Functional units in rainbow trout (Salmo gairdneri,Richardson) liver: III. Morphometric analysis of parenchyma,stroma, and component cell types

Hepatic stroma and parenchyma with its component cell types were quantitatively described in adult male and female actively-spawning 5-year-old rainbow trout (Salmo gairdneri, Richardson). Point-count morphometry of glycol methacrylate sections estimated volume compartments for stroma and parenchyma. Veins composed 85% of the stroma while arteries and bile ducts occupied approximately 6–7% each. Parenchyma accounted for 95% of hepatic volume. Point-count morphometry of transmission electron micrographs estimated volume compartments as well as numerical and surface density measurements for parenchymal components. Within the hepatic parenchymal compartment, hepatocytes occupied 85% and showed significant sex differences. Female hepatocytes were significantly more numerous but were smaller, only 60% of the volume of male hepatocytes. Since hepatocyte nuclear volume was equal in both sexes, differences were due to reduced cytoplasmic volume in females. Perisinusoidal macrophages of females occupied larger volumes of their respective parenchymal compartments, and their larger mean cytoplasmic volumes suggested activation. Biliary epithelial cells of preductules and ductules were numerous. Ratios of numerical density of hepatocytes to biliary epithelial cells were consistent with a tubular arrangement of hepatocytes. Factors possibly mediating the sexual dimorphism are discussed.     

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.     

Pathological changes induced byPomphorhynchus laevis Muller (Acanthocephala) in the gut of rainbow trout,Salmo gairdneri Richardson

The consequences of infection of rainbow trout,Salmo gairdneri Richardson, by an acanthocephalan,Pomphorhynchus laevis Muller, have been studied using light and electron microscopy. The mucosal epithelium, of the gut adjacent to the metasoma of the worm suffered compression and abrasion. The praesoma ofP. laevis penetrated the mucosal epithelium, lamina propria, stratum compactum, stratum granulosum, muscularis and serosa of the gut wall, and was invested by a fibrous capsule of inflammation tissue. This was composed of four principal cell types: eosinophilic granular cells (EGCs), type A cells, type B cells and fibroblasts. Some melanocytes and lymphocytes were also present. The role of the EGCs in the inflammation response is not known, but may not be an active one. The type A cells resembled neutrophils of other fish species, and have tentatively been placed in this category. The type B cells, with large cisternae formed by the RER, resembled plasma cells and may have been involved in a humoral response to the acanthocephalan. The integument ofP. laevis did not appear to be damaged by the cells of the inflammation tissue, and it seems likely that this host response was incapable of rejecting the parasite.Labels to figures C crater - EGC eosinophilic granular cell - G stratum granulosum - I inflammation tissue - L lamina propria - M muscularis - Nu nucleolus - P P. laevis - R rough endoplasmic reticulum cisterna - S stratum compactum - tA type A cell - tB type B cell     

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.     

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.     

Fish immunology. II. Morphologycal and cytochemical characterization and phagocytic activities of head kidney macrophages from rainbow trout (Salmo gairdneri Richardson)

In Salmo gairdneri Richardson trouts, a comparison was made between macrophages (MO) derived from head kidney and peripheral blood monocytes. Morphologically and cytochemically no differences were observed between these two types of mononucleated cells. On the other hand, in parallel studies the ability of trout erythrocytes to engulf Candida albicans (CA) was evaluated and compared to the MO phagocytosis. In erythrocytes, engulfment is preceded by binding to CA and cell membrane invagination, while this was not the case for MO. Finally, MO stimulated with lipopolysaccharides (LPS) did not modify their phagocytic capacities, thus suggesting a lack of LPS receptors or a tolerance to LPS.     

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.     

Fish immunology. I. Binding and engulfment of Candida albicans by erythrocytes of rainbow trout (Salmo gairdneri Richardson)

The role of fish erythrocytes (FE) as phagocytic cells has poorly been investigated, until now. Here, we have focussed our attention on the interplay between rainbow trout (Salmo gairdneri Richardson) erythrocytes and Candida albicans (CA). At the same time, the intervention of autologous head kidney macrophages (MO) in the CA processing by FE has been studied. Data show that CA particles bind to FE, which, in turn, are able to engulf but not kill them. In the presence of MO, a decrease of FE with bound CA occurs and, in some microscopic images, FE form rosettes with MO. Phagocytosis of CA is higher in rosetting MO than in non-rosetting ones. According to our findings, it appears that FE represent a reservoir of engulfed CA and rosetting is an efficacious phenomenon of presentation of pathogens to MO, where an effective clearance of them can take place.     

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.     

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.     

The effect of temperature and starvation on the clearance of bacteria from the bloodstream of rainbow trout (Salmo gairdneri Richardson)

The blood clearance of 51Cr-labeled heat-killed Salmonella pullorum was generally biphasic and exponential for each phase. Starvation had little significant effect on this pattern, although the rate of first phase clearance was probably slower. Raising the water temperature from 8 degrees C to 18 degrees C enhanced the rate of clearance of the second phase to almost exactly double that at 8 degrees C. At 18 hr postinoculation, the spleen contained much more radioactivity per gm than any other tissue. This finding is in marked contrast to earlier work that showed that at 1 hr postinoculation, the kidney contained the most, and it suggests that redistribution of bacteria occurred. The most distinct effect of temperature stress on tissue localization of bacteria was in the heart: A rising temperature stress caused increased numbers of bacteria to localize within the heart. Less clear-cut changes were also seen in other tissues with different treatments. With the possible exception of starvation effecting a slower first phase clearance rate, we have been unable to demonstrate that the vascular clearance mechanisms, including the reticuloendothelial system, are significantly compromised by raising the water temperature or by starvation.     

The rainbow trout (Salmo gairdneri Richardson) granular leukocytes and mast-cells system versus the human one. Cytomorphometrical and cytochemical characters in these two species philogenetically poles apart

In man, circulating granular leukocytes constitute a cellular system and are able to migrate in the tissues to take part in the immune reactions. This study was to characterize the granular leukocytes and the eventual existence of mast-cells of a low vertebrate, rainbow trout (Salmo gairdneri R.) in order to compare it with man. The blood smears and tissues sections have been tested whit panoptic methods, cytochemistry and toluidine blue. White blood cell count, leukocytes formula and cytomorphometric characterization was performed using an image analyser. Scanning of tissues sections in order to identify mast-cells has been also performed. In this model the granular leukocytes are all neutrophilic like; no eosinophilic, no basophilic no tissue mast-cells, basically existing in allergic and anaphylactic reactions, were found.     

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.     

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.     

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.     

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.