Asunto(s)
Enfermedades de los Peces/microbiología , Francisella/aislamiento & purificación , Infecciones por Bacterias Gramnegativas/veterinaria , Salmo salar/microbiología , Fosfatasa Alcalina/análisis , Animales , Secuencia de Bases , Chile , ADN Espaciador Ribosómico/genética , Francisella/enzimología , Francisella/genética , Infecciones por Bacterias Gramnegativas/microbiología , Datos de Secuencia Molecular , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADNRESUMEN
T-cell-mediated hypersensitivity could be central in soybean meal (SBM)-induced intestinal changes in salmon. However, tools for immunohistochemical detection of T cells have been lacking in teleosts, including Atlantic salmon. Application of a specific histochemical protocol allowed demonstration of T-cell-like reactivities in formalin-fixed, paraffin-embedded tissues using an antibody reacting to a conserved region of human CD3epsilon (Dako A0452). Characteristic staining was observed in cells of the thymus as well as distal intestine, skin, gills and spleen. These cells were negative for immunoglobulin M (IgM). Intestinal intraepithelial leucocytes were CD3epsilon positive. During the SBM-induced enteropathy, the mixed inflammatory infiltrate in the lamina propria of the distal intestine included many lymphocytes with a T-cell-like reactivity. Real-time polymerase chain reaction revealed significantly increased expression of a complex polypeptide (CD3pp), CD4 and CD8beta (P < 0.05) in the distal intestine of SBM-fed fish compared to fish meal-fed reference fish. Increased reactivity for extracellular IgM in the lamina propria and a positive material between the epithelial cells at the tips of the folds was observed, possibly due to leakage of IgM through an abrogated epithelial barrier. In conclusion, a T-cell-like response appears to be involved in this example of a food-sensitive enteropathy.
Asunto(s)
Antígenos CD/inmunología , Expresión Génica/inmunología , Glycine max/inmunología , Salmo salar/inmunología , Linfocitos T/inmunología , Alimentación Animal/análisis , Animales , Anticuerpos/análisis , Anticuerpos/metabolismo , Antígenos CD/genética , Antígenos CD/metabolismo , Secuencia de Bases , Cartilla de ADN/química , Inmunohistoquímica/veterinaria , Mucosa Intestinal/inmunología , Mucosa Intestinal/patología , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa/veterinaria , ARN/genética , ARN/aislamiento & purificaciónRESUMEN
We have investigated the initial steps in the interaction between infectious salmon anemia virus (ISAV) and cultured cells from Atlantic salmon (SHK-1 cell line). Using radioactively or fluorescently labelled viral particles we have studied the binding and fusion kinetics and the effect of pH on binding, uptake, and fusion of ISAV to SHK-1 cells and liposomes. As pH in the medium was reduced from 7.5 to 4.5, the association of virus to the cells was nearly doubled. The same effect of pH was observed when fusion between ISAV and liposomes was analyzed. In addition, the binding of ISAV to intact SHK-1 cells and to cell membrane proteins blotted onto filters was neuraminidase sensitive. However, the increased binding induced by low pH was not neuraminidase sensitive, probably reflecting activation of a fusion peptide at low pH. By using confocal fluorescence microscopy, the increased fusion of fluorescently labelled ISAV with the plasma membrane due to low pH could be demonstrated. When vacuolar pH in the cells was raised during inoculation with chloroquine or ammonium chloride, both electron and confocal microscopy showed accumulation of ISAV in endosomes and lysosomes. Production of infectious virus could be increased by lowering the extracellular pH during infection. Furthermore, chloroquine present during virus inoculation also caused a reduction in the synthesis of viral proteins in ISAV-infected cells as well as in the production of infective virus. These results indicate that ISAV binds to sialic acid residues on the cell surface and that the fusion between virus and cell membrane takes place in the acid environment of endosomes. This provides further evidence for a high degree of similarity between ISAV and influenza virus and extends the basis for the classification of this virus as a member of the Orthomyxoviridae family.
Asunto(s)
Concentración de Iones de Hidrógeno , Gripe Humana/virología , Macrólidos , Orthomyxoviridae/fisiología , Cloruro de Amonio/farmacología , Antibacterianos/farmacología , Línea Celular , Cloroquina/farmacología , Frío , Efecto Citopatogénico Viral/efectos de los fármacos , Humanos , Fusión de Membrana , Microscopía Electrónica , Orthomyxoviridae/patogenicidad , Orthomyxoviridae/ultraestructura , Inhibidores de la Síntesis de la Proteína/farmacología , Proteínas Virales/antagonistas & inhibidores , Proteínas Virales/biosíntesisRESUMEN
In macrophages of higher vertebrates, Fc receptors and receptors for complement and other serum factors, are generally known to enhance the phagocytic process. In lower vertebrates like salmonid fishes, none of these or other phagocytic receptors have been thoroughly characterized. The purpose of this study was to elucidate to what extent these and other receptors are involved in the process of phagocytosis in rainbow trout (Oncorhynchus mykiss) head kidney macrophages. We used tosyl activated, paramagnetic dynabeads (2.8 microm in diameter), specifically coated with 125I labeled Atlantic salmon (Salmo salar) IgM or bovine serum albumin (BSA) as phagocytic probes. The effect of complement opsonization was also investigated by incubating the beads in serum. Our results indicate that neither the Fc- nor the complement-receptor(s) were important for phagocytosis of these beads. Our data support the idea that scavenger receptors are involved in phagocytosis in rainbow trout head kidney macrophages, as the use of a competitive scavenger receptor ligand extensively decreased degradation of the labeled protein coat on the beads.