Eirin Fausa Pettersen

Phagocytosis by B-cells and neutrophils in Atlantic salmon (Salmo salar L.) and Atlantic cod (Gadus morhua L.)

Phagocytosis by fish cells has mostly been studied using adherent leucocytes, excluding suspended cells such as the majority of B-cells and neutrophils, but a recent study describes professional phagocytosis of latex beads and bacteria by B-cells from rainbow trout. In the present study, phagocytosis by B-cells and neutrophils from salmon and cod was studied. Leucocytes were isolated from peripheral blood (PBL) and head kidney (HKL). By flow cytometry analyses, proportions of MAb labelled cell populations with internalized fluorescent beads, as well as the number of beads within each cell, could be determined. Phagocytic capacity and ability were demonstrated in B-cells and neutrophils from salmon and cod. In salmon, B-cells had higher phagocytic ability than neutrophils in HKL, but not in PBL. For cod the phagocytic ability of B-cells were lower than for neutrophils in both HKL and PBL, but the phagocytic capacity of cod B-cells were higher than for neutrophils in both HKL and PBL. For salmon B-cells the phagocytic capacity was lower than or similar to neutrophils in HKL and PBL. The total phagocytic ability of leucocytes was different in the species studied. The highest phagocytic ability was observed in cod, showing similar values for PBL and HKL. Salmon PBL displayed about twice the phagocytic ability of cod PBL. There seemed to be some major differences between the two fish species concerning phagocytosis. In salmon, a rather large proportion of phagocytic leucocytes were phagocytic B-cells, indicating that B-cells may have an important function in particle clearance in this species. In cod, phagocytic leucocytes in HKL and PBL were mostly neutrophils, and only a small proportion of B-cells were phagocytic, supporting the more prominent role of innate immune functions in cod neutrophils.

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‐1β, IL‐10, IFN‐α, IFN‐γ, Mx, MHC‐I, MHC‐II, TCR‐α, CD8‐α 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‐1β, IL‐10, MHC‐II, TCR‐α, CD8‐α 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‐γ and especially IFN‐α, 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.

A highly phagocytic cell line TO from Atlantic salmon is CD83 positive and M-CSFR negative, indicating a dendritic-like cell type

Leucocyte cell lines are valuable tools for immunological studies. In this study the TO cell line, originating from Atlantic salmon head kidney leucocytes, is described with respect to enzyme cytochemistry, functional studies, reactivity with leucocyte specific antibodies and immune gene expression. Pronounced characteristics of the TO cell line are the rapid adherence to the plastic growth surface, high phagocytic capacity and bactericidal functions. No respiratory burst activity, and little or no NO production were detected under the experimental conditions tested, and thus the TO cells appear to have other effective killing mechanisms. The cells are reactive with a leucocyte specific monoclonal antibody (MAb), but does not bind a neutrophil specific MAb or stain for myeloperoxidase. Real-time RT-PCR showed the expression in TO cells of several immune genes, some of which were significantly regulated following LPS stimulation. The expression of CD83 might indicate a dendritic cell (DC) origin of the TO cells, as this marker is considered a hallmark for DC. Expression of TCR-alpha or the macrophage marker M-CSFR was not detected. Based on the present analyses the TO cells display a mixture of known characteristics for macrophages and DCs. At the same time the TO cells lack some central functions of phagocytic/myeloid cells. As the TO cells are developed to a long-term culture one cannot exclude that some functions might have been lost in this process. Nevertheless, the features of the TO cells indicate their potential as a model system for immunological studies of salmon phagocytic cells.

Isolation of rainbow trout neutrophils with an anti-granulocyte monoclonal antibody

A magnetic cell sorting system has been optimised for the purification of rainbow trout neutrophils using a monoclonal antibody (E3D9) raised against Atlantic salmon neutrophils. The purified neutrophils have good viability (85%) and purity (approximately 92%), and were functional in respiratory burst and migration assays. The isolated neutrophils responded rapidly to PMA stimulation, producing levels of superoxide anion (4.85 nmols superoxide min-1/10(6) cells) approximately twice as high as macrophages from the same species. In the migration assay, there was a four-fold increase in migrating cells using the purified neutrophils compared with unfractionated blood leucocytes, and a relatively high neutrophil migratory activity was seen in the absence of serum.

The intracellular lifestyle of Francisella noatunensis in Atlantic cod (Gadus morhua L.) leucocytes.

Francisella noatunensis causes the systemic granulomatous inflammatory disease, francisellosis in cod. Little is known about the lifestyle of this facultative intracellular bacterium within cod leucocytes. We have examined the interaction of this bacterium with phagocytic cells isolated from cod with emphasis on monocytes, macrophages, neutrophils and phagocytic B-cells. It is clear from confocal microscopy sections through adherent cell preparations that numerous bacteria were located intracellularly following in vitro infection in monocytes and macrophages. In these sections bacteria were immunostained and cell actin was stained using Alexa Fluor® 488 phalloidin. Bacteria were observed in close association with neutrophils and intracellularly (low numbers) in B-cells. Bacteria were observed more frequently in head kidney- than in peripheral blood- and spleen- leucocytes. Following infection, bacteria were initially observed grouped together and located close to the nucleus. Later they were found spread within the cytoplasm. This indicates regression of F. noatunensis from the phagosome to the cytoplasm where replication possibly takes place. It may be hypothesised that the bacteria may alter maturation of the phagosome and thus, avoid the potent intracellular killing mechanisms of phagocytic cells. The intracellular lifestyle involving escape to cytoplasm prior to fusion with the lysosome may have consequences for vaccine development as well as antibiotic treatment of infected cod.

Leucocyte populations and responses to immunization and photoperiod manipulation in Atlantic salmon (Salmo salar L.) 0+ smolt

Abstract The immune status of season-independent Atlantic salmon smolt may be affected by changes in photoperiod and high rearing temperatures. Variations in different leucocyte subpopulations were measured in photo-manipulated out-of-season (0+) Atlantic salmon smolt by use of flow cytometry and specific monoclonal antibodies. 0+ smolts were produced by exposing parr to continuous light (LD 24:0) until June 10 followed by a “winter” photoperiod (LD 12:12) for 6 weeks, and then continuous light. Three different groups were studied; untreated smolt (UT), smolt injected with a commercial multivalent oil-adjuvanted vaccine (VA), and smolt injected with the saline/oil-adjuvant used in the vaccine (AD). The fish were injected in the “winter” photoperiod. From introduction of “winter” photoperiod until 2 weeks after seawater transfer, fractions of B-cells (identified by G2H3), neutrophiles (identified by E3D9) as well as polymorphonuclear and B-cells (identified by C4B6) were monitored. From introducing continuous photoperiod and until seawater transfer, there were variations for all groups within the leucocyte subpopulations identified by the monoclonal antibodies (MAb) used. Neutrophiles had a minor increase, while the number of B-cells decreased. C4B6, identifying both types of cells recognised by E3D9 and G2H3 remained stable, indicating that the leucocyte fraction identified by C4B6 was not changed, but there was redistribution with respect to the numbers of B-cells and neutrophiles. After seawater transfer, another redistribution of leucocytes took place. A minor reduction for the neutrophiles was observed, while at the same time, both B-cells and the polymorphonuclear cells increased. It was not possible to identify any effect of vaccine and/or adjuvant on the leucocytes studied. The specific serum antibody levels measured by enzyme-linked immunosorbent assay (ELISA) were low during the experimental period. A low immune response was observed measured as antibody-producing cells (APC) by ELISPOT. The low levels of specific antibodies supported the findings of low APC numbers. There were indications that UT had lower numbers of APC compared to AD and VA. The results show that changes with respect to fractions of leucocyte populations take place during parr–smolt transformation, and these changes may affect the cellular and humoral immune response to vaccination. Different photoperiod regimes may have different effects on leucocyte populations and general conclusions should not be made from the single photoperiod studied.

Flow cytometry assays of respiratory burst in Atlantic salmon (Salmo salar L.) and in Atlantic cod (Gadus morhua L.) leucocytes.

The oxidation of dihydrorhodamine 123 (DHR) to the fluorescent rhodamine 123 (RHO) was detected using flow cytometry. This assay for detection of respiratory burst activity was established in peripheral blood leucocytes (PBL) and head kidney leucocytes (HKL) of Atlantic salmon and Atlantic cod. The leucocytes were stimulated by phorbol 12-myristate 13-acetate (PMA). For cod cells 10 times lower concentration of PMA had to be used compared to salmon cells, as higher concentrations were toxic and resulted in considerable cell death. The cells found to be RHO-positive were monocytes/macrophages and neutrophils based on the scatter dot plots, but for salmon also some small cells were found to have high fluorescence intensity both in the flow cytometry analyses and by fluorescence microscopy of cytospin preparations. The nature of these cells is not known. For cod leucocytes, such cells were not obvious. The instrument settings are a bit more demanding for cod, as cod cells die more easily compared to salmon cells. In both assays the limit between negative and positive cells has to be carefully considered. The presented flow cytometry protocols for measurements of respiratory burst in salmon and cod leucocytes can be applied in various studies where respiratory burst functions are involved, such as to verify if it is activated or suppressed in connection with infections and immunostimulation.

Flow cytometry assay for intracellular detection of Infectious Pancreatic Necrosis virus (IPNV) in Atlantic salmon (Salmo salar L.) leucocytes

Infectious Pancreatic Necrosis virus (IPNV) is traditionally detected in adherent leucocytes using immunofluorescence labelled specific antibodies, PCR or by further cultivation of infected cell material in cell lines. We present a flow cytometry (FCM) assay for detection of intracellular IPNV in salmon leucocytes, where each single cell is analysed for presence of virus. The method is established using in vitro challenge of salmon leucocytes and CHSE-214 cells. For detection of intracellular virus antigen the Cytofix/Cytoperm kit from BD is optimal compared with paraformaldehyde or acetone/methanol for cell permeabilisation. This is combined with labelling procedures allowing both internal virus antigen labelling and external antibody labelling of cell markers to identify B-cells and neutrophils. The secondary antibodies were Alexa Fluor 647 for the internal labelling and RPE for the external labelling of bound cell subtype specific antibodies. The presences of virus within cells are also demonstrated by confocal and light microscopy of infected cells. IPNV is successfully detected in blood and head kidney leucocyte samples. IPNV is found both in B-cells and neutrophils as well as in other types of leucocytes that could not be identified due to lack of cell-specific antibodies. Serial samples from cultivation of in vitro infected leucocytes and CHSE-214 cells analysed by flow cytometry showed that number of infected cells increased with increasing number of days. The flow cytometry protocol for detection of intracellular IPNV is verified using CHSE-214 cells persistently infected with IPNV. These analyses are compared with virus titre and virus infected naive CHSE-214 cells. The detection of IPNV in persistently infected cells indicates that carrier fish can be analysed, as such cells are considered to have virus titres similar to carriers.

Immunostaining of Atlantic salmon (Salmo salar L.) leucocytes

Different salmon leucocyte subpopulations were identified by immunostaining using rabbit antiserum raised against the salmonid cell line TO derived from head kidney leucocytes in combination with other available immunoglobulins. The rabbit anti-TO cell line serum immunostained all isolated leucocytes from head kidney, peripheral blood and spleen, as shown by analyses of these leucocytes by flow cytometry and by fluorescence microscopy. In cytospin preparations, the staining of salmon leucocytes using rabbit anti-TO serum as the primary antibody revealed greater morphological details compared to conventional staining procedures, especially among isolated spleen leucocytes where cells with a morphology usually limited to dendritic cells were seen. Other cells of various shapes and protrusions were also stained although the anti-TO serum did not stain protrusions on all cell types. Among the immunoglobulin positive cells, the thin protrusions were only seen when immunostained using anti-IgM antibody. The same was observed for neutrophils stained using the monoclonal E3D9 antibody. The double staining of cells using rabbit anti-TO serum and monoclonal antibodies specific for IgM positive cells or neutrophils clearly show how the morphology of these cells can be compared with the rest of the leucocyte population. The staining of salmon leucocytes by antiserum to a salmon leucocyte cell line TO provides a tool for staining the total population of salmon immune cells, and can be used in immunofluorescence or confocal microscopy in combinations with labelling of cellular components or pathogens. The detailed morphological characteristics, such as cell protrusions, visualized by the presented staining have not been observed on fish leucocytes by conventional cell staining procedures.