The production of a macrophage-activating factor from rainbow trout Salmo gairdneri leucocytes.

Rainbow trout head kidney and blood leucocytes are shown to be capable of secreting a soluble macrophage-activating factor (MAF) after stimulation with concanavalin A (Con A). The presence of phorbol myristate acetate (PMA) as a co-stimulant increased the production of MAF. Both respiratory burst activity (nitroblue tetrazolium, NBT, reduction and H2O2 production) and bactericidal activity were enhanced after incubation of resident or elicited macrophages with the MAF-containing supernatants for 48-72 hr. The target culture period before the addition of MAF did not affect their responsiveness, but a continuous presence of MAF was necessary for maximal stimulation.     

Evidence for a cutaneous secretory immune system in rainbow trout (Salmo gairdneri)

Serum and mucus of untreated rainbow trout did not contain detectable agglutinating antibody to sheep red blood cells. Intraperitoneal injections of sheep erythrocytes resulted in production of specific antibody in both serum and mucus. Immuno-diffusion and absorption studies confirmed that mucus and serum antibodies are antigenically similar. Indirect fluorescent antibody techniques demonstrated the presence of immunoglobulin-containing plasma cells in cutaneous dermis and mucus, supporting the hypothesis that an independent mammalian-like secretory immune system is operative in fish.     

Opsonic effect of rainbow trout (Salmo gairdneri) antibody on phagocytosis of Yersinia ruckeri by trout leukocytes

Partly purified peripheral blood leukocytes from normal rainbow trout (Salmo gairdneri) were used to study the influence of specific antibody on phagocytic uptake and intracellular killing of Yersinia ruckeri, a bacterial pathogen of trout. Specific antibody exerted a significant opsonic effect on the rate of phagocytic ingestion of the bacteria but did not affect the rate of intracellular killing. The results are discussed with reference to the current understanding of fish antibody function and phagocytosis by fish leukocytes.     

Accessory cells in the gill epithelium of the freshwater rainbow trout Salmo gairdneri

Two types of mitochondria-rich cells were identified in the gill epithelium of the freshwater-adapted rainbow trout, Salmo gairdneri, after selective impregnation of their tubular system with reduced osmium. A first type consisted of large cells with a poorly developed and loosely anastomosed tubular system; thus, that resembled the chloride cells commonly encountered in the gill epithelium of freshwater-adapted euryhaline fishes. A second type comprised smaller cells with an extensively developed and tightly anastomosed tubular system. These never reached the basal lamina of the gill epithelium and were adjacent to chloride cells, to which they were linked by shallow apical junctions (100-200 nm); thus, they resembled accessory cells, which are currently found in the gill epithelium of sea-water-adapted fishes but are usually lacking in freshwater living fishes. Transfer of the freshwater-adapted trout into seawater induced the proliferation of the tubular system in the chloride cells and the formation of lateral plasma membrane interdigitations between accessory cells and the apical portion of the chloride cells. The length of the apical junction sealing off this ex tended intercellular space was reduced to 20-50 nm. The tubular system of the accessory cells was not modified. The extension of the tubular system in the chloride cells of the seawater-adapted fishes indicated that, as in most euryhaline fishes, these cells have a role in the adaptation of the rainbow trout to seawater. In contrast, the function of the presumptive accessory cells in fresh water trout remains to be established.     

Macrophage activation during experimental allergic orchitis in rainbow trout (Salmo gairdneri)

Macrophages isolated from fish undergoing an experimentally induced autoimmune response against the testis were found to have been activated using several morphological and functional criteria. They exhibited increases in cell spreading, phagocytosis, reduction of nitroblue tetrazolium and acid phosphatase activity. In addition they possessed larger and more numerous cytoplasmic organelles and peripheral processes compared with control cells. Culture of the autoimmunologically elicited cells for 1 or 2 days resulted in their return to a non-activated state.     

Mixed Lymphocyte Reactions (MLR) in rainbow trout (Salmo gairdneri) sibling

Suitable conditions for 2 way MLR were determined in random combinations of commercially cultured trout and were then applied to full siblings in two families reared for experimental purposes. The primary aim of the investigation was to determine whether the immunogenetic predictions from mammalian and avian experiments would apply, i.e. (i) that all full sibs, short of rare intra-MHC recombinants, can be assigned to a maximum of four different MHC genotypes, and (ii) that individuals of the same genotype are mutually histocompatible as tested in MLR. If both predictions apply, five or more siblings tested in all pairwise combinations should invariably display compatible pairs, so that a maximum of four groups of mutually incompatible individuals should emerge. The findings did not fit this model, and two alternative immunogenetic models are discussed.     

The thymus of the rainbow trout (Salmo gairdneri). Light and electron microscopic study

The rainbow trout thymus is a paired organ, organized in 3 adjacent zones. The gland lying on a thick connective tissue layer, is covered by a thin epithelial capsule. Cellular components of thymus are essentially the thymocytes and the epithelial cells. Thymocytes occur mainly in the inner and outer zones but neither cortex nor medulla are clearly delimited. Septa represent a typical epithelial structure associated with thymocytes and blood vessels. Thymus is well vascularized and electron microscopy demonstrates a characteristic relationship between vascular system and lymphoid tissue.     

Susceptibility and immunity to Vibrio anguillarum in post-hatching rainbow trout fry, Salmo gairdneri Richardson 1836

Rainbow trout fry were tested for their susceptibility to experimental infection with Vibrio anguillarum, and their ability to mount an immune response against it, from the age of 2 weeks post-hatch onwards. Bath challenges were ineffective in inducing vibriosis until 6-8 weeks post-hatch, and then only low levels of specific mortality ensued, even at very high doses. However, at the earliest age tested, 7 weeks post-hatch, the fry were susceptible to infection by an intraperitoneal injection with live organisms. Protective immunity was evident in fry vaccinated by direct immersion as early as 2 weeks post-hatch (at an average weight of 0.14g), when tested by intraperitoneal challenge. By the time the fry reached 0.5g (10 weeks after hatching), protection levels had reached 50% for direct immersion vaccination and 100% for intraperitoneal vaccination. An oral vaccination, from first feeding onwards, proved ineffective in inducing immunity. The results are discussed in relation to the onset and maturation of immunological competence in rainbow trout.     

Role of autoantibodies in the autoimmune response to testis in rainbow trout (Salmo gairdneri).

Rainbow trout, Salmo gairdneri, passively immunized with anti-sperm antibodies developed lesions in the testis characteristic of autoimmune attack. Only animals pre-treated with Freund's complete adjuvant developed these lesions. Investigations into the possible role of autoantibodies during autoimmune attack, carried out in vitro, showed that sperm antibodies were not cytotoxic but did have an opsonic effect on macrophage phagocytosis of sperm in the presence of complement.     

The cellular immune response of rainbow trout (Salmo gairdneri Richardson) to sheep red blood cells

The cellular immune response of rainbow trout, Salmo gairdneri, to sheep erythrocytes was investigated. Both the primary and secondary responses were measured using the migration inhibition factor (MIF), antigen-binding (ABC), and plaque-forming cell (PFC) assays. These immune function assays provide measures of both T and B cell activity. The kinetics of these three responses at 16 degrees C were determined by sampling fish over an 18 day period for the primary response and a ten day period for the secondary response. The peak MIF response occurred two days after injection, while the primary peak PFC response was observed 14 days post-injection. Two ABC peaks were observed in the primary response, one at four days and one at ten days after injection. In the secondary response the peak ABC response was observed four days and the peak PFC response six days post-inoculation. The possible interrelationships of the various cell populations are discussed.     

Post-hatching development of the thymic epithelial cells in the rainbow trout Salmo gairdneri: An ultrastructural study

This study reports the ultrastructure of subpopulations of epithelial cells of the thymic parenchyma during the post-hatching development of the rainbow trout, Salmo gairdneri, kept at 14°C. At hatching, the thymus contained a small number of medium and large thymocytes interspersed among three different types of epithelial cells: (1) epthelial cells adjacent to the connective tissue capsule; (2) ramified dark epithelial cells with electron-dense cytoplasm; and (3) pale electron-lucent epithelial cells displaying secretory-like features. All these cell types were anchored to one another by desmosomes and had apparently differentiated from the pharyngeal epithelium. At 4 days after hatching, the thymus enlarged, and numerous gaps occurred between the cell processes of contiguous epithelial cell processes of contiguous epithelial cells adjacent to the capsular connective tissue. In 21-day-old trout, thymic trabeculae developed carrying blood vessels, and a subcapsular zone became evident containing lymphoblasts and large subcapsular epithelial cells. In 30-day-old trout, an outer thymic zone developed consisting of spindle-shaped epithelial cells which formed a dense network. At this stage, scattered cystic cells, which apparently differentiated from the pale epithelial cells, were present.     

Iron resistance of hepatic lesions and nephroblastoma in rainbow trout (Salmo gairdneri) exposed to MNNG

Histochemical markers are important for the early detection of chemically initiated neoplasia in experimental animal studies. The marker, iron resistance, was evaluated in the Shasta strain of rainbow trout (Salmo gairdneri). Twenty-one-day-old trout embryos were exposed to 100 ppm aqueous N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) for 30 min in a static water bath. Fish were fed a semipurified diet, and sampled monthly from the 4th to the 9th month. Two days before sampling, fish were iron-loaded with a single ip dose of 0.30 mg iron dextran/100 g body weight. Livers and kidneys were conventionally processed to paraffin sections for iron, or hematoxylin and eosin (H&E) staining. Normal hepatocytes accumulated iron in pericanalicular locations, but in hepatocytes from carcinogen-altered foci and tumors, iron staining was clearly reduced or absent. Normal renal tubule cells exhibited slight to moderate iron staining, while those from nephroblastoma were iron resistant. These results establish iron resistance as a property of preneoplastic and neoplastic trout hepatocytes and nephroblastoma cells for the first time. Iron resistance may offer a practical histochemical marker in experimental fish models of hepatocellular carcinoma and nephroblastoma.     

Substance P acts by releasing 5-hydroxytryptamine from enteric neurons in the stomach of the rainbow trout, Salmo gairdneri

The effect and mode of action of substance P was studied in a perfused stomach preparation and on isolated strip preparations of the stomach wall from the rainbow trout, Salmo gairdneri. Substance P was excitatory on the stomach muscle wall in a dose-dependent manner. Two other tachykinins, physalaemin and eledoisin, excited the preparations in a similar manner and in the same dose range. The effect of substance P was not antagonized by the substance P analogues [D-Pro2, D-Phe7, D-Trp9]substance P and [D-Pro2, D-Trp7,9]substance P (both 10(-5) M). Tetrodotoxin reduced or abolished the effect of substance P, while no reduction of the response was obtained after atropine, chlorisondamine or phentolamine (all 10(-6) M). 5-Hydroxytryptamine excited the stomach and this effect was not antagonized by tetrodotoxin, suggesting that the action of 5-hydroxytryptamine was direct on the smooth muscle. The 5-hydroxytryptamine antagonist methysergide, in a concentration which selectively blocked the response to 5-hydroxytryptamine, also blocked the response to substance P (10(-9)-10(-8) M). The outflow of 5-[3H]hydroxytryptamine from a preloaded perfused stomach was clearly increased by substance P, and this release was blocked by tetrodotoxin. Immunohistochemistry revealed the presence of nerve fibres and ganglion cells showing 5-hydroxytryptamine-like immunoreactivity in the myenteric plexus and smooth muscle layers of the stomach wall. The immunoreactive cells and nerve fibres were particularly abundant in the pyloric part of the stomach. It is concluded that the main effect of substance P on the stomach wall of the rainbow trout is indirect, via activation of a non-adrenergic, non-cholinergic neuron. The results are compatible with the view that this neuron exerts its action by release of 5-hydroxytryptamine. Supramaximal concentrations (greater than or equal to 10(-7) M) of substance P may in addition have a direct effect on the gastric smooth muscle.     

Pathogenicity factors and virulence for rainbow trout (Salmo gairdneri) of motile Aeromonas spp. isolated from a river

Ninety-seven motile Aeromonas strains were isolated over a period of a year from samples of water and sediment collected at different sites along a river. Strains were regularly recovered from all samples, regardless of the source of isolation or seasonal conditions. Isolates were biochemically characterized by the API 20NE system (Analytab Products, Plainview, N.Y.) and classified as Aeromonas hydrophila (74 strains), Aeromonas sobria (11 strains), and Aeromonas caviae (12 strains). Despite the high level of homogeneity observed in their biochemical patterns, they displayed different degrees of virulence for fish; 72.02% of A. hydrophila isolates and 63% of A. sobria isolates were virulent for fish by intramuscular challenge, but lower frequencies of virulence were observed when intraperitoneal injections were used. All A. caviae strains proved to be avirulent. Caseinases, hemolysins, and Vero cytotoxins were produced by 100, 91, and 94.59%, respectively, of A. hydrophila strains and with lower frequencies and lower caseinase activities by A. sobria isolates. No correlation was found between these activities and the degree of virulence of the strains for fish. Most hydrophobic strains seem to be concentrated in A. caviae, A. sobria, and avirulent A. hydrophila groups. Known virulence markers commonly associated with virulent strains (acriflavine negative and self-pelleting negative and precipitation after boiling positive phenotypes) had a low representation in the total of strains studied and were not associated with virulence.     

Enzyme- and immuno-histochemical study of the thymic stroma in the rainbow trout, Salmo gairdneri, Richardson.

Owing to the lack of data about thymic non-lymphoid cells in fish we decided to perform a histochemical characterization of these cells in order to ascertain their relationships to other thymic components. In the present study we analyze the enzyme-histochemical patterns for acid phosphatase, alkaline phosphatase, non-specific sigma-naphthyl acetate esterase and 5' nucleotidase activities, as well as the presence of keratin demonstrated by immunoperoxidase staining, in the non-lymphoid cell populations of the thymus of the rainbow trout, Salmo gairdneri. According to their location in the organ, morphology and histochemical reactivities, we were able to define seven different subpopulations of keratin-positive epithelial cells: 1) Epithelial cells limiting with the capsular and septal connective tissues; 2) Subcapsular epithelial cells; 3) Stellate epithelial cells of the inner thymic zone; 4) Large, ovoid epithelial cells of the inner thymic zone; 5) Acidophilic epithelial cells of the outer thymic zone; 6) Cystic cells; and 7) Goblet cells. The significance of the heterogeneity of the epithelial cell (EC) population, its specific distribution in the organ, which apparently conforms distinct cell microenvironments, as well as the possible phylogenetical relationships between these microenvironments and the classical cortex and medulla of the mammalian thymus, are discussed.