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Effects of husbandry on prevalence of amoebic gill disease and performance of reared Atlantic salmon (Salmo salar L.)
Aquaculture 241 (2004) 21 – 30 www.elsevier.com/locate/aqua-online
Effects of husbandry on prevalence of amoebic gill disease and performance of reared Atlantic salmon (Salmo salar L.) G.M. Douglas-Heldersa,*, I.J. Weirb, D.P. O’Brienb, J. Carsonc, B.F. Nowaka a
School of Aquaculture, University of Tasmania, Locked Bag 1-370, Launceston 7250, Tasmania, Australia b Huon Aquaculture Pty Ltd., P.O. Box 1, Dover, Tasmania, Australia c Fish Health Unit of the Tasmanian Aquaculture and Fisheries Institute, Aquafin Cooperative Research Centre, University of Tasmania, Locked Bag 1-370, Launceston 7250, Tasmania, Australia Received 10 November 2003; accepted 26 July 2004
Abstract Improved husbandry has been identified as an area that may alleviate amoebic gill disease (AGD) on Tasmanian salmon farms. We report results of three trials that aimed to reduce AGD prevalence and/or minimise losses associated with AGD. In the first trial, cages were rotated between different sites and data compared to stationary cages that remained on a reference site; this arrangement was repeated over two consecutive years. The second trial studied the effect of prophylactic freshwater bathing, while the third trial considered the effects of sea cage size. All trials evaluated the effect of treatment on AGD prevalence, fish biomass gain, and the percentage of mortalities. No significant reduction of AGD prevalence was detected in terms of Neoparamoeba presence on the gills as measured by the immuno-dot blot assay. However, fish from the rotated cages showed a significant longer period between freshwater baths ( P=0.037), and the mean biomass in the rotated cages ( P=0.038 in year 1 and P=0.041 in year 2), and the non-prophylactic bathed cages ( P=0.048) was significantly higher at the end of the trials. The mortality rate was not affected by any of the treatments. The results of these trials
* Corresponding author. Tel.: +61 3 63243807; fax: +61 3 63243804. E-mail address: [email protected] (G.M. Douglas-Helders). 0044-8486/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2004.07.026
22
G.M. Douglas-Helders et al. / Aquaculture 241 (2004) 21–30
suggest that impact of AGD on salmon industry can be offset by adjustment of husbandry methods. D 2004 Elsevier B.V. All rights reserved. Keywords: Amoebic gill disease; AGD; Husbandry; Atlantic salmon
1. Introduction Disease is a major risk factor in commercial aquaculture, with millions of dollars lost annually (Shariff, 1998). Survival of pathogens depends on, among others, host susceptibility, and environmental factors influencing reproduction, growth and spread of the pathogen (Bakke and Harris, 1998). Husbandry is an important factor in reducing the chance of survival and spread of pathogens and hence reducing the incidence of diseases (Menzies et al., 1998). Salmon farms employ a range of management practices to control AGD, such as reducing stocking densities and frequent freshwater baths (Parsons et al., 2001). Amoebic gill disease (AGD), caused by the protozoan Neoparamoeba pemaquidensis, is the main disease affecting the salmon industry in Tasmania, Australia. AGD not only results in high treatment costs, but can also cause significant fish mortalities (Munday et al., 1990; Parsons et al., 2001). Freshwater bathing is the main treatment method used for AGD infections in Tasmania. In a bath treatment, fish are exposed to oxygenated freshwater in a liner for 2–4 h after which they are released back into sea water (Parsons et al., 2001). The effect of prophylactic bathing or the optimal timing of the first freshwater bath after transfer to seawater is unknown (Nowak, 2001). Major disadvantages associated with the treatment include: the need for additional labour and bottlenecks in farm operations; the need to handle the fish causing stress, and the requirement for large volumes of freshwater (Howard and Carson, 1991). These are all factors that add to the total cost for managing AGD (Parsons et al., 2001). Since the time when AGD outbreaks were first reported, an increased frequency of freshwater bathing has been required during the summer months, and the need for freshwater bathing has been extended through most of the year (Parsons et al., 2001). Alternative methods to minimise AGD and its related costs to salmon farms are critical for the industry in Tasmania. In this paper, three husbandry methods are described which aim to reduce AGD prevalence and/or minimise the losses associated with AGD infections in salmonids.
2. Materials and methods 2.1. Trial design Trials were performed to test the effects of three different husbandry options on AGD prevalence and general fish performance as measured by mean weight gain and mortality rates. The first trial (rotation trial) used four replicate sea cages for each treatment and was
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repeated in two consecutive fish growing seasons (December to March 2000/2001 and December to April 2001/2002, see Table 1). The trial studied the effects of the placement of stocked cages onto sites that were fallowed for a period of time, ranging from 4 to 97 days. Data for these cages were compared with cages that remained on one site (first year) or sites (second year), which had not been fallowed during the trial. Samples were taken monthly for 4 months (first year) and 5 months (second year). In order to determine if any treatment effect was due to the movement of cages to the fallowed sites, the direct effect of towing on AGD prevalence was tested. In the short towing trial, 20 fish from five towed cages were sampled directly before and after a short tow. The towing speed was on average 2.8 km/h for all towed cages, and the towing time never exceeded 5 h. To assess the effect of time between the two samples for each towed cage, five stationary control cages were sampled at the same time as the towed cages, with the same time interval between the two samples. The second trial, the prophylactic bath trial, studied the effects of freshwater bath treatment after introduction to seawater, but before any gross signs appeared of paramoeba infection on the gills (see Table 1). This trial used three replicate cages and monthly samples were taken from October 2000 till March 2001. Data obtained from these cages were compared against data from cages that did not receive this freshwater bath in this point of time and will be called the unbathed group throughout the manuscript. The third trial, the cage size trial, studied the effects of cage size when stocked with a similar biomass (see Table 1). Two cages with a diameter of 60 m and three of 80 m diameter were used for the trial and monthly samples were taken from August 2000 till November 2000. All trials were conducted at one salmon farm in southeast Tasmania, containing several farming sites. 2.2. Fish Out-of-season Atlantic salmon (Salmo salar L.) smolt with similar mean weight between treatment groups within each trial were introduced to a salmon farm in the Huon Estuary, southeast Tasmania. Times of introduction, number of cages per treatment, sampling period, biomass and stocking densities per cage at the start of the trials are shown
Table 1 Time of introductions, average biomass per pen at the start of the trials, and sampling durations for each treatment groups for the three trials Trial
Time of introduction
Sampling period
Treatment groups
No. of trial cages
Mean biomass per pen, kg (S.E.)
Site rotation 1 Site rotation 2 Bath
April/May 2000
Dec. 2000–March 2001
November 2001
Dec. 2001–April 2002
February 2000
Oct. 2000–March 2001
Cage size
April 2000
Aug., Oct., Nov. 2000
Stationary Rotation Stationary Rotation Yes No 60 m F cages 80 m F cages
4 4 4 4 3 3 2 3
15,026 (2195) 17,115 (3530) 20,304.3 (259.8) 21,000.9 (856.6) 11,663 (3255) 20,929 (2409) 2337 (123) 2806 (119)
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in Table 1. All trial fish were fed commercial salmon pellets (Skretting, Australia) of various sizes according to fish size, using the Aquasmartk demand feeding system. 2.3. Disease assessment Signs of clinical disease were assessed monthly using the routine Tasmanian salmon farmers gill assessment method by examining at least 20 fish for the presence of AGD related mucous patches (Munday et al., 1993). These signs range from a slight discolouration of a part of a gill filament to white mucoid patches on one or more filaments of the gill arches, depending on the severity of the infection. A score of severity of infection was estimated for each sea cage based on the number of fish examined that were infected and the degree of infection for each fish (A. Steenholdt, personal communication). This scoring system was consistently used during the trial, and determined the need of freshwater bath treatment for all cages. At an overall moderate to heavy infection level in a cage, freshwater bath treatments were administered and all cages within one treatment group bathed in succession. Fish were transferred into cages with clean nets after freshwater bathing at all times. The number and timing of freshwater baths were recorded within internal farm data management systems for each trial treatment group. The gross gill score is routinely used on Tasmanian salmon farms, but is a non-specific detection method. In order to determine AGD prevalence for each cage, monthly samples of gill mucus from 20 fish per cage were collected for detection of N. pemaquidensis by immuno-dot blot (Douglas-Helders et al., 2001). In short, fish were caught by crowd and dip netting and anaesthetised in 0.5% Aqui-SR. Gill mucus was either scraped off visible AGD lesions or the second gill arch on the left hand side of the fish, using a wooden toothpick, suspended in a 1.5 ml microfuge tube containing 400 Al, 0.22 Am filtered and autoclaved (121 8C, 15 min) natural seawater and kept on ice during sampling. The gill mucus samples were processed and analysed as previously described (Douglas-Helders et al., 2001). The mucus was digested, decolourised and cells lysed with 40 Al of 0.21% v/v sodium hypochlorite and 0.045% v/v sodium hydroxide and 10 Al of 2 N hydrochloride, frozen at 20 8C, thawed rapidly at 37 8C and re-frozen. Just prior to use, the samples were centrifuged and the supernatant used for dot blotting. AGD prevalence per cage was determined as percentage of fish that tested dot blot positive. The effect of each treatment on general fish performance was determined by comparing weight gain and mortality data from farm records. Weight gain data were obtained either by manual weight checks or using the Vicass system (SIGMA Technologies, Canada). For manual weight checks 40–60 fish were used. The mean biomass for the sea cage was estimated by dividing the total biomass by the number of fish sampled and multiplying the figure by the approximate total number of fish in the cage. 2.4. Statistical analysis Due to logistical problems, it was decided to remove one of the four stationary cages of the repeat rotation trial (year 2) from analysis. AGD prevalence, weight gain data and mortality data for each trial from the first and the last sampling points were analysed for
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treatment differences. Data were checked for homogeneity of variance and normality before performing a Student’s t-test to determine differences. Treatment effects were examined comparing data from the two treatments within each trial at the final sampling, and between the 2 years within each treatment for the rotation trial. If within-treatment effects were statistically not significant, data of the two consecutive years were pooled for analysis. Mortality data were expressed in percentages by dividing the cumulative number of mortalities at completion of the trials by the initial number of fish in the cages at commencement of the trials. Weight gain data were analysed as the cumulative biomass of each cage from which the biomass of the cage at the start of the trial was subtracted. Results of all statistical analysis were considered significant when PV0.05.
3. Results The mean values of AGD prevalence, number of days between baths, and numbers of freshwater baths that were required for each trial are shown in Table 2. Mean values of the final cage biomass (adjusted for initial cage biomass) and percentages of cumulative mortalities for each trial are shown in Table 3. 3.1. Rotation trial AGD prevalences in the rotated cages were below those of the stationary cages at all times in both years. However, no statistical difference in AGD prevalence between the two treatment groups was detected in either year ( P=0.276 in year 1, P=0.072 in year 2). Maximum AGD prevalence occurred in January for both treatment groups (Fig. 1A). The time between freshwater baths in the rotated cages was significantly longer than stationary cages when data of the 2 years were pooled ( P=0.037, Table 2). Also, the weight gained in the rotated cages was significantly greater at completion of the trial than in the stationary cages ( P=0.038 in year 1, P=0.041 in year 2, Table 3). Using pooled data, the cumulative mortality rate of the rotated cages was not affected by treatment ( P=0.436, Table 3). The cumulative mortality at the end of the trials was 2.06% (S.E. 0.68) for the rotated cages Table 2 Mean values (S.E.) of AGD prevalence at the start and finish of the three trials, the number of freshwater baths required for AGD treatment during the trials, and the number of days between freshwater baths for each treatment group Trial name
Treatment groups
Initial AGD prevalence (%)
Final AGD prevalence (%)
No. of freshwater baths
No. of days between freshwater baths in days
Site rotation 1
Stationary Rotation Stationary Rotation Yes No 60 m 80 m
51.3 43.5 31.3 35.0 17.5 23.3 40.0 21.7
66.3 45.0 60.1 28.4 36.7 33.3 47.5 25.0
4.5 3.8 2.5 2.0 5.3 4.3 2.0 2.0
28.6 55.9 29.2 35.0 53.9 66.9 46.8 43.8
Site rotation 2 Bath Cage size
(9.7) (4.9) (6.6) (6.6) (8.0) (12.0) (0.002) (3.3)
(3.8) (15.1) (14.3) (6.1) (6.0) (6.7) (17.5) (10.4)
(0.3) (0.6) (0.3) (0.0) (0.3) (0.3) (0.0) (0.0)
(2.6) (26.7) (2.6) (3.5) (11.8) (16.9) (16.2) (13.3)
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Table 3 Average weight gain (S.E.) and cumulative percentage of mortalities per cage Trial name
Treatment groups
Weight gain/cage, kg (S.E.)
Cumulative mortalities/cage (S.E.)
Site rotation 1
Stationary Rotation Stationary Rotation Yes No 60 m 80 m
13,569.3 19,568.3 18,988.8 22,476.1 18,388.3 24,996.0 19,731.5 24,355.0
4.16 2.99 1.36 1.13 0.94 0.60 0.77 1.75
Site rotation 2 Bath Cage size
(1113.5) (1972.7) (1041.3) (789.9) (1538.5) (623.0) (2536.5) (3039.3)
(0.90) (1.26) (0.32) (0.14) (0.16) (0.07) (0.01) (0.68)
and 2.88% (S.E. 0.76) for the stationary cages. Towing of the cages from the short towing trial did not directly affect the AGD prevalence ( P=0.111). The mean AGD prevalence at commencement and completion of the short towing trial was 61.7% (S.E. 14.2) and 71.2% (S.E. 16.0) for the towed cages, and 44.6% (S.E. 11.3) and 42.3% (S.E. 15.8) for the nontowed control cages.
Fig. 1. Average AGD prevalence (A) over time and weight gained (B) for the rotation trial cages.
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3.2. Bath trial AGD prevalence increased to a maximum in December for the unbathed cages group and continued to rise till January for the prophylacticly bathed cages (Fig. 2A). At completion of the trial, AGD prevalence levels of the bathed cages did not differ significantly from those of the unbathed cages ( P=0.897). In this trial, the unbathed cages required on average less frequent freshwater baths than the pre-clinical bathed cages, but this difference was statistically not significant ( P=0.101, Table 2). At completion of the trial, the mean weight gain in the unbathed cages was significantly higher than in the bathed cages ( P=0.048, Table 3), while there was no significant difference at commencement of the trial ( P=0.313). The difference between growth rates in the cages of the two treatments was greatest from early December to the end of February, with a
Fig. 2. Average AGD prevalence (A) and cumulative weight gain (B) of the bath trial cages over time.
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Fig. 3. Average AGD prevalence (S.E.) of the cage size trial over time.
higher growth rate in the unbathed cages (Fig. 2B). Prophylactic bathing did not affect the mortality rate, and the cumulative mortality percentage at the completion of the trial did not significantly differ between the two treatment groups ( P=0.215, Table 3). 3.3. Cage size trial At commencement of this trial, a significantly higher AGD prevalence was found in the 60-m cages ( P=0.032), which reflects the effect of cage size at the time of stocking to when first sampled. The lowest AGD prevalence level was found in October for both cage sizes. In November, the final sampling time, AGD prevalence levels between the two cage sizes were not significant ( P=0.316, Fig. 3). The mean number of days between freshwater baths ( P=0.849) and the freshwater bathing frequency ( P=1.000) were similar or equal for both cage sizes (Table 2). No significant differences in weight gain were detected with the different cage sizes ( P=0.366), nor in the cumulative percentage of mortalities at the completion of the trial ( P=0.286).
4. Discussion The results of the trials suggest that losses due to AGD could be reduced by movement of cages to fallowed sites and avoidance of prophylactic bathing. The cage movement trial showed that the rotated cages had a significantly longer period between freshwater baths, and that fish in these cages grew significantly heavier. The prophylactic bath trial showed no statistically significant difference in either AGD prevalence or length of time between freshwater baths. This showed that prophylactic bathing was not needed, and therefore when no prophylactic bath is given the total cost due to AGD could be reduced. Freshwater bathing is a very costly procedure (Parsons et al., 2001), and any reduction in the number of freshwater baths would reduce the overall cost of managing AGD. AGD prevalence at commencement of the cage size trial was significantly higher in the 60-m cages compared to the 80-m cages, which may be due to the fact that the 60-m
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cages were stocked at a higher density. However, no significant difference was found at the completion of this trial, implying that the effect of cage size and/or initial stocking density did not affect the fish after a period of 6 months (from stocking to finish of the trial). Several factors could have influenced the results of the reduced freshwater bathing frequency in the site rotation trial. Neoparamoeba abundance on fallowed sites could be lower due to the lack of infected fish, a suggested source for N. pemaquidensis (Munday et al., 2001). A dflush-effectT due to a higher impact from tides and currents when a site lacks barriers such as sea cages and their netting could have had an influence on the results in this study. Also, the sites on which the rotated cages were moved to had been only recently used for fish culture. Therefore the accumulation of N. pemaquidensis in the aquatic environment, such as sediments (Crosbie et al., in press), might have been lower. The tows of the rotation cages were on average of a similar frequency, but of a longer mean duration. This was not likely to influence the AGD prevalence since no effect of towing on AGD prevalence was found in the towing trial, suggesting that the results were mostly due to placement onto fallowed sites. Pre-disposing factors for AGD such as structural gill changes due to seawater acclimation, poor gill health and cage hygiene have been suggested (Nowak and Munday, 1994; Dykova´ et al., 1998; Munday et al., 1993). However, this was not seen in the prophylactic bath trial with lack of a statistical difference between the bathed and nonprophylactic bathed groups. This suggests that prophylactic bathing may not act as a predisposing factor for AGD occurrence. The mean weight gained in the rotated cages and the non-pre-clinical bathed cages were significantly higher than the non-rotated and pre-clinical bathed cages at the conclusion of these trials. The significant weight gain was observed in those cages with an on average lower prevalence of AGD. This is in agreement with the findings of a decreased feeding and/or growth rate due to AGD in previous studies (Rodger and McArdle, 1996; Dykova´ et al., 1998). Also, cages with a lower AGD prevalence would have been handled less due to the lower requirement for freshwater bathing, causing less stress to the fish.
5. Conclusion The results of the trials suggest that the cost of AGD management for reared Atlantic salmon can be ameliorated by adjustment of husbandry methods. Fewer freshwater baths were required and fish grew faster when cages were rotated to fallowed sites and costs reduced when prophylactic bathing did not occur.
Acknowledgments The authors thank Huon Aquaculture for their ready participation in sample collection and logistical support; Bruce McCorkell for his useful comments, and the Co-operative Research Centre for Aquaculture for the PhD scholarship to M. Douglas-Helders. Last rotation trial and preparation of this manuscript were supported by Aquafin CRC.
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References Bakke, T.A., Harris, P.D., 1998. Diseases and parasites in wild Atlantic salmon (Salmo salar) populations. Can. J. Fish. Aquat. Sci. 55, 247 – 266. Crosbie, P.B.B., Nowak, B.F., Carson, J., 2003. Isolation of Neoparamoeba pemaquidensis Page, 1987 from Marine Sediments in Tasmania. Bull. Eur. Assoc. Fish Pathol. 23, 241 – 244. Douglas-Helders, G.M., Carson, J., Howard, T., Nowak, B.F., 2001. Development and validation of a new dot blot test for the detection of Paramoeba pemaquidensis (Page) in fish. J. Fish Dis. 24, 273 – 280. Dykova´, I., Figueras, A., Novoa, B., Casal, J.F., 1998. Paramoeba sp., an agent of AGD of turbot, Scophthalmus maximus L. Dis. Aquat. Org. 33, 137 – 141. Howard, T., Carson, J., 1991. AGD—in vitro studies of Paramoeba species. Proceedings of the Saltas Research Review Seminar, Tasmania. Salmon Enterprises of Tasmania, Dover, Tasmania, pp. 1 – 24. Menzies, F.D., Crockford, T., McLoughlan, M.F., Wheatley, S.B., Goodall, E.A., 1998. An epidemiological approach to investigating disease and production losses in farmed Atlantic salmon. In: Barnes, A., Davidson, G., Hiney, M., McIntosh, D. (Eds.), Methodology in Fish Diseases Research. Fisheries Research Services, Aberdeen, Scotland, pp. 289 – 294. Munday, B.L., Foster, C.K., Roubal, F.R., Lester, R.J.G., 1990. Paramoebic gill infection and associated pathology of Atlantic salmon, Salmo salar, and rainbow trout, Salmo gairdeneri, in Tasmania. In: Perkins, F.O., Cheng, T.C. (Eds.), Pathology in Marine Science. Academic Press, San Diego, CA, pp. 215 – 222. Munday, B.L., Lange, K., Foster, C., Lester, R.J.G., Handlinger, J., 1993. Amoebic gill disease of sea-caged salmonids in Tasmanian waters. Tasman. Mar. Fish. Res. 28, 14 – 19. Munday, B.L., Zilberg, D., Findlay, V., 2001. Gill disease of marine fish caused by infection with Neoparamoeba pemaquidensis. J. Fish Dis. 24, 497 – 507. Nowak, B.F., Munday, B.L., 1994. Histology of gills of Atlantic salmon during the first few months following transfer to sea water. Bull. Eur. Assoc. Fish Pathol. 14, 77 – 81. Nowak, B.F., 2001. Qualitative evaluation of risk factors for Amoebic Gill Disease in Atlantic salmon. In: Rodgers, C.J. (Ed.), Proceedings of the OIE International Conference on Risk Analysis in Aquatic Animal Health, Paris, France, 8–10 February 2000. Office International des Epizooties (OIE), Paris, France, pp. 148 – 155. 18 pp. Parsons, H., Nowak, B., Fisk, D., Powell, M., 2001. Effectiveness of commercial freshwater bathing as a treatment against amoebic gill disease in Atlantic salmon. Aquaculture 195, 205 – 210. Rodger, H.D., McArdle, J.F., 1996. An outbreak of amoebic gill disease in Ireland. Vet. Rec. 139, 348 – 349. Shariff, M., 1998. Impact of diseases on aquaculture in the Asia–Pacific region as exemplified by epizootic ulcerative syndrome (EUS). J. Appl. Ichthyol.-Z. Angew. Ichthyol. 14, 139 – 144.
Effects of husbandry on prevalence of amoebic gill disease and performance of reared Atlantic salmon (Salmo salar L.) G.M. Douglas-Heldersa,*, I.J. Weirb, D.P. O’Brienb, J. Carsonc, B.F. Nowaka a
School of Aquaculture, University of Tasmania, Locked Bag 1-370, Launceston 7250, Tasmania, Australia b Huon Aquaculture Pty Ltd., P.O. Box 1, Dover, Tasmania, Australia c Fish Health Unit of the Tasmanian Aquaculture and Fisheries Institute, Aquafin Cooperative Research Centre, University of Tasmania, Locked Bag 1-370, Launceston 7250, Tasmania, Australia Received 10 November 2003; accepted 26 July 2004
Abstract Improved husbandry has been identified as an area that may alleviate amoebic gill disease (AGD) on Tasmanian salmon farms. We report results of three trials that aimed to reduce AGD prevalence and/or minimise losses associated with AGD. In the first trial, cages were rotated between different sites and data compared to stationary cages that remained on a reference site; this arrangement was repeated over two consecutive years. The second trial studied the effect of prophylactic freshwater bathing, while the third trial considered the effects of sea cage size. All trials evaluated the effect of treatment on AGD prevalence, fish biomass gain, and the percentage of mortalities. No significant reduction of AGD prevalence was detected in terms of Neoparamoeba presence on the gills as measured by the immuno-dot blot assay. However, fish from the rotated cages showed a significant longer period between freshwater baths ( P=0.037), and the mean biomass in the rotated cages ( P=0.038 in year 1 and P=0.041 in year 2), and the non-prophylactic bathed cages ( P=0.048) was significantly higher at the end of the trials. The mortality rate was not affected by any of the treatments. The results of these trials
* Corresponding author. Tel.: +61 3 63243807; fax: +61 3 63243804. E-mail address: [email protected] (G.M. Douglas-Helders). 0044-8486/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2004.07.026
22
G.M. Douglas-Helders et al. / Aquaculture 241 (2004) 21–30
suggest that impact of AGD on salmon industry can be offset by adjustment of husbandry methods. D 2004 Elsevier B.V. All rights reserved. Keywords: Amoebic gill disease; AGD; Husbandry; Atlantic salmon
1. Introduction Disease is a major risk factor in commercial aquaculture, with millions of dollars lost annually (Shariff, 1998). Survival of pathogens depends on, among others, host susceptibility, and environmental factors influencing reproduction, growth and spread of the pathogen (Bakke and Harris, 1998). Husbandry is an important factor in reducing the chance of survival and spread of pathogens and hence reducing the incidence of diseases (Menzies et al., 1998). Salmon farms employ a range of management practices to control AGD, such as reducing stocking densities and frequent freshwater baths (Parsons et al., 2001). Amoebic gill disease (AGD), caused by the protozoan Neoparamoeba pemaquidensis, is the main disease affecting the salmon industry in Tasmania, Australia. AGD not only results in high treatment costs, but can also cause significant fish mortalities (Munday et al., 1990; Parsons et al., 2001). Freshwater bathing is the main treatment method used for AGD infections in Tasmania. In a bath treatment, fish are exposed to oxygenated freshwater in a liner for 2–4 h after which they are released back into sea water (Parsons et al., 2001). The effect of prophylactic bathing or the optimal timing of the first freshwater bath after transfer to seawater is unknown (Nowak, 2001). Major disadvantages associated with the treatment include: the need for additional labour and bottlenecks in farm operations; the need to handle the fish causing stress, and the requirement for large volumes of freshwater (Howard and Carson, 1991). These are all factors that add to the total cost for managing AGD (Parsons et al., 2001). Since the time when AGD outbreaks were first reported, an increased frequency of freshwater bathing has been required during the summer months, and the need for freshwater bathing has been extended through most of the year (Parsons et al., 2001). Alternative methods to minimise AGD and its related costs to salmon farms are critical for the industry in Tasmania. In this paper, three husbandry methods are described which aim to reduce AGD prevalence and/or minimise the losses associated with AGD infections in salmonids.
2. Materials and methods 2.1. Trial design Trials were performed to test the effects of three different husbandry options on AGD prevalence and general fish performance as measured by mean weight gain and mortality rates. The first trial (rotation trial) used four replicate sea cages for each treatment and was
G.M. Douglas-Helders et al. / Aquaculture 241 (2004) 21–30
23
repeated in two consecutive fish growing seasons (December to March 2000/2001 and December to April 2001/2002, see Table 1). The trial studied the effects of the placement of stocked cages onto sites that were fallowed for a period of time, ranging from 4 to 97 days. Data for these cages were compared with cages that remained on one site (first year) or sites (second year), which had not been fallowed during the trial. Samples were taken monthly for 4 months (first year) and 5 months (second year). In order to determine if any treatment effect was due to the movement of cages to the fallowed sites, the direct effect of towing on AGD prevalence was tested. In the short towing trial, 20 fish from five towed cages were sampled directly before and after a short tow. The towing speed was on average 2.8 km/h for all towed cages, and the towing time never exceeded 5 h. To assess the effect of time between the two samples for each towed cage, five stationary control cages were sampled at the same time as the towed cages, with the same time interval between the two samples. The second trial, the prophylactic bath trial, studied the effects of freshwater bath treatment after introduction to seawater, but before any gross signs appeared of paramoeba infection on the gills (see Table 1). This trial used three replicate cages and monthly samples were taken from October 2000 till March 2001. Data obtained from these cages were compared against data from cages that did not receive this freshwater bath in this point of time and will be called the unbathed group throughout the manuscript. The third trial, the cage size trial, studied the effects of cage size when stocked with a similar biomass (see Table 1). Two cages with a diameter of 60 m and three of 80 m diameter were used for the trial and monthly samples were taken from August 2000 till November 2000. All trials were conducted at one salmon farm in southeast Tasmania, containing several farming sites. 2.2. Fish Out-of-season Atlantic salmon (Salmo salar L.) smolt with similar mean weight between treatment groups within each trial were introduced to a salmon farm in the Huon Estuary, southeast Tasmania. Times of introduction, number of cages per treatment, sampling period, biomass and stocking densities per cage at the start of the trials are shown
Table 1 Time of introductions, average biomass per pen at the start of the trials, and sampling durations for each treatment groups for the three trials Trial
Time of introduction
Sampling period
Treatment groups
No. of trial cages
Mean biomass per pen, kg (S.E.)
Site rotation 1 Site rotation 2 Bath
April/May 2000
Dec. 2000–March 2001
November 2001
Dec. 2001–April 2002
February 2000
Oct. 2000–March 2001
Cage size
April 2000
Aug., Oct., Nov. 2000
Stationary Rotation Stationary Rotation Yes No 60 m F cages 80 m F cages
4 4 4 4 3 3 2 3
15,026 (2195) 17,115 (3530) 20,304.3 (259.8) 21,000.9 (856.6) 11,663 (3255) 20,929 (2409) 2337 (123) 2806 (119)
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in Table 1. All trial fish were fed commercial salmon pellets (Skretting, Australia) of various sizes according to fish size, using the Aquasmartk demand feeding system. 2.3. Disease assessment Signs of clinical disease were assessed monthly using the routine Tasmanian salmon farmers gill assessment method by examining at least 20 fish for the presence of AGD related mucous patches (Munday et al., 1993). These signs range from a slight discolouration of a part of a gill filament to white mucoid patches on one or more filaments of the gill arches, depending on the severity of the infection. A score of severity of infection was estimated for each sea cage based on the number of fish examined that were infected and the degree of infection for each fish (A. Steenholdt, personal communication). This scoring system was consistently used during the trial, and determined the need of freshwater bath treatment for all cages. At an overall moderate to heavy infection level in a cage, freshwater bath treatments were administered and all cages within one treatment group bathed in succession. Fish were transferred into cages with clean nets after freshwater bathing at all times. The number and timing of freshwater baths were recorded within internal farm data management systems for each trial treatment group. The gross gill score is routinely used on Tasmanian salmon farms, but is a non-specific detection method. In order to determine AGD prevalence for each cage, monthly samples of gill mucus from 20 fish per cage were collected for detection of N. pemaquidensis by immuno-dot blot (Douglas-Helders et al., 2001). In short, fish were caught by crowd and dip netting and anaesthetised in 0.5% Aqui-SR. Gill mucus was either scraped off visible AGD lesions or the second gill arch on the left hand side of the fish, using a wooden toothpick, suspended in a 1.5 ml microfuge tube containing 400 Al, 0.22 Am filtered and autoclaved (121 8C, 15 min) natural seawater and kept on ice during sampling. The gill mucus samples were processed and analysed as previously described (Douglas-Helders et al., 2001). The mucus was digested, decolourised and cells lysed with 40 Al of 0.21% v/v sodium hypochlorite and 0.045% v/v sodium hydroxide and 10 Al of 2 N hydrochloride, frozen at 20 8C, thawed rapidly at 37 8C and re-frozen. Just prior to use, the samples were centrifuged and the supernatant used for dot blotting. AGD prevalence per cage was determined as percentage of fish that tested dot blot positive. The effect of each treatment on general fish performance was determined by comparing weight gain and mortality data from farm records. Weight gain data were obtained either by manual weight checks or using the Vicass system (SIGMA Technologies, Canada). For manual weight checks 40–60 fish were used. The mean biomass for the sea cage was estimated by dividing the total biomass by the number of fish sampled and multiplying the figure by the approximate total number of fish in the cage. 2.4. Statistical analysis Due to logistical problems, it was decided to remove one of the four stationary cages of the repeat rotation trial (year 2) from analysis. AGD prevalence, weight gain data and mortality data for each trial from the first and the last sampling points were analysed for
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treatment differences. Data were checked for homogeneity of variance and normality before performing a Student’s t-test to determine differences. Treatment effects were examined comparing data from the two treatments within each trial at the final sampling, and between the 2 years within each treatment for the rotation trial. If within-treatment effects were statistically not significant, data of the two consecutive years were pooled for analysis. Mortality data were expressed in percentages by dividing the cumulative number of mortalities at completion of the trials by the initial number of fish in the cages at commencement of the trials. Weight gain data were analysed as the cumulative biomass of each cage from which the biomass of the cage at the start of the trial was subtracted. Results of all statistical analysis were considered significant when PV0.05.
3. Results The mean values of AGD prevalence, number of days between baths, and numbers of freshwater baths that were required for each trial are shown in Table 2. Mean values of the final cage biomass (adjusted for initial cage biomass) and percentages of cumulative mortalities for each trial are shown in Table 3. 3.1. Rotation trial AGD prevalences in the rotated cages were below those of the stationary cages at all times in both years. However, no statistical difference in AGD prevalence between the two treatment groups was detected in either year ( P=0.276 in year 1, P=0.072 in year 2). Maximum AGD prevalence occurred in January for both treatment groups (Fig. 1A). The time between freshwater baths in the rotated cages was significantly longer than stationary cages when data of the 2 years were pooled ( P=0.037, Table 2). Also, the weight gained in the rotated cages was significantly greater at completion of the trial than in the stationary cages ( P=0.038 in year 1, P=0.041 in year 2, Table 3). Using pooled data, the cumulative mortality rate of the rotated cages was not affected by treatment ( P=0.436, Table 3). The cumulative mortality at the end of the trials was 2.06% (S.E. 0.68) for the rotated cages Table 2 Mean values (S.E.) of AGD prevalence at the start and finish of the three trials, the number of freshwater baths required for AGD treatment during the trials, and the number of days between freshwater baths for each treatment group Trial name
Treatment groups
Initial AGD prevalence (%)
Final AGD prevalence (%)
No. of freshwater baths
No. of days between freshwater baths in days
Site rotation 1
Stationary Rotation Stationary Rotation Yes No 60 m 80 m
51.3 43.5 31.3 35.0 17.5 23.3 40.0 21.7
66.3 45.0 60.1 28.4 36.7 33.3 47.5 25.0
4.5 3.8 2.5 2.0 5.3 4.3 2.0 2.0
28.6 55.9 29.2 35.0 53.9 66.9 46.8 43.8
Site rotation 2 Bath Cage size
(9.7) (4.9) (6.6) (6.6) (8.0) (12.0) (0.002) (3.3)
(3.8) (15.1) (14.3) (6.1) (6.0) (6.7) (17.5) (10.4)
(0.3) (0.6) (0.3) (0.0) (0.3) (0.3) (0.0) (0.0)
(2.6) (26.7) (2.6) (3.5) (11.8) (16.9) (16.2) (13.3)
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Table 3 Average weight gain (S.E.) and cumulative percentage of mortalities per cage Trial name
Treatment groups
Weight gain/cage, kg (S.E.)
Cumulative mortalities/cage (S.E.)
Site rotation 1
Stationary Rotation Stationary Rotation Yes No 60 m 80 m
13,569.3 19,568.3 18,988.8 22,476.1 18,388.3 24,996.0 19,731.5 24,355.0
4.16 2.99 1.36 1.13 0.94 0.60 0.77 1.75
Site rotation 2 Bath Cage size
(1113.5) (1972.7) (1041.3) (789.9) (1538.5) (623.0) (2536.5) (3039.3)
(0.90) (1.26) (0.32) (0.14) (0.16) (0.07) (0.01) (0.68)
and 2.88% (S.E. 0.76) for the stationary cages. Towing of the cages from the short towing trial did not directly affect the AGD prevalence ( P=0.111). The mean AGD prevalence at commencement and completion of the short towing trial was 61.7% (S.E. 14.2) and 71.2% (S.E. 16.0) for the towed cages, and 44.6% (S.E. 11.3) and 42.3% (S.E. 15.8) for the nontowed control cages.
Fig. 1. Average AGD prevalence (A) over time and weight gained (B) for the rotation trial cages.
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3.2. Bath trial AGD prevalence increased to a maximum in December for the unbathed cages group and continued to rise till January for the prophylacticly bathed cages (Fig. 2A). At completion of the trial, AGD prevalence levels of the bathed cages did not differ significantly from those of the unbathed cages ( P=0.897). In this trial, the unbathed cages required on average less frequent freshwater baths than the pre-clinical bathed cages, but this difference was statistically not significant ( P=0.101, Table 2). At completion of the trial, the mean weight gain in the unbathed cages was significantly higher than in the bathed cages ( P=0.048, Table 3), while there was no significant difference at commencement of the trial ( P=0.313). The difference between growth rates in the cages of the two treatments was greatest from early December to the end of February, with a
Fig. 2. Average AGD prevalence (A) and cumulative weight gain (B) of the bath trial cages over time.
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Fig. 3. Average AGD prevalence (S.E.) of the cage size trial over time.
higher growth rate in the unbathed cages (Fig. 2B). Prophylactic bathing did not affect the mortality rate, and the cumulative mortality percentage at the completion of the trial did not significantly differ between the two treatment groups ( P=0.215, Table 3). 3.3. Cage size trial At commencement of this trial, a significantly higher AGD prevalence was found in the 60-m cages ( P=0.032), which reflects the effect of cage size at the time of stocking to when first sampled. The lowest AGD prevalence level was found in October for both cage sizes. In November, the final sampling time, AGD prevalence levels between the two cage sizes were not significant ( P=0.316, Fig. 3). The mean number of days between freshwater baths ( P=0.849) and the freshwater bathing frequency ( P=1.000) were similar or equal for both cage sizes (Table 2). No significant differences in weight gain were detected with the different cage sizes ( P=0.366), nor in the cumulative percentage of mortalities at the completion of the trial ( P=0.286).
4. Discussion The results of the trials suggest that losses due to AGD could be reduced by movement of cages to fallowed sites and avoidance of prophylactic bathing. The cage movement trial showed that the rotated cages had a significantly longer period between freshwater baths, and that fish in these cages grew significantly heavier. The prophylactic bath trial showed no statistically significant difference in either AGD prevalence or length of time between freshwater baths. This showed that prophylactic bathing was not needed, and therefore when no prophylactic bath is given the total cost due to AGD could be reduced. Freshwater bathing is a very costly procedure (Parsons et al., 2001), and any reduction in the number of freshwater baths would reduce the overall cost of managing AGD. AGD prevalence at commencement of the cage size trial was significantly higher in the 60-m cages compared to the 80-m cages, which may be due to the fact that the 60-m
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cages were stocked at a higher density. However, no significant difference was found at the completion of this trial, implying that the effect of cage size and/or initial stocking density did not affect the fish after a period of 6 months (from stocking to finish of the trial). Several factors could have influenced the results of the reduced freshwater bathing frequency in the site rotation trial. Neoparamoeba abundance on fallowed sites could be lower due to the lack of infected fish, a suggested source for N. pemaquidensis (Munday et al., 2001). A dflush-effectT due to a higher impact from tides and currents when a site lacks barriers such as sea cages and their netting could have had an influence on the results in this study. Also, the sites on which the rotated cages were moved to had been only recently used for fish culture. Therefore the accumulation of N. pemaquidensis in the aquatic environment, such as sediments (Crosbie et al., in press), might have been lower. The tows of the rotation cages were on average of a similar frequency, but of a longer mean duration. This was not likely to influence the AGD prevalence since no effect of towing on AGD prevalence was found in the towing trial, suggesting that the results were mostly due to placement onto fallowed sites. Pre-disposing factors for AGD such as structural gill changes due to seawater acclimation, poor gill health and cage hygiene have been suggested (Nowak and Munday, 1994; Dykova´ et al., 1998; Munday et al., 1993). However, this was not seen in the prophylactic bath trial with lack of a statistical difference between the bathed and nonprophylactic bathed groups. This suggests that prophylactic bathing may not act as a predisposing factor for AGD occurrence. The mean weight gained in the rotated cages and the non-pre-clinical bathed cages were significantly higher than the non-rotated and pre-clinical bathed cages at the conclusion of these trials. The significant weight gain was observed in those cages with an on average lower prevalence of AGD. This is in agreement with the findings of a decreased feeding and/or growth rate due to AGD in previous studies (Rodger and McArdle, 1996; Dykova´ et al., 1998). Also, cages with a lower AGD prevalence would have been handled less due to the lower requirement for freshwater bathing, causing less stress to the fish.
5. Conclusion The results of the trials suggest that the cost of AGD management for reared Atlantic salmon can be ameliorated by adjustment of husbandry methods. Fewer freshwater baths were required and fish grew faster when cages were rotated to fallowed sites and costs reduced when prophylactic bathing did not occur.
Acknowledgments The authors thank Huon Aquaculture for their ready participation in sample collection and logistical support; Bruce McCorkell for his useful comments, and the Co-operative Research Centre for Aquaculture for the PhD scholarship to M. Douglas-Helders. Last rotation trial and preparation of this manuscript were supported by Aquafin CRC.
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