Evolutionary, comparative, and functional analyses of STATs and regulation of the JAK-STAT pathway in lumpfish upon bacterial and poly(I:C) exposure

BackgroundThe Janus kinase/signal transducers and activators of transcription (JAK-STAT) system regulates several biological processes by affecting transcription of genes as a response to cytokines and growth factors. In the present study, we have characterized the STAT genes in lumpfish (Cyclopterus lumpus L.), belonging to the order Perciformes, and investigated regulation of the JAK-STAT signaling pathway upon exposure to bacteria (Vibrio anguillarum) and poly(I:C), the latter mimicking antiviral responses.MethodsCharacterization and evolutionary analyses of the STATs were performed by phylogeny, protein domain, homology similarity and synteny analyses. Antibacterial and antiviral responses were investigated by performing KEGG pathway analysis.ResultsWe observed that lumpfish have stat1a, 2, 3, 4, 5a, 5b, and 6. Transcriptome-wide analyses showed that most components of the JAK-STAT pathway were present in lumpfish. il-6, il-10, il-21, iκBα and stat3 were upregulated 6 hours post exposure (hpe) against bacteria while type I interferons (IFNs), irf1, irf3, irf10, stat1 and 2 were upregulated 24 hpe against poly(I:C).ConclusionsOur findings shed light on the diversity and evolution of the STATs and the data show that the STAT genes are highly conserved among fish, including lumpfish. The transcriptome-wide analyses lay the groundwork for future research into the functional significance of these genes in regulating critical biological processes and make an important basis for development of prophylactic measure such as vaccination, which is highly needed for lumpfish since it is vulnerable for both bacterial and viral diseases

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

The C4B6⁻ cells change morphology upon stimulation with various mitogens.

<p>Left panels, inverted microscope pictures. Middle panels, fluorescence microscopy pictures of cells stained with anti-TO antiserum, captured with 40× objective. Right panels, fluorescence microscopy pictures of cells stained with anti-TO antiserum, captured with 63× objective. (A) Cells without mitogen, 24 hrs incubation. (B) Cells stimulated with Con A and PMA for 18 hrs. (C) Cells stimulated with LPS for 24 hrs.</p

Antibiotic uptake by cultured Atlantic cod leucocytes and effect on intracellular Francisella noatunensis subsp. noatunensis replication

The granuloma disease caused by Francisella noatunensis subsp. noatunensis in farmed Atlantic cod has not been successfully treated by use of antibacterials, even when antibacterial resistance testing indicates a sufficient effect. The reason for this treatment failure may be the intracellular existence of the bacteria within immune cells, mainly macrophages. To investigate the effect of antibacterials on intracellular Francisella replication, we established a protocol for the detection of drugs within Atlantic cod immune cells using high-performance liquid chromatography (HPLC). When the uptake and intracellular concentrations of oxolinic acid and flumequine were analysed in isolated adherent head kidney leucocytes (HKLs) by HPLC, we found that uptake was rapid and the intracellular concentrations reflected the extracellular exposure concentrations. To investigate the effect of the antibacterial compounds on intracellular bacterial replication, adherent HKLs experimentally infected with the bacteria were analysed using flow cytometry and intracellular labelling of bacteria by specific antibodies. We found that flumequine did not inhibit intracellular bacterial replication. Unexpectedly, the results indicated that the intracellularly effiacy of the drug was reduced. The HPLC method used proved to be highly applicable for accurate determination of intracellular drug concentrations. When combined with sensitive and specific flow cytometry analyses for identification and measurement of intracellular bacterial replication, we suggest that this approach can be very valuable for the design of antibacterial treatments of intracellular pathogens

The C4B6⁻ cells are negative for lymphocyte-, neutrophil and monocyte/macrophage markers, but express CD83 and MHC class II.

<p>(A) Flow cytometry analyses of the unbound fraction after MACS show the C4B6⁻ cells' reactivity with MAb C4B6 (anti-leukocytes), C7G7 and G2H3 (B-cells), E3D9 (neutrophils) and the polyclonal anti-human CD3 antiserum (T- and IgM⁺ cells). Representative histograms are shown. Grey filled curves are negative controls. Positive cells are shown as red dots. The markers represent positive cells. The antibodies reactivity against PBL prior to MACS is shown in the lowest panels. (B) A silver stained SDS- polyacrylamide gel, left panel, and an immunoblot using polyclonal IgM serum developed with ECL, right panel. Lane 1, molecular mass markers; lane 2, PBL; lane 3, unbound fraction after MACS (C4B6⁻ cells); lane 4, bound fraction after MACS (C4B6⁺ cells); lane 5, salmon IgM. The 66 KDa band (*) in lane 3 and 4 is most likely BSA present in the MACS buffer. (C) qRT-PCR analysis of the C4B6⁻ cells. Gene expression of the following genes; IgM (B-cell marker), CD3, CD8 and TCRα (T-cell markers), MCSF-R (monocyte/macrophage marker), CD86 (involved in antigen presentation), CD83 (DC marker), MHC class II (APC), GATA-1 and G6F (thrombocyte/erythrocyte markers) is presented as mean normalized expression (MNE) using EF1α as reference gene (n = 4). The average of triplicates from four fish with standard error is shown. Note different scale in the inserted histogram.</p