No description available.

Singular or dual dietary depletions of vitamins C and E in Atlantic salmon parr increased susceptibility to Aeromonas salmonicida challenge. An array of immune parameters were evaluated to identify the involvement of these vitamin depletions on the immune response. Dietary vitamin E levels in salmon had no impact on haematological parameters, total serum protein or lysozyme levels. Similarly, dietary vitamin E levels did not affect leucocyte antibody production, macrophage activating factor (MAF) release and respiratory burst (RB) phenomena. However, haemolytic and opsonic properties of complement were compromised in vitamin E depleted salmon. Parenteral administration of vitamin E to vitamin E depleted carp did not elevate phytohaemagglutinin (PHA) lymphocyte proliferation responses or complement activity. In vitro additions of vitamin E to lymphocytes from carp fed a commercial diet did not elevate PHA proliferation responses either. The increased disease susceptibility provoked by dietary vitamin C restriction in Atlantic salmon was not correlated with serum protein levels, differential leucocyte numbers or phagocyte functions as tested by RB activity or phagocytosis by macrophages. Lymphocyte functions were operational in these fish as examined by MAF secretion and antibody production. Analogous to vitamin E depletion, dietary vitamin C restriction in salmon compromised complement haemolytic activity. Elevating the vitamin C content of diets above normal levels enhanced complement activity in salmon. Vitamin C was a potent modulator of rainbow trout leucocyte functions. In vitro supplementation of vitamin C in the sodium (NaAsc) or polyphosphate (PPAsc) form was required for PHA-proliferation responses. MAF secretion was also augmented by in vitro additions of NaAsc to leucocytes obtained from vitamin C depleted trout. Injecting NaAsc into depleted fish elevated PHA proliferation responses compared with saline-injected controls. Leucocytes from the latter group could recover proliferative responses to levels associated with NaAsc-injected fish with in vitro additions of 1x10-3 and 1x10-4M PPAsc. Increased disease susceptibility, reduced complement activity and haematocrit values were symptomatic of combined dietary vitamin C and E depletion in salmon parr. Total and differential leucocyte numbers, or serum parameters including antiprotease activity and total protein level were unaffected by this dietary regime. Although poorly adherent, macrophages obtained from dually depleted salmon had similar RB values and expressed greater responsiveness to a MAF-containing supernatant than vitamin sufficient counterparts. Lymphocyte functions were impervious to dual vitamin depletion as antibody and MAF production responses were intact. In vitro additions of NaAsc and PPAsc were shown to elevate RB responses in macrophages from vitamin-restricted and -adequate salmon. MAF secretion was demonstrated to be a temperature-dependent phenomenon in rainbow trout leucocytes. After 48h acclimation at low in vitro temperatures (6 C), leucocytes obtained from trout at 14C expressed impoverished MAF production. Acclimation of trout to 7 C did riot rescue MAF production. Also, normal RB activity was reduced in macrophages obtained from fish at 14 C after 48h at 6 C in vitro. However, if allowed to acclimate, these macrophages become more responsive to MAF and recovered RB activity to levels associated with macrophages held at 10 and 18C. RB responses of macrophages from fish at 7 C functioned equally well across a wide range of in vitro temperature regimes. Phagocytic activity of macrophages obtained from fish at 14 C and placed at 6 C in vitro for 48h expressed temperature sensitivity. This was especially apparent in fish fed a dietary ?-3/?-6 fatty acid ratio of 2.0. However, this dietary regime also elevated phagocytic activity of these macrophages to such an extent, that their response surpassed that of macrophages from fish fed commercial diets or ?-3/?-6 ratios of 0.5 and 1 even at low in vitro temperatures.

Given the widespread concern that chemical contaminants may be associated with infectious disease outbreaks in marine fish populations, work has been undertaken with the aim of developing a suite of non-specific and specific assays of marine fish immune function and the application of these techniques in a variety of field and laboratory investigations. Most of the work focused on dab, Limanda limanda (L.) in view of the importance of this species in several North Sea fish disease monitoring programmes, and was also supplemented with investigations of specific immune function in turbot, Scopthalmus maximus (L.). Initial field studies examined non-specific immune function in terms of lymphoid organ morphology in dab sampled along a North Sea gradient of chemical contamination during the March 1990 ICES/IOC Bremerhaven workshop. Significant differences were observed in the kidney and spleen cell populations from dab, and these observations were considered in view of the various other physico-chemical and biological results generated during the Bremerhaven workshop. Following the valuable experience gained of the practical aspects of the field monitoring approach, laboratory investigations were initiated with the aim of developing a suite of immune function assays for deployment in either laboratory or field studies of marine fish health. Assays for non-specific immune functions were considered, including serum protein and lysozyme levels, methods of phagocyte collection, phagocyte chemiluminescence, calorimetric detection of individual reactive oxygen species and in vitro cell migration assays. Additional field work was undertaken, with the monitoring of serum total protein levels and lysozyme activity in dab sampled from Lyme Bay, UK This study provided evidence of a marked seasonal variation in non-specific immune function, which appeared to be associated with environmental factors (e.g. water temperature) and the reproductive cycle. Selected non-specific assays were applied to dab and turbot exposed under controlled laboratory conditions to a variety of important marine contaminants, including cadmium, polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs), and the biological data integrated with appropriate chemical characterisation of the exposure matrix. Using cultured turbot as a test species, the non-specific assays were also supplemented with the assay of specific immune function in fish exposed to PCB contaminated sediments. In brief, there was evidence of significant impairment of immune function in fish exposed to either individual contaminants (viz. cadmium and PAHs) or contaminant mixtures (viz. PAHs and PCBs) under the laboratory conditions described. In summary, the project was successful in its primary aims of developing a suite of techniques for evaluating both cellular and humoral immune functions in marine flatfish, and applying these techniques in the laboratory to assess the impact of important classes of environmental contaminants on fish health. Selected techniques were also used in field monitoring studies of marine fish immune function, illustrating the potential of such techniques for use in future laboratory and field studies of fish health.

No description available.

One of the greatest challenges in finfish aquaculture is combating losses caused by infectious bacterial diseases, and a better understanding of the interactions between the host immune system and pathogens is essential for developing new methods to manage infections and outbreaks. Extracellular traps (ETs) are decondensed nuclear chromatin released by neutrophils into the extracellular matrix that can ensnare and kill microbes. Since the discovery of ETs in humans, these innate immune effectors have been characterised across the animal kingdom, including in some fish species, though their existence the salmonids has yet to be confirmed. Therefore, the aim of this thesis was to confirm and characterise the release of ETs in the rainbow trout (Oncorhynchus mykiss) and investigate the interaction of these structures with fish pathogenic bacteria. To do this, a triple-layer Percoll gradient technique was employed to give highly enriched cell suspensions of polymorphonuclear cells (PMNs) derived from head-kidney tissue preparations. Treatment of PMN-enriched cell suspensions with the nucleic-acid-specific stain, SYTOX Green, revealed the presence of ET-like structures that had been released without stimulation. These ET-like structures were confirmed by immunostaining techniques to contain the diagnostic proteinaceous markers of ETs: neutrophil elastase, myeloperoxidase and the H2A histone. Previously characterised inhibitors and inducers of ET release from phagocytic immune cells in other animals confirmed that calcium ionophore (CaI), flagellin, and cytochalasin D shared similar activities for ET-release by rainbow trout PMNs. However, interestingly, as the common ET-inducer phorbol-myristate acetate (PMA) and ET-inhibitor diphenyleneiodonium (DPI) did not exert their expected potency in ET release assays with the PMNs, perhaps indicating that these fish cells are less dependent on NADPH oxidase signalling for ET release compared to mammals and most invertebrate species. The PMN-derived ETs were demonstrated to bind to and trap the extracellular nuclease-deficient bacterial fish pathogen, Vibrio anguillarum (Vib 87) when co-cultured. Finally, extracellular nuclease activity produced by a V. anguillarum isolate (Vib 6) during culture was able to degrade ETs released by rainbow trout PMNs in a dose-dependent manner. Moreover, viable colony counts, fluorescent and phase contrast microscopy demonstrated that V. anguillarum Vib 6 eluded trapping by ETs, while an extracellular nuclease-deficient isolate did not. These observations are consistent with the suggestion that nucleases are a microbial virulence factor during host infection. Confirming the existence and antimicrobial potential of extracellular traps released by rainbow trout PMNs may provide a platform towards the development of novel therapeutics to reduce mortalities in finfish aquaculture caused by infectious microbial pathogens.