Francisella noatunensis in Atlantic cod (Gadus morhua L.); waterborne transmission and immune responses.

This is the first report that confirms waterborne transmission of francisellosis in Atlantic cod. To investigate the transmission of disease, particle reduced water was transferred from a tank with intraperitoneally infected cod to a tank with healthy cod. Waterborne transmission of Francisella noatunensis was confirmed in the effluent group using immunohistochemistry and real-time quantitative PCR (RT-qPCR). The bacteria were located inside the accumulated macrophage-like cells. Specific and high antibody responses against live and inactivated bacteria were observed. Oil adjuvant had no effect on the antibody responses against inactivated F. noatunensis compared to saline formulation. The antigen epitope was a 20-25 kDa component of F. noatunensis suggested to be lipopolysaccharide detected by Western blot, Sypro Ruby and Silver staining. Systemic immune reactions were investigated by measuring the expression of IFN-γ, IL-1ß and IL-10 genes with RT-qPCR. After i.p. injection of live bacteria, a significant up-regulation of IFN-γ and IL-1ß expression was observed from 15 to 60 days post infection in spleen and head kidney. In intestine, IFN-γ was significantly up-regulated after 30 days whereas rectum showed no significant differences in expression. Elevated expression of IL-10 was observed in all the organs tested but was only significantly up-regulated at 60 days post infection in intestine from i.p. infected fish. For the cohabitant group, IL-1ß and IFN-γ was up-regulated in spleen whereas intestine and rectum showed a down-regulation after 60 days. IL-10 was up-regulated in intestine of cohabitant fish from day 30 to day 60. These results indicate that F. noatunensis infection provokes both specific antibody responses and long term inflammatory responses in cod. The present study provides new knowledge about infection routes and shows that both humoral and cellular defence mechanisms are triggered by F. noatunensis in cod.

Vibriosis vaccines based on various sero-subgroups of Vibrio anguillarum O2 induce specific protection in Atlantic cod (Gadus morhua L.) juveniles.

The purpose of this study was to investigate the efficacy of three monovalent and a trivalent vibriosis dip vaccines in juvenile Atlantic cod (Gadus morhua L.), examine whether the responses were specific and study the expression of selected immune genes after dip vaccination. In addition, the study addressed whether the deviating isolates of Vibrio anguillarum serotype O2 belongs to another sero-subgroup than the previously established sero-subgroups O2a, O2b and O2c. Rabbit V. anguillarum serotype O2 antiserum adsorbed with V. anguillarum O2a O-antigen was shown, by both ELISA and immunoblotting, to still contain serotype O2 specific antibodies. Cod V. anguillarum serotype O2 antiserum reacted only with isolate of homologous serotype and not with heterologous sero-subgroups. This indicates that the deviating V. anguillarum O2 isolates represent a new sero-subgroup differing from sero-subgroup O2a. The monovalent vaccines included formalin inactivated cultures of V. anguillarum sero-subgroup O2a, O2b or serotype O2, while the trivalent vaccine contained all three sero-subgroups. Cod mounted high protection 7 weeks post dip vaccination with monovalent vaccines when challenged with homologous isolates and significantly lower when challenged with heterologous isolates, regardless of sero-subgroups. The trivalent vaccine resulted in efficient protection against all sero-subgroups tested. Dip vaccination of cod juveniles did not result in detectable antibody production or alteration in gene expression of the heavy chain of IgM and IgD. In the trivalent vaccine group expression of IFNγ and IL-12p40 were significantly up-regulated 3 days post vaccination. However, in groups vaccinated against V. anguillarum sero-subgroups O2b or O2, IL-12p40 and IFNγ gene expression were slightly increased 3 and 55 days post vaccination, respectively.

Cathepsin D from Atlantic cod (Gadus morhua L.) liver. Isolation and comparative studies.

The isolated cathepsin D-like enzyme from Atlantic cod (Gadus morhua L.) liver was shown to be a monomer with a molecular mass of approximately 40 kDa. It was inhibited by Pepstatin A and had an optimum for degradation of haemoglobin at pH 3.0. The purified enzyme had lower temperature stability than bovine cathepsin D. Antibodies raised against the purified enzyme and against two C-terminal peptides of cod cathepsin D recognized a 40 kDa protein in immunoblotting of the samples from the purification process. Both antisera showed cross reactivity with a similar sized protein in liver from cod, saithe (Pollachius virens L.), Atlantic herring (Clupea harengus L.) and Atlantic salmon (Salmo salar L.). A protein of same size was detected in wolffish (Anarhichas lupus L.) liver with the antibody directed against the purified enzyme. This antibody also recognized the native enzyme and detected the presence of cathepsin D in muscle of cod, saithe, herring and salmon. These antibodies may be useful in understanding the mechanisms of post mortem muscle degradation in fish by comparing immunohistochemical localization and enzyme activity, in particular in cod with different rate of muscle degradation. They may also be used for comparing muscle degradation in different fish species.

Atlantic salmon ISG15: Expression and conjugation to cellular proteins in response to interferon, double-stranded RNA and virus infections.

ISG15 is one of the earliest and most predominant proteins to be induced in mammals following IFN-alpha/beta stimulation, which suggests that it has an important function in the interferon system. Similar to ubiquitin, ISG15 forms covalent conjugates with its target proteins, but free ISG15 is released from human lymphocytes and monocytes during IFN-alpha/beta stimulation. In this work we describe a 17.3 kDa ISG15 orthologue in Atlantic salmon (AsISG15) with characteristic features of ISG15 proteins including tandem ubiquitin-homology domains and a conserved carboxy-terminal conjugating motif (LRLRGG). Furthermore, Northern blot analysis revealed strong induction by polyinosinic polycytidylic acid (poly I:C) and by viral infections, while Western blot analysis using a specific antibody generated against AsISG15 confirmed induction mediated by recombinant Atlantic salmon IFN-alpha/beta and demonstrated conjugation of AsISG15 to cellular proteins. Interestingly, the pattern of AsISG15 modified target proteins differed during ISAV infection compared to direct IFN-alpha/beta stimulation. Immunoprecipitation experiments demonstrated extracellular, free AsISG15 in supernatants of leucocytes stimulated with poly I:C. Moreover, immunoprecipitation of an about 65 kDa ISAV protein from infected TO cells using anti-AsISG15 antiserum suggests that binding between the AsISG15 and the ISAV protein occurred. Taken together, the results suggest that AsISG15 has a role in the antiviral interferon response of Atlantic salmon.

Inhibition of infectious pancreatic necrosis virus replication by atlantic salmon Mx1 protein.

Mx proteins form a family of interferon (IFN)-induced GTPases with potent antiviral activity against various single-stranded RNA viruses in mammals and chickens. In fish, alpha/beta IFN has been reported to inhibit the replication of infectious pancreatic necrosis virus (IPNV), but the mode of action has not been elucidated. A correlation between the inhibition of IPNV and Mx protein expression has, however, been observed. To examine whether Atlantic salmon Mx1 protein (ASMx1) possesses antiviral activity against IPNV, CHSE-214 cells constitutively expressing ASMx1 were established. ASMx1 appeared to be localized in the cytoplasm. The ASMx1-expressing clone selected showed a severely reduced IPNV-induced cytopathic effect, which was confirmed by a 500-fold reduction in virus yield. The antiviral activity against IPNV was further confirmed by the inhibition of virus protein synthesis and the reduced accumulation of virus transcripts. The present work further adds to the body of evidence which suggests that antiviral activity is a major functional role of vertebrate Mx proteins. Moreover, the list of viruses inhibited by Mx proteins is extended to include double-stranded RNA viruses.

Atlantic salmon interferon genes: cloning, sequence analysis, expression, and biological activity.

In this work, we report cDNA cloning of two type I interferons (IFNs) from the head kidney of Atlantic salmon, called SasaIFN-alpha1 (829 bp) and SasaIFN-alpha2 (1290 bp). Both translate into 175 amino acid precursor molecules showing 95% amino acid sequence identity. The precursors have a putative 23 amino acid signal peptide, which suggests that the mature Atlantic salmon IFNs contain 152 amino acids (18.2 kDa). Salmon IFN appears to have five alpha-helices, similar to mammalian and avian type I IFNs, and showed 45% sequence identity with zebrafish IFN, up to 29% identity with mammalian IFN-alpha sequences, and 17%-18% sequence identity with mammalian IFN-beta and chicken type I IFNs. Human embryonic kidney 293 cells transfected with the SasaIFN-alpha1 cDNA gene produced high titers of acid-stable antiviral activity, which protected salmonid cells against infectious pancreatic necrosis virus (IPNV) and also induced Mx protein in the cells. Poly(I)-poly(C) induced two IFN transcripts in head kidney of Atlantic salmon. Genomic IFN sequences contained four introns and five exons, which is different from the intronless type I IFN genes of birds and mammals.

An antiviral state induced in Chinook salmon embryo cells (CHSE-214) by transfection with the double-stranded RNA poly I:C.

Type I interferons (IFN alpha and beta) convert vertebrate cells into an antiviral state by inducing expression of proteins that inhibit virus replication. In humans and mice, Mx proteins constitute one family of interferon-induced antiviral proteins. Mx genes have recently been cloned from Atlantic salmon and rainbow trout. Moreover, double-stranded RNA (dsRNA) and type I IFN-like activity have been shown to induce Mx protein in salmonid cells. Chinook salmon embryo cells (CHSE-214 cells) have been suggested to have a defect in the IFN-system because the dsRNA polyinosinic polycytidylic acid (poly I:C) failed to induce an antiviral state in the cells. We have studied this phenomenon more closely in the present work. CHSE-214 cells were either transfected with poly I:C or incubated with poly I:C without transfection reagent. The cells were then studied for Mx protein expression and protection against infectious pancreatic necrosis virus (IPNV) infection. The results showed that cells transfected with poly I:C were protected from IPNV infection, whilst cells incubated with poly I:C were not protected. Cells transfected with the double-stranded DNA poly dI:dC were also not protected against IPNV. Mx protein was expressed in CHSE-214 cells upon transfection with poly I:C, but not after incubation with poly I:C alone. Stimulation of CHSE-214 cells with supernatants from cells transfected with poly I:C, induced protection against IPNV, indicating production of type I IFN-like activity. These results suggest that CHSE-214 cells in fact are able to produce type I IFN, but may have defects in the mechanisms mediating uptake of poly I:C or may degrade unprotected poly I:C.