Immune response to a recombinant capsid protein of striped jack nervous necrosis virus (SJNNV) in turbot Scophthalmus maximus and Atlantic halibut Hippoglossus hippoglossus, and evaluation of a vaccine against SJNNV.

Immunisation by intraperitoneal injection of an oil-emulgated recombinant partial capsid protein (rT2) from striped jack nervous necrosis virus (SJNNV) was performed on adult turbot Scophthalmus maximus and Atlantic halibut Hippoglossus hippoglossus. A specific humoral immune response was recorded in both species, and the levels of rT2-specific antibodies increased markedly in all groups during the 20 wk experiment. A challenge model for SJNNV was established by intramuscular injection of juvenile turbot. The turbot developed viral encephalopathy and retinopathy (VER), also known as viral nervous necrosis (VNN), with cumulative mortality in the range of 25 to 66%, after intramuscular inoculation with SJNNV propagated in the striped snake head cell line (SSN-1). Although neither clinical signs nor mortality were registered, SJNNV was neuroinvasive after bath exposure. The infection after both modes of challenge was verified by means of immunohistochemistry and RT-PCR, and SJNNV was reisolated in cell culture. The results indicate that SJNNV may have entered the central nervous system (CNS) by axonal transport through motor nerves after intramuscular inoculation. A vaccine efficacy test was performed on juvenile turbot, employing oil emulsified rT2 as a test vaccine and intramuscular inoculation of SJNNV. Significant protection was observed when the challenge was performed 10 wk post-vaccination.

Uptake and processing of a Vibrio anguillarum bacterin in Artemia franciscana measured by ELISA and immunohistochemistry.

Nauplii of Artemia franciscana were incubated in two different concentrations (undiluted and 1:9 in autoclaved sea water) of a divalent bacterin composed of two different serovars of Vibrio anguillarum. In order to investigate uptake and further processing of a bacterin in the live feed organism A. franciscana, immunohistochemistry was applied, visualising the presence of whole bacterial cells and antigens from the bacterin in individual nauplii. By using ELISA, it was shown that approximately 1.5-2-5 x 10(5) cells were incorporated into each Artemia under the conditions used. Maximum incorporation of cells was measured after 30 min, whereas after 60 min there was a decline to levels of 0.9-1.6 x 10(5) cells per Artemia. Immediately after incubation in the bacterin solution, the nauplii were transferred to a culture of the alga Isochrysis galbana, in order to simulate transfer of the nauplii to rearing tanks for fish larvae. From the ELISA, it could be concluded that the incorporated bacterial cells were excreted from the Artemia nauplii rapidly, however a large variation among different nauplii could be visualised by immunohistochemistry.

Classification of fish-pathogenic vibrios based on comparative 16S rRNA analysis.

No systematic classification of fish-pathogenic vibrios has been accomplished previously despite the use of serological, physiological, and genetical classification systems. In this study, a comparative 16S rRNA analysis of 34 strains (representing seven species) of fish-pathogenic vibrios was performed. The 16S rRNA sequences were obtained by using reverse transcriptase. Nearly complete sequences were obtained for nine strains. On the basis of the results of this analysis, the remaining strains were investigated by analyzing selected stretches containing a total of 560 nucleotides. With the exception of a few strains, including ATCC 43313 (serovar O9), our comparative 16S rRNA analysis confirmed that strains preliminarily identified as Vibrio anguillarum were phylogenetically closely related. Strains of V. anguillarum could be divided into groups, with the main group containing serotype O1 and O2 strains isolated from Atlantic salmon, rainbow trout, turbot, cod, and saithe. The other distinctive group was represented by type strain NCMB 6. This strain was nearly indistinguishable from the type strains of Vibrio ordalii and Vibrio damsela on the basis of the 16S rRNA stretches compared. The results of a comparative 16S rRNA analysis justified the status of Vibrio salmonicida as a distinct species. Originally, this species was characterized biochemically as a very homogeneous species. However, two strains, which were isolated from diseased halibut and from the intestines of healthy cod, could not be distinguished from V. salmonicida strains phylogenetically, although they differed from the original species description in several phenotypic traits. Our results indicate that V. salmonicida and Vibrio fischeri form a cluster that is clearly separated from the cluster that includes V. anguillarum.