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handle: 10261/251187
[EN] Three serological techniques (indirect immunofluorescence test, flow cytometry, and indirect dot - blot immunoenzymatic assay) have been evaluated for the detection of lymphocystis viral antigens using a gilt-head seabream cell line, SAF-1, and fish leukocytes. Six lymphocystis disease virus (LCDV) isolates from gilt-head seabream, and one reference strain (ATCC VR 342), were tested. Detection of viral LCDV antigens in SAF-1 cells and fish leukocytes by indirect immunofluorescence test occurs at similar periods (5-7 d post-inoculation), and viral antigens were detected as cytoplasmic inclusions located at the periphery of inoculated cells. The percentages of cells with LCDV antigens obtained by flow cytometry were very low, ranging between 0.9% at 5 d post-inoculation and 19.7% at 10 d post-inoculation. The optimal concentration of viral stocks detected by indirect dot - blot immunoenzymatic assay was 0.5 μg ml-1, when purified viral stocks were used as antigens. Inoculated and uninoculated SAF-1 cells could not be distinguished using LCDV antiserum binding. On the basis of these results, indirect immunofluorescence and flow cytometry tests appear to be the best serological methods to detect LCDV antigens in both SAF-1 cells and fish leukocytes. [ES] Se han evaluado tres técnicas serológicas (inmunofluorescencia indirecta, citometría de flujo y ensayo inmunoenzimático indirecto dot–blot) para la detección de antígenos del virus de linfocistis usando una línea celular procedente de dirada, SAF-1, y leucocitos de peces. En total se han utilizado 6 aislados de virus de linfocistis (LCDV) procedentes de doradas y una cepa de referencia del virus (ATCC VR 342). La detección de los antígenos LCDV en células SAF-1 y leucocitos de peces se producía en períodos similares (a los 5–7 días post-inoculación) usando la técnica de inmunofluorescencia indirecta, y los antígenos virales se detectaban como inclusiones citoplasmáticas localizadas en la periferia de las células inoculadas. Los porcentajes de células con antígenos de LCDV obtenidos por citometría de flujo fueron muy bajos, comprendidos entre el 0,9% a los 5 días post-inoculación y el 19,7% a los 10 días post-inoculación. La concentración óptima de los aislados virales detectados por el ensayo inmunoenzimático indirecto dot–blot fue 0,5 μg ml–1, usando aislados virales purificados como antígenos. No se pudo distinguir células SAF-1 inoculadas y sin inocular usando el antisuero contra el LCDV. En base a los resultados obtenidos, las técnicas de inmunofluorescencia indirecta y citometría de flujo fueron los mejores métodos serológicos para la detección de antígenos de LCDV tanto en células SAF-1 como en leucocitos de peces. This study was supported by a grant of CICYT of the Spanish Government (MAR99-0609). We greatly appreciate the assistance of Ms. M. Jose Navarrete for her help with the English revision.
Detection methods, Dot - blot, Immunofluorescence, Viral fish diseases, Flow cytometry, Lymphocystis
Detection methods, Dot - blot, Immunofluorescence, Viral fish diseases, Flow cytometry, Lymphocystis
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