Amoebic Gill Disease (AGD) in Atlantic Salmon (Salmo Salar) Farmed in Chile

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Aquaculture 310 (2011) 281–288

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Amoebic gill disease (AGD) in Atlantic salmon (Salmo salar) farmed in Chile

Patricio A. Bustos a,1, Neil D. Young b,c,1, Marco A. Rozas a,1, Harry M. Bohle a, Ricardo S. Ildefonso a, Richard N. Morrison b,d, Barbara F. Nowak b,⁎ a ADL Diagnostic Chile, Diagnostic and Biotechnology Laboratory, Puerto Montt, Chile b National Centre for Marine Conservation and Resource Sustainability, University of Tasmania, Launceston, Tasmania, Australia c Department of Veterinary Science, The University of Melbourne, 250 Princes Highway, Werribee, Victoria, Australia d Fish Health Unit, Department of Primary Industries, Parks, Water & Environment, 165 Westbury Road, Prospect, Tasmania, Australia article info abstract

Article history: Between May and November 2007, three marine Atlantic salmon farms around Chiloé Island, Chile, reported Received 5 September 2010 mortalities in which affected fish presented with Caligus rogercresseyi infections and gross gill lesions Received in revised form 31 October 2010 characteristic of amoebic gill disease (AGD). Histological examination of the gills from affected fish confirmed Accepted 1 November 2010 the presence of AGD lesions. Trophozoites possessing one or more endosymbiotic Perkinsela amoeba-like organisms (PLOs) were observed in association with hyperplastic tissue. Further analyses were undertaken Keywords: fi Atlantic salmon using a combination of PCR and in situ hybridization and the trophozoites were identi ed as Neoparamoeba Amoebic gill disease perurans. Thus, our data indicate that N. perurans is a causal agent of AGD in Chile. However, it is possible that Neoparamoeba perurans AGD was not the single cause of mortalities in the epizootics investigated here. The exceptionally high level of Sealice co-infection with Caligus rogercresseyi (maximum mean intensity 34, prevalence 100%), could have contributed to the production losses. © 2010 Elsevier B.V. All rights reserved.

1. Introduction samples obtained from Atlantic salmon (Salmo salar) that were presumptively diagnosed with AGD in Chile. Our data show that fish Amoebic gill disease (AGD) is a parasitic condition affecting some were indeed affected by AGD and that Neoparamoeba perurans was species of fish farmed in the marine environment (Munday, 1986; the causal agent. However, fish were also co-infected with Caligus Kent et al., 1988; Dyková et al., 1995). AGD is caused by Neoparamoeba rogercresseyi and a causal relationship between either of the parasites perurans (see Young et al., 2007), which is the confirmed aetiological and ongoing mortalities could not be established. agent of this disease in cases reported from Australia, Ireland, Japan, New Zealand, Norway, USA, Scotland and Spain (Young et al., 2008a; 2. Materials and methods Crosbie et al., 2010; Steinum et al., 2008; Nylund et al., 2008). AGD is grossly characterised by the presence of multifocal lesions in the gills 2.1. Epidemiology, clinical signs and fish sampling (Munday et al., 1990; Rodger and McArdle, 1996). The presence of these gross lesions allows presumptive diagnoses of AGD in the areas Between May and November 2007, three marine Atlantic salmon where AGD is enzootic. The final diagnosis is based on histopathology farms in Chiloe Island, Chile (Fig. 1), reported epizootics in which when amoebae that possess one or more endosymbiotic Perkinsela gross lesions typical of AGD (Adams et al., 2004) were observed in amoeba-like organisms (PLOs) (Dyková et al., 2003, 2008; Adams and affected fish. Fish were sampled and processed for histopathological Nowak, 2003) are detected in close association with hyperplastic examination from three farms over the course of one year. Fish from epithelial-like cells (Dyková and Novoa, 2001). Molecular tools, Farm 1 were sampled in May 2007 (20 fish, 10 per cage), August 2007 including PCR and in situ hybridization (ISH) have been developed (16 fish, 8 per cage) and November 2007 (20 fish, 10 per cage). Fish and used to confirm the identity of the parasite (Young et al., 2007, from Farm 2 were sampled in May 2007 (12 fish, 6 per cage), August 2008b). In this study, we applied these new diagnostic tools to 2007 (12 fish, 6 per cage) and November 2007 (12 fish, 6 per cage). Salmon from Farm 3 were sampled in August 2007 (10 fish from 1 cage) and November 2007 (31 fish from 3 cages). Fish that displayed signs of lethargy, respiratory stress or surface swimming were ⁎ Corresponding author. National Centre for Marine Conservation and Resource targeted for sampling. In addition to gill histology, all these fish Sustainability, University of Tasmania, Locked Bag 1370, Launceston, Tasmania, were sampled to confirm the presence of common pathogens in the Australia. Tel.: +61 3 63243814; fax: +61 3 63 243804. E-mail address: [email protected] (B.F. Nowak). region, including Piscirickettsia salmonis, Vibrio ordalli and Infectious 1 Those authors equally contributed to this manuscript. Pancreatic Necrosis Virus (IPNV). To detect Vibrio spp., samples from

Fig. 1. A map of Chiloé Island, Chile. The three affected salmon farms are shown on the map. P.A. Bustos et al. / Aquaculture 310 (2011) 281–288 283

Table 1 Environmental conditions during the AGD outbreak on salmon farms on Chiloe Island, Chile in 2007. Monthly means are shown for salinity, dissolved oxygen and temperature and monthly total is shown for rainfall. Salinity and dissolved oxygen were measured at 10 m depth. Average rainfall for 1993–2006 is also included. Rainfall information was provided by Department of Hydrology, Subdepartment of Meteorology and Snow.

Month Average Average Average Monthly Average monthly salinity dissolved temperature rainfall rainfall (mm) (ppt) oxygen (°C) (mm) (1993–2006) (mg/L)

May 27.9 7.1 11.6 36.2 218.2 June 31.9 11.8 11.0 197.5 278.6 July 32.1 11.9 9.6 54.5 203.6 August 31.8 5.9 9.0 154.5 212.0 September 30.4 6.5 9.5 115.0 145.6 October 31.4 5.9 10.8 113.5 110.1 November 32.8 6.3 12.0 57.3 96.4 anterior kidney were cultured on Tryptone-Soy Agar (TSA) (Difco) supplemented with 1% NaCl and then incubated at 20 °C for 3 days. To detect P. salmonis samples from kidney were cultured in the CHSE-214 cell line (without antibiotics added) in the presence of neutralizing anti-IPNV antibodies and incubated at 15–18 °C for 28 days (Lannan and Fryer, 1991). The presence of IPNV was determined by culturing CHSE-214 cells in the presence of extracts from the kidneys, spleens and livers from affected fish (Agius et al., 1982). Additionally, reverse transcription PCR (RT-PCR) of RNA from anterior kidney tissues was used to amplify a P. salmonis-specific amplicon (Mauel et al., 1996) was used to determine if the fish were positive for P. salmonis. Forty fish from each farm were monitored for sea lice infection every two weeks. The intensity of the infection with the copepod, C. rogercresseyi, was quantified as intensity and prevalence of infection (Bush et al., 1997). Fish had been treated for Caligus infections using Alphamax (PHARMAQ) in September 2007 on Farm 1 and in August 2007 on Farms 2 and 3. From July 2007 compulsory quarterly ISA testing was implemented Fig. 2. Clinical symptoms of amoebic gill disease affecting Atlantic salmon cultured in Chile. (A) Gross morphology of Atlantic salmon gills affected by amoebic gill disease. on all salmon farms in Chile (minimum 30 fish, Sernapesca, National (B) Gill lesion from an amoebic gill disease-affected fish (A) showing epithelial Fisheries Service, Chile). Moribund individuals were sampled and hyperplasia, lamellar fusion and amoebae, containing one or more endosymbiotic analyzed by ISAV-specificRT-PCR(Devold et al., 2000; Mjaaland et al., Perkinsela amoeba-like organisms (arrow), associated with pathological changes to the 2002). gill epithelium (arrowhead). Haematoxylin-eosin staining. Scale bar 50 μm.

2.2. Environmental conditions

Salinity (at 10 m depth), dissolved oxygen concentration (at 0 and collection. Information on rainfall was provided by Department of 10 m depth) and temperature were monitored during this study. Water Hydrology, Sub-department of Meteorology and Snow, Ministry of quality was measured inside cages on farms at the time of sample Public Works (Chile).

Table 2 Monthly mortality. AGD prevalence and intensity of Caligus rogercresseyi infection in Atlantic salmon at seawater farms with outbreaks of amoebic gill disease (AGD). Prevalence of AGD was determined on the basis of histology. At all farms, the prevalence of C. rogercresseyi infection was 100% at all times. NA – data not available.

Month Temperature Farm 1 monthly Farm 1 AGD Farm 1 monthly Farm 2 monthly Farm 2 AGD Farm 2 monthly Farm 3 monthly Farm 3 AGD Farm 3 monthly (°C) mortality (%) prevalence intensity mortality prevalence intensity mortality prevalence intensity (%) (Caligus/fish) (%) (%) (Caligus/fish) (%) (%) (Caligus/fish)

Mean (SD) (n) (Range) Mean (SD) (n) (Range) Mean (SD) (n) (Range)

May 11.6 3.8 65 15.7 10.7 67.7 11.1 1.2 NA 15.0 (5.0) (20) (3–51) (3.4) (12) (4–41) (0.3) (3–63) June 11.0 4.8 NA 5.0 6.3 NA 26.2 1.7 NA 16.1 (6.4) (1–19) (2.9) (5–69) (0.7) (8–64) July 9.6 4.2 NA 15.7 3.8 NA 19.4 1.9 NA 18.2 (3.8) (3–48) (4.66) (8–58) (1.5) (5–54) August 9.0 7.6 62.5 15.7 3.0 58.3 2.4 2.2 60 8.5 (5.6) (16) (2–29) (2.9) (12) (1–10) (2.1) (10) (2–17) September 9.5 4.4 NA 6.5 2.0 NA 2.4 1.0 NA 4.7 (2.2) (2–21) (3.9) (2–16) (0.6) (2–15) October 10.8 8.1 NA 13.6 3.5 NA 8.6 3.5 NA 6.1 (4.8) (2–36) (5.8) (5–24) (2.8) (2–18) November 12.0 20.9 75 26.1 8.5 75 34.0 0.8 64.8 7.7 (10.5) (20) (8–64) (7.7) (12) (6–79) (0.6) (31) (3–22) 284 P.A. Bustos et al. / Aquaculture 310 (2011) 281–288

2.3. Histology and in situ hybridisation (ISH) digoxigenin (DIG)-labeled 18S rRNA oligonucleotide probes specificfor closely related Neoparamoeba species, N. perurans, N. pemaquidensis and Gill arches from moribund fish with typical signs of AGD (gross N. branchiphila as previously described (Young et al., 2007). Positive and lesions as described by Adams et al., 2004) were fixed in 10% negative (no probe) controls were run in parallel with each ISH phosphate-buffered formalin for at least 24 h, dehydrated through a experiment by hybridizing each probe with a section containing graded alcohol series and processed for histological examination. representative isolates of each Neoparamoeba species termed an Sections (5 μm) of the gill arch were stained with haematoxylin and “amoebae array” as previously described (Young et al., 2007). Tissue eosin (H&E) and observations were made under a light microscope sections were incubated for up to 1 h with premixed BCIP/NBT solution (Leica DM1000, Hamburg, Germany). The prevalence of AGD was (Sigma-Aldrich, Castle Hill, New South Wales, Australia) for color determined by calculating the percentage of fish which were AGD development. positive on the basis of histology. AGD positive fish were defined as those which had amoebae containing PLOs in close association with 2.4. PCR of N. perurans DNA from salmon clinical samples hyperplastic epithelial-like cells in histological sections of gills. For in situ hybridization (ISH), serial sections (7 μm) were placed A 636 bp fragment of the N. perurans 18S rRNA gene was amplified onto Polysine glass slides (Menzel–Gläser, Braunschweig, Germany) from 20 ng of genomic DNA isolated from the gills of Atlantic salmon and dried overnight at 37 °C. Sections were hybridized with one of three presumptively diagnosed with AGD as previously described (Young

Fig. 3. Neoparamoeba perurans is associated with gill lesions from AGD-affected Atlantic salmon, Salmo salar L. sampled during an epizootic in Chile. Initially haematoxylin and eosin stained gill tissue sections from three replicate fish (A–C) were examined for histopathological evidence of AGD. Serial sections of the same AGD gill lesions with amoebae attached to the lesion surface (arrow heads) were probed using species-specific oligonucleotide probes that hybridise to 18S rRNA of N. perurans (D–E), Neoparamoeba pemaquidensis (G–I), Neoparamoeba branchiphila (J–L). Probes that hybridised to amoebae are blue. Inset boxes are magnifications of amoebae. Immunological detection using DIG-labelled oligonucleotide probes and BCIP/NBT, counterstained using 0.1% fast red. Low magnification scale bar represents 100 μm, high magnification represents 20 μm. Non-specific signal was not observed on the no-probe control sections (data not shown). P.A. Bustos et al. / Aquaculture 310 (2011) 281–288 285 et al., 2008b). PCR reactions containing either no template or plasmid rately for each farm using Pearson's correlation, all data were analysed DNA housing the full-length 18S rRNA gene of N. perurans (EF216901) using SPSS 16.0 statistical software (SPSS Inc, USA). A P-valueb0.05 was were used as negative and positive controls, respectively. PCR reactions considered a significant difference for all cases. were electrophoresed through 2% agarose/tris-borate EDTA buffer and visualized by staining with ethidium bromide. 3. Results

2.5. PCR product ampliﬁcation and sequencing 3.1. Epidemiology

Universal oligonucleotide primers complementary to conserved The highest monthly mortality on Farm 1 coincided with highest regions of the eukaryotic 18S rRNA gene were used to PCR amplify and temperature and highest salinity (Tables 1 and 2). However, overall sequence the entire N. perurans 18S rRNA gene from genomic DNA mortality patterns were inconsistent between farms (Table 2). The isolated from AGD-affected Atlantic salmon gill tissues collected in this fish were of poor condition, displaying signs of lethargy, respiratory study. Briefly, genomic DNA was extracted from gill tissues using a distress and surface swimming. Food intake was low leading to the Tissue DNA Kit (Omega-Biotek, USA) as per the manufacturer's reduction in growth rate (up to 25%) and increased feed conversion instructions. All amplification was performed as previously described rate (information provided by the salmon farms, data not shown). On (Dyková et al., 2005), except annealing temperatures were raised to 60° the basis of gross gill lesions AGD prevalence was estimated as 40– C and extension times were reduced to 30 s. PCR products were purified 60%. Concurrently, the fish were affected by a heavy C. rogercresseyi with EZNA® Extraction Gel Kit (Omega-biotek, USA) as per the infection (prevalence 100% at each sampling time and farm, manufacturer's instructions and sequenced using the ABI PRISM 310 maximum mean intensity 34), and subsequent to their removal as DNA analyzer (Applied Biosystem, USA) as per manufacturer's instruc- mortalities, a new recruitment of non-performing fish (runts) was tions. The 18S gene sequence from this study was deposited in GenBank observed. However, the monthly mortality rates were not directly (National Center for Biotechnology Information, US National Library of related to the mean intensity of C. rogercresseyi infection except for Medicine, Bethesda, MD) with the Accession Number GQ407108. Farm 1, where both the highest monthly mortality rate and highest mean intensity of C. rogercresseyi infection occurred within the month 2.6. Neoparamoeba phylogeny using the 18S rRNA gene of November 2007 (r=0.77, P=0.04, Table 2). On Farm 2 the highest mortality was recorded in May but the highest mean intensity of C. The Chilean 18S rRNA gene sequence was assembled using ContigEx- rogercresseyi infection was in November and there was no significant press (Vector NTi Advance v11, Invitrogen, USA) and compared with 18S relationship with monthly mortality (r=0.542, P=0.209, Table 2). rRNA gene sequences from 46 isolates of Neoparamoeba and an outgroup Similarly, there was no relationship on Farm 3, where the highest consisting of Korotnevella hemistylolepis (AY121850), Korotnevella stella mortality was in October but the peak of C. rogercresseyi infection was (AY686573, AY183893) and Pseudoparamoeba pagei (AY686576, reported in July (r=−0.108, P=0.818, Table 2). There was a AY183891) obtained from GenBank. All 18S rRNA gene sequences were significant positive correlation between monthly mortalities and aligned using AlignX Software (Vector NTi Advance v11, Invitrogen, USA) temperature on Farm 2 (r=0.852, P=0.015), however there was no (gap opening/gap extension penalty=8/2) and exported to NEXUS relationship between mortality and water temperature on the other format using BioEdit (Hall, 1999). Genetic distances among 18S rRNA two farms. The cumulative mortalities from May to November 2007 gene sequences were calculated as mean character differences using were 11.38% on Farm 3 and 37.85% on Farm 2, and it reached 53.82% at PAUP*4 Version 4.0b10 (Swofford, 2002). Maximum likelihood (ML) Farm 1 leading to grading and removal of the non-performing fish analysis was conducted on the described aligned dataset using PAUP* after the last sampling in November 2007. Version 4.0b10 (Swofford, 2002) incorporating the GTR+G+I model Alphamax (PHARMAQ) became available only in September 2007 selected using Akaike information criteria (AIC) in Modeltest 3.7 (Posada and has been used since then by Chilean salmon industry. Its use on and Crandall, 1998). Maximum likelihood analysis bootstrap support Farm 1 did not result in the decline in the intensity of infection. The (1,000 replicates) for each tree node was obtained using PHYML treatment with Alphamax in August 2007 on the other two farms (Guindon and Gascuel, 2003) with the GTR+G+I model. The consensus seemed to cause a decrease in the infection intensity in September, tree was visualised using Molecular Evolutionary Genetics Analysis however the intensity increased again in October and reached its peak (MEGA) software version 4.0 (Tamura et al., 2007). in November so there was no longer term effect. The gills of affected fish were pale and multifocal, diffuse white 2.7. Statistical analyses AGD-like lesions were observed (Fig. 2A). No Piscirickettsia salmonis, IPNV or Vibrio ordalli were detected in any of the fish sampled and all The relationship between monthly percentage mortality and Caligus the farms remained negative for ISAV until 2008 (Sernapesca, rogercresseyi intensity, salinity and temperature were assessed sepa- National Fisheries Services, Chile).

Fig. 4. Gel electrophoresis of PCR products produced by N. perurans-specific oligonucleotide (Young et al., 2008a). Lane M, Smartladder marker; Reactions containing either no template (lane 1) or plasmid DNA housing the full-length 18S rRNA gene of N. perurans (EF216901, lanes 8 and 9) were used as negative and positive controls, respectively. PCR amplicons were amplified from two fish from farm 1 (lanes 2 and 3), farm 2 (lanes 4 and 5) and farm 3 (lanes 6 and 7). 286 P.A. Bustos et al. / Aquaculture 310 (2011) 281–288

3.2. Environmental conditions replicate fish (Fig. 3D–E). In serially-sectioned gills, neither the N. pemaquidensis nor the N. branchiphila-specific probes hybridised with During the outbreak the salinity ranged from 27.9‰ in August to any trophozoites (Fig. 3F–L). Neoparamoeba species-specific probes 32.8‰ in November and water temperature ranged from 9.0 °C in hybridised with the corresponding Neoparamoeba species on the August to 11.9 °C in November (Table 1). The rainfall was lower than amoebae array while no signal was detected in trophozoites on the the fifteen year average, in particular in May 2007 it was only amoebae array when the probes were omitted from the hybridisation 36.2 mm, while May average for 1993–2007 was 206.4 mm (Table 1). procedure. This occurred in all hybridisation experiments (results not shown). 3.3. Histology 3.5. Diagnostic PCR Changes in the gills were characterized by hyperplasia of epithelial- like cells across the gill filaments. This resulted in the fusion of lamellae Employing the N. perurans-specific diagnostic PCR, similar sized and the development of round to oval interlamellar vesicles. Multiple PCR amplicons were generated from all gill tissue samples from farms rows of attached amoebae, each containing at least one PLO were 1, 2 and 3 of the expected size (636 bp) (Fig. 4). associated with the affected tissue (Fig. 2B). Additionally, hyperplasia of mucous cells was observed in the gills. The severe and diffuse hyperplasia 3.6. Phylogenetic analysis of Neoparamoeba 18S rRNA gene sequences of epithelial-like cells with the presence of amoebae containing PLO and the development of interlamellar vesicles is recognized as characteristic The 18S rRNA gene amplified from amoebae isolates derived from for AGD (Fig. 2B). The overall prevalence of AGD defined as the presence the gills of AGD-affected Atlantic salmon in Chile clustered with N. of amoebae in histological sections was 67.9% for Farm 1, 66.7% for Farm 2 perurans isolates globally with strong bootstrap support (Fig. 5). The and 63.4% for Farm 3. While the lesion severity was not quantified (for Chilean 18S rRNA gene sequence (GQ407108) has 98.4–99.2 example as percentage of filaments affected), the lesions were severe and similarity with N. perurans sequences from Australia and 99.6% characteristic of untreated AGD outbreak with most of filaments affected similarity with N. perurans from Norway (EU326494) (Table 3). by extensive hyperplasia (Figs. 2 and 3). 4. Discussion 3.4. In situ hybridisation This is the first confirmed record of an AGD outbreak in Atlantic The N. perurans-specific probe hybridized with all trophozoites in salmon farmed in Chile, caused by Neoparamoba perurans. Previous AGD-affected Atlantic salmon gill sections prepared from three records from Chile were incidental findings of amoebae on the gills of

Fig. 5. Tree from the phylogenetic analysis of Neoparamoeba 18S rRNA gene sequences and position of N. perurans from Chile with related to marine amoebae. Pseudoparamoeba pagei, Korotnevella hemistylolepis and K. stella were used as the outgroup. All sequences are presented with their GenBank accession numbers. The topology was inferred using maximum likelihood analysis incorporating the GTR+G+I model and values at nodes represent bootstrap support by fast stepwise addition analysis with 100 replicates and re- sampling of all characters using the PAUP software. All branches are to scale and the bar represents 0.1 substitutions per site. P.A. Bustos et al. / Aquaculture 310 (2011) 281–288 287

Table 3 Percent similarity among aligned 18S rRNA gene sequences of Neoparamoeba used for phylogenetic analysis calculated by pairwise alignment. The outgroup consists of sequences representative of Korotnevella hemistylolepis (AY121850), Korotnevella stella (AY686573, AY183893) and Pseudoparamoeba pagei (AY686576, AY183891). Isolates of Neoparamoeba perurans from Tasmania (T), Norway (N) and Chile (Ch) were examined individually. The sequence length for which these sequence similarities were obtained was 2079 bp.

N. perurans (T) N. perurans (N) N. perurans (Ch) N. pemaquidensis N. aestuarina N. branchiphila Outgroup

N. perurans (T) 97.94–98.97 98.63–99.26 98.38–99.17 90.54–91.52 88.53–89.72 88.01–88.89 75.36–80.68 N. perurans (N) 100.00 99.61 90.78–91.65 88.78–90.00 88.35–88.94 75.83–80.64 N. perurans (Ch) 100.00 90.49–91.32 88.49–89.70 88.21–88.79 75.73–80.54 N. pemaquidensis 96.63–100.00 92.22–95.10 88.38–89.78 75.04–81.69 N. aestuarina 94.30–97.91 87.44–88.98 74.98–81.48 N. branchiphila 95.82–98.60 75.40–78.59 Outgroup 75.59–99.63

fish suffering from other epizootics (Nowak et al., 2002) and an coordinated by INTESAL (Instituto Tecnológico del Salmón). The immunofluorescence antibody test (IFAT) previously used to confirm monitoring of ISA followed “ISA Contingency Plan” developed by the identity of the agent (Nowak et al., 2002) was shown to lack Sernapesca (National Fisheries Service) in August 2007 (Resolution species specificity (Morrison et al., 2004). Our study has linked the Number 1670) and then included in the Sanitary Specific Program to characteristic AGD gill histopathology with the presence of N. ISA Surveillance and Control. perurans trophozoites in close association with the AGD-affected gills from Chilean farmed Atlantic salmon. This extends the known References geographical range of N. perurans and is consistent with previous fi Adams, M.B., Nowak, B.F., 2003. Amoebic gill disease: sequential pathology in cultured ndings that N. perurans is the only known aetiological agent of AGD. 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