water at a rate of 6-7 L/min. Water volume in all tanks was 400 L and loading
density was about 6-7 g/L. Beginning on 28 July, we increased water temperature to
12°C over the course of 2 d. Fish were fed twice daily with a moist pellet (at a ration of
about 2-3% body weight per day) and kept under an ambient photoperiod simulated
with lights and timers that produced a gradual intensity dusk and dawn.
Bacterial immersion challenges.--On 31 July, groups of fish in duplicate tanks
were randomly assigned to one of four treatments: (1) control, no stress (CNS): fish that
were not challenged with Rs and did not receive the exogenous stressors; (2) control,
stressed (CS): fish that were not challenged with Rs but did receive the exogenous
stressors; (3) BKD, no stress (BKDNS): fish that received the Rs challenge and did not
receive the exogenous stressors; and (4) BKD, stress (BKDS): fish that received the Rs
challenge and also were exposed to the exogenous stressors.
To create the treatment groups, fish to be given BKD were experimentally
infected with Rs using an immersion challenge procedure similar to those described by
Murray et al. (1992) and Elliott and Pascho (1995). Control fish were subjected to a
95
sham challenge. The bacterium used for the challenge was Rs, isolate DWK 90. A 3mL aliquot of the isolate was grown in 1 L of KDM-2 broth media with 100 mL of fetal
bovine serum at 15°C for 10 d. Log phase cells were then harvested by centrifugation
and re-suspended in cold, sterile peptone-saline (0.1% w/v peptone + 0.85% NaC1) to a
total volume of 480 mL. The concentration of Rs cells in the final suspension was
determined by spectrophotometry and culture on KDM-2 agar media.
The immersion challenge procedure was as follows. The water volume in all
tanks was first lowered to 100 L by inserting new standpipes. Inflow water was shut off
and air from a compressor was added to each tank via airstones. All BKD treatment
tanks received 100 mL of broth culture containing about 4.92 x 108 bacteria/mL, thus
yielding a challenge dose of about 4.92 x 105 bacteria/mL. Control tanks each received
100 mL of sterile peptone-saline. The liquid was poured into the tank and the challenge
continued for 24 h, after which time water was partially drained from each tank, the
original standpipes replaced, and flow re-established.
Holding and sampling.--After the 24-h challenges had ended, fish were
maintained as described above (at 12-13°C) for 16 weeks. To monitor progression of
the disease and physiological condition during the holding period and to estimate the
time when exposure to the exogenous stressors should commence, we removed 7 fish
per tank (14 per treatment) for tissue and blood samples just before the challenge and at
2, 4, 6, 8, 10, 12, 14 and 16 weeks after the challenge ended. Fish from each tank were
rapidly removed by dip netting and placed in a lethal dose (200 mg/L) of buffered
tricaine methanesulfonate. After recording the length and weight of each fish, blood
96
samples were collected by bleeding fish into ammonium-heparinized capillary tubes
after severance of the caudal peduncle. Plasma and hematocrits were obtained by
centrifugation and the plasma stored at -80°C for future analysis. Plasma cortisol was
determined by an ELISA modified from procedures described by Munro and
Stabenfeldt (1984). Plasma glucose and lactate were measured using commercial kit
assays (Sigma Diagnostics, St. Louis, MO) that we modified for use with microplates.
Finally, the kidney was removed aseptically and frozen at -80°C for future analysis by
ELISA as described by Pascho and Mulcahy (1987) and modified by Elliott and Pascho
(1991).
Stress experiment.--We subjected two groups of fish (CS and BKDS) to a series
of exogenous stressors 8 weeks after the challenge. The stressor consisted of partially
draining the water out of a tank, netting all fish out of the tank and transferring them to
a perforated bucket inside a second bucket filled with water, and then removing the
perforated bucket and subjecting the fish to hypoxia, struggling, and mild agitation for
60 s. Fish were exposed to the stressor three times, with the first two applications
spaced by 72 h and the third occurring 48 h after the second; the stressor was applied at
0700 on each day. The spacing of the stressors was meant to simulate representative
travel times between dams for fish migrating in the Columbia River system. Blood
samples (N = 10 per time period; pooled samples from 5 fish from each duplicate tank)
were collected from stressed fish as described previously just before each stressor (time
0) and at 1, 3, 6, 12, and 24 h after the stressor. For unstressed groups (i.e., CNS and
BKDNS fish) we collected blood samples only at times 0, 12, and 24 h but did remove
97
fish during the other time periods to maintain similar densities in all tanks during the
experiment. Blood samples were centrifuged to obtain plasma and analyzed for cortisol,
glucose, and lactate as described previously. Kidneys were collected from fish only just
prior to application of the first stressor. Fish sampled just prior to application of the
first stressor served as our 8 week sample for infection monitoring and also as the time 0
sample for the first stressor.
Data analysis.--For all of our data collected bi-weekly, we found few or no
differences between mean values from fish in replicate tanks so data were pooled for all
analyses (t-tests, P > 0.05). We then calculated mean values for all data (lengths,
weights, ELISA absorbances, hematocrit, and concentrations of cortisol, glucose, and
lactate), plotted them over time, and compared means over time within a treatment and
on each sampling day among treatments using one-way analysis of variance (ANOVA)
followed by Tukey's Honestly Significant Difference (HSD) test. Where necessary, we
used logarithmic and inverse transformations to stabilize the variance before
comparison. In some analyses, transformations were ineffective so we conducted a oneway ANOVA by ranks followed by the HSD test on the ranked values. The positivenegative threshold absorbance values for the ELISA were calculated as described by
Pascho et al. (1987). Fish with a positive Rs infection with absorbance between the
positive-negative threshold (mean absorbance + SD = 0.074 + 0.002; N = 8) and 0.199
were considered to have low infection levels, fish with absorbance between 0.200 and
0.999 were considered to have moderate infections, and those with absorbance > 1.000
were considered to have high infection levels (Pascho et al. 1991; Pascho et al. 1993).
98
Mortalities during the experiment were tallied daily, plotted cumulatively, and the mean
number of days to death compared among treatments using ANOVA. For data collected
during the stress experiment, we calculated mean concentrations of metabolites within
each sample period for the CS and BKDS groups and tested them for homogenous
variances. Within a group, we then subjected the means to ANOVA and, when the Ftest was significant (P < 0.05), compared the means at 1, 3, 6, 12, and 24 h to the mean
at time 0 using Dunnett's multiple comparison procedure. For the CNS and BKDNS
groups, we calculated overall mean concentrations of metabolites from samples taken at
0, 12, and 24 h and compared them using two sample t-tests. For all tests, the level of
significance was 0.05 and analyses were conducted using SAS statistical software (SAS
Institute 1990).
Results
Length and weight.--Over the course of the study, mean length and weight of all
groups of fish increased significantly (Figures 4.1A and 4.1B; P < 0.0001). There were
no significant differences in mean length among the groups at any sample period except
for week 12 when BKDS fish were smaller than fish in the other groups (P = 0.0107).
The only differences in mean weight of fish occurred at weeks 4 and 6 when BKDNS
fish weighed less than fish in the other groups (P < 0.05).
99
Figure 4.1. Mean (and SE) fork lengths and weights for spring chinook salmon
during the 16-week holding and monitoring period of our BKD /stress response
experiments. See methods section for definitions of the four treatment groups. Means
within a time period with no letters in common are significantly different (P < 0.05);
time intervals with no letters shown indicate no significant difference among the means.
Figure 4.2. Mean (and SE) ELISA absorbances at selected time intervals after
spring chinook salmon had received a waterborne challenge with Renibacterium
salmoninarum and for unchallenged control fish during our BKD /stress response
experiments. See methods section for definitions of the four treatment groups. Means
within a time period with no letters in common are significantly different (P < 0.05).
100
0-- Control No Stress
---m- Control Stress
A BKD No Stress
120 -
-
-g 115
BKD Stress
E
. c 110
C)
105
0
LL
100
95 -
0
2
4
6
8
10
12
Week after Rs challenge
Figure 4.1
14
16
2.00
Control No Stress
Control Stress
BKD No Stress
BKD Stress
1.75
0
t.)
c 1.50
ca
sa
8
cn
co
1.25
1.00
(1)
Iw0.75
c
cz
2
a)
0.50
0.25
0.00
0
2
4
6
8
10
12
Week after Rs challenge
14
16
102
Disease progression and physiological monitoring.--The immersion challenge
produced fish with an established infection of Rs (Figure 4.2). During the study,
average ELISA absorbance in the CNS and CS groups was low and stable. For the
BKDNS and BKDS groups, mean ELISA absorbance increased significantly at 2 weeks
and continued to increase to maximal values at week 10 before decreasing for the rest of
the experiment. Both BKD groups had significantly (P < 0.0001) higher infection levels
than control fish at every sample period except for the first and last. Mean ELISA
absorbance did not differ between the two control groups at any sample period except
the first (P < 0.05). Between the two BKD groups, mean ELISA absorbance differed
significantly only at weeks 2 and 16 (P < 0.05). Fish in the BKD groups had low to
moderate infection levels during the first 6 weeks and high infection levels thereafter
except for BKDS fish during weeks 14 and 16. Control fish had very low infection
levels throughout the experiment.
Hematocrit in both control groups differed significantly over time, but only due
to changes observed at 2 and 12 weeks (Figure 4.3A; P < 0.0102). In contrast,
hematocrit in the BKD groups displayed a dramatic decrease (P < 0.0001) that occurred
at 10 weeks and persisted through 14 weeks before increasing at week 16. For the first
two sample periods, only CNS and BKDS fish had significantly different hematocrits (P
< 0.0347), but these differences were not apparent during weeks 4-6. Hematocrit was
significantly lower in both BKD groups when compared to control groups from week 10
on (P < 0.0017), except for week 14 when hematocrit in BKDNS fish did not differ
from that of CNS fish. Hematocrit never differed between the control groups nor
between the BKD groups.
103
Figure 4.3. Mean (and SE) hematocrit (A), and concentrations of plasma
cortisol (B), glucose (C), and lactate (D) for spring chinook salmon that had received a
waterborne challenge with Renibacterium salmoninarum and for unchallenged control
fish during the 16-week holding and monitoring period of our experiment. See methods
section for definitions of the four treatment groups. Means within a time period with no
letters in common are significantly different (P < 0.05); time intervals with no letters
shown indicate no significant difference among the means.
104
0 Control No Stress
100
80
60
40
20
----m Control Stress
- A. BKD No Stress
v- BKD Siress
0
9070 70-
60-a
50
40
30
60
50
40
30
20
10
0
2
4
6
8
10
12
14
Week after Rs challenge
Figure 4.3
16
105
Concentrations of plasma cortisol were low and similar in the CNS and CS
groups during the experiment but increased significantly (P < 0.05) after week 8 in both
BKD groups before declining during the last two samples (Figure 4.3B). Plasma
cortisol titers were low and similar among groups for the first 4 weeks, slightly elevated
(P < 0.0046) in CNS fish at week 6, and started to increase in both BKD groups at week
8. During weeks 10-16, because of extreme variance in cortisol concentrations in both
BKD groups, we conducted these intergroup comparisons using non-parametric rank
procedures. For weeks 10 and 12, plasma cortisol concentration in both BKD groups
was substantially higher than the control groups, but only significantly so when
compared to the CNS group (P < 0.006). At week 14, we were unable to detect any
statistical differences in cortisol concentration among the groups, although the BKDNS
fish had mean levels of more than twice that of the next lowest group. At week 16, only
BKDNS fish had significantly higher levels of cortisol than the control groups (P <
0.0062). Throughout the experiment, cortisol titers between the two control or two
BKD groups at each sample period never differed.
Circulating levels of plasma glucose differed significantly in all groups over
time (Figure 4.3C; P < 0.0001). In the control groups, this difference was due to the
elevated (but unexplained) levels of glucose observed at week 8, whereas in the BKD
groups differences were manifested primarily in the decreases observed during weeks
10-14. There were significant differences in glucose concentration among the groups at
all sample periods except the second and last (P < 0.0282). Concentrations of glucose
were significantly lower at weeks 0 and 4 in BKDS fish when compared to fish in the
106
other groups. At week 6, glucose levels were lower in the BKD groups than in the
control groups, but this difference was not evident at week 8 because BKDS fish
showed an unexpected increase in plasma glucose. From weeks 10-14, fish in both
BKD groups had significantly lower glucose concentrations than fish in the control
groups. Fish in the two control groups never differed from one another in levels of
plasma glucose, whereas fish in the BKD groups had different glucose levels at weeks
0, 4, and 8.
Concentrations of plasma lactate differed significantly in all groups over time
(Figure 4.3D; P < 0.0035), generally showing a steady increase in all groups through
week 8 before decreasing to various levels thereafter. During the first 8 weeks, plasma
lactate titers did not differ among the groups, except for week 2 when CNS fish had
lower levels than fish in two other groups (P < 0.02). During weeks 10-16,
concentrations of lactate in both control groups decreased to levels observed initially,
whereas fish in the BKD groups maintained elevated levels of lactate. However,
extreme variance heterogeneity led to non-significant statistical results for comparisons
made on data from weeks 10-14, and our analysis of data from week 16 indicated that
BKDNS fish had significantly higher levels of lactate than CS fish (P< 0.0031)
Mortality.--Only fish in the BKD groups died during the study, and we present
the data from each replicate tank in the two BKD-treatments separately in Figure 4.4.
Mortalities started in one of the BKDS replicates around the eighth week, which was
about 2 weeks earlier than the start of mortality in the other tanks. There were no
differences in mean time to death among fish in the four tanks (Table 4.1; P > 0.2030).
107
Figure 4.4. Cumulative mortality of spring chinook salmon after receiving a
waterborne challenge with Renibacterium salmoninarum during the 16-week holding
and monitoring period of our experiment. Data shown are from replicate tanks of two
treatments; no control fish died during the experiment. Data represent only fish that
died in rearing tanks during the period of disease progression.
60
. BKD No Stress - Tank #1
BKD No Stress - Tank #2
P
mt
o
50 - -A-- BKD Stress - Tank #1
v BKD Stress - Tank #2
40
2
> 30
(I)
61:.
:.,
4,
co
g 20z
0
100
I
.
I
1
9/22 /\
(8)
10/6 A
(10)
10/20 A
(12)
I
11/3 A
(14)
Date (Week) after Rs challenge
I t.
11/17 A
(16)
.
1
109
Table 4.1. Mean (and SD) number of days to death and total number of fish that
died during the 16 week holding and monitoring period of our BKD/stress response
experiment. Data are from fish in replicate tanks of two treatment groups that were
subjected to a waterborne challenge of Renibacterium salmoninarum; no control fish
died during the experiment.
Days to death
Tank
Mean
SD
BKDNS #1
82.5
12.4
36
BKDNS #2
83.8
11.7
43
BKDS #1
79.4
14.3
53
BKDS #2
84.5
12.4
43
Total mortality
110
Figure 4.5. Mean (and SE) plasma cortisol concentrations of Rs-challenged and
unchallenged juvenile spring chinook salmon subjected to three 60-s periods of
handling and mild agitation and for fish not exposed to the stressors. The first stressor
(A) occurred at the 8 week point of the experiment; the second stressor (B) occurred 72
h after the first; and the third stressor (C) occurred 48 h after the second. Asterisks
denote mean values that differ significantly (P < 0.05) from the initial (time 0) value of
that group. Bars represent the mean + SE of pooled samples taken at 0, 12, and 24 h
from Rs-challenged and unchallenged fish that were not exposed to the stressors.
111
240
200
ro
160
Control Stress
BKD Stress
Control No Stress
BKD No Stress
120
80
40
0
240
200
160
O
O
120
80
40
0
240
200
160
120
-
80
40
0
0
5
10
15
Hours after application of stressor
Figure 4.5
25
112
Total mortality was highest in the BKDS group that experienced the first mortalities,
and lowest in one of the tanks containing BKDNS fish (Table 4.1).
Stress experiment.--Exposure of fish to the exogenous stressors commenced 8
weeks after the Rs challenge. Just prior to application of the first stressor, cortisol
concentration was significantly higher in the BKDS group than in the CS group (P =
0.002; Figure 4.5A). Cortisol titers in both groups peaked 1 h after the stress and the
magnitude of this maximal response was about 160 ng/mL in both groups. Also, both
groups had cortisol concentrations that were significantly (P < 0.0001) elevated at 3 h
but returned to initial levels by 6 h and remained stable thereafter. Before application of
the second stressor, which occurred 72 h after the first, cortisol concentration did not
differ between CS and BKDS fish (P = 0.382) but was more than twice as high as levels
observed prior to the first stressor (Figure 4.5B). The magnitude of the maximum
response, which occurred 1 h after the stressor, was about 100 ng/mL in CS fish and 130
ng/mL in BKDS fish. These values were about 40-70 ng/mL less than those observed
during the first stressor. For both groups, cortisol returned to levels not significantly
different from initial levels by 3 h after the stressor. Just prior to application of the third
stressor, which occurred 48 h after the second, plasma cortisol titer between CS and
BKDS fish did not differ significantly (P = 0.138; Figure 4.5C). The initial cortisol
level in CS fish was about 50 ng/mL, a value similar to that observed in fish just prior to
the second stressor, whereas in BKDS fish, initial levels of cortisol were about 90
ng/mL, which is more than twice that observed in fish just prior to the second stressor
and more than four times that observed in fish just prior to the first stressor. Cortisol
113
levels in both groups of fish again peaked at 1 h after the stressor (P < 0.0001) and the
magnitude of maximal response was about 80 ng/mL in CS fish and 120 ng/mL in
BKDS fish, a difference of about 33%. Cortisol concentration in both groups returned
to initial baseline levels by 3 h, dropped below initial levels by 6 h, and then showed a
gradual, but non-significant, increase through 24 h. For all three applications of the
stressor, pooled data from the time 0, 12, and 24 h samples revealed that BKDNS fish
had higher levels of cortisol than did CNS fish (P < 0.05).
Plasma glucose concentration in BKDS fish was lower than in CS fish just prior
to application of the first stressor, but not significantly so (Figure 4.6A). Thereafter,
plasma glucose levels in both groups increased mildly--around 20-25 mg/dL--and there
were no significant differences between samples at time 0 and any subsequent samples
in either group. Just prior to application of the second stressor, plasma glucose levels
were significantly lower in BKDS fish when compared to CS fish (P = 0.002; Figure
4.6B). Glucose concentrations in CS fish were significantly elevated at 1 and 3 h after
the stressor (P < 0.0002) and returned to levels not different from the initial level by 6 h.
In contrast, BKDS fish were unable to mount a significant hyperglycemia following the
second stressor. Although glucose levels in BKDS fish did increase for up to 3 h after
the stressor, the magnitude of this change (ca. 25 mg/dL) combined with increased
variability led to consistently non-significant statistical comparisons. Glucose
concentrations just prior to the third stressor were again significantly lower in BKDS
fish than in CS fish (P = 0.001; Figure 4.6C). After the stressor, plasma glucose titers
increased significantly in CS fish and did not return to initial levels until 12 h (P <
114
Figure 4.6. Mean (and SE) plasma glucose concentrations of Rs-challenged and
unchallenged juvenile spring chinook salmon subjected to three 60-s periods of
handling and mild agitation and for fish not exposed to the stressors. The first stressor
(A) occurred at the 8 week point of the experiment; the second stressor (B) occurred 72
h after the first; and the third stressor (C) occurred 48 h after the second. Asterisks
denote mean values that differ significantly (P < 0.05) from the initial (time 0) value of
that group. Bars represent the mean + SE of pooled samples taken at 0, 12, and 24 h
from Rs-challenged and unchallenged fish that were not exposed to the stressors.
Figure 4.7. Mean (and SE) plasma lactate concentrations of Rs-challenged and
unchallenged juvenile spring chinook salmon subjected to three 60-s periods of
handling and mild agitation and for fish not exposed to the stressors. The first stressor
(A) occurred at the 8 week point of the experiment; the second stressor (B) occurred 72
h after the first; and the third stressor (C) occurred 48 h after the second. Asterisks
denote mean values that differ significantly (P < 0.05) from the initial (time 0) value of
that group. Bars represent the mean ± SE of pooled samples taken at 0, 12, and 24 h
from Rs-challenged and unchallenged fish that were not exposed to the stressors.
115
110
100
90
80
70
60
50
40
30
-
--m Control Stress
y BKD Stress
ezz Control No Stress
BKD No Stress
110
100
90
80
70
60
50
40
30
110
100
90
80
70
60
50
40
------li
30
i
0
5
10
15
Hours after application of stressor
Figure 4.6
25
-'
116
100
90
80
EZ21
Control Stress
BKD Stress
Control No Stress
BKD No Stress
70
60
50
40
rictirder7f.1";:,"2104.1743104,355,3,"s4iir7rWrirityrair
30
20
100
J 90
-0
E
80
70
60
50
ti)
J
40
30 MAiV
20
%PSI:
-
Aw"247
100
90
80
70
60
50
40
30
20
...............................
0
5
10
//
4I4V/MIVAW AI
15
Hours after application of stressor
Figure 4.7
25
117
0.0049). In BKDS fish, glucose levels showed a significant increase 1 h after the
stressor (P < 0.0160) but did not differ from the initial level at any sampling time
thereafter. For all applications of the stressor, BKDNS fish had significantly lower
glucose concentrations than CNS fish (P < 0.05).
During the stress exposure phase of the experiment, both BKDS and CS groups
had remarkably similar lactate responses (Figure 4.7). Just prior to the first stressor,
BKDS fish had slightly elevated lactate levels when compared to CS fish, but not
significantly so (P = 0.128; Figure 4.7A). After the stressor, lactate concentration
increased significantly in both groups to a peak at 1 h before returning to initial levels
by 6 h (P < 0.0001). Both groups also showed significantly lower lactate levels 24 h
after the stressor. Lactate levels were significantly higher in BKDS fish than in CS fish
just prior to application of the second stressor (P = 0.009; Figure 4.7B), increased
substantially 1 h after the stressor, and decreased to levels not different from initial
concentrations by 3 h. Prior to the third stressor, lactate was again significantly elevated
in BKDS fish when compared to CS fish (P = 0.021; Figure 4.7C) and thereafter
displayed a response similar to that observed in fish exposed to the second stressor. For
all three stressors, the magnitude of the maximum response in BKDS and CS fish
ranged from 35 to 45 mg/dL. During the experiment, the only difference in mean
lactate concentrations observed between BKDNS and CNS fish was during exposure to
the second stressor (P < 0.007).
118
Discussion
Few studies have examined physiological stress responses--and their possible
consequences--in diseased fish, despite the high probability in nature of diseased fish
being exposed to a variety of exogenous stressors. Information on the stress responses
of diseased fish increases our understanding of the stress physiology of fishes and the
potential ecological impact of diseases in the wild. Because juvenile salmonids
migrating to the ocean are commonly infected with Rs and exposed to various stressors,
we surmised that exposure to exogenous stress might worsen infections, preclude the
possibility for recovery from disease, and lead to increased and more rapid mortality.
However, our results indicate that the imposition of multiple, physical stressors on fish
with well established Rs infections did not lead to higher infection levels or increased
mortality when compared to diseased fish that did not receive the stressors.
Furthermore, our fish with moderate to high BKD were able to show compensatory
physiological responses to the imposed stressors in a manner similar to fish that had low
or no detectable infection of Rs. This information should be important to our
understanding of the impact BKD has on the survival of downstream migrants but, as
will be evident throughout this section, should also be viewed with caution.
The physiological changes observed in our fish during the chronic progression
of BKD indicate that this disease is stressful, particularly during the later stages. As the
disease worsened, fish developed decreased hematocrits and blood glucose levels and
increased levels of cortisol and lactate. These results are consistent with other BKD
119
studies conducted by us and the literature on general physiological changes in salmonids
during the progression of BKD (Wedemeyer and Ross 1973; Donaldson 1981; Bruno
and Munro 1986; Iwama et al. 1986). Such physiological changes during the
progression of BKD are probably contributory to mortality, and the reader is referred to
Mesa et al. (1998) for a detailed discussion of the reasons for, and consequences of, the
physiological changes associated with BKD.
We observed no significant differences in the mean infection level or mean time
to death between Rs-infected fish that received multiple handling stressors and those
that did not. We also observed no significant increase in infection level or mortality in
our control (i.e., not Rs challenged) fish that received the stressors. This is a significant
finding because an average of 71% (range = 41-89%) of these fish had ELISA
absorbances above the positive-negative cutoff and were therefore classified as having a
low level of infection. In essence, then, our study actually assessed the effects of
exogenous stress on fish with two different Rs infection levels, high and low. That fish
with low infection levels remained healthy after receiving the stressors may be
especially relevant since this infection level is the most common among downstream
migrants in the Columbia River basin (Maule et al. 1996; Elliott et al. 1997). Our
results are similar to those of Specker and Schreck (1980) who found that various
stressful transport regimes did not cause increased expression of latent BKD in juvenile
coho salmon 0. kisutch. In contrast to the above results, Barton et al. (1986) observed
50% mortality after exposure of juvenile chinook salmon with chronic fin rot and an
infection of the coldwater disease bacterium Cytophaga psychrophila to repeated
120
handling stressors. Furthermore, other studies have shown that exposure of Rs-infected
fish to other stressors such as seawater challenges and predation have led to an
increased rate of mortality in the diseased fish (Banner et al. 1983; Elliott et al. 1995;
Moles 1997; Mesa et al. 1998). Discrepancies between studies are probably due to a
variety of factors, including stock differences, relative degree of "sickness" in the test
fish, type of stressor, and the interstressor interval used (e.g., 48-72 h in our study v. 3 h
in Barton et al. [1986]).
Given the well documented role of environmental stressors in predisposing fish
to disease and the immunosuppressive effects of stress on fish, we surmised that the
potential for increased infection levels and mortality in our stressed (BKDS) fish was
high. However, information from studies showing that various types of environmental
stressors can predispose fish to disease (Wedemeyer 1970; Snieszko 1974; Pickering
1993) may not be applicable to studies addressing the effects of stress on fish that were
already diseased. In addition, although several studies have documented some
immunosuppressive effects of stress on fish (e.g., Maule et al. 1989; Salonius and
Iwama 1991; Mazur and Iwama 1993), others have found that fish may actually show
enhancement of some components of the immune system (e.g., Mock and Peters 1990;
Demers and Bayne 1997 ) or show no significant changes in total immunological status
(Espelid et al. 1996) after exposure to stress. Similar findings are replete in the
mammalian literature (for a review see Biondi and Zannino 1997). This discrepancy
arises in part because: (1) the immune response to stress is dependent on the type of
stressor; (2) the stressor may affect different components of the immune system
121
differently; and (3) the underlying mechanisms are not yet well understood (Espelid et
al. 1996). Thus, it appears that enhancement, suppression, and no change in status of
the immune system are common responses of fish to stress. Although we did not study
the immune system in our fish, we believe it must have been involved in preventing the
established infections in our fish from becoming worse. At the least, our BKDS fish
probably showed no significant suppression of immune system components that may be
relevant to BKD following stress because they showed no accelerated pathogenesis of
BKD or increased mortality. Whether the immune system of our BKDS fish showed
enhancement of some components following stress is unknown, but evidence from the
literature suggests this is a likely possibility. Enhancement of the immune system,
although transitory, usually occurs after fish are exposed to acute, rather than chronic,
stress. Acute stress has been found to lead to a redistribution and altered function of
leucocytes, and increases in complement component C3 and lysozyme activity (Peters et
al. 1991; Fevolden et al. 1994; Dhabhar et al. 1996; Demers and Bayne 1997).
Furthermore, it is well known that acute stress causes the release of cortisol which,
although it has been linked to immunosuppression, has recently been shown to prevent
depressed phagocytic activity in the rainbow trout 0. mykiss (Narnaware and Baker
1996). Together, the various components contributing to enhancement of the immune
system following acute stress could have, at the least, prevented established infections
in our fish from becoming worse. Clearly, more research on the interactions of disease,
immune function, and exogenous stress is warranted.
122
In general, the physiological responses of our healthy and diseased fish to
multiple stressors were similar and show that each group was able to achieve some
degree of physiological compensation after each application of the stressor. Of course,
their ability to achieve physiological compensation is also reflected in the lack of a
significant increase in post-stress mortality. We were primarily interested in three
aspects of the physiological responses: (1) the initial levels of the physiological factors
prior to application of a stressor; (2) the magnitude and timing of the maximum change;
and (3) the time required for the physiological factors to return to initial levels. Thus,
although in general the physiological responses between groups was similar, there are
several nuances in the data that require discussion
The cortisol kinetics of BKDS and CS fish following each application of the
stressor are typical responses of salmonids to acute stress. This type of physiological
response has been observed in many species of fish subjected to a variety of acute
stressors (e.g., Pickering et al. 1982; Thomas and Robertson 1991; Mesa 1994). We
noted that initial concentrations of cortisol increased with each application of the
stressor--particularly in BKDS fish just prior to the third stress--suggesting that fish did
not achieve ideal physiological compensation during the interstressor intervals. This
could reflect liver dysfunction and altered metabolic clearance of cortisol (especially in
BKDS fish) or perhaps a hypersensitivity of the interrenal leading to a low-level
secretion of cortisol. The magnitude of the maximum change in cortisol decreased with
each application of the stressor, particularly after the first stress, suggesting that fish
became conditioned to respond in this manner with each subsequent stressor, the
123
capacity of the interrenal to secrete cortisol was diminished, or that the plasma clearance
rate of cortisol increased. Indeed, Barton et al. (1986) suggested that the reason for the
failure of their diseased fish to elicit a corticosteroid response after exposure to the third
in a series of three consecutive stressors was due to interrenal exhaustion. However,
their interstressor interval was only 3 h compared to 48-72 h in this study. Although it
appeared as though BKDS fish required more time for cortisol to return to initial levels,
particularly for the first two stressors, the differences observed (i.e., cortisol levels at 3
and 6 h v. initial levels) were not statistically significant. Variability in cortisol titers
was higher in BKDS fish than in CS fish, which probably accounts for the lack of
significant differences in some of our data. We have noted substantial variability in
cortisol concentrations of fish in other BKD studies at our laboratory and attribute this
at least partially to variation in infection levels and the consistent finding that cortisol
tends to become significantly elevated only during the later stages of BKD.
Resting plasma glucose concentrations in our Rs-infected fish were always lower
than control fish during the experiment and the BKDS fish displayed a significant, but
short lived, hyperglycemia only after the third stressor. Control fish showed no
significant hyperglycemia after application of the first stressor, a significant elevation of
glucose levels after the second stressor that lasted 3 h, and a similar elevation of glucose
titers after the third stressor that lasted at least 6 h. This indicates that CS fish were
more capable of showing a metabolic, secondary response to the stressors than were
BKDS fish. In all cases, the magnitude of the maximum change in glucose ranged from
20-35 mg/dL, which is similar to values observed in other studies assessing the effects
124
of acute handling or other physical stressors (e.g., Barton and Schreck 1987; Barton et
al. 1987; Mesa 1994). That our Rs-infected fish were often unable to elicit a significant
hyperglycemia to the stressors may be an artifact of some statistical considerations such
as the relatively high variability in these samples and the presumably low statistical
power of our tests. On the other hand, the results could be due to an altered adrenergic
response to the stressors combined with a low level of energy reserves (i.e., hepatic
glycogen). In teleosts, stress induces an elevation of plasma catecholamines from
chromaffin cells of the posterior cardinal vein and head kidney which in turn induce
hyperglycemia via glycogenolytic or gluconeogenic pathways (Mazeaud and Mazeaud
1981). If, because of its affinity for the kidney and chronic pathogenesis, BKD causes
damage to the chromaffin tissue, it is possible that the biosynthesis and release of
catecholamines could be compromised. Coupling this idea with the excess use of
carbohydrates by Rs-infected fish to help combat the chronic infection (Mesa et al.
1998) indicates that fish with moderate to advanced BKD may not be able to elicit a
significant hyperglycemia following physical stress. We are currently examining liver
glycogen levels and the activities of key metabolic enzymes in Rs-infected fish to learn
more about the metabolic effects of BKD.
The trends in plasma lactate concentrations of our CS and BKDS fish were
remarkably similar in terms of the magnitude of maximum change and the time required
to return to initial levels and are consistent with results of other studies (e.g., Pickering
et al. 1982; Lackner et al. 1988; Mesa 1994). Our BKDS and BKDNS fish always had
higher (sometimes significantly) initial or resting levels of lactate than control fish.
125
This is consistent with other results from our laboratory and probably reflects a general
hypoxia and poor oxygen transport in fish with BKD (Mesa et al. 1998). The rapid
post-stress decrease in lactate levels in our fish may be due to complete oxidation by the
tissues, excretion, or gluconeogenic conversion of lactate to glycogen. Because lactate
levels tend to become chronically elevated in more advanced cases of BKD, such fish
may have more difficulty dealing metabolically with a surge of lactate in response to an
exogenous stressor. Chronically elevated lactate combined with sporadic increases due
to stress would probably place severe demands on the acid-base balance of fish (Olsen
et al. 1992; Mesa et al. 1998).
In summary, we have examined some aspects of what is a very common
scenario among downstream migrating juvenile salmonids, namely fish being exposed
to various stressors while they have BKD. We subjected fish with low and high levels
of Rs infection to a series of acute handling stressors and hypoxia and found that
infection levels did not worsen nor did mortality increase following application of the
stressors. Our results should stimulate comparative research into the physiological and
immunological aspects of: (1) disease-free fish that are subjected to stress and
subsequently challenged with a pathogen; and (2) fish that are already diseased and
subjected to exogenous stress. We caution, however, that our results represent the
combined effects of one type of stressor and one disease only and probably cannot be
applied to other scenarios. The physiological effects of disease and the responses of
diseased fish to exogenous stress are probably dependent on a multitude of factors,
including the pathogen itself, the type of stressor (particularly acute v. chronic
126
stressors), fish species, size, and condition, environmental conditions, and the
interstressor interval. Thus, the interactions of disease and stress appears to be a fruitful
area for further research and, until we gain more information, our understanding of the
ecological impacts of fish diseases will remain limited.
Acknowledgments
We thank Karen Hans, Jack Hotchkiss, Sally Sauter, Stan Smith, Scott
Vanderkooi, and Joe Warren for laboratory assistance; Lisa Weiland for laboratory,
editorial, and graphical assistance; and personnel at the Little White Salmon National
Fish Hatchery for kindly providing fish for this study.
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132
SUMMARY AND CONCLUSIONS
Diseases are an integral, natural part of life for all animals. The direct impacts
of disease on individuals and animal populations can generally be divided into two
types, lethal and sublethal. By far, most study of diseases has focused on aspects
involved with their lethal effects, and this is deservedly so. After all, the most obvious
and attention-getting impact of disease is mass mortality caused by an epidemic. The
resurgence of old diseases of humans, such as tuberculosis, malaria, and cholera and the
appearance of new diseases such as AIDS, Ebola, and Lassa fever are killing people by
the thousands each year and presenting new and difficult challenges to medical science
(Reuter Information Service 1996). Disease is important as a mortality factor in many
animal populations and may be the predominant factor in bird populations (Davis et al.
1971). For example, epidemic disease is likely responsible for the catastrophic decline
of Australian rain forest frogs (Laurence et al. 1996) and is probably the main cause of
death among bighorn sheep in North America (Forrester 1971). The seminal papers by
Anderson and May (1979) and May and Anderson (1979) discussed the population
biology of infectious diseases and indicated that diseases are likely as important as, or at
least complimentary to, predation or resource limitation in constraining the growth of
plant and animal populations. Diseases of cultured fish are well known to cause
substantial mortality and there are numerous reports of disease-related mortality in wild
fish (Goede 1986; Warren 1991; Bucke 1993). Thus, because mortality is the most
133
overt outcome of diseases and can have emotional and economic impacts, it is easy to
understand why prevention and control are still the backbone of disease research today.
A more insidious but important impact of diseases or disease organisms upon
animals is general reduction in performance, which forms the underlying theme of the
research presented in my thesis. What is performance? Schreck (1981) provides a
useful definition: "...the ability or capacity of fish to maintain homeostasis or do,
physiologically or behaviorally, those things they must do to persist, grow, and
reproduce...". Schreck argues that performance capacity, which defines the totality of
all possible individual, discrete performances, is delimited ultimately by the organism's
genotype and proximately by environmental influences and stress (which, I believe,
includes disease). Thus, the organism is left with a "realized" performance capacity,
which changes with ontogeny, that defines the true performance capability of the
individual (Schreck 1981). My research targets the limits of realized performance
capacity and, in some ways, tests the concept by asking a simple question: does
infection with Renibacterium salmoninarum (Rs) significantly reduce the performance
of juvenile salmonids?
The sublethal impacts of disease or disease organisms on fish have received
some attention as evidenced by references cited in this thesis. The sublethal effects of
disease have been, and are today, often mentioned by fish health professionals as
important to our understanding of the impacts of diseases. For example, in the recent
American Fisheries Society symposium "Pathogens and Diseases of Fish in Aquatic
Ecosystems: Implications for Fisheries Management" published in the Journal of
134
Aquatic Animal Health, both Hedrick (1998) and Reno (1998) discussed, albeit briefly,
some sublethal or indirect impacts of diseases. The reason why it is important to study
the sublethal effects of diseases stems from the notion that, from the perspective of
disease dynamics and the infectious process, most individuals in a population will have
subclinical infections or are carriers of disease organisms. In other words, diseases do
not always result in direct mortality. This importance of subclinical cases and carriers
in human medicine is discussed by Anderson et al. (1962): "...subclinical cases and
carriers constitute the ultimate reservoir responsible for the greater portion of the spread
and for the perpetuation of certain human diseases." Furthermore, Dubos (1955),
referring to situations in which disease organisms are a constant and ubiquitous
component of the environment, states: "theories of disease must account for the
surprising fact that in any community, a large percentage of healthy and normal
individuals continually harbor potentially pathogenic microbes without suffering any
symptoms or lesions." In fish, examples of subclinical infections or carrier states are
evident in populations with BKD (Sanders et al. 1992; Maule et al. 1996) and fish with
viral diseases (Warren 1991). Thus, if fish with subclinical infections represent a large
proportion of a population, investigating the effects of such infections would be
important to fully understanding the potential impacts of disease.
In my research, I chose to study the effects of Rs infections on three aspects of
juvenile salmonid performance capacity: predator avoidance ability, smoltification, and
responses to exogenous stressors. While obviously not all inclusive, these measures of
performance are critical to the survival of fish in the wild. In the following discussion, I
135
will summarize the results from each study and show how they have contributed to our
understanding of the ecology of BKD.
Juvenile chinook salmon with either a low to moderate or high infection level of
Rs were significantly more vulnerable to predation than fish with very low or no
detectable infection. This result was consistent across two types of predators (northern
pikeminnow and smallmouth bass) with distinctly different predatory tactics. I believe
these results have a high degree of ecological relevance because they occurred over a
wide range of infection levels, used two types of predators, and represent a highly
integrative level of behavior with potential consequences at the population and
community levels (Scherer 1992).
A progressively worsening infection with Rs did not alter the normal changes in
gill Na, IC-ATPase and condition factor associated with smoltification in juvenile
chinook salmon in freshwater. This suggests that the role ATPase serves in preparing
fish for seawater entry would remain intact in fish with bacterial kidney disease (BKD)
and the widely reported poor osmocompetence of Rs-infected fish would not result from
reduced ATPase activity. Furthermore, in one instance, a dramatic proliferation of BKD
was associated with the maximal response of ATPase (and decreased condition factor),
suggesting that the process of smoltification itself can trigger outbreaks of disease.
The imposition of multiple, acute handling stressors on fish with low, moderate,
or high Rs infection levels did not lead to higher infection levels or increased mortality
when compared to diseased fish that did not receive the stressors. Also, the kinetics of
plasma cortisol, glucose, and lactate over a 24-h period following each application of
136
the handling stressor were similar between fish with moderate to high Rs infection
levels and those that had low or no detectable infection. These results indicate that fish
with well-established BKD were able to compensate physiologically to physical
stressors in a manner similar to fish that, at worst, had low levels of the infection. These
results challenge the well reported notion that stress (i.e., acute stress) predisposes fish
to disease by indicating that increased infection levels, disease pathogenesis, and
mortality are not a universal consequence of exposure to exogenous acute stress.
In all my studies, there were consistent physiological changes in fish during
disease progression that indicate BKD is stressful, particularly during the later stages.
During disease progression, plasma cortisol and lactate concentrations typically
increased whereas plasma glucose levels and hematocrits decreased. The cortisol data
indicate that the hypothalamic-pituitary-interrenal axis responds as the Rs infection
progresses. Together, these physiological changes in fish with BKD may present
problems with immunocompetence, osmoregulation, acid-base balance, and
intermediary metabolism, among other things.
Fish used in these studies fed well and grew, indicating that fish with relatively
high Rs infection levels feed and can channel some energy into somatic growth. That
Rs-infected fish feed and grow during disease progression is important in at least two
aspects related to this research: (1) larger fish are relatively less vulnerable to predation;
and (2) growth rate and size are known to be critical factors in successful smoltification.
Collectively, my results offer evidence of the functional and ecological
significance of BKD and the role it plays in affecting the survival of fish in the wild.
137
The importance of BKD, and diseases in general, as mortality factors in animal
populations may be significantly greater in toto than simply mortality directly attributed
to the pathogen.
138
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