Bacteriolytic activity of rainbow trout (Oncorhynchus mykiss) complement

The total bacteriolytic activity comprising of the classical, alternative and possible lectine pathways as well as the bacteriolytic activity of the alternative pathway (AP) of rainbow trout (Oncorhynchus mykiss) complement was assessed in temperatures ranging from 0 to 35 °C against a recombinant strain Escherichia coli containing two reporter genes gfp and lucFF. At 35 °C there was no difference between the total (TC) activity and the activity of the AP, but at 10 °C the TC was notably higher than the AP. Total activity peaked at 30 °C and gradually grew smaller towards 0 °C. The activity of the AP was similarly temperature-dependent, but CB50 value was found to be beyond measurable range at temperatures below 10 °C. When compared to human serum complement, the peak human TC activity at 37 °C was four times higher than the TC of rainbow trout at 30 °C. Human TC activity was 10.1-fold lower at 25 °C when compared to the activity at 37 °C. At 37 °C the human AP bacteriolytic activity was 4.5-fold less effective than human TC, but at 25 °C there was no difference between human TC and AP. In contrast to previous reports where AP activity of fish was assayed as hemolytic activity our study showed that the bacteriolytic activity of AP was lower than that of TC and very low at temperatures below 10 °C suggesting that the earlier proposed particular importance of AP in fish should be reconsidered.     

In vitro leukocyte-encapsulation model in rainbow trout (Oncorhynchus mykiss)

We developed an in vitro model to study the cellular and molecular mechanisms of granulomatous inflammation in response to invading pathogens. Ichthyophonus hoferi was used as a target for encapsulation by cultivated leukocytes from the kidney of the rainbow trout (Oncorhynchus mykiss). The encapsulation process was observed over 1 week. The leukocytes were identified as either macrophages in the inner layer, or neutrophils and lymphocytes in the outer layer. The encapsulation response was inhibited by treatment with heat, but not formalin or methanol. The recognition of heat-unstable molecules on the pathogen surface could induce encapsulation. Increased expression of pro-inflammatory cytokines, such as interleukin (IL)-1beta, IL-8 and tumor necrosis factor-alpha2, was observed during encapsulation. These cytokines might play crucial roles in the encapsulation process. In particular, IL-8, which was expressed at a late phase, might recruit specific cell populations, such as the lymphocytes comprising the outer cellular layer around the target.     

The effect of social interactions on circadian self-feeding rhythms in rainbow trout Oncorhynchus mykiss Walbaum

Circadian feeding rhythms have been revealed in several fish species, but whether or not social interactions influence the expression of the rhythms remains largely unexplored. This paper reported such an exploration in rainbow trout. The experiment was conducted in two consecutive stages in two adjacent insulated rooms. In Stage 1, 40 fish (146.0+/-21.7 g, mean+/-S.D.) were held individually in Room 1. In Stage 2, those 40 fish from Room 1 were distributed into eight groups of five fish according to their previous circadian properties and placed in Room 2. Isolated trout were generally diurnal feeders, but variability among the individuals was evident, with five types of diurnal self-feeding pattern being detected. The influence of social interactions on diel self-feeding pattern in a group was found to be temporary and reversible. The grouping process did not necessarily enhance the expression of circadian self-feeding rhythms, and the dominant individual(s) did not determine the properties of circadian self-feeding rhythms in the group.     

ADP-ribosylation of rainbow trout (Oncorhynchus mykiss) actin by botulinum C2 toxin.

Intracellular actin of rainbow trout macrophages was ADP-ribosylated by botulinum C2 toxin, which is composed of two nonlinked protein components, component I and trypsinized component II. The actin in the supernatants of various tissue homogenates of the trout was also directly ADP-ribosylated by component I of C2 toxin, indicating that fish actin other than those of land vertebrates is susceptible to enzymatic modification by component I of C2 toxin.     

Effect of environmental temperature on rainbow trout (Oncorhynchus mykiss) innate immunity

Phagocytosis, complement lytic activity and opsonization capacity of rainbow trout plasma as well as the ability of phagocytes to recognize foreign particles were studied at different temperatures. Respiratory burst (RB) activity and opsonization capacity were assessed as chemiluminescence emission from diluted whole blood of fish which were acclimatized for 57 days at temperatures between 5 and 20 degrees C. RB activity was higher at higher acclimatization and in vitro assay temperatures. The peak time of RB was significantly delayed in fish kept at lower temperatures (5-10 degrees C) as compared to fish kept at 15 or 20 degrees C temperatures. Opsonization capacity of plasma decreased in fish acclimatized at low temperatures and was also affected by in vitro assay temperature. The importance of glucan receptors in RB activity increased in fish kept at higher temperatures and was also affected by the in vitro assay temperature. The higher acclimation temperatures increased the lytic activity of both total and alternative complement pathways.     

CK-1, a putative chemokine of rainbow trout (Oncorhynchus mykiss)

Summary: Chemokines arc small inducible proteins that direct the migration of leukocytes. While chemokines are well characterised in mammals, they have yet to be identified in fish. We have isolated a cDNA clone from rainbow trout (Oncorhynchus mykiss) which encodes a protein (CK-1) having structural features typical of chemokines. Amino-acid residues that define the β-chemokines of mammals are conserved in CK-1, including the paired cysteine motif, CC. Further similarities are shared with the C6 subfamily of β-chemokines. In contrast, the organisation of the CK-f gene is closer to that of mammalian α-chemokine genes than β-chemokine genes. The CK-1 gene is present in all four salmonid species examined and the nucleotide sequences of the exons are highly conserved. CK-1 has characteristics in common with mammalian α and β-chemokine suggesting that this salmonid chemokine gene preserves traits once present in the ancestral chemokine gene from which modern mammalian chemokine genes evolved.     

The biological effects of rainbow trout (Oncorhynchus mykiss) recombinant interleukin-8

In this report, recombinant interleukin-8 (rIL-8) was produced and its activity tested for the first time in fish. The rainbow trout rIL-8 was produced in Escherichia coli and purified using a 6xHis tag at the N-terminus. The rIL-8 induced a dose-dependent migration of head kidney leukocytes at concentrations from 0.1 to 10 ng/ml, with a peak response at 1 ng/ml. Trout rIL-8 also had a significant effect on superoxide production by head kidney cells, with maximal activity at 0.1 and 1 ng/ml. When injected intraperitoneally into trout, rIL-8 had a clear effect on total leukocyte number in the peritoneal cavity, with increasing doses (up to 5 microg) eliciting more cells. Of three leukocyte types distinguished, neutrophils were the dominant cell type, especially at higher rIL-8 concentrations. In contrast, the proportion of macrophages and lymphocytes decreased with rIL-8 administration, suggesting that they were not attracted at the same rate as neutrophils.     

In vitro immunostimulation of rainbow trout (Oncorhynchus mykiss) spleen cells with levamisole.

An in vitro immunization and cultivation method for fish spleen organ sections was used to investigate the effects of levamisole on the immune response. After 10 days of culture with either 50 micrograms/ml, 25 micrograms/ml, 5 micrograms/ml or no levamisole in the media, the nonspecific defense reactions were measured by determining the metabolic activity of neutrophils by using the nitroblue tetrazolium test, and phagocytic and adherence indexes by incubating the fish cells with suspensions of formalin-killed Staphylococcus aureus. The specific immune system activity was shown by the passive hemolytic plaque assay demonstrating the numbers of antibody-producing cells. Elevations were found in nonspecific defense and specific immune response in the 5 micrograms/ml levels of levamisole. The 25 micrograms/ml levels instigated a slight elevation in some nonspecific levels but showed suppression in the specific immune response. The 50 micrograms/ml levels suppressed all indicators whether levamisole was given alone or combined with Yersinia ruckeri O-antigen or DNP-Ficoll. These antigens are themselves nonspecific defense stimulators, and a synergistic effect was evident when combined with 25 micrograms/ml and 5 micrograms/ml levels of levamisole.     

Cortisol reduces cell proliferation in the telencephalon of rainbow trout (Oncorhynchus mykiss)

The fish brain grows throughout life, and new cells are added continuously in all major brain areas. As in mammals, the rate of adult brain cell proliferation in fish can be regulated by external factors including environmental complexity and interaction with conspecifics. We have recently demonstrated that the stress experienced by subordinate rainbow trout in social hierarchies leads to a marked suppression of brain cell proliferation in the telencephalon, and that this is accompanied by an increase in plasma levels of cortisol. Corticosteroid hormones are known to suppress adult neurogenesis in mammals, and to investigate whether this is also the case in fish, rainbow trout were fed feed containing either a low or a high dose of cortisol for 6 days. Compared to control animals receiving regular feed, both cortisol treated groups had significantly elevated cortisol levels 24 h after the last feeding, with the high group having levels comparable to those previously reported in socially stressed fish. To quantify cell proliferation, immunohistochemistry for proliferating cell nuclear antigen (PCNA) was performed to identify actively cycling cells. The density of PCNA-positive nuclei in the telencephalon was reduced by about 50% in both cortisol treated groups. The effect of cortisol on brain cell proliferation did not reflect a general down regulation of growth, as only the high cortisol group had reduced growth rate, and there was no correlation between brain cell proliferation and growth rate in any group. These results indicate that the reduced proliferative activity seen in brains of socially stressed fish is mediated by cortisol, and that there is a similar suppressive effect of cortisol on brain cell proliferation in the teleost forebrain as in the mammalian hippocampus.     

Ubiquitin-proteasome-dependent proteolysis in rainbow trout (Oncorhynchus mykiss): effect of food deprivation

The ubiquitin-proteasome proteolytic pathway is a major route of protein degradation and of particular importance in muscle proteolysis in mammals. In this study, the beta proteasome subunit N3 and polyubiquitin genes of the rainbow trout, Oncorhynchus mykiss, were sequenced and tissue distribution of gene expression was examined. The effects of 14-day food withdrawal were assessed on the N3 subunit and polyubiquitin gene expression in terms of mRNA, 20S proteasome proteolytic activity and ubiquitin protein abundance in trout liver and muscle. Both sequences are highly conserved, and the rainbow trout ubiquitin amino acid sequence is identical to the mammalian protein. The proteasome beta subunit N3 has 92% similarity to the Xenopus sequence. Starvation halved the polyubiquitin mRNA level in liver but had no effect on muscle levels. No significant effect of food withdrawal was observed on the proteasome mRNA in liver or muscle. Food withdrawal decreased the 20S proteasome proteolytic activity and the abundance of ubiquitin protein in both muscle and liver. Co-regulation of the proteasome and ubiquitin was indicated by the high correlation ( R=0.924) between 20S activity and ubiquitin abundance. Overall, this study demonstrates that starvation down-regulates the ubiquitin-proteasome pathway, possibly highlighting differences in the regulation of protein turnover in poikilothermic and endothermic animals.     

Immunoregulatory activities of eicosanoids in the rainbow trout (Oncorhynchus mykiss).

Eicosanoids such as prostaglandins (PG), leukotrienes (LT) and lipoxins (LX) have been shown to be potent immunoregulatory molecules in mammals. To determine if they have similar roles in 'lower' animals, rainbow trout (Oncorhynchus mykiss) were immunized with either sheep erythrocytes or Aeromonas salmonicida in the presence or absence of the stable analogue of PGE2, 16,16-dimethyl-PGE2, and the number of plaque-forming cells (PFC) or specific antibody levels determined. The higher dose of 16,16-dimethyl-PGE2 (200 micrograms/kg body weight) caused a significant reduction in both PFC number and antibody titre compared with the control. The effect of PGE2, PGE3, 16,16-dimethyl-PGE2, LTB4, LTB5, LXA4, 12-HETE and 12-HEPE on PFC generation following the in vitro challenge of trout splenocytes with sheep erythrocytes was also determined. All of the prostaglandins tested showed a dose-dependent inhibition of PFC after 11 days in culture, while of the remaining eicosanoids only LXA4 had any effect on PFC number, with a dose-dependent stimulatory effect. The cyclo-oxygenase inhibitor, indomethacin, also caused a stimulation in the number of PFC generated, with a maximal effect at c. 25 microM, while the lipooxygenase inhibitors, esculetin and nordihydroguaiaretic acid (5-100 microM), had no significant effect on PFC generation at all concentrations tested. The present results show that, as in mammals, prostaglandins and the cyclo-oxygenase pathway are also important in the regulation of the piscine humoral immune response. Of the lipoxygenase products tested, however, only LXA4 had any significant effect on PFC generation, suggesting that these compounds have only a limited role to play in immune regulation in this organism. Overall this work shows that eicosanoids have a long evolutionary history in immunoregulation, probably dating back at least to the appearance of bony fish some 400 million years ago.     

In vitro effect of levamisole on the neutrophil activity in rainbow trout (Oncorhynchus mykiss).

An in vitro immunization and cultivation method for fish spleen organ section was used to investigate the effects of levamisole on the neutrophil activity. After 10 days of culture with either 50 micrograms/ml, 25 micrograms/ml, 5 micrograms/ml or no levamisole in the media, the nonspecific defense reactions were measured by determining the metabolic activity of neutrophils by using the nitroblue tetrazolium test, and phagocytic and adherence indexes by incubating the fish cells with suspensions of formalin-killed Staphylococcus aureus. Elevations were found in nonspecific cellular defense in the 5 micrograms/ml levels of levamisole. The 25 micrograms/ml levels instigated a slight elevation in nonspecific cellular defense and the 50 micrograms/ml levels suppressed all indicators, whether levamisole was given alone or combined with Yersinia ruckeri O-antigen or DNP-Ficoll. These antigens are themselves nonspecific cellular defense stimulators, and a synergistic effect was evident when combined with 25 micrograms/ml and 5 micrograms/ml levels of levamisole.     

Genomic organisation of rainbow trout, Oncorhynchus mykiss TGF-beta

The genomic organisation of Oncorhnchus mykiss TGF-beta has been determined through the generation of contiguous clones by PCR. The O. mykiss TGF-beta gene is approximately 3.4 Kb in length and consists of 7 coding exons with no introns in the 5'-UTR. Whilst this is the same number of exons found in TGF-beta genes of amphibians, birds and mammals, in the O. mykiss gene intron 2 of other vertebrates is absent and an additional intron is present at the 3' end of the molecule, splitting exon 7 of the other known TGF-beta genes into two exons (trout exons 6 and 7). Comparison of exon sizes in the coding region support the suggestion that the Xenopus TGF-beta5 and trout TGF-beta sequences are the forerunners of TGF-beta1. Conservation of exons coding for the mature TGF-beta peptide is relatively high (63-73% identity) but other exons show lower identities (37-58%). Comparison of the TGF-beta intron sequences reveals that in general the O. mykiss introns are considerably shorter than the avian homologs. The impact of the teleost TGF-beta gene organisation on theories of the gene evolution of this cytokine family are discussed.     

Indoleamine 2,3-dioxygenase: First evidence of expression in rainbow trout (Oncorhynchus mykiss)

The role of enzymes as active antimicrobial agents of the innate immunity in teleost fish is proposed in diverse works. Secretion of Indoleamine 2,3-dioxygenase (IDO) has been described in higher vertebrates; it degrades l-tryptophan in extracellular environments associated mainly with mucosal organs. The effect of IDO on decreasing amino acid concentration may inhibit the growth of potential pathogens. In fish the study of this molecule is still. Here we report the identification of an Onchorhyncus mykiss IDO homologue (OmIDO). IDO was cloned, sequenced, and the primary structure shows conservation of key functional sites. The constitutive expression is altered when the fish is challenged with LPS as a pathogen-associated molecular pattern (PAMPs). Up-regulation of IDO was shown preferentially in the fish’s mucosal cells. In order to obtain evidence of a possible regulation mechanism, an in vitro cell model was used for to show that OmIDO is induced by rIFN. These study has identified a Indoleamine 2,3-dyoxigenase in O. mykiss will contribute to expands our knowledge of the function this protein in fish immune response. These findings allow to propose the use of OmIDO as a molecular indicator of strength of the animal’s immune response and wellbeing.     

The Gyrodactylidae fauna of rainbow trout Oncorhynchus mykiss Walbaum 1792 in the Rogg breeding pound in Bavaria, Germany

In July 2005, 107 rainbow trout in age 1+ from a salmonid farm in Southern Germany situated in the southern tributary area of the Danube river were examined. The aim of this study was to determine the gyrodactylid species found on rainbow trout and to identify their location on the host’s body. In total, 291 specimens from genus Gyrodactylus were collected. The most abundantly occurring species was Gyrodactylus truttae (181 specimens), whilst the others were less abundant. For the first time in Germany, Gyrodactylus teuchis and Gyrodactylus derjavinoides on rainbow trout were found. Most parasites occurred on the pectoral and ventral fins. Few specimens were found on the anal or caudal fins, in the oral cavity or on the gills. The only uninfected place was the nasal cavity.     

Characterization of early molecular sex differentiation in rainbow trout, Oncorhynchus mykiss.

Early differentiation in rainbow trout gonads was investigated by expression profiling and in situ hybridization (ISH). Expression of cyp19a1 and fst in females and sox9a1 in males were sexually dimorphic between 32 to 35 days post-fertilization (dpf). After 35 dpf, the differentiation proceeded with sexually dimorphic profiles for sox9a2, dmrt1, cyp11b2.1, amh in males and foxl2a, foxl2b, hsd3b1, inha in females. cyp17a1, cyp11a1, star, nr5a1b increased only after 40 dpf in both sexes with a slightly higher expression in females. cyp19a1 expression was localized in a cluster of somatic cells in the ventral side of female gonads, and sox9a2 and amh in somatic cells surrounding the germ cells, at 28 dpf and thereafter, both in male and female gonads. cyp11b2.1, cyp17a1, and cyp11a1 expressions were only detected in scattered somatic cells in males after 46 dpf. This confirms the early implication of cyp19a1 in trout ovarian differentiation and suggests that early testicular differentiation does not need androgen production.     

Antibody-antigen kinetics following immunization of rainbow trout (Oncorhynchus mykiss) with a T-cell dependent antigen.

Enhancement of the immune response through affinity maturation of the antibody response is a feature of the mammalian immune system and has important implications with respect to development of vaccination strategies. However, an absence of germinal centres and apparent lack of somatic hypermutation of immunoglobulin V genes suggests that this phenomenon does not occur in fish. We investigated the question of affinity maturation in rainbow trout (Oncorhynchus mykiss) by measuring antibody-antigen binding kinetics using a BIAcore biosensor. Following immunization with a T-cell dependent antigen (FITC-KLH), relative binding affinities of serum and mucosal antibodies were assessed based on their dissociation rate constants (k(diss.)). A detectable serum anti-FITC response developed by 4 weeks post-immunization, and a consistent shift to higher affinity antibody production (i.e. a decrease in k(diss.)) was observed over the ensuing course of the immune response. An average k(diss.) of 3.5 x 10(-4)+/-0.27 x 10(-4)sec(-1) was observed during early stages of the response (4 weeks), while by 6 weeks this decreased significantly (p<0.05). Further reduction in k(diss.) was observed, with a low of 1.2 x 10(-4)+/-0.06 x 10(-4)sec(-1) being observed by week 12. Analysis of the anti-FITC response in skin-derived mucus revealed a similar pattern of decreasing k(diss.) as the immune response progressed. While these data clearly demonstrate a 2-3 fold increase in antibody-antigen binding during the course of the immune response in trout, the magnitude of this increase is much less than that seen in the mammalian immune response. This may reflect differences in the mechanisms underpinning this phenomenon in divergent species.     

Leukocytes of rainbow trout (Oncorhynchus mykiss) pronephros: cell types producing superoxide anion

Fish pronephric and blood leukocytes yield a chemiluminescent response (CL) when stimulated appropriately. This response reflects the production of highly reactive oxygen derivatives which contribute to oxygen-dependent killing of targets such as pathogens and parasites. From suspensions of pronephric cells of the Rainbow trout, Oncorhynchus mykiss, we have obtained populations enriched for CL-positive cells. Four bands of cells were obtained using continuous gradients generated with 60% Percoll. The leukocytes of band II showed a very strong PMA-induced CL response, the magnitude of which was several times higher than that observed with equivalent numbers of cells form unseparated pronephric cell suspensions. Cells present in other bands were not significantly chemiluminescent. Flow cytometric analysis showed that band II contained large granular cells and small granular cells. Cytochemical analysis showed that this subpopulation was greatly enriched with neutrophils. Many band II cells adhere to glass whereas few band III cells do so. The glass-adherent cells loose their CL potential after a few days in vitro, whereas the nonadherent cells retain their CL responsiveness for at least a week in vitro.