Lumpfish (Cyclopterus lumpus L.) develop amoebic gill disease (AGD) after experimental challenge with Paramoeba perurans and can transfer amoebae to Atlantic salmon (Salmo salar L.)

    Lumpfish (Cyclopterus lumpus L.) develop amoebic gill disease (AGD) after experimental challenge with Paramoeba perurans and can transfer amoebae to Atlantic salmon (Salmo salar L.) Gyri T. Haugland, Anne-Berit Olsen, Anita Rønneseth, Linda Andersen PII: DOI: Reference:

S0044-8486(16)30174-0 doi: 10.1016/j.aquaculture.2016.04.001 AQUA 632092

To appear in:

Aquaculture

Received date: Revised date: Accepted date:

11 January 2016 2 April 2016 4 April 2016

Please cite this article as: Haugland, Gyri T., Olsen, Anne-Berit, Rønneseth, Anita, Andersen, Linda, Lumpfish (Cyclopterus lumpus L.) develop amoebic gill disease (AGD) after experimental challenge with Paramoeba perurans and can transfer amoebae to Atlantic salmon (Salmo salar L.), Aquaculture (2016), doi: 10.1016/j.aquaculture.2016.04.001

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ACCEPTED MANUSCRIPT Lumpfish (Cyclopterus lumpus L.) develop amoebic gill disease (AGD) after experimental challenge with Paramoeba perurans and can transfer amoebae to

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Atlantic salmon (Salmo salar L.)

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Gyri T. Haugland1, Anne-Berit Olsen2, Anita Rønneseth1, Linda Andersen3* 1

Department of Biology, University of Bergen, P.O.Box 7803, N-5006 Bergen, Norway Norwegian Veterinary Institute, Bontelabo 8b, 5003 Bergen, Norway 3 The Industrial and Aquatic Laboratory (ILAB), Thormøhlensgate 55, N-5008 Bergen, Norway

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*

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Corresponding author

E-mail addresses:

Anita.Rø[email protected]

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[email protected]

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[email protected]

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[email protected]

Keywords: lumpsucker, gill scoring, transmission, carrier, cleaner fish, cohabitant challenge

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ACCEPTED MANUSCRIPT Abstract Cleaner fish such as lumpfish (Cyclopterus lumpus L.) and ballan wrasse (Labrus bergylta

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A.) are increasingly used to delouse farmed Atlantic salmon (Salmo salar L.). In 2014, more than

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20 million cleaner fish were placed into net-pens with farmed salmon in Norway. Amoebic gill

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disease (AGD), caused by the opportunistic, parasitic amoeba Paramoeba perurans, is emerging in salmon farming in Northern Europe. The amoeba displays low host specificity as it has been

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isolated from a range of fish species in addition to salmonids, such as wrasse and lumpfish

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cohabitating with farmed salmon. It is, however, not known to which degree lumpfish respond to P. perurans challenge, to which extent lumpfish may develop AGD, and if they can function as a

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vector for the spread of P. perurans to salmon. The present study shows that lumpfish can be

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infected with P. perurans under experimental conditions and develop AGD. However, lumpfish are more resistant and the development of pathology is slower compared to salmon. It is also

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shown that lumpfish can act as carriers and transmit parasitic amoebae to Atlantic salmon. Importantly, it is demonstrated that the gill lesion score system extensively used for evaluating AGD in Atlantic salmon is less suitable for lumpfish infected with P. perurans as the disease develops more slowly in lumpfish and because lumpfish may be non-symptomatic carriers.

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ACCEPTED MANUSCRIPT 1.

Introduction Cleaner fish such as lumpfish (Cyclopterus lumpus L.) and ballan wrasse (Labrus bergylta

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Ascanius, 1767) are increasingly used to delouse farmed Atlantic salmon (Salmo salar L.)

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infested with sea lice (Lepeophtheirus salmonis Krøyer, 1837) (Skiftesvik et al., 2013; Imsland et

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al., 2014). In 2014, more than 20 million cleaner fish were put into net-pens with farmed Atlantic salmon in Norway (http://www.fiskeridir.no/English/Aquaculture/Statistics/Atlantic-salmon-and-

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rainbow-trout, Use of clean fish 1998-2014). Of these, around five million were farmed lumpfish,

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whereas the largest proportion was wild-caught cleaner fish, mostly wrasse. Extensive movement of wild-caught fish, often over long distances, calls for increased awareness of the health status of

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the cleaner fish involved, especially to avoid introduction of pathogens into new areas and to new

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hosts. Moreover, the cleaner fish species involved may display a different susceptibility to pathogens common to salmon. Thus they may either be more severely affected than salmon

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causing welfare issues for the cleaner fish, or if less susceptible, possibly act as non-symptomatic carriers that will facilitate spread of the infectious agent involved. An emerging disease condition in Norway is amoebic gill disease (AGD) caused by the parasitic amoeba Paramoeba perurans Feehan, 2013 (syn. Neoparamoeba perurans). AGD has caused large problems in Tasmania, Australia, for several decades (Mitchell and Rodger, 2011; Nowak et al., 2014). The disease was first reported from Norway in 2006 (Steinum et al., 2008), but did not cause severe problems until 2013, when the number of outbreaks increased ten-fold from the previous year (five outbreaks diagnosed in 2012 versus 58 in 2013). In 2014, AGD was reported from 69 fish farms in Norway (Bornø and Lie Linaker, 2014).

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ACCEPTED MANUSCRIPT The opportunistic, free-living parasitic amoeba P. perurans is ubiquitous and has been reported from several fish species around the world; turbot (Scophthalmus maximus L.), ayu

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(Plecoglossus altivelis Temminck & Schlegel, 1846), sea bass (Dicentrarchus labrax L.),

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Sharpsnout sea bream (Diplodus puntazzo Walbaum, 1792), wrasse (L. bergylta L.) together with several salmonid species (Salmo salar L., Oncorhynchus mykiss Walbaum, 1792, Oncorhynchus

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kisutch Walbaum, 1792, Oncorhynchus tshawytscha Walbaum, 1792) (Kent et al., 1988; Dykova

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et al., 1995, 1998; Dykova and Novoa, 2001; Young et al. 2007, 2008; Crosbie et al 2010; Karlsbakk et al., 2013). An overview of marine invertebrate and vertebrate species that are

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susceptible to Paramoeba/Neoparamoeba ssp. infection is given by Feehan et al. (2013). P. perurans has also been detected from lumpfish cohabitating with farmed Atlantic salmon with

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AGD (Karlsbakk et al., 2014, Karlsbakk 2015). However, to our knowledge this is the first report

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where it is shown that lumpfish develop AGD after experimental challenge and that they can be

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vectors for the spread of P. perurans to Atlantic salmon. In this study lumpfish was challenged with P. perurans in order to study the susceptibility to P. perurans and disease development in this species. Moreover, disease development for lumpfish and Atlantic salmon was compared, and if the lumpfish can function as carriers and transmit parasitic amoebae to salmon was examined.

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ACCEPTED MANUSCRIPT 2.

Materials and methods

2.1.

Fish

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Farmed lumpfish C. lumpus L. were supplied by Fjord Forsk Sogn AS, a commercial

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breeder in Sogn & Fjordane County, Norway. The fish were around seven cm (maximum

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standard length) at start-up of the experiment (mean length was 7.3 ± 2 cm), which is comparable to the size they have when they are put into net-pens with farmed Atlantic salmon (Schaer and

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Vestvik, 2012). The fish experiments were conducted at the Aquatic and Industrial Laboratory

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(ILAB), Bergen, Norway. The water temperature throughout the experiment was 12°C and the light regime 12 hours light: 12 hours dark. The water flow, 34 PSU, was 300-400 L per h per tank

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during the experimental period. The outlet water had a minimum of 77 % oxygen saturation. The

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fish were fed with the commercial dry feed Amber Neptune (three mm), which is a marine feed

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developed for gadoids, obtained from Skretting Norway. The Atlantic salmon smolts, 60-70 g, were hatched and bred at ILAB. The rearing conditions for salmon were similar to those described for lumpfish. There were no signs of infections and no mortality among the lumpfish or the salmon. The animal experiments were approved by the Norwegian Animal Research Authority (NARA) in 2013 under the identification code 5865.

2.2.

Paramoeba perurans- isolate The P. perurans (Amoebozoa) isolate used in this study originated from farmed Atlantic

salmon with AGD sampled in October 2013 in Hordaland County, Western Norway. The amoeba isolate had been kept in continuous culture in malt yeast broth, MYB, (0.01 % malt extract, 0.01 5

ACCEPTED MANUSCRIPT yeast extract, 34 PSU saltwater) at 15°C for six months when the isolate was used in this study to challenge lumpfish and Atlantic salmon. For preparation of the inoculum, the number of viable

Experiment I: Bath-challenge of lumpfish with P. perurans

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2.3.

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amoebae was determined in a CASY Model TT Cell counter (Innovatis, Roche Diagnostics).

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After an acclimatization period of 28 days the fish (n=90) were transferred to two containers (45 fish in each) with 10 L of seawater (34 PSU). Two hundred viable amoebae were

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added to one container and an equivalent volume of MYB medium to the second container as a control/non-infected group. The exposure baths with fish were aerated and oxygenated during the

2.4.

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challenge. After exposure for 1 h the fish were transferred back into their respective 150 L tanks.

Experiment II: Bath-challenge with P. perurans to compare disease development in lumpfish and Atlantic salmon In order to compare disease development in lumpfish and Atlantic salmon, lumpfish

(n=15) and salmon (n=15) were bath challenged together in 60 L of seawater (34 PSU) for 1 h (1000 amoebae L-1). After 1 h of exposure to amoebae, each fish species was separated and put into separate 150 L tanks.

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ACCEPTED MANUSCRIPT 2.5. Experiment III. Challenge from infected lumpfish to cohabitating salmon To examine if infected lumpfish could transmit amoebae to salmon, 15 uninfected salmon

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of 60-70 g were transferred to the 150 L tank containing 15 lumpfish previously exposed to P.

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perurans (36 d post bath-infection described in Experiment I, section 2.3). Gills from five salmon

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and five lumpfish were sampled at each time point.

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2.6. Sampling regime

At each time point (as indicated in Figs. 2-4), sampling was performed from fish from the

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challenged group and the uninfected control group in Experiment I (n = 10 per group) and from

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lumpfish and salmon in Experiment II and III ( n = five per group). Length and weight of the fish was measured. Gill scores for each individual were assessed using the system of Taylor et al

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(2009). The gill arch with the most severe lesions was used for gill scoring. From each fish, reisolation of amoebae from gills was made by scraping gill tissues onto malt yeast agar (MYA) (Crosbie et al 2012) and the presence of amoebae was examined using light microscopy. The second gill arch from each individual was placed in 10 % neutral buffered formalin solution (Sigma-Aldrich) for histology, whereas a piece of gill sample (second gill arch) was placed in RNAlater (Ambion) for real-time RT-PCR analyses. Any macroscopic changes in internal organs were also noted. Water samples (one L) were collected from the tanks at several time points after infection (as indicated in Figs. 2-4) in order to quantify Paramoeba RNA levels in water.

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ACCEPTED MANUSCRIPT 2.7. Re-isolation of amoebae on MYA Re-isolation of amoebae was performed at each sampling point by scraping gill samples

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directly onto MYA and adding autoclaved saltwater according to the method described by

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Crosbie and coworkers (Crosbie et al., 2012). The MYA were then incubated at 15°C and

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evaluated for the presence of amoebae after at least seven days in a Leica DM IL LED inverted microscope (Leica Microsystems) at 40-400 x magnification. The presence of amoebae was then

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Light microscopy

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2.8.

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evaluated as a +/- assay.

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Mucus and gill tissues were scraped off gills and placed onto slides and cover slipped (wet mounts using a drop of autoclaved salt water, 34 PSU). The presence of amoebae was then

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evaluated with a Leitz Laborlux S microscope (Tamro Medlab AS) as a +/- assay. The slides (only one slide from each individual fish was prepared) were evaluated at 100-400 x magnification using bright field.

2.9. Histopathological examination Tissues were fixed in 10 % neutral buffered formalin solution (Sigma), and kept at 4°C until processing. Formalin-fixed tissues were embedded in paraffin wax, sectioned (two-three µm) and stained with haematoxylin and eosin (H&E) according to standard histological methods (Culling et al., 1985) at the Norwegian Veterinary Institute, Oslo. Coverslips were mounted onto slides using PERTEX mounting media (Histolab Products). One gill section per fish was 8

ACCEPTED MANUSCRIPT examined by light microscopy. Microscopic gill lesions were assessed using a modified protocol after Adams and Nowak (2001; 2003) for each individual. Each gill was examined for the

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presence of findings typical for AGD. Moreover, the severity of lesions was assessed as the

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percentage of gill epithelium affected: 0 = no lesions, 1 = 1-10 %, 2 = 11-30 %, 3 = 31-60 %, 4 = 61-100 %. The presence of amoebae in the sections was also evaluated: 0 = no amoebae, 1= 1-4

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amoebae, 2 = 5-10 amoebae, 3 = >10 amoebae.

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2.10. Real-time RT-PCR to detect Paramoeba in gill samples from lumpfish and salmon Real-time RT-PCR analysis of total RNA extracted from gill tissues was performed by a

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commercial real-time RT-PCR diagnostic company, PHARMAQ Analytiq AS, Bergen, Norway. Briefly, tissue samples were conserved in RNAlater®Stabilization Solution (Ambion) according

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to manufacturer’s instructions. The tissue samples were processed using a biorobot (BioRobot Universal system; Qiagen), and the corresponding RNeasy 96 BioRobot 8000 Kit (Qiagen), following the supplier`s recommendations. The real-time RT-PCR analysis was performed using a

Paramoeba

sp.

specific

assay,

TTGTCAGAGGTGAAATTCTTGGATT-3`;

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(NEOuni-Fwd:

NEOuni-Rev:

5´5´-

TGAAAACATCTTTGYCAAATGC-3´; NEOuni-Probe: 6FAM- ATGAAAGACGAACTTCTGMGBFNQ) (made available by A. Nylund, University of Bergen, Norway) for detection of the amoeba, with the housekeeping gene elongation factor as an internal control from salmon (Olsvik et al., 2005) and lumpfish (S. Nylund, PHARMAQ Analytiq, personal communication). Primer and probe concentrations were optimised by the commercial company. A standard curve was generated using a 10-fold serial dilution of RNA in three parallels. Regression analysis, standard

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ACCEPTED MANUSCRIPT curve slopes s of Ct versus log quantity, and amplification efficiency E where E = [101/ (-slope)] were calculated in Q-Gene (Müller et al., 2002) (Table 1). All real-time RT-PCR reactions were

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run in an Applied Biosystems 7900 HT real-time system (Applied Biosystems, ThermoFischer

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Scientific, Massachusetts, USA) under standard conditions. The obtained threshold Ct-values

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were normalised against elongation factor using the method described in Andersen et al. (2010).

2.11. Real-time RT-PCR to detect Paramoebae in water samples

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Water samples were filtrated directly through electropositive 1 MDS Virosorb filters according to the method described in Andersen et al. (2010). Lysis buffer was added to the filters

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and the lysates were collected and stored at -20C. Total RNA was isolated from thawed lysates using E.Z.N.A. Total RNA Kit I (Omega

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Bio-Tek, Norcross, USA) according to manufacturer’s instructions. After thawing, 10 L infectious salmon anaemia virus (ISAV) supernatant (TCID50 of 1x 106) produced in the salmonid TO-cells (Wergeland and Jakobsen, 2001) was added to the lysate (350 L), mixed with an equal amount (360 L) of 70 % ethanol before loading onto the column. The RNA was eluted using 40 L of DEPC water preheated to 70C. The RNA was treated with DNaseI (Sigma, Saint Louis, USA), quantified in NanoDrop (ND-1000 UV-Vs Spectrophotometer, Saveen & Werner, Malmö, Sweden) and further transcribed into cDNA using qScript cDNA synthesis kit (Quanta BioSciences) according to the manufacturer’s instructions. Real-time RT-PCR was performed in a CFX96 (BioRad) using SYBR green Jumpstart Taq ready mix kit for quantitative PCR (Sigma, Saint Louis, USA), and HPLC purified primers

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ACCEPTED MANUSCRIPT (Sigma-Genosys company Ltd.). The AGD-Peru assay is described in Fringuelli et al (2012) and the ISAV assay in Rimstad et al (2001). The PCR reactions (25 L) and cycling conditions were

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as described previously (Rønneseth et al., 2013) using 5 L of cDNA (diluted 1:5), 12.5 L 2 X

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SYBR green jumpstart Taq Ready Mix, 1 L (0.4 mM) forward primer, 1 L (0.4 mM) reverse

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primer and 5.5 L nuclease-free water (Sigma, Saint Louis, USA). Two-fold dilution curves of cDNA were made for efficiency calculations (Table 1). Three parallels were performed for each

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dilution. Negative controls: without template (non-template control, NTC) and cDNA reactions

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without reverse transcriptase (-RT) were included for all master mixes. Also, the Paramoeba assay was tested on cDNA from a pure ISAV sample and the ISAV assay were tested on cDNA

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from a pure Paramoebae sample to ensure that the PCR-primers did not bind to the non-infected

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materials. As positive controls, a pure Paramoebae sample and a pure virus samples were included (Fig. 1). Three replicates were performed for all samples. After the run, melting curve

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analyses were performed of each amplicon to ensure the specificity of the primers and the realtime RT-PCR products were visualised on an agarose gel for size determination (Fig. 1). Ctvalues for the target gene were normalised against ISAV by using the Microsoft Excel based computer software Q-Gene (Müller et al., 2002).

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ACCEPTED MANUSCRIPT 3.

Results

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3.1. Validation of PCR-assays used to quantify P. perurans To quantify P. perurans RNA levels from gill tissue and water, real-time RT-PCR was

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performed. The efficiencies of the assays are given in Table 1. For relative quantification of

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amoebae RNA levels from gill tissues, elongation factor was used as internal control for both

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lumpfish and salmon, while for quantification of amoebae in the water, ISAV was added to the samples prior to RNA isolation and used as an external control for quantification. Validation of

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the assays used for quantification of P. perurans in water is shown in Fig. 1. The positive control, a P.perurans sample, and a water sample from experiment III, were positive for the P.perurans-

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assay, giving a real-time RT-PCR-product of 139 bp, while product (no Ct-values) was detected

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in the negative controls (Fig. 2, lane 2-6). For the ISAV-assay, an ISAV-sample was included as

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the positive control. The positive control and a water sample from experiment III, in which ISAV had been added as a exogenous spike, were positive giving a band of 117 bp, while no product was detected in the negative controls (Fig. 1, lane 7-11).

3.2. Experiment I. Lumpfish are susceptible to P. perurans and develop AGD In the challenged group, macroscopic gill lesions were observed at five dpi (Fig. 2A, B). At 12 and 26 dpi, both the prevalence and gill score were similar to five dpi, but at 26 dpi one fish had a gill score of 2 (Fig. 2B, Supplementary Table 1). Lesions were observed as pale patches on the gill filaments, often at the base of the filaments (Fig. 2A). Histopathology revealed typical AGD lesions in 10% of the examined fish at day 12 and 20% of the fish at day 26 (Supplementary Table 1). Typical hyperplasia of poorly differentiated epithelial cells and fusion 12

ACCEPTED MANUSCRIPT of lamellae, and in the more advanced cases, also formation of interlamellar spaces, were observed. Mild leukocyte infiltration and some mucus cell hyperplasia were present. Presence of

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amoebae was determined by bright field microscopy. The amounts of amoebae correlated with

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the histopathology-results, being 10% positive samples at day five, 10% at day 12 and 0% at day 26. To verify presence of amoebae, they were re-isolated from gills at all time-points

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(Supplementary Table 1)

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The presence of amoebae was also detected by real-time RT-PCR (Fig. 2C). The Ct-

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values obtained with Paramoebae-specific PCR assay on gill tissues were normalised using the lumpfish EF1. After five dpi, 10% of the fish was positive, while at 12 and 26 dpi, 60% and

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80% of the lumpfish analysed, were positive, respectively (Fig. 2C). No lumpfish died during the

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time course of the infection. Amoebae were detected in the water from seven dpi and throughout the experimental period (Fig. 2D). The highest level of amoebae RNA in the water was seen at

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the last sampling point, which was at 26 dpi. No amoebae were detected in lumpfish by real-time RT-PCR, cultivation on MYA or by microscopic or histological examination prior to start-up of the experiment or in the uninfected control fish during the experiment.

3.3. Experiment II. Comparison of disease development in lumpfish and salmon exposed to P. perurans. Gill lesions in lumpfish in Experiment I were less prominent than what is usually seen with P. perurans-challenge in Atlantic salmon given similar concentrations of amoebae. Thus, both lumpfish and salmon were challenged with amoebae simultaneously in the same container to directly compare the disease development between the two species. Both lumpfish and salmon 13

ACCEPTED MANUSCRIPT challenged with P. perurans became infected and developed AGD, but salmon became more severely affected than lumpfish (Fig. 3A, B, C and Supplementary Table 2). The prevalence of

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fish with macroscopic lesions during the experimental period was higher for salmon, 87% of the

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salmon versus 27% of the lumpfish. Moreover, salmon had more severe gill lesions than lumpfish at each time point and the disease developed more quickly in salmon. Macroscopic lesions were

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seen in salmon from 12 dpi and at 61 dpi all the salmon had a score of four or five (Fig. 3B,

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Supplementary Table 3). At 61 dpi, only 20% of the lumpfish had gill lesions. Amoebae could not be re-isolated from any of the lumpfish examined at day 12, 20% at day 12 and 100% at day

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61. The percentages for salmon will gill lesions were 40% at day 12 and 100% at day 26 and 61. Amoebae were not observed by microscopic examination from lumpfish, but were observed from

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20% and 100% of the salmon at day 26 and day 61, respectively. The presence of Paramoebae RNA in gill samples and water was detected by real-time RT-

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PCR (Fig. 3B and C) using a Paramoebae-specific PCR assay. Amoebae were detected from salmon gill tissues in 40% of the fish at 12 dpi and in 100% of the fish sampled at 26 and 61 dpi, respectively (Fig. 3C). No lumpfish were positive by real-time RT-PCR prior to the sampling at 61 dpi. At this time-point, amoebae were detected in 80% of the lumpfish, but at lower levels compared with salmon (Fig 3C). Amoebae in the water were only detected from the tank with salmon (Fig. 3D).

3.4.

Experiment III: Lumpfish can transfer P. perurans to Atlantic salmon To investigate whether lumpfish can act as carriers/vectors and transfer P. perurans to

Atlantic salmon, uninfected salmon were transferred to a tank with infected lumpfish at 36 dpi. 14

ACCEPTED MANUSCRIPT Twelve days after salmon were exposed to infected lumpfish through cohabitation (48 d post bath-infection for the lumpfishes), gross lesions (score 1-2) were observed for 80% of the salmon

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(Fig. 4A). At this time point high gill scores were detected in all lumpfish sampled; 20% had

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score two whereas 80% of the lumpfish had gill score three (Fig. 4A, B). Histopathological examination revealed lesions as described in experiment I, but for most affected gills, more

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extensively distributed, and typically located at the proximal end of the filaments. Amoebae were

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observed occasionally in interlamellar spaces (Fig. 4C). In some gills hyperplasia of chloride cells was seen, as well as the recruitment of eosinophilic granular cells. Spongiosis of

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hyperplastic tissue was also recorded (Fig. 4D), as reported for Atlantic salmon (Adams and Nowak, 2003). At 26 dpi for salmon/ 62 dpi for the lumpfish, the disease had progressed for both

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species (Fig. 4A, Supplementary Table 3), as all sampled salmon and lumpfish had scores

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between three and five. No clinical signs or mortality were seen in lumpfish during the trial,

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whereas three salmon died suddenly at 45-55 days post exposure to infected lumpfish. The three salmon had a gill score of ≥ four. Experiment II showed that the disease developed more slowly and at a lower prevalence in lumpfish compared to salmon. However, 48 days after infection, some individual lumpfish had developed severe gill lesions (Fig. 4A). Surprisingly, not all lumpfish were affected by the amoeba infection. At 93 dpi two individuals had score five, whereas the others had no lesion score, whereas all the salmon sampled were severely affected. At this time point, sudden mortality had occurred among salmon and the two salmon remaining in the tank had score five when examined (Fig. 4A). Re-isolation of amoebae on MYA was successful from 80%, 100% and 60% of the lumpfish at 48, 62 and 93 dpi, and from all examined salmon at 12 and 26 dpi, respectively. Re-isolation on agar was not attempted from salmon with score five at 57 dpi. The presence of amoebae observed microscopically correlated with the results from the re-isolation. 15

ACCEPTED MANUSCRIPT The presence of amoebic RNA in gill tissues from lumpfish and cohabitating salmon and water was detected by real-time RT-PCR (Fig. 4E and F). Amoebae were detected in gill tissues

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from all examined lumpfish at 48, 62 and 93 dpi and in gills from all but one cohabitating salmon

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sampled at 12, 26 and 57 dpi post exposure (Fig. 4E). The P. perurans levels were similar in gill tissues from both lumpfish and salmon at the first two sampling points, whereas higher RNA

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levels were detected in salmon compared to lumpfish at the last sampling point (Fig. 4E).

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Increasing levels of amoebic RNA were detected in the water after salmon were added to the tank with infected lumpfish and throughout the experimental period (Fig. 4F). The highest level of

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4. Discussion

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amoebic RNA in the water could be seen 29 d after the salmon were transferred to the tank.

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Lumpfish are now farmed and together with different species of wrasse, increasingly used as cleaner fish in the fight against salmon louse infestations in farming of Atlantic salmon in Europe (Skiftesvik et al., 2013; Imsland et al., 2014). Amoebic gill disease (AGD) caused by the parasitic amoeba P. perurans occurs world-wide in all of the major salmon producing countries, except Canada (Nowak et al 2014). In addition to a vast number of other fish species, P. perurans has also been reported from lumpfish cohabitating with AGD-affected salmon (Karlsbakk et al., 2014, Karlsbakk 2015), but no pathogenesis studies that have examined the susceptibility of lumpfish to P. perurans infection have been published to date. In the present study it is shown that lumpfish are susceptible to infection with P. perurans and can develop AGD after experimental challenge. The histopathological findings in lumpfish during the experimental challenge in this study were consistent with the pathology previously described for AGD in

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ACCEPTED MANUSCRIPT Atlantic salmon (Adams and Nowak, 2001, 2003), ballan wrasse (Karlsbakk et al., 2013) and lumpfish from net pens with amoebic gill diseased Atlantic salmon (Karlsbakk 2015).

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The presence and severity of AGD in farmed populations of salmon are usually evaluated

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based upon a macroscopic gill lesion scoring system, where the proportions of the gill arch

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affected is evaluated (Taylor et al., 2009). This is a system used by fish farmers and fish health services in several countries, including Norway. This is a subjective, but valuable tool and is to

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some extent used to evaluate if it is necessary to treat the fish population. In Norway, gill scoring

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together with histology and real-time RT-PCR analysis are used to evaluate and to monitor AGD in salmon.

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Since a high number of cleaner fish, including lumpfish, are put into net pens for

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delousing of salmon, it is important to gain more knowledge of the susceptibility of lumpfish to

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P. perurans, the disease development and to evaluate if gill scoring is a useful tool for this species. From our study it was apparent that lumpfish could be P. perurans PCR-positive and amoebae could be re-isolated and/or be observed by direct microscopy without any macroscopic lesions or visible mucoid patches on the gills. This is in contrast to salmon, where if a gill score is seen, the fish are highly likely to be PCR-positive and amoebae possible to re-isolate. From experimental infection trials for salmon with a virulent P.perurans strain, a divergence between gill score and PCR may be seen only shortly after infection as at later time points there is a consistency between methods. For lumpfish, however, fish that were PCR positive without gill score could be seen as late as 90 days after infection, showing that not all individuals are affected. In lumpfish, where gill scores were present, the patches were smaller, less protruded and less mucoid in appearance. Gill scores were also harder to spot due to the natural paler appearance of lumpfish gills and fewer of the gill arches being affected compared to salmon. The time lag 17

ACCEPTED MANUSCRIPT before gill score developed in lumpfish was also later than seen for salmon, and most individuals did not develop severe gill scores. Based upon this, it is suggested that gill score is not a method

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that can be applied for evaluating the presence of P. perurans or the progression of AGD in

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lumpfish. The gill scoring system has also been shown not to be reliable for less severe cases of AGD in salmon (Clark & Nowak, 1999). Instead, real-time RT-PCR and cultivation of amoebae

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on malt yeast agar are sensitive methods that should be applied for evaluating the status of

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cleaner fish.

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Our findings show that lumpfish are more resistant than Atlantic salmon against infection with P. perurans and that lumpfish may act as asymptomatic carriers of amoebae, thus posing a

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However, during an outbreak of AGD, Atlantic salmon will probably be the main reservoir as these are highly susceptible to a P. perurans infection and become severely affected. Our

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findings indicate that lumpfish should be screened using sensitive molecular methods such as real-time RT-PCR prior to transfer to net-pens and that the water intake at the cleaner fish farming facilities should be monitored for amoebae.

5. Conclusions The present study has shown that lumpfish may be infected by P. perurans and develop AGD after experimental challenge. However, the lumpfish seem less susceptible than salmon. Fewer fish are affected by the disease and the gill lesions and RNA levels were lower. In addition, the disease develops more slowly in lumpfish and no mortality occurred in P. perurans challenged lumpfish. Moreover, it is shown that the gill scoring system may not be used for lumpfish as

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ACCEPTED MANUSCRIPT these may be asymptomatic carriers, harboring amoebae without visible gill scores. Thus, the lumpfish population should be screened, not scored, before they are put into net-pens with

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salmon.

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References

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Adams, M.B., Nowak, B.F., 2001. Distribution and structure of lesions in the gills of Atlantic salmon, Salmo salar L., affected with amoebic gill disease. J. Fish Dis. 24,

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Atlantic salmon, Salmo salar L. J. Fish Dis. 26, 601-614. Andersen, L., Hodneland, K., Nylund, A., 2010. No influence of oxygen levels on pathogenesis

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and virus shedding in Salmonid alphavirus (SAV)-challenged Atlantic salmon (Salmo salar L.). Virology J. 7, 198 Bornø, G., Lie Linaker, M., 2014. The health situation in Norwegian aquaculture 2014 (Fiskehelserapporten 2014). In: The Norwegian Veterinary Instiute, p. 38. 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 Crosbie, P.B., Ogawa, K., Nakano, D., Nowak, B.F., 2010. Amoebic gill disease in hatcheryreared ayu, Plecoglossus altivelis (Temminck & Schlegel), in Japan is caused by Neoparamoeba perurans. J. Fish Dis. 33, 455-458.

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ACCEPTED MANUSCRIPT Crosbie, P.B., Bridle, A.R., Cadoret, K., Nowak, B.F., 2012. In vitro cultured Neoparamoeba perurans causes amoebic gill disease in Atlantic salmon and fulfils Koch's postulates. Int.

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Culling, C.F.A., Allison, R.T., Barr, W.T., 1985. Haematoxylin and its counterstains. In: Cellular Pathology Technique. Butterworth & Co Ltd., pp. 157-160.

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Dykova, I., Novoa, B., 2001. Comments on diagnosis of amoebic gill disease (AGD) in turbot,

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Scophthalmus maximus. Bull. Eur. Assoc. Fish Pathol. 21, 40-44.

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Dykova, I., Figueras, A., Novoa, B., 1995. Amoebic gill infection of turbot Scophthalmus maximus. Folia Parasitol. 42, 91-96.

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Dykova, I., Figueras, A., Novoa, B., Casal, J.F., 1998. Paramoeba sp., an agent of amoebic gill

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disease of turbot Scophthalmus maximus. Dis. Aquat. Org. 33, 137-141. Feehan, C.J., Johnson-Mackinnon, J., Scheibling, R.E., Lauzon-Guay, J.S., Simpson, A.G., 2013.

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Validating the identity of Paramoeba invadens, the causative agent of recurrent mass mortality of sea urchins in Nova Scotia, Canada. Dis. Aquat. Org. 103, 209-227. Fringuelli, E., Gordon, A.W., Rodger, H., Welsh, M.D., Graham, D.A., 2012. Detection of Neoparamoeba perurans by duplex quantitative Taqman real-time PCR in formalin-fixed, paraffin-embedded Atlantic salmonid gill tissues. J. Fish Dis. 35, 711-724. Imsland, A.K., Reynolds, P., Eliassen, G., Hangstad, T.A., Foss, A., Vikingstad, E., Elvegård, T.A., 2014. The use of lumpfish (Cyclopterus lumpus L.) to control sea lice (Lepeophtheirus salmonis Krøyer) infestations in intensively farmed Atlantic salmon (Salmo salar L.). Aquaculture 424–425, 18-23.

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ACCEPTED MANUSCRIPT Karlsbakk, E., 2015. Amøbisk gjellesykdom (AGD)-litt om den nye plagen (in Norwegian). www.imr.no/filarkiv/2015/03/amobisk_gjellesykdom_agd.pdf/nb-no.

In,

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oppdrettet

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(in

Norwegian).

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www.imr.no/filarkiv/2014/03/sykdom_og_parasitter_i_vill_og_oppdrettet_rognkjeks.pdf/

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In, Havforskningsrapporten p. 37-39.

Karlsbakk, E., Olsen, A.B., Einen, A.-C.B., Mo, T.A., Fiksdal, I.U., Aase, H., Kalgraff, C., Skår,

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S.-Å., Hansen, H., 2013. Amoebic gill disease due to Paramoeba perurans in ballan wrasse (Labrus bergylta). Aquaculture 412–413, 41-44. M.L.,

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kisutch reared in sea water. Dis. Aquat. Org. 5, 163-169. Mitchell, S.O. and Rodger, H.D., 2011. A review of infectious gill disease in marine salmonid fish. J. Fish Dis. 34, 411-432 Muller, P.Y., Janovjak, H., Miserez, A.R., Dobbie, Z., 2002. Processing of gene expression data generated by quantitative real-time RT-PCR. Biotechniques 32, 1372-78. Nowak, B., Valdenegro-Vega, V., Crosbie, P., Bridle, A., 2014. Immunity to amoeba. Dev. Comp. Immunol. 43, 257-67. Olsvik, P.A., Lie, K.K., Jordal, A.E., Nilsen, T.O., Hordvik, I., 2005. Evaluation of potential reference genes in real-time RT-PCR studies of Atlantic salmon. BMC Mol. Biol. 6, 21. Rimstad, E., Mjaaland, S., Snow, M., Mikalsen, A.B., Cunningham, C.O., 2001. Characterization of the infectious salmon anemia virus genomic segment that encodes the putative hemagglutinin. J. Virol. 75, 5352-6. 21

ACCEPTED MANUSCRIPT Rønneseth, A., Haugland, G.T., Wergeland, H.I., 2013. Flow cytometry detection of infectious pancreatic necrosis virus (IPNV) within subpopulations of Atlantic salmon (Salmo salar

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Skiftesvik, A.B., Bjelland, R.M., Durif, C.M.F., Jahansen, I.S., Browman, H.I., 2013. Delousing

Aquaculture 402-403, 113-118.

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of Atlantic salmon (Salmo salar) by cultured vs. wild ballan wrasse (Labrus bergylta).

Steinum, T., Kvellestad, A., Ronneberg, L.B., Nilsen, H., Asheim, A., Fjell, K., Nygard, S.M.,

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Olsen, A.B., Dale, O.B., 2008. First cases of amoebic gill disease (AGD) in Norwegian

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seawater farmed Atlantic salmon, Salmo salar L., and phylogeny of the causative amoeba

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using 18S cDNA sequences. J. Fish Dis. 31, 205-14. Taylor, R.S., Muller, W.J., Cook, M.T., Kube, P.D., Elliott, N.G., 2009. Gill observations in Atlantic salmon (Salmo salar L.) during repeated amoebic gill disease (AGD) field exposure and survival challenge. Aquaculture 290, 1-8. Wergeland, H.I., Jakobsen, R.A., 2001. A salmonid cell line (TO) for production of infectious salmon anaemia virus (ISAV). Dis. Aquat Organ. 44, 183-90. Young, N.D., Crosbie, P.B., Adams, M.B., Nowak, B.F., Morrison, R.N., 2007. Neoparamoeba perurans n. sp., an agent of amoebic gill disease of Atlantic salmon (Salmo salar). Int J Parasitol. 37, 1469-1481.

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78, 217-223.

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Figure legends

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Figure 1. Validation of the PCR assays used to quantify P. perurans in the water. Positive and negative controls for the Paramoeba assay (Lane 2-6) and the ISAV assay (Lane 7-11). Lane

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1 and 12, 50 bp DNA Ladder (Invitrogen); lane 2, re-isolated P. perurans; lane 3, water sample

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from experiment III; lane 4, - RT control; lane 5, non-template control (NTC); lane 6, ISAV supernatant; lane 7, ISAV supernatant; lane 8, water sample from experiment III; lane 9, - RT

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control; lane 10, NTCand lane 11, re-isolated P. perurans. Teoretical size of the real-time RTPCR-product generated by the Paraemoeba assay is 139 bp and for the ISAV assay 117 bp.

Figure 2. Examination of AGD in lumpfish and quantification of P. perurans from gill tissues and water samples (Experiment I). A) Gill lesions were observed as white patches on the gills, often located basally on the filaments. B) Gill score of individual fish during the time course of infection. C) Quantification of amoeba in gill tissues and D) Quantification of amoeba in the water using real-time RT-PCR.

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ACCEPTED MANUSCRIPT Figure 3. Gill lesion in lumpfish and comparison of disease development in lumpfish and salmon (Experiment II). A) Gill lesion in lumpfish and salmon at 26 and 61 dpi. B) Gill score of

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individual fish during the time course of infection. L= lumpfish, S= salmon. C) Quantification of

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P. perurans within gills using real-time RT-PCR. White dots= salmon samples and black dots= lumpfish samples. C) Quantification of amoebae in the water using real-time RT-PCR. White

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bars= level of P. perurans in the tank with salmon. Black bars = level of P. perurans in the tank

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with lumpfish.

Figure 4. Salmon develop AGD after cohabitant challenge from lumpfish (Experiment III).

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A) Gill scoring of individual fish during the time course of infection. x= not available for

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examination. B) Macroscopic lesions of lumpfish with score 2. C) Histopathological findings

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showing hyperplasia of epithelial cells and the formation of interlamellar spaces with amoebae (arrows). In the inserted picture in right panel, amoeba with the characteristic junxtanuclear parasome is shown (arrow). D) Histopathological findings showing extensive proliferation of epithelial cells and spongiosis (*). Amoebae are present on the gill surface (arrow head in the middle panel). In the right panel, neutrophils (blue arrow) and eosinophilic granular cells (EGC) are seen in the filament vessel and EGC also in hyperplastic tissue (long arrow). E) Quantification of P. perurans within gill tissues from lumpfish (black dots) and salmon (white dots) using real-time RT-PCR. F) Quantification of amoebae in the water using real-time RTPCR. Black bars= level of P. perurans in the tank with lumpfish only. White bars = level of P. perurans in the tank with both lumpfish and salmon.

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E 1.96 1.80 1.78 2.00 2.02

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y -3.43 -3.91 -3.98 -3.32 -3.27

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Target Use P. perurans EF1A_Lumpfish Quantify amoeba in gills EF1A_Salmon P. perurans Quantify amoeba in water ISAV segm 6

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ACCEPTED MANUSCRIPT Statement of Relevance

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1) The last few years, lumpfish have been used as cleaner fish in order to delouse farmed Atlantic salmon. However, nothing is yet known about its susceptibility to the parasitic amoeba P. perurans.

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2) Currently, nothing is known about amoebic gill disease (AGD) development in lumpfish and the possibility that amoeba may be transferred from lumpfish to salmon.

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3) There is currently little information about the pathology (macroscopic and histological changes) in lumpfish.

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4) We have found that the commonly used gill scoring system for salmon is not applicable for lumpfish, and we recommend that the lumpfish is screened, not scored before transfer to net-pens with salmon.

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Lumpfish develop AGD after experimental challenge Lumpfish are less susceptible to P. perurans compared to Atlantic salmon Lumpfish can transfer amoebae to Atlantic salmon The commonly used gill scoring system for salmon is not applicable for lumpfish, and we recommend that lumpfish have to be screened, not scored before transfer to net-pens with salmon

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1) 2) 3) 4)

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