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Influence of bacterial kidney disease on smoltification in salmonids: is it a case of double jeopardy?
Aquaculture 174 Ž1999. 25–41
Influence of bacterial kidney disease on smoltification in salmonids: is it a case of double jeopardy? Matthew G. Mesa
a,b,)
, Alec G. Maule a , Thomas P. Poe a , Carl B. Schreck c
a
c
U.S. Geological SurÕey, Biological Resources DiÕision, Columbia RiÕer Research Laboratory, 5501A Cook-Underwood Road, Cook, WA 98605, USA b Oregon CooperatiÕe Fishery Research Unit, Department of Fish and Wildlife, Oregon State UniÕersity, CorÕallis, OR 97331-3803, USA Oregon CooperatiÕe Fish and Wildlife Research Unit, Oregon State UniÕersity, CorÕallis, OR 97331-3803, USA Accepted 22 December 1998
Abstract We investigated the effects of a chronic, progressive infection with Renibacterium salmoninarum ŽRs., the causative agent of bacterial kidney disease ŽBKD., on selected aspects of smoltification in yearling juvenile spring chinook salmon Ž Oncorhynchus tshawytscha.. After experimentally infecting fish with Rs using an immersion challenge, we sampled them every two weeks to monitor changes in gill Naq, Kq-ATPase ŽATPase., cortisol, infection level, mortality, growth, and other stress-related physiological factors during the normal time of parr–smolt transformation in fresh water Ži.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
)
Corresponding author. Tel.: q1-509-538-2299 ext. 246; fax: q1-509-538-2843; e-mail: [email protected] 0044-8486r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 4 4 - 8 4 8 6 Ž 9 9 . 0 0 0 1 2 - 5
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trigger outbreaks of disease. Our results suggest mechanisms that probably influence the reported inability of Rs-infected fish to successfully adapt to sea water. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Bacterial kidney disease; Renibacterium salmoninarum; Oncorhynchus tshawytscha; Smoltification; Physiological stress
1. 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 Žsee 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 ŽSheridan et al., 1985., and aquaculturists are recognizing the importance of understanding factors influencing the quality of smolts Že.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 ŽWedemeyer 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 ŽBKD. is one of the most serious and prevalent health problems affecting hatchery-reared and wild salmonids today ŽFryer and Sanders, 1981; Sanders et al., 1992.. The disease is caused by Renibacterium salmoninarum ŽRs., a slow-growing Gram-positive diplobacillus that produces a chronic, systemic infection that is often lethal. Efforts to control BKD have had little success ŽElliott et al., 1989. and the disease is considered a major obstacle to the successful culture of salmonids ŽFryer 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 ŽFryer 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 ŽFryer 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
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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 Ž Oncorhynchus tshawytscha.. After experimentally infecting fish with Rs using an immersion challenge, we monitored changes in gill Naq, Kq-ATPase ŽATPase., cortisol, infection level, mortality, growth, and other stress-related physiological factors during the normal time of parr–smolt transformation in fresh water Ži.e., from winter through spring..
2. Materials and methods 2.1. Test fish 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. On 12 January 1993, we obtained about 2000 fish Žaverage length " S.E.s 123.8 " 2.3 mm; average weights 21.0 " 1.2 g. from Leavenworth National Fish Hatchery ŽWA., whereas on 19 January 1994 we obtained the same number of fish Žaverage lengths 121.5 " 2.4 mm; average weights 21.7 " 1.5 g. from Little White Salmon National Fish Hatchery ŽWA.. All fish were offspring from adults with a negative or low Rs infection level as determined by enzyme-linked immunosorbent assay ŽELISA; 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 4–58C well water at a rate of about 4 lrmin. 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 grl. Fish were fed twice daily with moist pellets Žat a ration of about 2% body weight per day. and kept under an ambient photoperiod simulated with lights and timers that produced a gradual intensity dusk and dawn. 2.2. Bacterial immersion challenges The experimental design involved creating two groups of fish, one group Žtreatment fish. that received a 24-h waterborne challenge with Rs and another group Žcontrol 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. Ž1992. and Elliott and Pascho Ž1995.. The bacterium used for the challenges was Rs, isolate DWK 90. This isolate was originally cultured from a spring chinook salmon at Dworshak National Fish Hatchery in 1990 and is maintained at our Western Fisheries Research Center in Seattle, WA. A 1–2 ml aliquot of the isolate was grown in 1–2 l of KDM-2 broth media with serum ŽEvelyn, 1977. at 158C for 7–11 d. Cells in their log phase of
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growth were then harvested by centrifugation Ž1700 = g for 40 min at 158C. and re-suspended in cold, sterile peptone-saline Ž0.1% wrv peptoneq 0.85% NaCl.. The concentration of Rs cells in the final suspension was determined by a membrane filtration-fluorescent antibody test ŽElliott and Barila, 1987., 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 = 10 8 bacteriarml, thus yielding a challenge dose of about 7.8 = 10 5 bacteriarml. 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 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 Ži.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 = 10 8 bacteriarml into each bucket containing treatment fish, thus yielding a challenge dose of 4.5 = 10 5 bacteriarml. 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. 2.3. Holding and sampling of fish After the 24-h challenges, fish were maintained as described above 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 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
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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 Ž200 mgrl. 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 y808C for future analysis. Plasma cortisol was determined by an ELISA modified from procedures described by Munro and Stabenfeldt Ž1984.. Plasma glucose and lactate were measured with a biochemistry analyzer ŽYellow Springs Instruments, model 2700D.. Next, whole kidneys were removed aseptically and frozen at y808C for future analysis by ELISA as described by Pascho and Mulcahy Ž1987. and modified by Elliott and Pascho Ž1991.. 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. Ž1994.. 2.4. 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 ŽANOVA, P ) 0.05.. We calculated mean lengths and weights of fish over time and plotted mean condition factor for each group of fish using the formula K s weightrlength3 = 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. Ž1987.. Fish with a positive Rs infection with absorbance between the positive–negative threshold 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 ŽPascho et al., 1991, 1993.. Mortalities during the holding period were tallied 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.
3. Results 3.1. Body length, weight and condition factor 3.1.1. 1993 experiments Over the course of the study, mean fork length and weight of both groups was similar and increased significantly from about 120 mm and 21 g to about 140 mm and 26 g
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during the experiment Ždata not shown.. Condition factor dropped markedly after week 8 in both groups ŽFig. 1A; P s 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. 3.1.2. 1994 experiments Mean fork length and weight of both groups changed in a manner similar to that described for our 1993 experiments Ždata not shown.. Condition factor dropped signifi-
Fig. 1. Mean Ž"S.E.. condition factor for spring chinook salmon that had received a waterborne challenge with Renibacterium salmoninarum and for unchallenged control fish during 1993 ŽA. and 1994 ŽB.. Asterisks denote mean values within a time period that differ significantly Ž P - 0.05..
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cantly Žrelative to earlier samples. in both groups of fish by 8–10 weeks ŽFig. 1B; P s 0.0001. before increasing Žrelative to the sample at week 8. in treatment fish during weeks 14 and 16 Ž 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 Ž P - 0.02. in treatment fish during weeks 10–16. 3.2. Rs-infection leÕels 3.2.1. 1993 experiments The immersion challenge was successful in producing fish with an established infection of Rs ŽFig. 2A.. Over the course of the study, average ELISA absorbance increased slightly during the first 12 weeks in treatment fish before increasing significantly Ž P s 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 Ž P s 0.0001., but this was probably due to the exceptionally low variance in the samples. Treatment fish had significantly Ž 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. 3.2.2. 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 ŽFig. 2B.. After 4 weeks, mean ELISA values in treatment fish were significantly higher than the initial sample Ž P s 0.0001. and continued to increase to a peak at 12 weeks before decreasing slightly during weeks 14–16. Control fish showed a steady, but slight, increase in average infection level during the first 12 weeks before infection level rose dramatically Ž P s 0.0001. and unexpectedly during weeks 14–16. Average ELISA absorbance did not differ between 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 Ž 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. 3.3. Physiological data 3.3.1. 1993 experiments Over the course of the experiment, all physiological factors changed significantly in both groups of fish. Gill ATPase activity increased during weeks 12–14 to levels that were significantly higher than earlier samples ŽFig. 3A; P s 0.0001.. Gill ATPase
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Fig. 2. Mean Žand S.E.. 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 ŽA. and 1994 ŽB.. Asterisks denote mean values within a time period that differ significantly Ž P - 0.05..
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 ŽFig. 3A.. Cortisol levels in treatment fish during weeks 16–20 were higher than earlier samples, but significantly so only at week 18 ŽFig. 3B.. Concentrations of cortisol in control fish were low and stable during the first 4 weeks, increased significantly Ž P s 0.0001. at week 8, and decreased steadily thereafter. The peak in cortisol titer occurred about 6 weeks prior to the peak in ATPase. Cortisol concentrations were significantly Ž P - 0.05. higher in control than in treatment fish at week 8, but then the reverse was true during weeks 16–20.
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Fig. 3. Mean Žand S.E.. gill Naq, Kq-ATPase activity ŽA., and concentrations of plasma cortisol ŽB., glucose ŽC., and lactate ŽD. 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 Ž P - 0.05..
Plasma glucose concentrations were significantly elevated Žrelative to all other samples. at the first sample period in both groups ŽFig. 3C; P s 0.0001.. Thereafter, glucose titers in treatment fish were stable during weeks 2–16 and then declined significantly Žrelative 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 in treatment fish were significantly elevated, relative to all other samples, at weeks 0 and 18 ŽFig. 3D; P s 0.0001.. Lactate concentrations in control fish
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were significantly higher in the first sample and at week 14 when compared to all other samples. The increase in lactate concentration in control fish 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 ŽFig. 3D; P - 0.05.. 3.3.2. 1994 experiments As in 1993, all physiological factors examined changed significantly in both groups over the course of the experiment. Gill sodium, potassium-activated ATPase increased to
Fig. 4. Mean Žand S.E.. gill Naq, Kq-ATPase activity ŽA., and concentrations of plasma cortisol ŽB., glucose ŽC., and lactate ŽD. 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 Ž P - 0.05..
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peak values during weeks 6–8 in both groups ŽFig. 4A; P s 0.0001.. There were no differences in mean ATPase activity between treatment and control fish during any sample period. In treatment fish, cortisol concentrations were significantly higher at weeks 8 and 12 than samples taken during the first four weeks and at week 16 ŽFig. 4B; P s 0.0001.. In control fish, 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. In treatment fish, glucose levels declined significantly after 8 weeks ŽFig. 4C; P s 0.0001.. In control fish, glucose levels increased significantly to a peak at 8 weeks, which coincided with the maximal activity in ATPase. Glucose concentrations then declined significantly relative to this peak during weeks 10–14. Mean glucose concentrations were substantially lower Ž P - 0.05. in treatment than in control fish at weeks 4, 8, 10, 12, and 14. Plasma lactate levels in treatment fish increased steadily to a peak at 12 weeks and were erratic thereafter ŽFig. 4D; P s 0.0001.. Lactate concentrations in control fish rose significantly Žrelative 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 Ž P - 0.05. between treatment and control fish at weeks 12 and 16.
4. Discussion This research investigated a common, yet essentially unstudied, scenario among anadromous juvenile salmonids in freshwater, namely fish undergoing the process of 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. The trends of ATPase and condition factor associated with smoltification in our fish were not affected by a worsening infection with Rs. In addition, all of our fish fed well and grew during these experiments, which is important because growth rate and size have been shown to be critical factors in successful smoltification ŽClarke and Shelbourne, 1985; McCormick and Saunders, 1987; Saunders et al., 1994; Shrimpton, 1996.. The mortality attributed solely to BKD upon exposure of Rs-infected fish to seawater has been highly variable ŽBanner et al., 1983; Sanders et al., 1992; Elliott et al., 1995; Moles, 1997. and indicates that Rs-infected fish may be dying of other proximate causes besides disease during seawater challenges, such as reduced osmocompetence ŽMoles, 1997.. 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
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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 Že.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. Several studies have reported a rise in plasma cortisol concentrations during smoltification Že.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 is not precisely defined; some reports have shown cortisol to increase ATPase activity and cause cellular differentiation and proliferation of chloride cells Že.g., Richman and Zaugg, 1987; Madsen, 1990; Shrimpton et al., 1994., while others have reported little or no effect ŽLangdon et al., 1984; Langhorne and Simpson, 1986; Richman et al., 1987.. 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 Rs-infected fish is consistent with other experiments at our laboratory and probably reflects a generalized stress response to disease ŽMesa et al., 1998.. The implications of such chronic elevations in cortisol titers for fish undergoing smoltification might include reduced osmoregulatory ability Že.g., Maule and Schreck, 1991; Shrimpton and Randall, 1994. and immunocompetence Že.g., Maule et al., 1987; Maule et al., 1989; Mazur and Iwama, 1993; Pickering, 1993; Espelid et al., 1996.. 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 Že.g., Bjornsson ¨ et al., 1989.. Evidence of a change in blood glucose concentration during smoltification is mixed, with some reports of an increase ŽWendt and Saunders, 1973; Woo et al., 1978; Plisetskaya et al., 1988. and other reports of a decrease ŽSweeting 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 notion of a hyperglycemia during smoltification. The declines in glucose concentrations we observed in Rs-infected fish Ži.e., both treatment groups and the 1994 control fish after the period of smoltification. were probably due 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 ŽWedemeyer and Ross, 1973; Iwama et al., 1986. and are probably due to excessive use of this energy substrate to help combat the infection ŽMesa et al., 1998.. Although changes in plasma lactate concentrations are commonly used as an indicator of stress in fish, less is known about changes in lactate levels due to disease or
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smoltification. In both years of our study, lactate levels in control fish rose to a peak coincident with the peak in ATPase and declined slowly thereafter, indicating that elevations in plasma lactate may be involved in the parr–smolt transformation. In our treatment fish Žand our 1994 control fish at week 16. BKD apparently altered any smoltification-related changes in lactate concentrations due most likely to the presumed hypoxemia elicited by BKD ŽMesa et al., 1998.. Increases in plasma lactate during smoltification might be related to the increased restlessness and migratory behavior of smolting fish or to the ontogenetic changes in complexity of the hemoglobin system that reflects preadaptations to changes in oxygen transfer, CO 2 transfer, and pH regulation ŽGiles and Vanstone, 1976; Hoar, 1988.. Increased levels of circulating lactate could be used by smolting fish to: Ž1. replenish or maintain blood glucose and glycogen stores via liver gluconeogenesis; Ž2. serve as a glucose precursor in the Cori cycle; or Ž3. serve as a preferred oxidative substrate for liver, erythrocytes, heart, and red muscle Že.g., Suarez and Mommsen, 1987; Milligan and Girard, 1993; Eros and Milligan, 1996.. Because of our limited understanding, it appears that lactate metabolism during smoltification and its potential influence on this transformation are topics in need of further study. Our results from 1993 offer the intriguing possibility that the ‘stress’ associated with smoltification ŽSimpson, 1985; Franklin et al., 1992. may have triggered a proliferation of BKD. In 1993, mean ELISA absorbance values in treatment fish increased dramatically to values above 1.0, indicating fish had progressed from a low or moderate 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 reasoning have lead us to conclude that the association of smoltification and BKD progression is more than just coincidence. First, we are unable to attribute the increased infection levels at week 14 to other stressors such as density Žwhich was low. or water temperature Žwhich was stable.. Second, Maule et al. Ž1987. reported that the salmonid immune system is depressed during smoltification, which could allow an established but latent infection to proliferate. Third, several reports indicate that Rs and its major surface antigen, p57, are immunosuppressive ŽTuraga et al., 1987; Fredricksen et al., 1997; Siegel 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. That we did not observe similar changes in infection level in our other groups of fish that underwent smoltification could be related to: Ž1. the amount of antigen Žor numbers of bacteria. necessary for BKD proliferation in response to other stressors or smoltification; or Ž2. stock differences in the relative resistance to BKD, as indicated by several studies evaluating genetic variation in resistance of salmonids to various diseases ŽSuzumoto et al., 1977; McIntyre and Amend, 1978; Winter et al., 1980; Withler and Evelyn, 1990; Beacham and Evelyn, 1992.. Considerable evidence shows that various forms of environmental stress can be an important factor in outbreaks of infectious disease in fishes ŽSnieszko, 1974; Pickering, 1993.. We believe our results substantiate this notion by implicating smoltification as a possible influence on disease outbreaks.
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5. Conclusions Ž1. A progressively worsening infection with Renibacterium salmoninarum did not alter the normal changes in gill ATPase activity and condition factor associated with smoltification in juvenile chinook salmon. Ž2. Bacterial kidney disease did, however, alter trends in plasma cortisol, glucose, and lactate indicating that the disease is stressful, particularly in the later stages. Ž3. 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. Ž4. Our results should emphasize the importance of considering fish health and stock differences in the conduct and interpretation of physiological studies.
Acknowledgements We thank Ron Pascho, Diane Elliott, Connie McKibben, Marne 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.
References Banner, C.R., Rohovec, J.S., Fryer, J.L., 1983. Renibacterium salmoninarum as a cause of mortality among chinook salmon in salt water. J. World. Maricul. Soc. 14, 236–239. Beacham, T.D., Evelyn, T.P.T., 1992. Population and genetic variation in resistance of chinook salmon to vibriosis, furunculosis, and bacterial kidney disease. J. Aquat. Anim. Health 4, 153–167. Bjornsson, B.Th., Young, G., Lin, R.J., Deftos, L.J., Bern, H.A., 1989. Smoltification and seawater adaptation ¨ in coho salmon Ž Oncorhynchus kisutch.: plasma calcium regulation, osmoregulation, and calcitonin. Gen. Comp. Endocrinol. 74, 346–354. Clarke, W.C., Shelbourne, J.E., 1985. Growth and development of seawater adaptability by juvenile fall chinook salmon Ž Oncorhynchus tshawytscha. in relation to temperature. Aquaculture 45, 21–31. Elliott, D.G., Barila, T.Y., 1987. Membrane filtration-fluorescent antibody staining procedure for detecting and quantifying Renibacterium salmoninarum in coelomic fluid of chinook salmon Ž Oncorhynchus tshawytscha.. Can. J. Fish. Aquat. Sci. 44, 206–210. Elliott, D.G., Pascho, R.J., Bullock, G.L., 1989. Developments in the control of bacterial kidney disease of salmonid fishes. Dis. Aquat. Org. 6, 201–215. Elliott, D.G., Pascho, R.J. 1991. Juvenile fish transportation: Impact of bacterial kidney disease on survival of springrsummer chinook salmon stocks. Annual Report, 1989, prepared by the U.S. Fish and Wildlife Service, Seattle, WA, for the U.S. Army Corps of Engineers, Walla Walla, WA. Elliott, D.G., Pascho, R.J., 1995. Juvenile fish transportation: Impact of bacterial kidney disease on survival of
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springrsummer chinook salmon stocks. Annual Report, 1993, prepared by the U.S. Fish and Wildlife Service, Seattle, WA, for the U.S. Army Corps of Engineers, Walla Walla, WA. Elliott, D.G., Pascho, R.J., Palmisano, A.N., 1995. Brood stock segregation for the control of bacterial kidney disease can affect mortality of progeny chinook salmon Ž Oncorhynchus tshawytscha. in seawater. Aquaculture 132, 133–144. Eros, S.K., Milligan, C.L., 1996. The effect of cortisol on recovery from exhaustive exercise in rainbow trout Ž Oncorhynchus mykiss .: potential mechanisms of action. Phys. Zool. 69, 1196–1214. Espelid, S., Løkken, G.B., Steiro, K., Bøgwald, J., 1996. Effects of cortisol and stress on the immune system in Atlantic salmon Ž Salmo salar L... Fish Shellfish Immunol. 6, 95–110. Evelyn, T.P.T., 1977. An improved growth medium for the kidney disease bacterium and some notes on using the medium. Bull. Off. Int. Epizoot. 87, 511–513. Folmar, L.C., Dickhoff, W.W., 1980. The parr–smolt transformation Žsmoltification. and seawater adaptation in salmonids. Aquaculture 21, 1–37. Franklin, C.E., Davison, W., Forster, M.E., 1992. Seawater adaptability of New Zealand’s sockeye Ž Oncorhynchus nerka. and chinook salmon Ž O. tshawytscha.: physiological correlates of smoltification and seawater survival. Aquaculture 102, 127–142. ˚ Endresen, C., Wergeland, H.I., 1997. Immunosuppressive effect of a low molecular weight Fredricksen, A., surface protein from Renibacterium salmoninarum on lymphocytes from Atlantic salmon Ž Salmo salar L... Fish Shellfish Immunol. 7, 273–282. Fryer, J.L., Sanders, J.E., 1981. Bacterial kidney disease of salmonid fish. Annu. Rev. Microbiol. 35, 273–298. Fryer, J.L., Lannan, C.N., 1993. The history and current status of Renibacterium salmoninarum, the causative agent of bacterial kidney disease in Pacific salmon. Fish. Res. 17, 15–33. Giles, M.A., Vanstone, W.E., 1976. Ontogenetic variation in the multiple hemoglobins of coho salmon Ž Oncorhynchus kisutch. and effect of environmental factors on their expression. J. Fish. Res. Board Can. 33, 1144–1149. Hoar, W.S., 1976. Smolt transformation: evolution, behavior, and physiology. J. Fish. Res. Board Can. 33, 1234–1252. Hoar, W.S., 1988. The physiology of smolting salmonids. In: Hoar, W.S., Randall, D.J. ŽEds.., Fish Physiology, Vol. XIB. Academic Press, New York, NY, pp. 275–343. Iwama, G.K., 1992. Smolt quality: a special section. World Aquaculture 23, 38–39. Iwama, G.K., Greer, G.L., Randall, D.J., 1986. Changes in selected haematological parameters in juvenile chinook salmon subjected to a bacterial challenge and a toxicant. J. Fish. Biol. 28, 563–572. Ji, H., Bradley, T.M., Tremblay, G.C., 1996. Lactate-dependent gluconeogenesis and atractyloside-sensitive flux through pyruvate carboxylase are reduced during smoltification of Atlantic salmon Ž Salmo salar .. J. Exp. Zool. 276, 375–386. Langdon, J.S., Thorpe, J.E., Roberts, R.J., 1984. Effects of cortisol and ACTH on gill NaqrKq-ATPase, SDH and chloride cells in juvenile Atlantic salmon Salmo salar L. Comp. Biochem. Physiol. A 77, 9–12. Langhorne, P., Simpson, T.H., 1986. The interrelationship of cortisol, gill ŽNaq K. ATPase, and homeostasis during the parr–smolt transformation of Atlantic salmon Ž Salmo salar L... Gen. Comp. Endocrinol. 61, 203–213. Madsen, S.S., 1990. The role of cortisol and growth hormone in seawater adaptation and development of hypoosmoregulatory mechanisms in sea trout parr Ž Salmo trutta trutta.. Gen. Comp. Endocrinol. 79, 1–11. Maule, A.G., Schreck, C.B., Kaattari, S.L., 1987. Changes in the immune system of coho salmon Ž Oncorhynchus kisutch. during the parr-to-smolt transformation and after implantation of cortisol. Can. J. Fish. Aquat. Sci. 44, 161–166. Maule, A.G., Tripp, R.A., Kaattari, S.L., Schreck, C.B., 1989. Stress alters immune function and disease resistance in chinook salmon Ž Oncorhynchus tshawytscha.. J. Endocrinol. 120, 135–142. Maule, A.G., Schreck, C.B., 1991. Stress and cortisol treatment changed affinity and number of glucocorticoid receptors in leukocytes and gill of juvenile coho salmon. Gen. Comp. Endocrinol. 84, 83–93. Mazur, C.F., Iwama, G.K., 1993. Handling and crowding stress reduces number of plaque-forming cells in Atlantic salmon. J. Aquat. Anim. Health 5, 98–101. McCormick, S.D., Saunders, R.L., 1987. Preparatory physiological adaptations for marine life of salmonids: osmoregulation, growth, and metabolism. Am. Fish. Soc. Symp. 1, 211–229.
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McCormick, S.D., Dickhoff, W.W., Duston, J., Nishioka, R.S., Bern, H.A., 1991. Developmental differences in the responsiveness of gill Naq, Kq-ATPase to cortisol in salmonids. Gen. Comp. Endocrinol. 84, 308–317. McIntyre, J.D., Amend, D.F., 1978. Heritability of tolerance for infectious hematopoietic necrosis in sockeye salmon Ž Oncorhynchus nerka.. Trans. Am. Fish. Soc. 107, 305–308. Mesa, M.G., Poe, T.P., Maule, A.G., Schreck, C.B., 1998. Vulnerability to predation and physiological stress responses in juvenile chinook salmon Ž Oncorhynchus tshawytscha. experimentally infected with Renibacterium salmoninarum. Can. J. Fish. Aquat. Sci. 55, 1599–1606. Milligan, C.L., Girard, S.S., 1993. Lactate metabolism in rainbow trout. J. Exp. Biol. 180, 175–193. Moles, A., 1997. Effect of bacterial kidney disease on saltwater adaptation of coho salmon smolts. J. Aquat. Anim. Health 9, 230–233. Munro, C., Stabenfeldt, G., 1984. Development of a microtitre plate enzyme immunoassay for the determination of progesterone. J. Endocrinol. 101, 41–49. Murray, C.B., Evelyn, T.P.T., Beacham, T.D., Barner, L.W., Ketcheson, J.E., Prosperi-Porta, L., 1992. Experimental induction of bacterial kidney disease in chinook salmon by immersion and cohabitation challenges. Dis. Aquat. Org. 12, 91–96. Olsen, Y.A., Reitan, L.J., Røed, K.H., 1993. Gill Naq, Kq-ATPase activity, plasma cortisol level, and non-specific immune response in Atlantic salmon Ž Salmo salar . during parr–smolt transformation. J. Fish Biol. 43, 559–573. Pascho, R.J., Mulcahy, D., 1987. Enzyme-linked immunosorbent assay for a soluble antigen of Renibacterium salmoninarum, the causative agent of salmonid bacterial kidney disease. Can. J. Fish. Aquat. Sci. 44, 183–191. Pascho, R.J., Elliott, D.G., Mallett, R.W., Mulcahy, D., 1987. Comparison of five techniques for the detection of Renibacterium salmoninarum in coho salmon. Trans. Am. Fish Soc. 116, 882–890. Pascho, R.J., Elliott, D.G., Streufert, J.M., 1991. Brood stock segregation of spring chinook salmon Oncorhynchus tshawytscha by use of the enzyme-linked immunosorbent assay ŽELISA. and the fluorescent antibody technique ŽFAT. affects the prevalence and levels of Renibacterium salmoninarum infection in progeny. Dis. Aquat. Org. 12, 25–40. Pascho, R.J., Elliott, D.G., Achord, S., 1993. Monitoring of the in-river migration of smolts from two groups of spring chinook salmon, Oncorhynchus tshawytscha ŽWalbaum., with different profiles of Renibacterium salmoninarum infection. Aqua. Fish. Mgt. 24, 163–169. Pickering, A.D., 1993. Endocrine-induced pathology in stressed salmonid fish. Fish. Res. 17, 35–50. Plisetskaya, E.M., Swanson, P., Bernard, M.G., Dickhoff, W.W., 1988. Insulin in coho salmon Ž Oncorhynchus kisutch. during the parr to smolt transformation. Aquaculture 72, 151–164. Prunet, P., Boeuf, G., Bolton, J.P., Young, G., 1989. Smoltification and seawater adaptation in Atlantic salmon Ž Salmo salar .: plasma prolactin, growth hormone, and thyroid hormones. Gen. Comp. Endocrinol. 74, 355–364. Richman, N.H. III, Zaugg, W.S., 1987. Effects of cortisol and growth hormone on osmoregulation in pre- and desmoltified coho salmon Ž Oncorhynchus kisutch.. Gen. Comp. Endocrinol. 65, 189–198. Richman, N.H. III, Nishioka, R.S., Young, G., Bern, H.A., 1987. Effects of cortisol and growth hormone replacement on osmoregulation in hypophysectomized coho salmon Ž Oncorhynchus kisutch.. Gen. Comp. Endocrinol. 67, 194–201. Sanders, J.E., Long, J.J., Arakawa, C.K., Bartholomew, J.L., Rohovec, J.S., 1992. Prevalence of Renibacterium salmoninarum among downstream-migrating salmonids in the Columbia River. J. Aquat. Anim. Health 4, 72–75. Saunders, R.L., Duston, J., Benfey, T.J., 1994. Environmental and biological factors affecting growth dynamics in relation to smolting of Atlantic salmon, Salmo salar L. Aqua. Fish. Manage. 25, 9–20. Schrock, R.M., Beeman, J.W., Rondorf, D.W., Haner, P.V., 1994. A microassay for gill sodium, potassiumactivated ATPase in juvenile pacific salmonids. Trans. Am. Fish. Soc. 123, 223–229. Sheridan, M.A., Woo, N.Y.S., Bern, H.A., 1985. Changes in the rates of glycogenesis, glycogenolysis, lipogenesis, and lipolysis in selected tissues of the coho salmon Ž Oncorhynchus kistuch. associated with parr–smolt transformation. J. Exp. Zool. 236, 35–44. Simpson, T.H., 1985. Epilogue. Aquaculture 45, 395–398. Siegel, D.C., Congleton, J.L., 1997. Bactericidal activity of juvenile chinook salmon macrophages against
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Aeromonas salmonicida after exposure to live or heat-killed Renibacterium salmoninarum or to soluble proteins produced by R. salmoninarum. J. Aquat. Anim. Health 9, 180–189. Shrimpton, J.M., Randall, D.J., 1994. Downregulation of corticosteroid receptors in the gills of coho salmon due to stress and cortisol treatment. Am. J. Physiol. 267, R432–R438. Shrimpton, J.M., Bernier, N.J., Randall, D.J., 1994. Changes in cortisol dynamics in wild and hatchery-reared juvenile coho salmon Ž Oncorhynchus kisutch. during smoltification. Can. J. Fish. Aquat. Sci. 51, 2179–2187. Shrimpton, J.M., 1996. Relationship between size, gill corticosteroid receptors, Naq, Kq-ATPase activity and smolting in juvenile coho salmon Ž Oncorhynchus kisutch. in autumn and spring. Aquaculture 147, 127–140. Snieszko, S.F., 1974. The effects of environmental stress on outbreaks of infectious diseases of fishes. J. Fish Biol. 6, 197–208. Soengas, J.L., Fuentes, J., Otero, J., Andres, ´ M.D., Aldegunde, M., 1992. Seasonal changes in carbohydrate metabolism in the rainbow trout Ž Oncorhynchus mykiss . and their relationship to changes in gill Naq Kq-ATPase activity. Aquaculture 108, 369–380. Specker, J.L., Schreck, C.B., 1982. Changes in plasma corticosteroids during smoltification of coho salmon, Oncorhynchus kisutch. Gen. Comp. Endocrinol. 46, 53–58. Suarez, R.K., Mommsen, T.P., 1987. Gluconeogenesis in teleost fishes. Can. J. Zool. 65, 1869–1882. Suzumoto, B.K., Schreck, C.B., McIntyre, J.D., 1977. Relative resistances of three transferrin genotypes of coho salmon Ž Oncorhynchus kisutch. and their hematological responses to bacterial kidney disease. J. Fish. Res. Board. Can. 34, 1–8. Sweeting, R.M., Wagner, G.F., McKeown, B.A., 1985. Changes in plasma glucose, amino acid nitrogen and growth hormone during smoltification and seawater adaptation in coho salmon, Oncorhynchus kisutch. Aquaculture 45, 185–197. Turaga, P., Weins, G., Kaattari, S., 1987. Bacterial kidney disease: the potential role of soluble protein antigenŽs.. J. Fish. Biol. 31, 191–194, Supplement A. Wedemeyer, G.A., Ross, A.J., 1973. Nutritional factors in the biochemical pathology of corynebacterial kidney disease in the coho salmon Ž Oncorhynchus kisutch.. J. Fish. Res. Board Can. 30, 296–298. Wedemeyer, G.A., Saunders, R.L., Clarke, W.C., 1980. Environmental factors affecting smoltification and early marine survival of anadromous salmonids. Mar. Fish. Rev. 42, 1–14. Wendt, C.A.G., Saunders, R.L., 1973. Changes in carbohydrate metabolism in young Atlantic salmon in response to various forms of stress. In: Smith, M.W., Carter, W.H., ŽEds.., International Atlantic Salmon Symposium, Vol. 4. Atlantic Salmon Foundation, New York, NY, pp. 55–82. Winter, G.W., Schreck, C.B., McIntyre, J.D., 1980. Resistance of different stocks and transferrin genotypes of coho salmon, Oncorhynchus kisutch, and steelhead trout, Salmo gairdneri, to bacterial kidney disease and vibriosis. Fish. Bull. 77, 795–802. Withler, R.E., Evelyn, T.P.T., 1990. Genetic variation in resistance to bacterial kidney disease within and between two strains of coho salmon from British Columbia. Trans. Am. Fish. Soc. 119, 1003–1009. Woo, N.Y.S., Bern, H.A., Nishioka, R.S., 1978. Changes in body composition associated with smoltification and premature transfer to seawater in coho salmon Ž Oncorhynchus kisutch. and king salmon Ž O. tshawytscha.. J. Fish Biol. 13, 421–428. Young, G., Bjornsson, B.Th., Prunet, P., Lin, R.J., Bern, H.A., 1989. Smoltification and seawater adaptation in ¨ coho salmon Ž Oncorhynchus kisutch.: plasma prolactin, growth hormone, thyroid hormones, and cortisol. Gen. Comp. Endocrinol. 74, 335–345. Zaugg, W.S., Beckman, B.R., 1990. Saltwater-induced decreases in weight and length relative to seasonal gill Naq, Kq-ATPase changes in coho salmon Ž Oncorhynchus kisutch.: a test for saltwater adaptability. Aquaculture 86, 19–23.
Influence of bacterial kidney disease on smoltification in salmonids: is it a case of double jeopardy? Matthew G. Mesa
a,b,)
, Alec G. Maule a , Thomas P. Poe a , Carl B. Schreck c
a
c
U.S. Geological SurÕey, Biological Resources DiÕision, Columbia RiÕer Research Laboratory, 5501A Cook-Underwood Road, Cook, WA 98605, USA b Oregon CooperatiÕe Fishery Research Unit, Department of Fish and Wildlife, Oregon State UniÕersity, CorÕallis, OR 97331-3803, USA Oregon CooperatiÕe Fish and Wildlife Research Unit, Oregon State UniÕersity, CorÕallis, OR 97331-3803, USA Accepted 22 December 1998
Abstract We investigated the effects of a chronic, progressive infection with Renibacterium salmoninarum ŽRs., the causative agent of bacterial kidney disease ŽBKD., on selected aspects of smoltification in yearling juvenile spring chinook salmon Ž Oncorhynchus tshawytscha.. After experimentally infecting fish with Rs using an immersion challenge, we sampled them every two weeks to monitor changes in gill Naq, Kq-ATPase ŽATPase., cortisol, infection level, mortality, growth, and other stress-related physiological factors during the normal time of parr–smolt transformation in fresh water Ži.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
)
Corresponding author. Tel.: q1-509-538-2299 ext. 246; fax: q1-509-538-2843; e-mail: [email protected] 0044-8486r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 4 4 - 8 4 8 6 Ž 9 9 . 0 0 0 1 2 - 5
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trigger outbreaks of disease. Our results suggest mechanisms that probably influence the reported inability of Rs-infected fish to successfully adapt to sea water. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Bacterial kidney disease; Renibacterium salmoninarum; Oncorhynchus tshawytscha; Smoltification; Physiological stress
1. 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 Žsee 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 ŽSheridan et al., 1985., and aquaculturists are recognizing the importance of understanding factors influencing the quality of smolts Že.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 ŽWedemeyer 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 ŽBKD. is one of the most serious and prevalent health problems affecting hatchery-reared and wild salmonids today ŽFryer and Sanders, 1981; Sanders et al., 1992.. The disease is caused by Renibacterium salmoninarum ŽRs., a slow-growing Gram-positive diplobacillus that produces a chronic, systemic infection that is often lethal. Efforts to control BKD have had little success ŽElliott et al., 1989. and the disease is considered a major obstacle to the successful culture of salmonids ŽFryer 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 ŽFryer 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 ŽFryer 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
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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 Ž Oncorhynchus tshawytscha.. After experimentally infecting fish with Rs using an immersion challenge, we monitored changes in gill Naq, Kq-ATPase ŽATPase., cortisol, infection level, mortality, growth, and other stress-related physiological factors during the normal time of parr–smolt transformation in fresh water Ži.e., from winter through spring..
2. Materials and methods 2.1. Test fish 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. On 12 January 1993, we obtained about 2000 fish Žaverage length " S.E.s 123.8 " 2.3 mm; average weights 21.0 " 1.2 g. from Leavenworth National Fish Hatchery ŽWA., whereas on 19 January 1994 we obtained the same number of fish Žaverage lengths 121.5 " 2.4 mm; average weights 21.7 " 1.5 g. from Little White Salmon National Fish Hatchery ŽWA.. All fish were offspring from adults with a negative or low Rs infection level as determined by enzyme-linked immunosorbent assay ŽELISA; 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 4–58C well water at a rate of about 4 lrmin. 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 grl. Fish were fed twice daily with moist pellets Žat a ration of about 2% body weight per day. and kept under an ambient photoperiod simulated with lights and timers that produced a gradual intensity dusk and dawn. 2.2. Bacterial immersion challenges The experimental design involved creating two groups of fish, one group Žtreatment fish. that received a 24-h waterborne challenge with Rs and another group Žcontrol 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. Ž1992. and Elliott and Pascho Ž1995.. The bacterium used for the challenges was Rs, isolate DWK 90. This isolate was originally cultured from a spring chinook salmon at Dworshak National Fish Hatchery in 1990 and is maintained at our Western Fisheries Research Center in Seattle, WA. A 1–2 ml aliquot of the isolate was grown in 1–2 l of KDM-2 broth media with serum ŽEvelyn, 1977. at 158C for 7–11 d. Cells in their log phase of
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growth were then harvested by centrifugation Ž1700 = g for 40 min at 158C. and re-suspended in cold, sterile peptone-saline Ž0.1% wrv peptoneq 0.85% NaCl.. The concentration of Rs cells in the final suspension was determined by a membrane filtration-fluorescent antibody test ŽElliott and Barila, 1987., 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 = 10 8 bacteriarml, thus yielding a challenge dose of about 7.8 = 10 5 bacteriarml. 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 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 Ži.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 = 10 8 bacteriarml into each bucket containing treatment fish, thus yielding a challenge dose of 4.5 = 10 5 bacteriarml. 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. 2.3. Holding and sampling of fish After the 24-h challenges, fish were maintained as described above 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 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
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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 Ž200 mgrl. 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 y808C for future analysis. Plasma cortisol was determined by an ELISA modified from procedures described by Munro and Stabenfeldt Ž1984.. Plasma glucose and lactate were measured with a biochemistry analyzer ŽYellow Springs Instruments, model 2700D.. Next, whole kidneys were removed aseptically and frozen at y808C for future analysis by ELISA as described by Pascho and Mulcahy Ž1987. and modified by Elliott and Pascho Ž1991.. 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. Ž1994.. 2.4. 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 ŽANOVA, P ) 0.05.. We calculated mean lengths and weights of fish over time and plotted mean condition factor for each group of fish using the formula K s weightrlength3 = 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. Ž1987.. Fish with a positive Rs infection with absorbance between the positive–negative threshold 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 ŽPascho et al., 1991, 1993.. Mortalities during the holding period were tallied 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.
3. Results 3.1. Body length, weight and condition factor 3.1.1. 1993 experiments Over the course of the study, mean fork length and weight of both groups was similar and increased significantly from about 120 mm and 21 g to about 140 mm and 26 g
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during the experiment Ždata not shown.. Condition factor dropped markedly after week 8 in both groups ŽFig. 1A; P s 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. 3.1.2. 1994 experiments Mean fork length and weight of both groups changed in a manner similar to that described for our 1993 experiments Ždata not shown.. Condition factor dropped signifi-
Fig. 1. Mean Ž"S.E.. condition factor for spring chinook salmon that had received a waterborne challenge with Renibacterium salmoninarum and for unchallenged control fish during 1993 ŽA. and 1994 ŽB.. Asterisks denote mean values within a time period that differ significantly Ž P - 0.05..
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cantly Žrelative to earlier samples. in both groups of fish by 8–10 weeks ŽFig. 1B; P s 0.0001. before increasing Žrelative to the sample at week 8. in treatment fish during weeks 14 and 16 Ž 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 Ž P - 0.02. in treatment fish during weeks 10–16. 3.2. Rs-infection leÕels 3.2.1. 1993 experiments The immersion challenge was successful in producing fish with an established infection of Rs ŽFig. 2A.. Over the course of the study, average ELISA absorbance increased slightly during the first 12 weeks in treatment fish before increasing significantly Ž P s 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 Ž P s 0.0001., but this was probably due to the exceptionally low variance in the samples. Treatment fish had significantly Ž 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. 3.2.2. 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 ŽFig. 2B.. After 4 weeks, mean ELISA values in treatment fish were significantly higher than the initial sample Ž P s 0.0001. and continued to increase to a peak at 12 weeks before decreasing slightly during weeks 14–16. Control fish showed a steady, but slight, increase in average infection level during the first 12 weeks before infection level rose dramatically Ž P s 0.0001. and unexpectedly during weeks 14–16. Average ELISA absorbance did not differ between 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 Ž 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. 3.3. Physiological data 3.3.1. 1993 experiments Over the course of the experiment, all physiological factors changed significantly in both groups of fish. Gill ATPase activity increased during weeks 12–14 to levels that were significantly higher than earlier samples ŽFig. 3A; P s 0.0001.. Gill ATPase
32
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Fig. 2. Mean Žand S.E.. 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 ŽA. and 1994 ŽB.. Asterisks denote mean values within a time period that differ significantly Ž P - 0.05..
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 ŽFig. 3A.. Cortisol levels in treatment fish during weeks 16–20 were higher than earlier samples, but significantly so only at week 18 ŽFig. 3B.. Concentrations of cortisol in control fish were low and stable during the first 4 weeks, increased significantly Ž P s 0.0001. at week 8, and decreased steadily thereafter. The peak in cortisol titer occurred about 6 weeks prior to the peak in ATPase. Cortisol concentrations were significantly Ž P - 0.05. higher in control than in treatment fish at week 8, but then the reverse was true during weeks 16–20.
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Fig. 3. Mean Žand S.E.. gill Naq, Kq-ATPase activity ŽA., and concentrations of plasma cortisol ŽB., glucose ŽC., and lactate ŽD. 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 Ž P - 0.05..
Plasma glucose concentrations were significantly elevated Žrelative to all other samples. at the first sample period in both groups ŽFig. 3C; P s 0.0001.. Thereafter, glucose titers in treatment fish were stable during weeks 2–16 and then declined significantly Žrelative 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 in treatment fish were significantly elevated, relative to all other samples, at weeks 0 and 18 ŽFig. 3D; P s 0.0001.. Lactate concentrations in control fish
34
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were significantly higher in the first sample and at week 14 when compared to all other samples. The increase in lactate concentration in control fish 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 ŽFig. 3D; P - 0.05.. 3.3.2. 1994 experiments As in 1993, all physiological factors examined changed significantly in both groups over the course of the experiment. Gill sodium, potassium-activated ATPase increased to
Fig. 4. Mean Žand S.E.. gill Naq, Kq-ATPase activity ŽA., and concentrations of plasma cortisol ŽB., glucose ŽC., and lactate ŽD. 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 Ž P - 0.05..
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peak values during weeks 6–8 in both groups ŽFig. 4A; P s 0.0001.. There were no differences in mean ATPase activity between treatment and control fish during any sample period. In treatment fish, cortisol concentrations were significantly higher at weeks 8 and 12 than samples taken during the first four weeks and at week 16 ŽFig. 4B; P s 0.0001.. In control fish, 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. In treatment fish, glucose levels declined significantly after 8 weeks ŽFig. 4C; P s 0.0001.. In control fish, glucose levels increased significantly to a peak at 8 weeks, which coincided with the maximal activity in ATPase. Glucose concentrations then declined significantly relative to this peak during weeks 10–14. Mean glucose concentrations were substantially lower Ž P - 0.05. in treatment than in control fish at weeks 4, 8, 10, 12, and 14. Plasma lactate levels in treatment fish increased steadily to a peak at 12 weeks and were erratic thereafter ŽFig. 4D; P s 0.0001.. Lactate concentrations in control fish rose significantly Žrelative 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 Ž P - 0.05. between treatment and control fish at weeks 12 and 16.
4. Discussion This research investigated a common, yet essentially unstudied, scenario among anadromous juvenile salmonids in freshwater, namely fish undergoing the process of 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. The trends of ATPase and condition factor associated with smoltification in our fish were not affected by a worsening infection with Rs. In addition, all of our fish fed well and grew during these experiments, which is important because growth rate and size have been shown to be critical factors in successful smoltification ŽClarke and Shelbourne, 1985; McCormick and Saunders, 1987; Saunders et al., 1994; Shrimpton, 1996.. The mortality attributed solely to BKD upon exposure of Rs-infected fish to seawater has been highly variable ŽBanner et al., 1983; Sanders et al., 1992; Elliott et al., 1995; Moles, 1997. and indicates that Rs-infected fish may be dying of other proximate causes besides disease during seawater challenges, such as reduced osmocompetence ŽMoles, 1997.. 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
36
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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 Že.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. Several studies have reported a rise in plasma cortisol concentrations during smoltification Že.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 is not precisely defined; some reports have shown cortisol to increase ATPase activity and cause cellular differentiation and proliferation of chloride cells Že.g., Richman and Zaugg, 1987; Madsen, 1990; Shrimpton et al., 1994., while others have reported little or no effect ŽLangdon et al., 1984; Langhorne and Simpson, 1986; Richman et al., 1987.. 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 Rs-infected fish is consistent with other experiments at our laboratory and probably reflects a generalized stress response to disease ŽMesa et al., 1998.. The implications of such chronic elevations in cortisol titers for fish undergoing smoltification might include reduced osmoregulatory ability Že.g., Maule and Schreck, 1991; Shrimpton and Randall, 1994. and immunocompetence Že.g., Maule et al., 1987; Maule et al., 1989; Mazur and Iwama, 1993; Pickering, 1993; Espelid et al., 1996.. 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 Že.g., Bjornsson ¨ et al., 1989.. Evidence of a change in blood glucose concentration during smoltification is mixed, with some reports of an increase ŽWendt and Saunders, 1973; Woo et al., 1978; Plisetskaya et al., 1988. and other reports of a decrease ŽSweeting 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 notion of a hyperglycemia during smoltification. The declines in glucose concentrations we observed in Rs-infected fish Ži.e., both treatment groups and the 1994 control fish after the period of smoltification. were probably due 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 ŽWedemeyer and Ross, 1973; Iwama et al., 1986. and are probably due to excessive use of this energy substrate to help combat the infection ŽMesa et al., 1998.. Although changes in plasma lactate concentrations are commonly used as an indicator of stress in fish, less is known about changes in lactate levels due to disease or
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smoltification. In both years of our study, lactate levels in control fish rose to a peak coincident with the peak in ATPase and declined slowly thereafter, indicating that elevations in plasma lactate may be involved in the parr–smolt transformation. In our treatment fish Žand our 1994 control fish at week 16. BKD apparently altered any smoltification-related changes in lactate concentrations due most likely to the presumed hypoxemia elicited by BKD ŽMesa et al., 1998.. Increases in plasma lactate during smoltification might be related to the increased restlessness and migratory behavior of smolting fish or to the ontogenetic changes in complexity of the hemoglobin system that reflects preadaptations to changes in oxygen transfer, CO 2 transfer, and pH regulation ŽGiles and Vanstone, 1976; Hoar, 1988.. Increased levels of circulating lactate could be used by smolting fish to: Ž1. replenish or maintain blood glucose and glycogen stores via liver gluconeogenesis; Ž2. serve as a glucose precursor in the Cori cycle; or Ž3. serve as a preferred oxidative substrate for liver, erythrocytes, heart, and red muscle Že.g., Suarez and Mommsen, 1987; Milligan and Girard, 1993; Eros and Milligan, 1996.. Because of our limited understanding, it appears that lactate metabolism during smoltification and its potential influence on this transformation are topics in need of further study. Our results from 1993 offer the intriguing possibility that the ‘stress’ associated with smoltification ŽSimpson, 1985; Franklin et al., 1992. may have triggered a proliferation of BKD. In 1993, mean ELISA absorbance values in treatment fish increased dramatically to values above 1.0, indicating fish had progressed from a low or moderate 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 reasoning have lead us to conclude that the association of smoltification and BKD progression is more than just coincidence. First, we are unable to attribute the increased infection levels at week 14 to other stressors such as density Žwhich was low. or water temperature Žwhich was stable.. Second, Maule et al. Ž1987. reported that the salmonid immune system is depressed during smoltification, which could allow an established but latent infection to proliferate. Third, several reports indicate that Rs and its major surface antigen, p57, are immunosuppressive ŽTuraga et al., 1987; Fredricksen et al., 1997; Siegel 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. That we did not observe similar changes in infection level in our other groups of fish that underwent smoltification could be related to: Ž1. the amount of antigen Žor numbers of bacteria. necessary for BKD proliferation in response to other stressors or smoltification; or Ž2. stock differences in the relative resistance to BKD, as indicated by several studies evaluating genetic variation in resistance of salmonids to various diseases ŽSuzumoto et al., 1977; McIntyre and Amend, 1978; Winter et al., 1980; Withler and Evelyn, 1990; Beacham and Evelyn, 1992.. Considerable evidence shows that various forms of environmental stress can be an important factor in outbreaks of infectious disease in fishes ŽSnieszko, 1974; Pickering, 1993.. We believe our results substantiate this notion by implicating smoltification as a possible influence on disease outbreaks.
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5. Conclusions Ž1. A progressively worsening infection with Renibacterium salmoninarum did not alter the normal changes in gill ATPase activity and condition factor associated with smoltification in juvenile chinook salmon. Ž2. Bacterial kidney disease did, however, alter trends in plasma cortisol, glucose, and lactate indicating that the disease is stressful, particularly in the later stages. Ž3. 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. Ž4. Our results should emphasize the importance of considering fish health and stock differences in the conduct and interpretation of physiological studies.
Acknowledgements We thank Ron Pascho, Diane Elliott, Connie McKibben, Marne 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.
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