Microbial Communities in a Flow-Through Fish Farm for Lumpfish (Cyclopterus lumpus L.) During Healthy Rearing Conditions

Lumpfish can efficiently remove sea lice from Atlantic salmon in net-pens, and production of lumpfish in closed fish farms is a new, fast developing industry in Norway. However, periodic outbreaks of bacterial diseases in the fish farms represent a large problem, both economically and ethically. Therefore it is important to obtain a better understanding of how microbial communities develop in these production facilities. Knowledge on the characteristics of microbial communities associated with healthy fish could also enable detection of changes associated with disease outbreaks at an early stage. In this study we have monitored microbial communities in a fish farm for lumpfish during normal operational conditions with no disease outbreak by using 16S rRNA gene amplicon sequencing. The study involved weekly samplings of water and biofilms from fish tanks, and fish. The results revealed that the microbial communities in fish tank water were different from the intake water. The water and biofilm in fish tanks were highly similar in regards to microbial community members, but with large differences in relative abundances for some taxa. The sampled fish were associated with mostly the same taxa as in tank water and biofilm, but more variation in relative abundances of different taxonomic groups occurred. The microbial communities in the fish farm seemed stable over time, and were dominated by marine bacteria and archaea within Alphaproteobacteria, Epsilonproteobacteria, Flavobacteria, Gammaproteobacteria, Thaumarchaeota, Planctomycetes, Sphingobacteriia, and Verrucomicrobiae (>10% relative abundance). Bacterial genera known to include fish-pathogenic strains were detected in all types of sample materials, but with low relative abundances (<5%). Exceptions were some samples of fish, biofilm and water with high relative abundance of Tenacibaculum (<85.8%) and Moritella (<82%). In addition, some of the eggs had a high relative abundance of Tenacibaculum (<89.5%). Overall, this study shows that a stable microbial community dominated by various genera of non-pathogenic bacteria is associated with a healthy environment for rearing lumpfish. Taxa with pathogenic members were also part of the microbial communities during healthy conditions, but the stable non-pathogenic bacteria may limit their growth and thereby prevent disease outbreaks.publishedVersio

Susceptibility of Atlantic cod Gadus morhua, halibut Hippoglossus hippoglossus and wrasse (Labridae) to Aeromonas salmonicida subsp. salmonicida and the possibility of transmission of furunculosis from farmed salmon Salmo salar to marine fish

The possibility of transmission of Aeromonas salmonicida subsp. salmonicida from Atlantic salmon Salmo salar to cod Gadus morhua, halibut Hippoglossus hippoglossus, and wrasse (Labridae) was studied in both laboratory cohabitation and injection challenge trials, and in a field situation during an outbreak of furunculosis in Atlantic salmon in 2 sea farms containing both cod and salmon. A, salmonicida subsp, salmonicida was isolated from cod, halibut and wrasse, but only occasionally. The injection challenge studies with A, salmonicida subsp. salmonicida were carried out on cod and halibut. Despite challenge with 4.6 x 10(3), 4.6 x 10(4) and 4.6 x 10(5) CFU (colony-forming units) of the bacterium, only 1 halibut died, whereas no mortality occurred in the cod groups. In cohabitation challenge trials in which cod, halibut and wrasse were kept together with infected salmon, 1 cod and 1 halibut died, with subsequent isolation of A, salmonicida subsp. salmonicida from their kidneys. In a group of salmon challenged in the same way with infected salmon cohabitants, all the salmon died. In the field study, there were high mortalities of salmon due to furunculosis. However, A, salmonicida subsp. salmonicida was isolated from only 1 of the wrasse used as 'cleaner fish' for the salmon. A. salmonicida was not isolated from cod during the field study. The results suggest that A, salmonicida subsp. salmonicida can be transmitted from salmon and cause disease in cod, halibut and wrasse, but that this is a relatively rare event

Microbiome dataset from a marine recirculating aquaculture system (RAS) for salmon post-smolt production in Norway

A marine aquaculture recycling system (RAS) for the production of post-smolt was monitored for microbial community structures during the first year of operation. Sample material was obtained monthly from the biofilter biofilm carriers, the production water (tank 3), the fish skin (tank 3) and the tank 3 wall biofilm. Additional samples were taken during outbreaks of fish skin wounds, washing of the plant, UV filtration of the inlet water and from various wall biofilms. Samples for depth profiles from all fish tanks were also collected. The sampling tools were a ladle for capturing biofilter biofilm carriers, toothbrushes for wall biofilm capture, filters for capture of water microbes and scalpels for skin tissue slicing. The sampling times were indicated by the production cycle number (cycle 2-5) and the week number within the cycle (W). Prior to bacterial community analysis, the stored samples were exposed to cell lysis and extraction of environmental DNA by commercial kits. All samples were subjected for PCR amplification of 16S rDNA sequences for library formations and prepared for Ion Torrent technology, which sequences 250 bp fragments. A total of 1.1 million reads were obtained from the 100 RAS samples analysed. The process from Ion Torren analysis to library involved bioinformatics steps with sorting, filtering, adjustment and taxonomic identification, and the final output was shown in a table as operational taxonomic units (OTUs) and relative abundance at different sampling sites and sampling time points. Of a total of 450 taxonomically assigned OTUs, 45% were classified at genus level. The 16S library raw data are deposited in the Mendeley data repository and cited in this Data in Brief article co-submitted with the article “Microbial colonization and stability in a marine post-smolt RAS inoculated with a commercial starter culture.” [1]. So far, the raw data are referenced in four more publications in progress. These cover microbial shifts and enrichments between sampling times, sulfur cycling, “in vivo biofilm” and identification of relatives of fish pathogens in RAS. All library sequences are available in GenBank with accession numbers MN890148-MN891672.publishedVersio

Neutrophils in Atlantic salmon (Salmo salar L.) are MHC class II+ and secret IL-12p40 upon bacterial exposure

Antigen-presentation via major histocompatibility complex (MHC) to T cells is the key event to initiate adaptive immune responses. In teleosts, as in mammals, the main types of professional antigen-presenting cells (APCs) are dendritic cells (DCs), monocytes/macrophages, and B cells. In the current study, flow cytometry, immunostaining and qPCR have been used to show that neutrophils in the teleost fish Atlantic salmon (Salmo salar L.) have antigen-presenting properties. The neutrophils were positive for MHC class II, CD83 and CD80/86, and upon in vitro bacterial exposure, gene expression analysis of purified neutrophils showed that IL-12p40, which is essential for proliferation of naïve T cells, was highly upregulated at both 6 and 24 h post bacterial exposure. Based on presence of MHC class II and upregulation of molecules involved in antigen presentation and T cell activation, we suggest that neutrophils in Atlantic salmon have potential to function as professional APCs. This work makes an important basis for further exploring the potential of using neutrophils to develop new, targeted immunoprophylactic measures.publishedVersio

Antibacterial treatment of lumpfish (Cyclopterus lumpus) experimentally challenged with Vibrio anguillarum, atypical Aeromonas salmonicida and Pasteurella atlantica

Lumpfish is a novel farmed species used as cleaner fish for the removal of lice from farmed salmon. As often with new, farmed species, there are challenges with bacterial infections. The frequency of prescription of antibiotic agents to lumpfish is increasing, despite the lack of knowledge about appropriate doses, duration of treatment and application protocols for the various antibacterial agents. In the current study, we have tested the effect of medicated feed with florfenicol (FFC), oxolinic acid (OA) and flumequine (FLU) on lumpfish experimentally challenged with Vibrio anguillarum, atypical Aeromonas salmonicida and Pasteurella atlantica. We found that all three antibacterial agents efficiently treated lumpfish with vibriosis using 10 and 20 mg kg−1 day−1 of FFC, 25 mg kg−1 day−1 of OA and 25 mg kg−1 day−1 FLU, whereas only FFC (20 mg kg−1 day−1) had good effect on lumpfish with pasteurellosis. None of the antibacterial agents were efficient to treat lumpfish with atypical furunculosis. FFC 20 mg kg−1 day−1 showed promising results in the beginning of the experiment, but the mortality increased rapidly 14 days post-medication. Efficient treatment is important for the welfare of lumpfish and for reducing the risk of development of antibiotic-resistant bacteria. To our knowledge, this is the first study to establish protocols for antibacterial treatment of lumpfish.publishedVersio

Phaeobacter gallaeciensis Reduces Vibrio anguillarum in Cultures of Microalgae and Rotifers, and Prevents Vibriosis in Cod Larvae

Phaeobacter gallaeciensis can antagonize fish-pathogenic bacteria in vitro, and the purpose of this study was to evaluate the organism as a probiont for marine fish larvae and their feed cultures. An in vivo mechanism of action of the antagonistic probiotic bacterium is suggested using a non-antagonistic mutant. P. gallaeciensis was readily established in axenic cultures of the two microalgae Tetraselmis suecica and Nannochloropsis oculata, and of the rotifer Brachionus plicatilis. P. gallaeciensis reached densities of 107 cfu/ml and did not adversely affect growth of algae or rotifers. Vibrio anguillarum was significantly reduced by wild-type P. gallaeciensis, when introduced into these cultures. A P. gallaeciensis mutant that did not produce the antibacterial compound tropodithietic acid (TDA) did not reduce V. anguillarum numbers, suggesting that production of the antibacterial compound is important for the antagonistic properties of P. gallaeciensis. The ability of P. gallaeciensis to protect fish larvae from vibriosis was determined in a bath challenge experiment using a multidish system with 1 larva per well. Unchallenged larvae reached 40% accumulated mortality which increased to 100% when infected with V. anguillarum. P. gallaeciensis reduced the mortality of challenged cod larvae (Gadus morhua) to 10%, significantly below the levels of both the challenged and the unchallenged larvae. The TDA mutant reduced mortality of the cod larvae in some of the replicates, although to a much lesser extent than the wild type. It is concluded that P. gallaeciensis is a promising probiont in marine larviculture and that TDA production likely contributes to its probiotic effect

Exploring the Effect of Phage Therapy in Preventing <i>Vibrio anguillarum</i> Infections in Cod and Turbot Larvae

The aquaculture industry is suffering from losses associated with bacterial infections by opportunistic pathogens. Vibrio anguillarum is one of the most important pathogens, causing vibriosis in fish and shellfish cultures leading to high mortalities and economic losses. Bacterial resistance to antibiotics and inefficient vaccination at the larval stage of fish emphasizes the need for novel approaches, and phage therapy for controlling Vibrio pathogens has gained interest in the past few years. In this study, we examined the potential of the broad-host-range phage KVP40 to control four different V. anguillarum strains in Atlantic cod (Gadus morhua L.) and turbot (Scophthalmus maximus L.) larvae. We examined larval mortality and abundance of bacteria and phages. Phage KVP40 was able to reduce and/or delay the mortality of the cod and turbot larvae challenged with V. anguillarum. However, growth of other pathogenic bacteria naturally occurring on the fish eggs prior to our experiment caused mortality of the larvae in the unchallenged control groups. Interestingly, the broad-spectrum phage KVP40 was able to reduce mortality in these groups, compared to the nonchallenge control groups not treated with phage KVP40, demonstrating that the phage could also reduce mortality imposed by the background population of pathogens. Overall, phage-mediated reduction in mortality of cod and turbot larvae in experimental challenge assays with V. anguillarum pathogens suggested that application of broad-host-range phages can reduce Vibrio-induced mortality in turbot and cod larvae, emphasizing that phage therapy is a promising alternative to traditional treatment of vibriosis in marine aquaculture

Pharmacokinetics of florfenicol in lumpfish (Cyclopterus lumpus L.) after a single oral administration

Farming of lumpfish for biological removal of sea lice from farmed Atlantic salmon has expanded rapidly in Europe and Canada over the last 5–6 years and the lumpfish has become an economically important species. There are, however, health challenges associated with bacterial diseases. In recent years, there has been an increase in antibacterial treatments prescribed for this fish species despite a lack of knowledge regarding pharmacokinetics and effect of treatment with different antibiotics. The present study examined the uptake, tissue distribution, metabolism and elimination of the antibacterial agent florfenicol in lumpfish (Cyclopterus lumpus L.) following a single oral administration of 10 mg/kg fish given in feed. Plasma, head kidney, liver and muscle from six fish were sampled at each time point and analysed by liquid chromatography/mass spectrometry (LC-MS). Absorption was moderate for this drug characterised by a calculated peak plasma concentration (Cmax) of 3.55 μg/ml obtained after 21.2 hours (Tmax) and the elimination halflife (t1/2β) relatively extended in plasma at 30 hours. Area under curve (AUC) and AUC from 0 to 24 hours (AUC0-24h) were calculated to be 248 and 61 h μg/ml, respectively. Cmax was calculated to 2.99 μg/g in muscle, 2.54 μg/g in liver and 4.70 μg/g in head kidney with corresponding Tmax of 22.1, 26.4 and 19.4 h, respectively. The main metabolite, florfenicol-amine was found in low concentrations in plasma and all tissues examined. The minimum inhibition concentrations (MIC) for florfenicol of 28 of Aeromonas salmonicida isolates from diseased lumpfish ranged from 0.39 to 1.56 μg/ml. The pharmacokinetical data presented here make an important basis for efficient antibacterial treatment for lumpfish using florfenicol and for calculation of suitable withdrawal time. Knowledge of florfenicol pharmacokinetics, combined with determination of antibiotic resistance among fish pathogenic bacteria and the effect of antibacterial agents on diseased lumpfish in vivo are important for the welfare of lumpfish and prevention of resistant bacteria.publishedVersio

Pharmacokinetic Data Show That Oxolinic Acid and Flumequine Are Absorbed and Excreted Rapidly From Plasma and Tissues of Lumpfish

This study examined the uptake, tissue distribution and elimination of the antibacterial agents oxolinic acid and flumequine in lumpfish (Cyclopterus lumpus L.) by use of LC-MS/MS following a single oral administration of 25 mg/kg fish given in feed. Lumpfish are increasingly used as cleaner fish for removal of sea lice on commercially farmed salmon. The production of lumpfish is successful, but there are challenges with bacterial infections and the number of antibacterial treatments has increased in recent years. As the lumpfish is a novel species to farming, there is a need for pharmacokinetic data and establishment of protocols for efficient antibacterial treatment. The current study describes the pharmacokinetic properties of oxolinic acid and flumequine in lumpfish. Absorption of oxolinic acid was moderate and was characterized by a calculated peak plasma concentration (Cmax) of 2.12 μg/ml after 10.3 h (Tmax) and an elimination half-life (t1/2β) of 21 h. Area under curve (AUC) and AUC from 0 to 24 h (AUC0−24h) were calculated to be 60.9 and 34.0 h μg/ml, respectively. For flumequine, plasma Cmax was found to be 2.77 μg/ml after 7.7 h (Tmax) with t1/2β of 22 h. The area under the curve (AUC) and AUC from 0 to 24 h (AUC0−24) were calculated as 104.3 and 50.3 h μg/ml, respectively. Corresponding Cmax values in muscle, liver, and head-kidney for oxolinic acid were 4.01, 3.04, and, 4.68 μg/g, respectively and Tmax of 11.1, 9.2, and 10.0 h, respectively. For flumequine, Cmax values of 4.16, 4.01, and 7.48 μg/g were obtained in muscle, liver, and head kidney, respectively, with corresponding Tmax values of 10.2, 10.3, and 6.0 h. Antimicrobial susceptibility values as determined by minimum inhibitory concentration (MIC) analyses against 28 isolates of Aeromonas salmonicida isolated from diseased lumpfish ranged from 0.06 to 15 μg/ml for oxolinic acid and 0.024 to 6.25 μg/ml for flumequine. Bimodal distributions in susceptibility to both oxolinic acid and flumequine were observed. The combination of pharmacokinetic properties and MIC data make possible calculation of efficient treatment doses, which are needed to improve the welfare of lumpfish and minimize development of antibiotic resistant bacteria.publishedVersio