- Email: [email protected]
Effects of commercial freshwater bathing on reinfection of Atlantic salmon, Salmo salar, with Amoebic Gill Disease
Aquaculture 219 (2003) 135 – 142 www.elsevier.com/locate/aqua-online
Effects of commercial freshwater bathing on reinfection of Atlantic salmon, Salmo salar, with Amoebic Gill Disease Gemma Clark, Mark Powell, Barbara Nowak * School of Aquaculture, Tasmanian Aquaculture and Fisheries Institute, University of Tasmania, Locked Bag 1370, Launceston, Tasmania 7250, Australia Received 24 June 2002; received in revised form 16 December 2002; accepted 30 December 2002
Abstract Fish with Amoebic Gill Disease (AGD) were examined over a 10-day period following commercial freshwater bathing to assess the time to reinfection. Samples were taken from fish before freshwater bathing and then 1, 3, 5 and 10 days after bathing to determine the number of amoebae present on the gills. Freshwater bathing significantly reduced the number of amoebae on the gills, with an 86 F 9.1% reduction in the number of live amoebae found on the gills after freshwater bathing. However, amoeba numbers returned to pre-bath levels 10 days after bathing. There was no significant effect on number of AGD lesions/filament, the mean ranged from 0.08, 3 days after bathing, to 0.14, 5 days after bathing. However, the number of Neoparamoeba pemaquidensis dramatically dropped in histological sections from 0.53 per AGD lesion before the bath to 0 per AGD lesion 1 day after the bath and then remained significantly lower, reaching 0.08 per AGD lesion 10 days after the bath. The number of mucous cells changed, with Alcian blue (AB) (pH 1) positive cells decreasing immediately after bathing. Results of this study show that commercial freshwater bathing is effective at removing amoebae from the gills of fish, however, reinfection can occur within a week. D 2003 Elsevier Science B.V. All rights reserved. Keywords: Amoebic Gill Disease; Atlantic salmon; Freshwater bathing
* Corresponding author. Tel.: +61-3-6324-3814; fax: +61-3-6324-3804. E-mail address: [email protected] (B. Nowak). 0044-8486/03/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0044-8486(03)00020-6
136
G. Clark et al. / Aquaculture 219 (2003) 135–142
1. Introduction Amoebic Gill Disease (AGD) is a significant health problem in sea-pen salmonid culture in Tasmania (Munday et al., 1990; Nowak, 2001). The condition has been also reported in farmed salmonids from other countries, including Ireland (Rodger and McArdle, 1996) and the USA (Kent et al., 1988; Douglas-Helders et al., 2001 ) as well as in cultured marine fish species in the Mediterranean (Dykova et al., 1987). The causative agent of AGD is Neoparamoeba pemaquidensis, Page 1987 (Munday et al., 1990; Howard and Carson, 1993; Zilberg et al., 2001). N. pemaquidensis is considered a facultative amphizoic parasite and has not only been associated with the gills of salmonids, but it has also been found on sea-cage netting and in oceanic surface microlayers (Davis et al., 1978; Martin, 1985; Howard and Carson, 1993; Tan et al., 2002). The re-occurrence of AGD as indicated by bathing frequency has increased over the years (Nowak, 2001). The treatment of choice for the Tasmanian salmon industry to reduce impact of AGD is to bathe salmon in hyperoxic fresh water for 2 – 4 h (Parsons et al., 2001). This treatment reduces amoebae numbers on the gills of fish immediately after the bath as determined by histology (Parsons et al., 2001). However, it has been suggested that some amoebae could survive the treatment (Parsons et al., 2001). The aim of this study was to assess the efficacy of freshwater bathing and the time to reinfection following commercial bathing of Atlantic salmon affected by AGD.
2. Materials and methods 2.1. Commercial freshwater bathing Three successive commercial freshwater baths were investigated. Oxygen in each bath was maintained above 120% saturation and water temperatures ranged from 14.9 to 16.4 jC. The fish had been previously bathed once before, 3 weeks before this study. The need for the bath was on the basis of gross gill score, which reflects the number and size of white patches on fish gills. Each bath was 2 h in duration (from the transfer of the last fish), with each pen remaining at the bathing site to be towed back to respective grow-out sites on the second day post-bath. Bath biomass during bathing was 16 590 kg for cage one, 23 023 kg for cage two and 24 296 kg for cage three. All fish were of the same year class. As the experiment was run in a commercial setting and the samples were taken from commercial cages, no untreated controls could be included, due to animal welfare concerns and production issues. Thus, the results at different time points were compared to each other, with the main emphasis on the difference from the pre-bath means. The efficacy of commercial freshwater bathing was evaluated by monitoring changes in amoeba numbers on the gills and histological lesions over time after bath. Changes in the number of amoebae on the gills were monitored over a 10-day period with samples taken before the bath and then 1, 3, 5 and 10 days after bathing. Fish were collected using a 4-m box net in conjunction with a dip net. Ten fish were killed by anaesthetic overdose (0.04%) of clove oil and their second left gill arch was sampled for histology and fixed in seawater Davidson’s fixative. From five of the fish sampled for histology, all right gill arches were
G. Clark et al. / Aquaculture 219 (2003) 135–142
137
used for isolation of amoeba followed by amoeba count. The presence of N. pemaquidensis in gill isolates was confirmed by IFAT. 2.2. Histology Two stains were used to stain gill sections; haematoxylin and eosin (HE), and Periodic Acid Schiff’s (PAS) in conjunction with 1% Alcian blue (AB) at pH 1.0 and 2.5 (Bancroft and Cook, 1994). The number of lesions per filament, amoeba per lesion, and interlamellar vesicles (ILV) per lesion were counted at 400 magnification. Filaments from the ventral, medial and dorsal area of the second gill arch were chosen for counts of mucous cells. The number of cells stained PAS, Alcian blue at pH 1 (AB1), Alcian blue at pH 2.5 (AB2.5) or PAS + AB1 positive (PAS + AB1) was counted on 10 interlamellar units (Powell et al., 1995; Speare et al., 1991) and expressed per interlamellar unit. Only filaments sectioned correctly as indicated by equal length of lamellae on both sides of the filament were included in quantitative observations. 2.3. Amoeba isolation Amoebae were isolated from the gills using a technique modified from Howard and Carson (1994). All gill arches on the right side of the fish were removed from fish and placed in 50 ml plastic tubes. Sterile 2.5% ammonium –chloride and antibiotic (carbenicillin 0.1 g ml 1, penicillin 0.1 ml 1, streptomycin sulphate 0.1 ml 1, erythromycin 0.01 ml 1 and ampicillin 0.025 ml 1) solution was added to a total volume of 50 ml and kept at 4 jC for between 12 and 24 h. Gills were removed and mucus was collected into 50 ml plastic tubes. Mucus was re-suspended in sterile seawater and mixed. A 100 Al aliquot of solution was sampled and stained with 0.5% trypan blue. Live amoebae were counted using a haemocytometer (Neubauer, BS 748). A subsample of the solution was collected with a sterile loop, placed on a glass slide and air dried for IFAT (Howard and Carson, 1994). 2.4. Statistical analysis Analysis of variance (ANOVA) was used for data analysis. Homogeneity was tested using a Levene’s test. Results were considered significant if P V 0.05. Results for individual cages were pooled if P>0.25. Tukey’s test was used for comparison of means. Statistical analysis was conducted using SPSSR (version 8.0) software. Results are given as mean and standard error of the mean.
3. Results The number of live amoebae was significantly lower 1 day after freshwater bathing (Fig. 1). The number of live amoeba numbers began to increase 5 days after bathing and the counts of amoebae 10 days after bathing were not significantly different from pre-bath counts (Fig. 1). All subsamples collected from the isolates were IFAT positive. The
138
G. Clark et al. / Aquaculture 219 (2003) 135–142
Fig. 1. Average number of live amoebae present in isolation solution taken from the gills of fish ( F S.E.). Gill samples were taken before bathing, 1, 3, 5, and 10 days after bathing. Results pooled for three cages, five fish were sampled from each cage (n = 15). Common letters indicate treatments not significantly different ( P>0.05).
number of observed histological lesions did not change over the duration of the experiment (Table 1, P = 0.09). Multifocal hyperplastic lesions were common involving more than 10 lamellae along the full length of the lamellae. Lesions dominated in the proximal and medial region of the filaments. The numbers of N. pemaquidensis found on histological lesions were significantly reduced after the bath (Table 1). N. pemaquidensis numbers observed in association with lesions remained low throughout the experiment. A significant decrease in the number of ILV associated with lesions occurred on days 5 and 10 after bathing ( P < 0.05). There were no changes in mucous cell morphology or differences in the location of mucous cells (dorsal, medial, or ventral) observed. Similarly there was no effect of gill position on the number of PAS, PAS + AB, AB1 and AB2.5 positive mucous cells found per interlamellae unit ( P>0.05). Therefore, location on the gill was not considered to Table 1 Presence of gill lesions and amoebae in histology before and after freshwater bath Variable measured Number of AGD lesions/filament Number of Neoparamoeba/lesion ILV per lesions
Before
1 Day a
0.10(0.02) 0.53(0.27)a 0.69(0.24)a
3 Days a
0.11(0.02) 0(0)b 0.51(0.15)ab
5 Days a
0.08(0.02) 0.06(0.05)b 0.32(0.12)ab
10 Days a
0.14(0.05) 0(0)b 0.04(0.04)b
0.09(0.03)a 0.08(0.05)b 0.03(0.02)b
Common letters indicate no significant difference ( P >0.05). Results shown as mean and standard error. Results pooled for three bathings, n = 30 for each time.
G. Clark et al. / Aquaculture 219 (2003) 135–142
139
Table 2 The number of PAS1, PAS + AB1, AB1 and AB2.5 positive mucous cells per interlamellar unit found on the gills Stain PAS PAS + AB1 AB1 AB2.5 Total pH 1.0 Total pH 2.5
Before
1 Day a
0.18(0.05) 1.55(0.22)a 0.52(0.13)a 0.17(0.08)a 0.75(0.08)a 0.84(0.10)a
3 Days a
0.31(0.08) 1.82(0.22)ab 0.01(0)b 0.02(0.03)a 0.71(0.08)a 0.75(0.09)a
5 Days a
0.29(0.09) 2.40(0.24)bc 0.03(0)b 0.29(0.16)a 0.90(0.10)a 0.88(0.10)a
10 Days a
0.36(0.08) 2.13(0.03)ab 0.06(0.04)b 0.30(0.22)a 0.85(0.10)a 0.92(0.12)a
0.15(0.08)a 2.94(0.39)c 0.13(0.09)b 0.03(0.02)a 1.07(0.14)a 0.93(0.13)a
Results shown as mean and standard error. Common letters indicate no statistically significant effect ( P >0.05). PAS—Periodic Acid Schiff, AB—Alcian blue. Ten interlamellar units were counted for each fish, 30 fish were examined for each time (results pooled from three bathings).
influence counts and data were pooled by treatment. All mucous cell types were observed on the gills and were mainly located at the distal end of the lamellae. There was no significant difference between the number of PAS positive mucous cells per lamellar unit for the duration of the trial (Table 2). However, there was a significant decrease in the number of AB1 positive (sulphated mucins) mucous cells per lamellar unit after freshwater bathing on day 1. AB1 positive mucous cells remained low throughout the duration of the trial (Table 2). The number of AB2.5 positive mucous cells per interlamellar unit did not significantly change over the duration of the trial. Although the average number of AB2.5 positive cells was reduced on days 1 and 10 after bathing, the difference was not statistically significant, due to high variability between fish. The majority of mucous cells were PAS + AB1 positive in all regions of the gill. No significant change in the number of PAS + AB1 positive mucous cells in the gills occurred until 3 days after bathing when the number of these cells was slightly increased. There was no significant change in the total number of mucous cells found on the gills over the trial period at both pH 1.0 and 2.5.
4. Discussion Freshwater bathing was successful at reducing amoeba number on the gills of Atlantic salmon. This was confirmed by direct counts of amoeba isolated from the gills and counts from histological sections. An 86 F 9.1% reduction of number of amoebae present in the gill isolates after bathing was lower than the decrease reported previously (Parsons et al., 2001). However, the previous report was based on preliminary data using a different technique with a very low replication of samples (Parsons et al., 2001). However, the fact that freshwater bathing did not remove all N. pemaquidensis from the gills left a potential reservoir of the parasite on the gills for rapid proliferation. Numbers of amoebae increased 5 days after bathing and amoebae returned to pre-bath numbers by 10 days after bathing. The increase in the number of amoebae on the gills may be the result of rapid proliferation after bathing and/or reinfection from the surrounding environment. Proliferation on the gills was suggested by laboratory reinfection of recovered Atlantic salmon with AGD achieved without introducing the pathogen (Findlay and Munday, 1998), however, this result could be due to accidental introduction of N. pemaquidensis with water as the
140
G. Clark et al. / Aquaculture 219 (2003) 135–142
experimental sea water was not filtered. Environmental conditions have been shown to increase AGD prevalence with increasing temperature and salinity (Clark and Nowak, 1999). The conditions under which experimentation was conducted were within the optimal conditions established for N. pemaquidensis growth and clinical AGD in fish (Clark and Nowak, 1999; Nowak, 2001). This indicates that the amoeba load on the gills can increase rapidly when optimal environmental conditions are present. The number of amoebae per lesion decreased after bathing. This was in agreement with the previous research (Howard and Carson, 1992; Parsons et al., 2001). However, the number of amoebae isolated from the gills quickly returned to the pre-bath level. Furthermore, the counts from histological sections did not correlate with the amoebae counted in gill isolates and remained very low for the whole time after bathing. It is possible that after freshwater bathing, amoebae are not attached to the gills and could be lost during fixation. All isolates from which the amoebae were counted tested IFAT positive and while histology is very specific, its sensitivity is not high as only a small section of tissue is examined. However, trypan blue staining followed by haemocytometer count is not specific as a diagnostic method. Other amoebae or cells may be counted in addition to N. pemaquidensis. Unfortunately, this is the only quantitative method currently available. Further research is required to explore the discrepancy between histology results and trypan blue counts. There was no increase in the number of ILV following bathing and the ILV within AGD lesions did not contain amoeba after bathing. This was contrary to the previous findings of Parsons et al. (2001) of amoebae within ILV following bathing, suggesting that ILV formation and encystment of amoeba during bathing offered protection to the amoeba (Parsons et al., 2001). However, ILV formation is now believed to be a host response enclosing the parasite, resulting eventually in the degradation and removal of amoeba by host cellular processes (Adams and Nowak, 2001). Staining of mucous cells at different pH allows the identification of different glycoproteins (Ferguson et al., 1992). Two types of AB positive mucus are present on gills and are identified as sulphated at pH 1.0, carboxylated at pH 4.0 and combined at pH 2.5 (Ferguson et al., 1992). The observed decrease in AB1 positive mucous cells was not mirrored by an observed decrease in total AB cells at pH 2.5. In contrast, Powell et al. (2001) reported a decrease in AB 2.5 positive mucous cells in gills of Atlantic salmon following freshwater bathing. However, in that study, the sections were stained at only one pH and therefore, it is undetermined whether the decrease in AB positive mucous cells was a result of a decrease in sulphated or carboxylated mucins. The presence of high numbers of sulphated mucins (AB1 positive) was unusual. Sulphated mucins in high quantities are more often associated with freshwater adapted fish (Solanki and Benjamin, 1982). The hypersecretion of mucus is often associated with gill irritation (Powell et al., 1995, 1998) and may help to clear toxins or pathogens (Ferguson et al., 1992). Mucus may provide a barrier of protection at the interface between the amoeba and the gill epithelium. This would explain the increase in mucus observed on the gills during infection and the high numbers of amoeba isolated from the gills without the corresponding gill changes in histology. This investigation has shown that freshwater bathing is successful in reducing amoeba numbers on the gills of Atlantic salmon, however, reinfection occurred within 10 days.
G. Clark et al. / Aquaculture 219 (2003) 135–142
141
This shows that amoeba load on the gills can increase rapidly, however, the mechanism of the infection is unknown. The environmental conditions during the sampling were optimal for AGD outbreaks as defined by Nowak (2001) and Douglas-Helders et al. (2001), and may explain the relatively fast increase in amoeba numbers on the gills after freshwater bathing. It is likely that the time to reinfection would be longer under different environmental conditions. While this study focused on changes after second freshwater bath (the first one took place 3 weeks before this experiment), the pattern of reinfection is always similar in summer, regardless of the number of bath (M. Powell and B. Nowak, unpublished). The effect of multiple bathings on the properties of gill mucus is currently under investigation.
Acknowledgements The authors wish to acknowledge Huon Aquaculture Company for all their support during this trial. We thank Dr Rick Butler for the helpful comments.
References Adams, M., Nowak, B., 2001. Lesion distribution and structure in the gills of Atlantic salmon, Salmo salar affected with Amoebic Gill Disease. J. Fish Dis. 24, 535 – 542. Bancroft, J.D., Cook, H.C., 1994. Manual of Histological Techniques and Their Diagnostic Application. Churchill Livingstone, Singapore, p. 457. Clark, A., Nowak, B.F., 1999. Field investigations of Amoebic Gill Disease in Atlantic salmon, Salmo salar L., in Tasmania. J. Fish Dis. 22, 433 – 443. Davis, P.G., Caron, D.A., Sieburth, J.M., 1978. Oceanic amoebae from the North Atlantic: culture, distribution, and taxonomy. Trans. Am. Microsc. Soc. 97, 73 – 88. Douglas-Helders, M., Saksida, S., Raverty, S., Nowak, B.F., 2001. Temperature as a risk factor for outbreaks of Amoebic Gill Disease in farmed Atlantic salmon (Salmo salar). Bull. Eur. Assoc. Fish Pathol. 21, 114 – 116. Dykova, I., Figueras, A., Peric, Z., 2000. Neoparamoeba Page, 1987: light and electron microscopic observations on six strains of different origin. Dis. Aquat. Org. 43, 217 – 223. Ferguson, H.W., Morrison, D., Ostland, V.E., Lumsden, J., Bryne, P., 1992. Responses of mucus-producing cells in gill disease of rainbow trout (Oncorhynchus mykiss). J. Comp. Pathol. 106, 255 – 265. Findlay, V.L., Munday, B.L., 1998. Further studies on acquired resistance to Amoebic Gill Disease (AGD) in Atlantic salmon, Salmo salar L. J. Fish Dis. 21, 121 – 125. Howard, T., Carson, J., 1992. Studies of amoeba associated with Amoebic Gill Disease in Atlantic salmon. Barriers and Breakthroughs. Proceedings of the SALTAS Research Review Seminar 29th April 1992. SALTAS, Hobart, Australia, pp. 101 – 122. Howard, T., Carson, J., 1993. Verification that Paramoeba species are consistently associated with gill damage in fish affected with Amoebic Gill Disease. Proceedings of the Saltas Research and Development Review Seminar, Tasmania 1993. Salmon Enterprises of Tasmania, Dover, Tasmania, pp. 69 – 80. Howard, T., Carson, J., 1994. Amoebic Gill Disease laboratory research 1993/1994. Reports from the SALTAS 1993 – 1994. Research and Development Program. Salmon Enterprises of Tasmania, Dover, Tasmania, pp. 71 – 87. Kent, M.L., Sawyer, T.K., Hedrick, R.P., 1988. Paramoeba pemaquidensis (Sarcomastigophora: Paramoebidae) infestation of the gills of coho salmon Oncorhynchus mykiss reared in sea water. Dis. Aquat. Org. 5, 163 – 169. Martin, R.E., 1985. Population growth in stationary and suspension culture of Paramoeba pemaquidensis Page (Amoebida, Paramoebidae). J. Protozool. 32, 738 – 739.
142
G. Clark et al. / Aquaculture 219 (2003) 135–142
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 gairdneri, in Tasmania. In: Perkins, F.O., Cheng, T.C. (Eds.), Pathology in Marine Science. Academic Press, San Diego, CA, pp. 215 – 222. Nowak, B., 2001. Qualitative evaluation of risk factors for Amoebic Gill Disease in cultured 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 (O.I.E.), Paris, France, pp. 148 – 155. 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. Powell, M.D., Wright, G.M., Speare, D.J., 1995. Morphological changes in rainbow trout (Oncorhynchus mykiss) gill epithelia following repeated intermittent exposure to chloramine-T. Can. J. Zool. 73, 154 – 165. Powell, M.D., Haman, F., Wright, G.M., Perry, S.F., 1998. Response of rainbow trout (Oncorhynchus mykiss) to a graded hypoxia following repeated intermittent exposure to chloramine-T. Aquaculture 165, 27 – 39. Powell, M.D., Parsons, H.J., Nowak, B.F., 2001. Physiological effects of freshwater bathing of Atlantic salmon (Salmo salar) as a treatment for Amoebic Gill Disease. Aquaculture 199, 259 – 266. Rodger, H.D., McArdle, J.F., 1996. An outbreak of Amoebic Gill Disease in Ireland. Vet. Rec. 5, 348 – 349. Solanki, T.G., Benjamin, M., 1982. Changes in the mucous cells of the gills, buccal cavity and epidermis of the nine-spined stickleback, Pungitius pungitius L., induced by transferring the fish to seawater. J. Fish Biol. 21, 563 – 575. Speare, D.J., Ferguson, H.W., Beamish, F.W.M., Yager, J.A., Yamashiro, S., 1991. Pathology of bacterial gill disease: ultrastructure of branchial lesions. J. Fish Dis. 14, 1 – 20. Tan, C.K.F., Nowak, B.F., Hodson, S.L., 2002. Biofouling as a reservoir of Neoparamoeba pemaquidensis (Page 1970), the causative agent of Amoebic Gill Disease in Atlantic salmon. Aquaculture 210, 49 – 58. Zilberg, D., Gross, A., Munday, B.L., 2001. Production of salmonid Amoebic Gill Disease by exposure to Paramoeba sp. harvested from the gills of infected fish. J. Fish Dis. 24, 79 – 82.
Effects of commercial freshwater bathing on reinfection of Atlantic salmon, Salmo salar, with Amoebic Gill Disease Gemma Clark, Mark Powell, Barbara Nowak * School of Aquaculture, Tasmanian Aquaculture and Fisheries Institute, University of Tasmania, Locked Bag 1370, Launceston, Tasmania 7250, Australia Received 24 June 2002; received in revised form 16 December 2002; accepted 30 December 2002
Abstract Fish with Amoebic Gill Disease (AGD) were examined over a 10-day period following commercial freshwater bathing to assess the time to reinfection. Samples were taken from fish before freshwater bathing and then 1, 3, 5 and 10 days after bathing to determine the number of amoebae present on the gills. Freshwater bathing significantly reduced the number of amoebae on the gills, with an 86 F 9.1% reduction in the number of live amoebae found on the gills after freshwater bathing. However, amoeba numbers returned to pre-bath levels 10 days after bathing. There was no significant effect on number of AGD lesions/filament, the mean ranged from 0.08, 3 days after bathing, to 0.14, 5 days after bathing. However, the number of Neoparamoeba pemaquidensis dramatically dropped in histological sections from 0.53 per AGD lesion before the bath to 0 per AGD lesion 1 day after the bath and then remained significantly lower, reaching 0.08 per AGD lesion 10 days after the bath. The number of mucous cells changed, with Alcian blue (AB) (pH 1) positive cells decreasing immediately after bathing. Results of this study show that commercial freshwater bathing is effective at removing amoebae from the gills of fish, however, reinfection can occur within a week. D 2003 Elsevier Science B.V. All rights reserved. Keywords: Amoebic Gill Disease; Atlantic salmon; Freshwater bathing
* Corresponding author. Tel.: +61-3-6324-3814; fax: +61-3-6324-3804. E-mail address: [email protected] (B. Nowak). 0044-8486/03/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0044-8486(03)00020-6
136
G. Clark et al. / Aquaculture 219 (2003) 135–142
1. Introduction Amoebic Gill Disease (AGD) is a significant health problem in sea-pen salmonid culture in Tasmania (Munday et al., 1990; Nowak, 2001). The condition has been also reported in farmed salmonids from other countries, including Ireland (Rodger and McArdle, 1996) and the USA (Kent et al., 1988; Douglas-Helders et al., 2001 ) as well as in cultured marine fish species in the Mediterranean (Dykova et al., 1987). The causative agent of AGD is Neoparamoeba pemaquidensis, Page 1987 (Munday et al., 1990; Howard and Carson, 1993; Zilberg et al., 2001). N. pemaquidensis is considered a facultative amphizoic parasite and has not only been associated with the gills of salmonids, but it has also been found on sea-cage netting and in oceanic surface microlayers (Davis et al., 1978; Martin, 1985; Howard and Carson, 1993; Tan et al., 2002). The re-occurrence of AGD as indicated by bathing frequency has increased over the years (Nowak, 2001). The treatment of choice for the Tasmanian salmon industry to reduce impact of AGD is to bathe salmon in hyperoxic fresh water for 2 – 4 h (Parsons et al., 2001). This treatment reduces amoebae numbers on the gills of fish immediately after the bath as determined by histology (Parsons et al., 2001). However, it has been suggested that some amoebae could survive the treatment (Parsons et al., 2001). The aim of this study was to assess the efficacy of freshwater bathing and the time to reinfection following commercial bathing of Atlantic salmon affected by AGD.
2. Materials and methods 2.1. Commercial freshwater bathing Three successive commercial freshwater baths were investigated. Oxygen in each bath was maintained above 120% saturation and water temperatures ranged from 14.9 to 16.4 jC. The fish had been previously bathed once before, 3 weeks before this study. The need for the bath was on the basis of gross gill score, which reflects the number and size of white patches on fish gills. Each bath was 2 h in duration (from the transfer of the last fish), with each pen remaining at the bathing site to be towed back to respective grow-out sites on the second day post-bath. Bath biomass during bathing was 16 590 kg for cage one, 23 023 kg for cage two and 24 296 kg for cage three. All fish were of the same year class. As the experiment was run in a commercial setting and the samples were taken from commercial cages, no untreated controls could be included, due to animal welfare concerns and production issues. Thus, the results at different time points were compared to each other, with the main emphasis on the difference from the pre-bath means. The efficacy of commercial freshwater bathing was evaluated by monitoring changes in amoeba numbers on the gills and histological lesions over time after bath. Changes in the number of amoebae on the gills were monitored over a 10-day period with samples taken before the bath and then 1, 3, 5 and 10 days after bathing. Fish were collected using a 4-m box net in conjunction with a dip net. Ten fish were killed by anaesthetic overdose (0.04%) of clove oil and their second left gill arch was sampled for histology and fixed in seawater Davidson’s fixative. From five of the fish sampled for histology, all right gill arches were
G. Clark et al. / Aquaculture 219 (2003) 135–142
137
used for isolation of amoeba followed by amoeba count. The presence of N. pemaquidensis in gill isolates was confirmed by IFAT. 2.2. Histology Two stains were used to stain gill sections; haematoxylin and eosin (HE), and Periodic Acid Schiff’s (PAS) in conjunction with 1% Alcian blue (AB) at pH 1.0 and 2.5 (Bancroft and Cook, 1994). The number of lesions per filament, amoeba per lesion, and interlamellar vesicles (ILV) per lesion were counted at 400 magnification. Filaments from the ventral, medial and dorsal area of the second gill arch were chosen for counts of mucous cells. The number of cells stained PAS, Alcian blue at pH 1 (AB1), Alcian blue at pH 2.5 (AB2.5) or PAS + AB1 positive (PAS + AB1) was counted on 10 interlamellar units (Powell et al., 1995; Speare et al., 1991) and expressed per interlamellar unit. Only filaments sectioned correctly as indicated by equal length of lamellae on both sides of the filament were included in quantitative observations. 2.3. Amoeba isolation Amoebae were isolated from the gills using a technique modified from Howard and Carson (1994). All gill arches on the right side of the fish were removed from fish and placed in 50 ml plastic tubes. Sterile 2.5% ammonium –chloride and antibiotic (carbenicillin 0.1 g ml 1, penicillin 0.1 ml 1, streptomycin sulphate 0.1 ml 1, erythromycin 0.01 ml 1 and ampicillin 0.025 ml 1) solution was added to a total volume of 50 ml and kept at 4 jC for between 12 and 24 h. Gills were removed and mucus was collected into 50 ml plastic tubes. Mucus was re-suspended in sterile seawater and mixed. A 100 Al aliquot of solution was sampled and stained with 0.5% trypan blue. Live amoebae were counted using a haemocytometer (Neubauer, BS 748). A subsample of the solution was collected with a sterile loop, placed on a glass slide and air dried for IFAT (Howard and Carson, 1994). 2.4. Statistical analysis Analysis of variance (ANOVA) was used for data analysis. Homogeneity was tested using a Levene’s test. Results were considered significant if P V 0.05. Results for individual cages were pooled if P>0.25. Tukey’s test was used for comparison of means. Statistical analysis was conducted using SPSSR (version 8.0) software. Results are given as mean and standard error of the mean.
3. Results The number of live amoebae was significantly lower 1 day after freshwater bathing (Fig. 1). The number of live amoeba numbers began to increase 5 days after bathing and the counts of amoebae 10 days after bathing were not significantly different from pre-bath counts (Fig. 1). All subsamples collected from the isolates were IFAT positive. The
138
G. Clark et al. / Aquaculture 219 (2003) 135–142
Fig. 1. Average number of live amoebae present in isolation solution taken from the gills of fish ( F S.E.). Gill samples were taken before bathing, 1, 3, 5, and 10 days after bathing. Results pooled for three cages, five fish were sampled from each cage (n = 15). Common letters indicate treatments not significantly different ( P>0.05).
number of observed histological lesions did not change over the duration of the experiment (Table 1, P = 0.09). Multifocal hyperplastic lesions were common involving more than 10 lamellae along the full length of the lamellae. Lesions dominated in the proximal and medial region of the filaments. The numbers of N. pemaquidensis found on histological lesions were significantly reduced after the bath (Table 1). N. pemaquidensis numbers observed in association with lesions remained low throughout the experiment. A significant decrease in the number of ILV associated with lesions occurred on days 5 and 10 after bathing ( P < 0.05). There were no changes in mucous cell morphology or differences in the location of mucous cells (dorsal, medial, or ventral) observed. Similarly there was no effect of gill position on the number of PAS, PAS + AB, AB1 and AB2.5 positive mucous cells found per interlamellae unit ( P>0.05). Therefore, location on the gill was not considered to Table 1 Presence of gill lesions and amoebae in histology before and after freshwater bath Variable measured Number of AGD lesions/filament Number of Neoparamoeba/lesion ILV per lesions
Before
1 Day a
0.10(0.02) 0.53(0.27)a 0.69(0.24)a
3 Days a
0.11(0.02) 0(0)b 0.51(0.15)ab
5 Days a
0.08(0.02) 0.06(0.05)b 0.32(0.12)ab
10 Days a
0.14(0.05) 0(0)b 0.04(0.04)b
0.09(0.03)a 0.08(0.05)b 0.03(0.02)b
Common letters indicate no significant difference ( P >0.05). Results shown as mean and standard error. Results pooled for three bathings, n = 30 for each time.
G. Clark et al. / Aquaculture 219 (2003) 135–142
139
Table 2 The number of PAS1, PAS + AB1, AB1 and AB2.5 positive mucous cells per interlamellar unit found on the gills Stain PAS PAS + AB1 AB1 AB2.5 Total pH 1.0 Total pH 2.5
Before
1 Day a
0.18(0.05) 1.55(0.22)a 0.52(0.13)a 0.17(0.08)a 0.75(0.08)a 0.84(0.10)a
3 Days a
0.31(0.08) 1.82(0.22)ab 0.01(0)b 0.02(0.03)a 0.71(0.08)a 0.75(0.09)a
5 Days a
0.29(0.09) 2.40(0.24)bc 0.03(0)b 0.29(0.16)a 0.90(0.10)a 0.88(0.10)a
10 Days a
0.36(0.08) 2.13(0.03)ab 0.06(0.04)b 0.30(0.22)a 0.85(0.10)a 0.92(0.12)a
0.15(0.08)a 2.94(0.39)c 0.13(0.09)b 0.03(0.02)a 1.07(0.14)a 0.93(0.13)a
Results shown as mean and standard error. Common letters indicate no statistically significant effect ( P >0.05). PAS—Periodic Acid Schiff, AB—Alcian blue. Ten interlamellar units were counted for each fish, 30 fish were examined for each time (results pooled from three bathings).
influence counts and data were pooled by treatment. All mucous cell types were observed on the gills and were mainly located at the distal end of the lamellae. There was no significant difference between the number of PAS positive mucous cells per lamellar unit for the duration of the trial (Table 2). However, there was a significant decrease in the number of AB1 positive (sulphated mucins) mucous cells per lamellar unit after freshwater bathing on day 1. AB1 positive mucous cells remained low throughout the duration of the trial (Table 2). The number of AB2.5 positive mucous cells per interlamellar unit did not significantly change over the duration of the trial. Although the average number of AB2.5 positive cells was reduced on days 1 and 10 after bathing, the difference was not statistically significant, due to high variability between fish. The majority of mucous cells were PAS + AB1 positive in all regions of the gill. No significant change in the number of PAS + AB1 positive mucous cells in the gills occurred until 3 days after bathing when the number of these cells was slightly increased. There was no significant change in the total number of mucous cells found on the gills over the trial period at both pH 1.0 and 2.5.
4. Discussion Freshwater bathing was successful at reducing amoeba number on the gills of Atlantic salmon. This was confirmed by direct counts of amoeba isolated from the gills and counts from histological sections. An 86 F 9.1% reduction of number of amoebae present in the gill isolates after bathing was lower than the decrease reported previously (Parsons et al., 2001). However, the previous report was based on preliminary data using a different technique with a very low replication of samples (Parsons et al., 2001). However, the fact that freshwater bathing did not remove all N. pemaquidensis from the gills left a potential reservoir of the parasite on the gills for rapid proliferation. Numbers of amoebae increased 5 days after bathing and amoebae returned to pre-bath numbers by 10 days after bathing. The increase in the number of amoebae on the gills may be the result of rapid proliferation after bathing and/or reinfection from the surrounding environment. Proliferation on the gills was suggested by laboratory reinfection of recovered Atlantic salmon with AGD achieved without introducing the pathogen (Findlay and Munday, 1998), however, this result could be due to accidental introduction of N. pemaquidensis with water as the
140
G. Clark et al. / Aquaculture 219 (2003) 135–142
experimental sea water was not filtered. Environmental conditions have been shown to increase AGD prevalence with increasing temperature and salinity (Clark and Nowak, 1999). The conditions under which experimentation was conducted were within the optimal conditions established for N. pemaquidensis growth and clinical AGD in fish (Clark and Nowak, 1999; Nowak, 2001). This indicates that the amoeba load on the gills can increase rapidly when optimal environmental conditions are present. The number of amoebae per lesion decreased after bathing. This was in agreement with the previous research (Howard and Carson, 1992; Parsons et al., 2001). However, the number of amoebae isolated from the gills quickly returned to the pre-bath level. Furthermore, the counts from histological sections did not correlate with the amoebae counted in gill isolates and remained very low for the whole time after bathing. It is possible that after freshwater bathing, amoebae are not attached to the gills and could be lost during fixation. All isolates from which the amoebae were counted tested IFAT positive and while histology is very specific, its sensitivity is not high as only a small section of tissue is examined. However, trypan blue staining followed by haemocytometer count is not specific as a diagnostic method. Other amoebae or cells may be counted in addition to N. pemaquidensis. Unfortunately, this is the only quantitative method currently available. Further research is required to explore the discrepancy between histology results and trypan blue counts. There was no increase in the number of ILV following bathing and the ILV within AGD lesions did not contain amoeba after bathing. This was contrary to the previous findings of Parsons et al. (2001) of amoebae within ILV following bathing, suggesting that ILV formation and encystment of amoeba during bathing offered protection to the amoeba (Parsons et al., 2001). However, ILV formation is now believed to be a host response enclosing the parasite, resulting eventually in the degradation and removal of amoeba by host cellular processes (Adams and Nowak, 2001). Staining of mucous cells at different pH allows the identification of different glycoproteins (Ferguson et al., 1992). Two types of AB positive mucus are present on gills and are identified as sulphated at pH 1.0, carboxylated at pH 4.0 and combined at pH 2.5 (Ferguson et al., 1992). The observed decrease in AB1 positive mucous cells was not mirrored by an observed decrease in total AB cells at pH 2.5. In contrast, Powell et al. (2001) reported a decrease in AB 2.5 positive mucous cells in gills of Atlantic salmon following freshwater bathing. However, in that study, the sections were stained at only one pH and therefore, it is undetermined whether the decrease in AB positive mucous cells was a result of a decrease in sulphated or carboxylated mucins. The presence of high numbers of sulphated mucins (AB1 positive) was unusual. Sulphated mucins in high quantities are more often associated with freshwater adapted fish (Solanki and Benjamin, 1982). The hypersecretion of mucus is often associated with gill irritation (Powell et al., 1995, 1998) and may help to clear toxins or pathogens (Ferguson et al., 1992). Mucus may provide a barrier of protection at the interface between the amoeba and the gill epithelium. This would explain the increase in mucus observed on the gills during infection and the high numbers of amoeba isolated from the gills without the corresponding gill changes in histology. This investigation has shown that freshwater bathing is successful in reducing amoeba numbers on the gills of Atlantic salmon, however, reinfection occurred within 10 days.
G. Clark et al. / Aquaculture 219 (2003) 135–142
141
This shows that amoeba load on the gills can increase rapidly, however, the mechanism of the infection is unknown. The environmental conditions during the sampling were optimal for AGD outbreaks as defined by Nowak (2001) and Douglas-Helders et al. (2001), and may explain the relatively fast increase in amoeba numbers on the gills after freshwater bathing. It is likely that the time to reinfection would be longer under different environmental conditions. While this study focused on changes after second freshwater bath (the first one took place 3 weeks before this experiment), the pattern of reinfection is always similar in summer, regardless of the number of bath (M. Powell and B. Nowak, unpublished). The effect of multiple bathings on the properties of gill mucus is currently under investigation.
Acknowledgements The authors wish to acknowledge Huon Aquaculture Company for all their support during this trial. We thank Dr Rick Butler for the helpful comments.
References Adams, M., Nowak, B., 2001. Lesion distribution and structure in the gills of Atlantic salmon, Salmo salar affected with Amoebic Gill Disease. J. Fish Dis. 24, 535 – 542. Bancroft, J.D., Cook, H.C., 1994. Manual of Histological Techniques and Their Diagnostic Application. Churchill Livingstone, Singapore, p. 457. Clark, A., Nowak, B.F., 1999. Field investigations of Amoebic Gill Disease in Atlantic salmon, Salmo salar L., in Tasmania. J. Fish Dis. 22, 433 – 443. Davis, P.G., Caron, D.A., Sieburth, J.M., 1978. Oceanic amoebae from the North Atlantic: culture, distribution, and taxonomy. Trans. Am. Microsc. Soc. 97, 73 – 88. Douglas-Helders, M., Saksida, S., Raverty, S., Nowak, B.F., 2001. Temperature as a risk factor for outbreaks of Amoebic Gill Disease in farmed Atlantic salmon (Salmo salar). Bull. Eur. Assoc. Fish Pathol. 21, 114 – 116. Dykova, I., Figueras, A., Peric, Z., 2000. Neoparamoeba Page, 1987: light and electron microscopic observations on six strains of different origin. Dis. Aquat. Org. 43, 217 – 223. Ferguson, H.W., Morrison, D., Ostland, V.E., Lumsden, J., Bryne, P., 1992. Responses of mucus-producing cells in gill disease of rainbow trout (Oncorhynchus mykiss). J. Comp. Pathol. 106, 255 – 265. Findlay, V.L., Munday, B.L., 1998. Further studies on acquired resistance to Amoebic Gill Disease (AGD) in Atlantic salmon, Salmo salar L. J. Fish Dis. 21, 121 – 125. Howard, T., Carson, J., 1992. Studies of amoeba associated with Amoebic Gill Disease in Atlantic salmon. Barriers and Breakthroughs. Proceedings of the SALTAS Research Review Seminar 29th April 1992. SALTAS, Hobart, Australia, pp. 101 – 122. Howard, T., Carson, J., 1993. Verification that Paramoeba species are consistently associated with gill damage in fish affected with Amoebic Gill Disease. Proceedings of the Saltas Research and Development Review Seminar, Tasmania 1993. Salmon Enterprises of Tasmania, Dover, Tasmania, pp. 69 – 80. Howard, T., Carson, J., 1994. Amoebic Gill Disease laboratory research 1993/1994. Reports from the SALTAS 1993 – 1994. Research and Development Program. Salmon Enterprises of Tasmania, Dover, Tasmania, pp. 71 – 87. Kent, M.L., Sawyer, T.K., Hedrick, R.P., 1988. Paramoeba pemaquidensis (Sarcomastigophora: Paramoebidae) infestation of the gills of coho salmon Oncorhynchus mykiss reared in sea water. Dis. Aquat. Org. 5, 163 – 169. Martin, R.E., 1985. Population growth in stationary and suspension culture of Paramoeba pemaquidensis Page (Amoebida, Paramoebidae). J. Protozool. 32, 738 – 739.
142
G. Clark et al. / Aquaculture 219 (2003) 135–142
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 gairdneri, in Tasmania. In: Perkins, F.O., Cheng, T.C. (Eds.), Pathology in Marine Science. Academic Press, San Diego, CA, pp. 215 – 222. Nowak, B., 2001. Qualitative evaluation of risk factors for Amoebic Gill Disease in cultured 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 (O.I.E.), Paris, France, pp. 148 – 155. 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. Powell, M.D., Wright, G.M., Speare, D.J., 1995. Morphological changes in rainbow trout (Oncorhynchus mykiss) gill epithelia following repeated intermittent exposure to chloramine-T. Can. J. Zool. 73, 154 – 165. Powell, M.D., Haman, F., Wright, G.M., Perry, S.F., 1998. Response of rainbow trout (Oncorhynchus mykiss) to a graded hypoxia following repeated intermittent exposure to chloramine-T. Aquaculture 165, 27 – 39. Powell, M.D., Parsons, H.J., Nowak, B.F., 2001. Physiological effects of freshwater bathing of Atlantic salmon (Salmo salar) as a treatment for Amoebic Gill Disease. Aquaculture 199, 259 – 266. Rodger, H.D., McArdle, J.F., 1996. An outbreak of Amoebic Gill Disease in Ireland. Vet. Rec. 5, 348 – 349. Solanki, T.G., Benjamin, M., 1982. Changes in the mucous cells of the gills, buccal cavity and epidermis of the nine-spined stickleback, Pungitius pungitius L., induced by transferring the fish to seawater. J. Fish Biol. 21, 563 – 575. Speare, D.J., Ferguson, H.W., Beamish, F.W.M., Yager, J.A., Yamashiro, S., 1991. Pathology of bacterial gill disease: ultrastructure of branchial lesions. J. Fish Dis. 14, 1 – 20. Tan, C.K.F., Nowak, B.F., Hodson, S.L., 2002. Biofouling as a reservoir of Neoparamoeba pemaquidensis (Page 1970), the causative agent of Amoebic Gill Disease in Atlantic salmon. Aquaculture 210, 49 – 58. Zilberg, D., Gross, A., Munday, B.L., 2001. Production of salmonid Amoebic Gill Disease by exposure to Paramoeba sp. harvested from the gills of infected fish. J. Fish Dis. 24, 79 – 82.