No description available.

No description available.

No description available.

Peripheral blood leucocytes of the turbot, Scophthalmus maximus, were characterised into 4 distinct groups following morphological, morphometric and histochemical examination. Total and differential cell counts were determined. Thrombocytes, the most abundant leucocyte type (52%), were highly mobile and encountered in several morphological forms. Granulocytes, representing 5.6% of the leucocyte population, histochemically most resembled the mammalian neutrophil. Both large and small lymphocytes (40.8%), were encountered. Monocytes were rarely observed (1.6%). Thrombocytes and monocytes were phagocytic in vitro at 12oc and 22oc, showing increased phagocytic activity at the higher temperature. The thymus was paired and consisted of a well developed outer cortex and an inner meduallary region. The spleen was bounded by a fibrous tissue capsule and contained a large volume of blood. Diffuse areas of red and white pulp, ellipsoids and melanomacrophage centres were apparent. Lymphocytes, thrombocytes and mature erythrocytes made up the cellular components. The kidney, located beneath the vertebral column contained haemopoietic tissue throughout. Excretory tubules were evident posteriorly. Cellular elements included developing granulocytes, large and small lymphocytes and melanomacrophages. Investigation of ontogenic development of the lymphoid tissue, from 24h post-hatch to the completion of metamorphosis (Day 63) revealed thymic, splenic and kidney rudiments all present at Day 4 with the first lymphoid cells appearing in thymus and kidney by Day 8. Splenic lymphoid cells and the development of areas of white pulp were apparent by Day 28. Differentiation of the thymus had occurred and melanomacrophage centres were seen in the spleen, completing structural lymphoid development by Day 63. Critical stages of lymphoid ontogeny were correlated with easily recognisable external morphological features. A study of the kinetics of carbon clearance by the reticuloendothelial system, revealed a phagocytic capacity in the spleen, kidney and heart. Splenic carbon was seen at 20min post injection, accumulating around ellipsoids and rising to a maximum level at 24h. By Day 5 carbon levels within phagocytes, by now more distant from the ellipsoids, had begun to decrease and carbon was seen within melanomacrophages. Levels of kidney carbon, present within large macrophage-like cells which increased in size forming larger aggregations, increased to a maximum at Day 3. Clearance appeared more rapid in the posterior kidney. Low level uptake was seen within the epicardium. Carbon uptake was not observed in the liver or gill. Kidney leucocyte migration in vitro was examined to a range of chemoattractants using a number of assays. 24h bacterial culture supernatants of Vibrio alginolyticus induced significant cellular responses. The under agarose assay demonstrated migration inhibition to 100%, 50% and 40% supernatant dilutions. Enhanced migration was detected to dilutions of 5-50% in the microchemotaxis chamber, being optimal at 20%. The leucocyte polarisation assay demonstrated cell orientation in response to I 00% culture filtrate and the capillary tube migration assay revealed cellular inhibition at concentrations of 10% & SO%. Leukotriene B4 (LTB4) also induced migration in the filter-based assay, being optimal at to-7M. Cellular migration and orientation were observed in filter and polarisation assays to turbot serum, with normal and activated serum inducing elevated responses in the filter based assay. No response was detected by any of the assay systems to n-formylmethionyl-leucyl- phenylalanine (FMLP) or casein at any concentration tested. Results are discussed in relation to the cellular defence mechanisms of fish.

The prevalence and impact of proliferative kidney disease (PKD) and myxosporidiosis has been investigated in wild fish stocks in the UK, over 1,500 fish representing 17 species being examined. PKD was recorded in brown trout, grayling and pike, the causative agent, the PKX cell, being identified with the aid of light and electron microscopy. A further 27 myxosporean species were also noted, with Myxobolus cotti (syn. M. jiroveci), in the brain of bullheads Cottus gobio being recorded for the first time. Studies on the structure and development of Myxidium lieberkuehni in pike revealed several previously undescribed features. Comparative morphological studies were undertaken to assess affinities of PKX with known myxosporean species. Results indicated similarities with early presporogonic stages of several myxosporean species, especially those belonging to the genus Sphaerospora. The apparent rarity of spore formation associated with PKX infections in the hosts examined focussed attention on species of Sphaerospora as possible sources of infection to salmonids. Studies concentrated on the possible involvement of the 3-spined stickleback, Gasterosteus aculeatus and its renal parasite, Sphaerospora elegans, in PKD transmission. A re-description of this parasite (recently elevated to "type species" for the genus), was prepared. Laboratory experiments using rainbow trout PKX cells successfully transmitted the infection to rainbow trout, brown trout, brook trout and grayling; however sticklebacks challenged with PKX cells did not appear to become infected. Rainbow trout challenged with S. elegans spores and presporogonic stages showed no evidence of sphaerosporosis or PKD. Experiments designed to investigate the possible role of tubificid worms in PKD transmission provided inconclusive results. Field studies provided data on the pathogenesis of PKD in grayling and showed this species to be highly susceptible to the disease.

Mycobacterium spp. isolated from food and ornamental fish in Thailand (strains TB 1, TB40, TB267, TB268), and the type strains Mycobacterium marinum (NCIMB 1298), Mycobacterium fortuitum (NCIMB 1294), and Mycobacterium chelonae (NClMB 1474) were cultured in Long's medium, Eagle's minimum essential medium, Sauton's medium and modified Sauton's medium. The latter enabled excellent growth and production of extracellular products (ECP) from TB40, TB267, TB268 and M marinum in particular, whereas growth and production of ECP for all strains was limited in Long's medium. SDS-PAGE protein profiles of ECPs from 14 day culture supernatants showed major bands at 65, and <14 kDa. After 2 days culture at the higher temperature of 37°C (heat shock), the production of ECP from all mycobacteria strains except M marinum averaged approximately 4 to 10 fold higher than from strains cultured for 14 days at 28°C. The major fibronectin binding proteins from ECP of Mycobacterium spp. isolated from infected fish were identified at 21-25 kDa. Cross reactivity was detected between ECP from Mycobacterium spp. and MAb anti-heat shock protein (60 kDa) and MAb anti-M Tuberculosis. The 65 kDa antigen of TB267 is a strongly immunogenic protein eliciting antibodies in fish, rabbits and mice. Cross-reactivity was found between rabbit anti-65 kDa antibody and sonicated proteins from many other bacterial species. Therefore, the 65 kDa protein from Mycobacterium sp. isolated from snakehead fish may be a common protein in fish bacterial pathogens. Eighteen MAbs to TB 267 and M chelonae were produced. The epitopes to which the MAbs are against located on molecules susceptible to protease treatment. All MAbs recognized the 65 kDa protein. It is one of major proteins in the ECP, whole cell sonicates and lysates from Jv1ycobacterium spp. and is located in the peri plasmic space or cell wall, and is secreted in the medium during culture. A pnmary intraperitoneal (IP) immunisation of extracellular products (ECP) from Jv1ycobacterium spp., (strains TB40, TB267 or M marinum) mixed with Freund's incomplete adjuvant (FIA), followed by a secondary IP injection at 8 wks, resulted in the elevation of both the non-specific immune response (by measuring nitroblue tetrazolium, lysozyme and phagocytosis activity) and the specific immune responses of rainbow trout, Oncorhynchus mykiss (by measuring specific antibody levels). Nile tilapia were immunised by injecting extracellular products (ECP) of Jv1ycobacterium spp. (strain TB40, TB267 or the type strain M marinum) into their swimbladders and this resulted in the elevation of the non specific immune response. The cytological response of rainbow trout head kidney macrophages to ingested Mycobacterium spp was examined in vitro. The bacteria had previously been opsonised with either fresh rainbow trout serum (FS), or serum which had been heat-inactivated (IDS), or rainbow trout antiserum against the extracellular products (ECP) of Mycobacterium strains TB267 or M marinum. MAbs against the ECP were also used as opsonins. Opsonisation of the mycobacteria was found to greatly enhance the phagocytic and killing activity of the rainbow trout macrophage.

No description available.

No description available.

No description available.

This project investigated several factors that influence the virulence of the fish pathogen <I>Aeromonas salmonicida</I> subspecies <I>salmonicida</I>. In this study the effect of iron supplementation was investigated on whole cell bacterin used as a vaccine. It was shown that A-layer negative strains grown under iron supplementation do not confer high protection. A-layer positive strains though, do provide protection at levels similar to the A-layer negative iron restricted bacterin. Use of a combined vaccine appeared to compound the protection. Cells grown under iron normal and iron restricted conditions have previously been shown to produce, respectively, ferric superoxide dismutase (SOD) and manganese SOD to protect against superoxide anions. Analysis here followed the transfer from iron restricted culture to iron supplemented culture to investigate the change in superoxide dismutase (SOD) induction. It appeared that both ferric and manganese SOD were expressed even though only one form was active in either iron condition. This implies post-translational control of activity. Catalase in another enzyme with a protective role and is induced by hydrogen peroxide. Cells grown without the addition of hydrogen peroxide do not appear to express a functional catalase. Analysis of catalase induction has shown that iron normal and iron supplemented growth using hydrogen peroxide induces expression of a catalase enzyme. Iron restricted bacteria show sensitivity to normal induction doses of hydrogen peroxide. Lowering the induction concentration allows expression of the catalase. This suggests that the catalase is heme co-factored. The relationship between iron levels and catalase induction suggests that catalase has a high priority for any available iron. The <I>in vivo</I> expression of the serine proteinase and iron regulated outer membrane proteins (IROMP's) was investigated. Whilst the serine proteinase was induced <I>in vivo</I> in a normal virulent strain, a proteinase-negative strain did not appear to produce the proteinase. This suggests that the serine proteinase is not essential for virulence. The expression of IROMP's <I>in vivo</I> indicates an iron restricted environment. <I>A. salmonicada</I> secretes a glycerophospholipid: cholesterol acyl transferase (GCAT) that is often complexed with LPS and believed to be a major toxin of the ECP. Analysis of culture supernatants has identified a secreted extracellular polysaccharide that may also complex with GCAT.