Search

Background The most common assay used to detect natural killer (NK) and cytotoxic T-lymphocyte (CTL) activity is the 51Cr release assay. The numerous disadvantages of this method led us to evaluate cytotoxicity functions by flow cytometry. We described a flow cytometric assay to assess NK and CTL activity from different species. Methods This assay is based on a dual fluorescent staining of target cells. The dye, DIOC18(3) (3,3′-dioctadecyloxacarbocyanine perchlorate), is used to stain the membrane of different target cells. Propidium iodide (PI) is used to label dead target and effector cells. This labeling allows a clear discrimination between both cell populations. Results A good correlation was observed between the percentage of target lysis and the effector-to-target cell (E/T) ratios with human and porcine peripheral blood mononuclear cells (PBMC) as effector cells. The flow cytometric assay was shown to be as sensitive and as reliable as the 51Cr release performed with human cells. The assay was also applied successfully to measure NK cell activity in other animal species (pig, rabbit, hen, and mouse) and to measure murine CTL activity against the influenza virus. Conclusions We provide evidence that the flow cytometric assay using DIOC18(3) is highly reproducible and is suitable to measure different types of cell cytotoxicity. Cytometry 41:289–297, 2000. © 2000 Wiley-Liss, Inc.

International audience; The anatomy, histology and ultrastructure of the thymus of a dipnoan, the Australian lungfish, Neoceratodus forsteri , was studied by light and transmission electron microscopy. The thymic tissue showed clear demarcation into a cortex and medulla with ample vascularization. Large cells including foamy and giant multinucleated cells with periodic acid Schiff/Alcian blue positive staining properties were localized mainly in the medulla. The major cellular components were epithelial cells and lymphoid cells. The epithelial cells were classified by location and ultrastructure into six sub-populations: capsular cells, cortical and medullary reticular cells, perivascular endothelial cells, intermediate cells, nurse-like cells and Hassall-like corpuscles. Myoid cells were found mainly in the cortico-medullary boundary and medulla. Macrophages and secretory-like cells were also present. These findings will provide a base of knowledge about the cellular immune system of lungfish.

The most common assay used to detect natural killer (NK) and cytotoxic T-lymphocyte (CTL) activity is the (51)Cr release assay. The numerous disadvantages of this method led us to evaluate cytotoxicity functions by flow cytometry. We described a flow cytometric assay to assess NK and CTL activity from different species.This assay is based on a dual fluorescent staining of target cells. The dye, DIOC18((3)) (3, 3'-dioctadecyloxacarbocyanine perchlorate), is used to stain the membrane of different target cells. Propidium iodide (PI) is used to label dead target and effector cells. This labeling allows a clear discrimination between both cell populations.A good correlation was observed between the percentage of target lysis and the effector-to-target cell (E/T) ratios with human and porcine peripheral blood mononuclear cells (PBMC) as effector cells. The flow cytometric assay was shown to be as sensitive and as reliable as the (51)Cr release performed with human cells. The assay was also applied successfully to measure NK cell activity in other animal species (pig, rabbit, hen, and mouse) and to measure murine CTL activity against the influenza virus.We provide evidence that the flow cytometric assay using DIOC18((3)) is highly reproducible and is suitable to measure different types of cell cytotoxicity.

Chilmonczyk. S .. D. Monge: Cellular Immune Response in Rainbow Trout (Oncorhynchus mykiss): Flow Cytometric Study. Acta vet. Brno 1998.67: 207-213. Assays devoted to the study of non-specific cellular defense mechanisms of rainbow trout: phagocytosis. oxidative burst and nonspecitic cytotoxicity. were developed. Compared to conventional methodologies flow cytometry provides a rapid. suitable and reproducible method for analysing fish cellular immune functions. In addition to the assessment of the defense mechanisms interesting data are directly provided from the observation of unstained cells. Flow cytometric profiles of cells are representative of the organ from which the leukocytes originated and each lymphoid organ displays a characteristic cytogram pattern. Flow cytometry emerges as a useful tool to study immune reactions and pathogenic processes.

Trout leucocytes from most of the survivors of viral hemorrhagic septicemia virus (VHSV) infections were capable of in vitro proliferation (T-like response) when cultured in the presence of short synthetic peptides designed from the G and the N cDNA-derived protein sequences of VHSV, a virus with substantial economic impact in trout farms. In contrast, no significant proliferative responses were obtained for the above-mentioned peptides from leucocytes obtained from either noninfected or genetically VHSV-resistant trout. However, since the anamnestic recognition of particular peptides (epitopes) of the G and the N protein by trout leucocytes varies largely within the outbred trout population, larger VHSV protein fragments were also tested. The finding that recombinant G and N fragments carrying multiple epitopes are recognized by the majority of the individual trout surviving VHSV infections and with higher stimulation indexes suggests that recombinant viral proteins could be used as vaccines given the outbred nature of the fish. © 1995 Academic Press, Inc.

Leucocyte populations from rainbow trout subjected to experimental viral haemorrhagic septicaemia virus (VHSV) infections under in vivo and in vitro conditions were analysed by flow cytometry. Quantitative analysis shows that only a low percentage of the leucocytes support viral replication. Lymphocytes, compared with monocytes granulocytes and macrophages, are the least susceptible sub-population. A decrease in the amount of the phagocytic activity was observed after the cells were infected with VHSV. Obvious modifications were observed through dot plot profiles in the composition of the cell sub-populations and in the cell morphology. In addition to a direct effect of VHSV on the macrophages, a systemic effect is produced. This could also be related to the stress induced by the experimental infection process.

International audience

Summary A cell line, named Epithelioma papulosum cyprini (EPC) and originating from carp epidermal herpes virus-induced hyperplastic lesions, was esta blished and has now given rise to more than 80 subcultures. It grows within a wide temperature range (15–33°C), survives between 7 and 10°C for several months without alterations in its further virus susceptibility and growth characteristics, and degenerates at 37°C. Its karyotype is 2n=96. It propagates most of the systemic infection viruses of the fish families Salmonidae, Cyprinidae, Anguillidae and Esocidae, as well as Indiana-type vesicular stomatitis virus. Despite the origin of EPC cells, we were unable to demonstrate the presence of herpes virus in them. Their transformation was reflected in their karyotype (normally 2n=100) and by their growth ability in soft agar medium; monolayer growth, however, was inhibited at a density of 3×106 cells/cm2. The temperature growth range, good splitting ratio (1/10) and virus susceptibility make EPC cells a highly suitable material both for fish pathology and for comparative virology studies.

The nucleotide sequence of the gene that encodes for the structural viral protein VP1 of bovine rotavirus (RF strain) has been determined. The sequence data indicate that segment 1 contains 3302 by and is A+T rich (65.3%). The positive strand of segment 1 contains a single open reading frame that extends 1088 codons and possesses 5′- and 3′-terminal untranslated regions of 18 and 20 bp, respectively. The first AUG conforms to the Kozak consensus sequence and if utilized, would yield a protein having a calculated molecular weight of 124–847, very close to the apparent molecular weight of VP1 (M.W. 125,000). The deduced amino acid sequence presents significant similarities with RNA-dependent RNA polymerase of several RNA viruses. VP1 was also synthesized in baculovirus using two transfer vecors: pAC461 and pVL941. Following infection of Sf9 cells with a recombinant baculovirus, a full-length nonfusion protein was synthesised which shares properties with authentic VP1 made in monkey kidney cells. The level of VP1 synthesis was about 10-fold higher when the baculovirus recombinant was derived from the pVL941 transfer vector. In that case, VP1 was expressed in yields approximately equivalent to 10% of the cellular protein. The recombinant protein was immunoprecipitated by hyperimmune serum raised against purified rotavirus. It also was immunogenic; a hyperimmune serum made in guinea pigs reacted with VP1 using immunoprecipitation and Western blot. This serum did not possess neutralization activity.

Fish were irradiated with 60Co gamma rays at doses ranging from 10 to 50 Gy. Lethal doses were determined in fishes of different ages. For a given dose, fry and fingerlings were more susceptible than subadults. Whatever the irradiation dose was, the fish displayed a sharp decrease in blood leucocyte count. At the lowest doses, this acute leucopaenia was reversible. The cellular damage in the lymphoid organs was particularly obvious in the thymus. The depletion of lymphoid cells from immunocompetent organs decreased (viral hemorrhagic septicaemia, VHS) or increased (Y. ruckeri, A salmonicida) the susceptibility of trout to pathogens. The suppressive effect of radiation was age dependent. Irradiation appeared to be a reliable technique to detect asymptomatic carrier fish.

La Microsporidiose des Salmonides precedemment observee dans le Sud-Ouest de la France atteint egalement les Salmonides de la Bresie, fleuve cotier normand. Les truites de mer adultes (Salmo trutta fario) en periode de reproduction et les jeunes saumons mâles (Salmo salar) sont plus particulierement sensibles a cette parasitose. Les spores de microsporidies sont revelees sur des calques d'organes ou de tissus infestes.

The natural pathway of viral hemorrhagic septicemia (VHS) virus penetration in trout is unknown. Fish pathologists suspect the gill as a possible route for virus entry into the fish. Some results obtained with another rhabdoviral model, the spring viremia of carp, showed that the gill can play an important role in viral pathogeny [1].

Publisher Summary This chapter focuses on the evolution of the thymus in rainbow trout. Many studies have been devoted to the origin and the description of the fish thymus. In recent years, immunological studies have renewed interest in the thymus. With regard to salmonid fish, the histogenesis of the early thymic rudiment was examined by Ellis (1977) and Grace and Manning (1980). The thymus of rainbow trout is a typical lymphoid organ. During the whole life of the fish, the thymus fills an intraepithelial position inside the epithelium of the gill chamber. The main structural transformations that appear during the growth of the fish are a result of the epithelial tissue. The epithelial cells proliferate from the germinal layer and progressively migrate throughout the thymic tissue according to a classical polarity from the base to the apical part of the organ. The thymus of salmonid fish is devoid of Hassall's corpuscles. This observation is in agreement with other descriptions (Zapata, 1981). In histological preparations, cross sections of septa present a similar appearance to Hassall's corpuscles and may be mistaken for these structures. In view of the peripheral localization of the thymus, the epithelial capsule may play an important role. In addition to its protective function, the epithelial capsule is important in the relationships between the thymic tissue and the outside environment.

Peripheral blood lymphocytes of the rainbow trout can be stimulated by PHA, conA and LPS. Optimal concentrations for stimulation are 1-50 microgram/ml for PHA, 1-10 microgram/ml for ConA and 1 microgram/ml for LPS. Best stimulation is obtained after an incubation period of 72 h at a temperature of 28 degrees C. Thymidine incorporation into lymphocytes is enhanced when PHA and LPS (1 microgram/ml each) are given simultaneously. After a first stimulation by LPS the lymphocytes become less sensitive to a second exposure to this same mitogen. In contrast, LPS-primed lymphocytes are highly reactive to PHA. These results suggest that there exists (at least) two populations of peripheral blood lymphocytes, one sensitive to PHA and the other sensitive to LPS.

Une Microsporidiose provoque actuellement de fortes mortalités dans les populations de Salmonidés du Sud-Ouest de la France. Les lésions de cette maladie ont fait penser à une atteinte d'Ulcerative Dermal Necrosis ou de Syndrome mycosique. En fait, l'observation en microscopie photonique de coupes histologiques et de frottis d'oeufs et d'organes (fig. 3-4-5-6), montre qu'il s'agit d'une parasitose, dont l'agent pathogène, une Microsporidie, n'est pas encore déterminé spécifiquement. L'infection se localise surtout à la peau, au rein, à l'intestin et aux gonades. Au niveau des ovaires elle se traduit par la ponte soit d'oeufs abortifs soit d'oeufs féconds donnant naissance à des alevins parasités qui résorbent difficilement leur vitellus. Cette transmission verticale est un facteur de dissémination très important du fait du commerce des oeufs. Aussi des mesures prophylactiques visant à s'assurer de l'état sanitaire des géniteurs et des oeufs doivent-elles être prises pour éviter une extension géographique de la parasitose.