Rainbow Trout Head Kidney Macrophages Research Articles

Tenacibaculum dicentrarchi produce outer membrane vesicles (OMV) that are associated with the cytotoxic effect in rainbow trout head kidney macrophages.

Tenacibaculum dicentrarchi is the second most important pathogen in Chilean salmon farming. This microorganism causes severe skin lesions on the body surface of farmed fish. The bacterium can also adhere to surfaces and form biofilm, survive in fish skin mucus, and possess different systems for iron acquisition. However, the virulence mechanisms are still not fully elucidated. Outer membrane vesicles (OMV) are nanostructures released by pathogenic Gram-negative bacteria during growth, but none has been described yet for T. dicentrarchi. In this study, we provide the first reported evidence of the fish pathogen T. dicentrarchi producing and releasing OMV from 24 h after incubation, increasing thereafter until 120 h. Analyses were conducted with T. dicentrarchi TdCh05, QCR29, and the type strain CECT 7612T . The OMV sizes, determined via scanning electron microscopy, ranged from 82.25 nm to 396.88 nm as per the strain and incubation time point (i.e., 24 to 120 h). SDS-PAGE revealed that the number of protein bands evidenced a drastically downward trend among the T. dicentrarchi strains. In turn, the OMV shared five proteins (i.e., 22.2, 31.9, 47.7, 56.3, and 107.1 kDa), but no protein pattern was identical. A heterogeneous amount of protein, RNA, and DNA were obtained, depending on the time at which OMV were extracted. Purified OMV were biologically active and induced a cytotoxic effect in macrophage-enriched cell cultures from rainbow trout (Oncorhynchus mykiss) head kidneys. This is the first step towards understanding the role that OMV could play in the pathogenesis of T. dicentrarchi.

Protein Nanoparticles Made of Recombinant Viral Antigens: A Promising Biomaterial for Oral Delivery of Fish Prophylactics.

In the search for an eminently practical strategy to develop immunostimulants and vaccines for farmed fish, we have devised recombinant viral antigens presented as “nanopellets” (NPs). These are inclusion bodies of fish viral antigenic proteins produced in Escherichia coli. Soluble recombinant proteins are too labile to endure the in vivo environment and maintain full functionality, and therefore require encapsulation strategies. Yet when they are produced as nanostructures, they can withstand the wide range of gastrointestinal pH found in fish, high temperatures, and lyophilization. Moreover, these nanomaterials are biologically active, non-toxic to fish, cost-effective regarding production and suitable for oral administration. Here, we present three versions of NPs formed by antigenic proteins from relevant viruses affecting farmed fish: the viral nervous necrosis virus coat protein, infectious pancreatic necrosis virus viral protein 2, and a viral haemorrhagic septicemia virus G glycoprotein fragment. We demonstrate that the nanoparticles are taken up in vitro by zebrafish ZFL cells and in vivo by intubating zebrafish as a proof of concept for oral delivery. Encouragingly, analysis of gene expression suggests these NPs evoke an antiviral innate immune response in ZFL cells and in rainbow trout head kidney macrophages. They are therefore a promising platform for immunostimulants and may be candidates for vaccines should protection be demonstrated.

Characterisation of arginase paralogues in salmonids and their modulation by immune stimulation/ infection

In this study we show that four arginase isoforms (arg1a, arg1b, arg2a, arg2b) exist in rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar). We have characterised these molecules in terms of a) sequence analysis, b) constitutive expression in different tissues, and modulated expression following c) stimulation of head kidney macrophages in vitro, or d) vaccination/infection with Yersinia ruckeri and e) parasite infection (AGD caused by Paramoeba perurans and PKD caused by Tetracapsuloides bryosalmonae). Synteny analysis suggested that these arginase genes are paralogues likely from the Ss4R duplication event, and amino acid identity/similarity analyses showed that the proteins are relatively well conserved across species. In rainbow trout constitutive expression of one or both paralogues was seen in most tissues but different constitutive expression patterns were observed for the different isoforms. Stimulation of rainbow trout head kidney macrophages with PAMPs and cytokines also revealed isoform specific responses and kinetics, with arg1a being particularly highly modulated by the PAMPs and pro-inflammatory cytokines. In contrast the type II arginase paralogues were induced by rIl-4/13, albeit to a lesser degree. Vaccination and infection with Y. ruckeri also revealed isoform specific responses, with variation in tissue expression level and kinetics. Lastly, the impact of parasite infection was studied, where down regulation of arg1a and arg1b was seen in two different models (AGD in salmon and PKD in trout) and of arg2a in AGD. The differential responses seen are discussed in the context of markers of type II responses in fish and paralogue subfunctionalization.

Fish cell cultures as in vitro models of pro-inflammatory responses elicited by immunostimulants

We have tested the elicitation of innate defence-related responses in two stromal cell lines derived from the spleen (trout splenic stroma, TSS) and the pronephros (trout pronephric stroma-2, TPS-2) of rainbow trout (Oncorhynchus mykiss) after they were exposed to different concentrations of lipopolysaccharide (LPS), levamisole, or polyinosinic polycytidylic acid (poly-I:C). For comparison, cultures of rainbow trout head kidney macrophages were also included in the study, and the effect of the immunostimulants on the phagocytic activity, the intracellular and extracellular reactive oxygen species and nitric oxide production were assayed. Although the responses varied depending upon the concentration of the immunostimulants and the particular cell line, our results demonstrate that those activities were enhanced in the TSS and TPS-2 cell lines after exposure to any of the immunostimulants. These results indicate that the stromal cells of the main lympho-haemopoietic organs of O. mykiss develop innate defence responses, which are enhanced by well-known immunostimulants. In addition, such enhancement of the defence responses in the TSS and TPS-2 cell lines could be also elicited when they were exposed to conditioned supernatants from levamisole- or poly I:C-stimulated HK macrophage cultures, thus demonstrating that the haemopoietic stromal cells respond to macrophage-derived factors. Moreover, we demonstrate that the stromal cell lines constitutively expressed the Toll-like receptors TLR3, TLR5 and TLR9 genes. The results are discussed considering the role of the lympho-haemopoietic stromal cells in the innate immune responses, and the possibility of using histiotypic cell cultures of non-leucocyte cells of the haemopoietic organs to develop in vitro methods to select new immunostimulant candidates for aquaculture.

Persistence of Yersinia ruckeri in trout macrophages

Yersinia ruckeri is the etiological agent of enteric redmouth disease, a systemic infection which mainly affects salmonids. Although this important freshwater pathogen was discovered in 1966, little is known about its virulence mechanisms. In the present study, the interactions with rainbow trout head kidney macrophages were investigated. In vitro experiments were performed to measure uptake, intracellular survival, respiratory burst response and macrophage viability after exposure to Y. ruckeri. Additionally, the fate of Y. ruckeri in the head kidney after immersion infection was studied in vivo. Results show that Y. ruckeri induced the production of reactive oxygen species and that this response peaked at around 3 h after exposure. Despite these toxic substances, Y. ruckeri is able to survive in vitro inside trout macrophages for at least 24 h. Transmission electron microscopy demonstrated that Y. ruckeri bacteria are sequestered in autophagocytic compartments without fusion with primary lysosomes. Inside these compartments, bacteria were capable of replicating. Immersion infection of juvenile rainbow trout resulted in steadily increasing numbers of bacteria in the head kidney over time. As the infection progressed, Y. ruckeri shifted from a predominantly extracellular phase during the first week after infection to an intracellular phase inside the host macrophages from day 7 onwards. In conclusion, this study clearly demonstrates the capacity of Y. ruckeri to survive in rainbow trout macrophages in vitro as well as in vivo, confirming its facultative intracellular nature.

Survival and replication of Piscirickettsia salmonis in rainbow trout head kidney macrophages

Piscirickettsia salmonis is pathogenic for a variety of cultured marine fish species worldwide. The organism has been observed within host macrophages in natural disease outbreaks among coho salmon and European sea bass. In vitro studies, incorporating transmission electron microscopy (TEM) and ferritin loading of lysosomes, have confirmed that P. salmonis is capable of surviving and replicating in rainbow trout macrophages. Certain features of this intracellular survival underline its difference to other intracellular pathogens and suggest that a novel combination of defence mechanisms may be involved. Escape into the macrophage cytoplasm is not used as a means to avoid phago-lysosomal fusion and the organism remains at least partly enclosed within a vacuole membrane. While the piscirickettsial vacuole is often incomplete, survival and replication appear to require occupation of a complete, tightly-apposed, vacuolar membrane which does not fuse with lysosomes. Unlike some mammalian rickettsiae, actin-based motility (ABM) is not used as a means of intercellular spread. It is postulated that the presence of numerous small vesicles within vacuoles, and at gaps in the vacuolar membrane, may result from the blebbing of the piscirickettsial outer membrane seen early in the infection.

Antibody increases phagocytosis and killing of Lactococcus garvieae by rainbow trout (Oncorhynchus mykiss, L.) macrophages

The present study reports that specific antibody increased the bactericidal activity of rainbow trout head-kidney macrophages against virulent capsulated Lactococcus garvieae in the absence of complement. The observed increased bactericidal activity appeared to result from increased phagocytosis of capsulated L. garvieae in the presence of specific antibody and may in part explain the protective effect of antibody previously reported against this disease.

Immunostimulatory CpG oligodeoxynucleotides stimulate expression of IL-1β and interferon-like cytokines in rainbow trout macrophages via a chloroquine-sensitive mechanism

Synthetic oligodeoxynucleotides (ODNs) containing unmethylated CpG motifs are known to stimulate immune responses and are potent adjuvants in higher vertebrates, but so far the effects in fish are poorly described. We here report that CpG ODNs induce IL-1β expression and production of interferon-like cytokines in rainbow trout head-kidney macrophages, whereas ODNs with an inverted motif (GpC) have a much less stimulatory effect. We further demonstrate that endosomal maturation is essential for CpG signalling, as chloroquine, a compound known to block endosomal acidification, inhibits cytokine expression in the macrophages.

The immune response of rainbow trout (Oncorhynchus mykiss) againstAphanomyces invadans

Aphanomyces invadans, also referred toA. invaderisorA. piscicida, is the aetiological agent of epizootic ulcerative syndrome (EUS). Immune responses of rainbow trout (Oncorhynchus mykiss) against this fungus were examined, including both antibody and macrophage responses. The fish antibodies to the fungus, assessed by an enzyme-linked immunosorbent assay (ELISA) were generally non-specific, but sera from fish vaccinated withA. invadansreacted with bands at 55 and 40kDa by Western blot analysis. Macrophage activity was examinedin vitroby culturing rainbow trout head kidney macrophages with fungal spores. The macrophages were able to phagocytose sporesin vitroand, in some cases, hyphae were observed growing from germinated spores within the macrophage. When low concentrations of spores (1×102and 1×103spore ml−1) were cultured with macrophage monolayers, no germination of spores or growth of hyphae was observed. At concentrations of 1×104spore ml−1or higher, macrophages were unable to kill all the spores and fungal mycelium eventually overgrew the culture. Transmission electron microscopy (TEM) was used to examine the ultrastructural interaction between macrophages and the fungus. The possible role of rainbow trout as a model forA. invadansinfection in salmonids is also discussed.

A role for scavenger receptors in phagocytosis of protein-coated particles in rainbow trout head kidney macrophages

In macrophages of higher vertebrates, Fc receptors and receptors for complement and other serum factors, are generally known to enhance the phagocytic process. In lower vertebrates like salmonid fishes, none of these or other phagocytic receptors have been thoroughly characterized. The purpose of this study was to elucidate to what extent these and other receptors are involved in the process of phagocytosis in rainbow trout ( Oncorhynchus mykiss) head kidney macrophages. We used tosyl activated, paramagnetic dynabeads (2.8 μm in diameter), specifically coated with 12;I labeled Atlantic salmon ( Salmo salar) IgM or bovine serum albumin (BSA) as phagocytic probes. The effect of complement opsonization was also investigated by incubating the beads in serum. Our results indicate that neither the Fc- nor the complement-receptor(s) were important for phagocytosis of these beads. Our data support the idea that scavenger receptors are involved in phagocytosis in rainbow trout head kidney macrophages, as the use of a competitive scavenger receptor ligand extensively decreased degradation of the labeled protein coat on the beads.

In vitro activation of rainbow trout macrophages stimulates inhibition of Renibacterium salmoninarum growth concomitant with augmented generation of respiratory burst products

DAO Diseases of Aquatic Organisms Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsSpecials DAO 25:175-183 (1996) - doi:10.3354/dao025175 In vitro activation of rainbow trout macrophages stimulates inhibition of Renibacterium salmoninarum growth concomitant with augmented generation of respiratory burst products Hardie LJ, Ellis AE, Secombes CJ Rainbow trout head kidney macrophages pretreated for 48 h with a supernatant which contained macrophage activating factor (MAF) obtained from Concanavalin A/phorbol myristate acetate stimulated rainbow trout leucocytes express an enhanced ability to inhibit Renibacterium salmoninarum growth in vitro compared with untreated controls. Enhanced 'killing' capacity of these macrophages was detectable after 24 h exposure to R. salmoninarum in vitro. Addition of NG-methyl-L-arginine in vitro did not diminish bacterial growth inhibition by MAF-activated macrophages, suggesting that nitric oxide radicals were not involved in this event. However, MAF-treated macrophages expressed enhanced respiratory burst activity compared with control macrophages after a 3 d exposure to R. salmoninarum in vitro. In addition, it was demonstrated that R. salmoninarum was susceptible to hydrogen peroxide (H2O2) in vitro using a cell-free assay. Introduction of catalase to the in vitro macrophage assay abrogated the 'killing' activity, suggesting that H2O2 release from activated macrophages was involved in the inhibition of R. salmoninarum growth. The results are discussed in relation to other intracellular pathogens and the possible importance of cell-mediated immunity for protection against bacterial kidney disease. Renibacterium salmoninarum . Macrophage activation . Respiratory burst . Growth inhibition Full text in pdf format PreviousNextExport citation RSS - Facebook - Tweet - linkedIn Cited by Published in DAO Vol. 25, No. 3. Publication date: June 06, 1996 Print ISSN:0177-5103; Online ISSN:1616-1580 Copyright © 1996 Inter-Research.

The role of reactive oxygen species in the killing of the bacterial fish pathogen Aeromonas salmonicida by rainbow trout macrophages

The role of reactive oxygen species (ROS) in the killing of the bacterial pathogen Aeromonas salmonicida by in vitro cultured rainbow trout (Oncorhynchus mykiss) head kidney macrophages has been investigated. Using a microtitre plate bactericidal assay the respiratory burst pathway responsible for ROS generation was inhibited completely using the NADPH oxidase inhibitor trifluoperazine and partially by the use of ROS scavengers (formate and/or mannitol) and the antioxidative enzymes SOD and/or catalase. Increased bacterial survival of an avirulent strain (MT004) in the presence of trifluoperazine, catalase and formate confirmed that ROS such as hydrogen peroxide and hydroxyl radicals were indeed an important killing agent. This was further confirmed using sodium nitroprusside and aminotriazole, inhibitors of SOD and catalase respectively. The presence of aminotriazole caused a significantly increased bactericidal activity against a virulent strain (MT423) normally resistant to killing by cultured resident rainbow trout head kidney macrophages.

Fluid-phase endocytosis by rainbow trout head-kidney macrophages

Fluid-phase endocytosis in rainbow trout macrophages was studied using 125 I-poly (vinylpyrrolidone) (125I-PVP) as a marker. Uptake of 125I-PVP was measurable at 12° C using a high cell concentration (30-50 × 106 cell ml-1) and after extensive washing of the cells by centrifugation. The amount of extracellular fluid taken up after 30 min incubation with labelled PVP was estimated as 3.6±2.2 nl/106 cells, corresponding to a fluid uptake of approximately 1·4% of the cell volume per hour. Uptake of 125 I-PVP was temperature dependent and was not saturable after coincubation with excess unlabelled PVP. Fluid-phase endocytosis of labelled PVP did not follow a linear time course. This seemed to be caused by exocytosis of internalised 125I-PVP to the medium.