Vol. 10: 31-38, 1991 l DISEASES OF AQUATIC ORGANISMS Dis. aquat. Org. l Published March 8 Detection of rainbow trout antibody to Egtved virus by enzyme-linked immunosorbent assay (ELISA), immunofluorescence (IF), and plaque neutralization tests (50 %PNT) N. J. Olesen, N. Lorenzen, P. E. V. Jergensen National Veterinary Laboratory. Hangevej 2, DK-8200 hhus N. Denmark ABSTRACT. The humoral immune response to Egtved virus, the causative agent of viral haemorrhagic septicaemia (VHS) in rainbow trout Oncorhynchus mykiss, was investigated by means of the enzyme- linked immunosorbent assay (ELISA), immunofluorescence (IF), and the 50 'X plaque neutralization test (50°/"PNT). Sera from fish immunized with virus under aquarium conditions as well as sera collected from fish in farms with different VHS status were included In the experiments. ELISA proved to be more sensitive and less time- and material-consuming than either IF or 50°/~PNT Using Elisa, antibody to Egtved virus was found in 54 %I of fish from infected trout farms examined wh~le only 16 % were positive by 50°/r,PNT The IF test had a sensitivity close to that of ELISA, but was less suitable for examination of large numbers of sera. Non-neutralizing antibodies tend to perslst longer after VHS infection than neutralizing antibodies. The kinetics of the antibody response was close to that prev~ously published. No anamnestic immune response was observed with any of the tests. We conclude that our ELISA is well suited for VHS surveillance. INTRODUCTION The use of serological methods to survey for viral haemorrhagic septicaemia (VHS) in rainbow trout Oncorhynchus mykiss was made possible by the recent development of a sensitive plaque neutralization test (50%PNT) for detecting neutralizing trout antibodies to Egtved virus, the causative agent of VHS (Dorson & Torchy 1979, Olesen & Jsrgensen 1986a). Because the technique in question was relatively time-consuming (and thus unsuitable for examining large numbers of serum samples), attempts have been made to develop less time-consuming antibody detection methods of similar or higher sensitivity and specificity. The present report describes a modification of a previously devel- oped immunofluorescence (IF) test (Jsrgensen 1974) as well as a newly developed enzyme-linked immuno- sorbent assay (ELISA). The 3 techniques were com- pared by examining a number of sera from trout exposed to VHS under experimental or field conditions. MATERIALS AND METHODS Viruses and fish cell lines. Egtved virus, reference strain F1 (Jensen 1965), was cultivated and purified as previously described (Lorenzen et al. 1988). Stock preparations of 2 F1-related low-passage isolates of Egtved virus, Gelsbro and Voldbjerg, and of the heat-adapted F25 mutant strain of F1 (de Kinkelin et al. 1980) were prepared in bluegill fry (BF-2) cells (Wolf et al. 1966) grown in Eagles MEM containing 10 % foetal bovine serum and Tris-HCL buffer and antibiotics in standard concentrations. The spring viraemia of carp (SVC) virus (Fijan et al. 1971), a rhabdovirus, was included in the work to test the virus-specificity of the IF test. The SVC virus refer- ence strain received from N. Fijan, Zagreb, Yugoslavia, was grown in EPC cells (Fijan et al. 1983) at 15°C as described for Egtved virus. Antibodies and antibody conjugates. Antiserum to Egtved virus was prepared as follows: New Zealand O Inter-Research/Printed in Germany