Inflammatory and regenerative responses in salmonids following mechanical tissue damage and natural infection.

Locale responses in muscle tissue against either a sterile tissue damage or infection were compared in salmonid fish in order to examine the inflammatory responses and regeneration of tissue. From higher vertebrates both damage and infection are known to cause inflammation since DAMPs released from injured cells as well as PAMPs from the surface of pathogens are immunogenic. To examine this in salmonid fishes, Atlantic salmon (Salmo salar) were infected with Moritella viscosus, the causative agent of winter ulcer. Muscle tissue was sampled from infected fish at 4, 7 and 14 days post infection. Samples were obtained from site of lesions and from locations without clinical signs of disease and lesions. The tissue damage was performed in rainbow trout (Oncorhynchus mykiss) by applying sterile needles to skin and muscle tissue to one side of the fish. Samples were taken 7, 14, 21, 28 and 42 days post injury from the injured side and non-injured site (internal control). From both infected and damaged fish, samples were subject to real-time RT-PCR for measuring the expression of IL-1beta, IL-8, IL-10, Hsp70, iNOS, TGF-beta, TLR-5m, TLR-9, TLR-22, TGF-beta, MMP-2, CTGF, myostatin-1alphabeta and collagen-1alpha which are coding for immunological factors and tissue regeneration. Locale, inflammatory responses were seen as strong up-regulation of IL-1beta and IL-8 in both groups of fish, but it was more pronounced in infected fish. Expression of the toll-like receptors showed induction of TLR-5m following infection, but TLR-9 and TLR-22 following damage. Further, in both studies the regenerative genes TGF-beta, MMP-2, CTGF, myostatin-1alphabeta were induced, but showed different kinetics. Collagen-1alpha was only induced in infected fish, probably due to heavier tissue damage in these.

Differential expression of immune genes in Atlantic salmon (Salmo salar L.) challenged intraperitoneally or by cohabitation with IPNV.

Atlantic salmon smolts challenged intraperitoneally (ip) and by cohabitation with a highly virulent strain of infectious pancreatic necrosis virus showed strong activation of important immune genes in spleen, liver, head-kidney and gill measured by real-time quantitative PCR. The genes investigated were IL-1beta, IL-10, IFN-alpha, IFN-gamma, Mx, MHC-I, MHC-II, TCR-alpha, CD8-alpha and mIgM. A low final cumulative mortality of about 10% was seen in the ip-challenged group, while more than 40% of the cohabitants died in the sampling period. Sampling was performed at day 15, 24 and 37 post ip-challenge. Overall, the expression of the investigated genes varied highly. The expression of IL-1beta, IL-10, MHC-II, TCR-alpha, CD8-alpha and mIgM showed more or less the same patterns between the two groups of fish by being significantly upregulated at day 24 post ip-challenge. However, the degree of regulation varied a lot among the genes. A pattern showing differences between ip-challenged and cohabitants were seen for IFN-gamma and especially IFN-alpha, where the upregulation seemed to last longer for the cohabitants. The Mx gene was the most induced gene, but also the one with highest individual variance. Mx but also MHC-I were both still highly upregulated at the last sampling point within both groups of fish. The results seem to indicate that the differences in expression pattern(s) could reflect the different routes of entrance of the virus into the fish. This could maybe explain the different kinetics in the onset and the degree of mortality or the potential different molecular mechanisms used for combating the virus.

Cloning and expression of TNF-alpha, IL-1beta and COX-2 in an anadromous and landlocked strain of Atlantic salmon (Salmo salar L.) during the smolting period.

The parr-smolt transformation involves complex modulation of immune parameters, affecting both cell populations and humoral factors. The expression of cytokines was studied in salmon cells and tissues during this period using an anadromous and a landlocked freshwater resident dwarf strain of Atlantic salmon (Salmo salar L.). The constitutive activity of three immunoregulatory genes encoding the cytokines tumour necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) and the cyclo-oxygenase (COX) isoform COX-2 was investigated in head kidney, spleen and gill tissue from healthy, unvaccinated fish by real-time PCR. The TNF-alpha gene was generally lower expressed than COX-2 and IL-1beta1, which were approximately expressed at equal levels and constitutive expression was seen for COX-2 and IL-1beta1 in all tissues examined and at all sampling dates. The expression of all three genes in head kidney and spleen tissue seemed to be highest at the sampling in May for both strains around the time of seawater transfer suggesting an influence of smolting related hormones on cytokine expression. The gill tissue experienced the highest expression of IL-1beta1 and COX-2 at all sampling dates indicating that this organ is immunologically important.