Immunofluorescence

Immunofluorescence has been used to study fish viruses since 1972 (J0rgensen and Meyling, 1972) and is widely accepted for the detection and identification of IHNV in cell cultures. Either direct (DFAT) or indirect (IFAT) fluorescent antibody tests can be used. Routinely, DFAT uses fluorescein isothiocyanate (FITC)-labelled anti-IHNV polyclonal antibody or MAb and IFAT uses unlabelled primary antibody, which is detected using FITC-conjugated secondary antibody. Leong et al. (1983a) first reported IFAT for detecting IHNV in cell cultures and this method is rapid, specific and sensitive and can detect all five electropherotypes of IHNV (LaPatra et al., 1989a). A diagnosis can be made in 24 h using 50% end-point titration, if ovarian fluid inoculated on to cells has a virus titre of at least 102 9 pfu ml-1, and pretreatment of the cells with PEG improves the limit of detection to 102 5 pfu ml-1. A plaque assay is slightly more sensitive, detecting as low as 10 pfu ml-1, but requires up to 8 days to observe plaques and only provides a presumptive diagnosis (LaPatra et al., 1989a). Polyclonal and monoclonal antinucleoprotein and antiglycoprotein antibodies used in IFAT have equivalent sensitivity and time requirements for making a diagnosis, but preadsorption of polyclonal serum is required to prevent non-specific fluorescence (LaPatra et al., 1989b; Arnzen et al., 1991).

Used on cell culture samples, IFAT and DFAT have similar sensitivity (LaPatra et al., 1989a; Arnzen et al., 1991), but DFAT may be preferred because it is more rapid (Ristow and Arnzen, 1989; Arnzen et al., 1991). Cells should ideally be incubated for 16-24 h before using IFAT or DFAT, but, when rapid decisions are required, DFAT can be performed after only 10-12 h incubation (LaPatra et al., 1989b; Arnzen et al., 1991). Monoclonal antibody-based IFAT or DFAT can be used to type viral isolates, identify new viral types and study viral variants (Ristow and Arnzen, 1989; Ristow and Arnzen-de Avila, 1991; Roberti et al., 1991).

The two potential disadvantages of using fluorescent antibody tests (FATs) on cell cultures have been the use of cover glasses and detachment of cells after acetone fixation (LaPatra et al., 1989a). However, these problems have been overcome since LaPatra et al. (1989a) demonstrated that IFAT could be used directly on plaque assay plates and cells could be fixed with other fixatives. Kamei et al. (1991) confirmed that cover glasses were not needed by showing that IHNV could be detected in formalin-fixed cells in 96-well plates. Other disadvantages of FATs are that trained personnel are required and fluorescent microscopes are fairly expensive.

Direct detection of IHNV in fish tissues using IFAT or DFAT could substantially shorten the time for a diagnosis from several days to a few hours. Indirect FAT detected IHNV in blood smears and kidney imprints from clinically infected juvenile fish, but was unsuccessful in detecting virus in smears of seminal fluid (LaPatra et al., 1989a). The use of DFAT on fish tissues has yet to be investigated.

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