Distribution of red-spotted grouper nervous necrosis virus (RGNNV) antigens in nervous and non-nervous organs of European seabass(Dicentrarchus labrax) during the course of an experimental challenge

Abstract

The distribution of red-spotted grouper nervous necrosis virus (RGNNV) antigens was examined by immunohistochemistry in the nervous and non-nervous organs of juvenile European seabass (Dicentrarchus labrax) during the course of an intramuscular infection. Histological changes resulting from the infection were evaluated from 3 days to 2 months post-infection. The specific antibody response was also studied 2 months post-challenge. Viral proteins were present throughout the experimental period in the retina (inner nuclear layer, ganglion layer, outer limiting membrane, and outer plexiform layer), brain(cerebellum and tectum opticum), and liver (hepatocytes and endothelial cells). These proteins were also observed in the renal tubular cells, white pulp of spleen, and in fibroblasts and cartilage of caudal fin. This is the first report of RGNNV proteins appearing in these organs, where the immunostaining was only detected at certain sampling times after the onset of mortality. Brain and retina of virus-exposed fish showed high levels of vacuolation, while accumulation of fat vacuoles was observed in the liver. RGNNV infection also induced a specific antibody response as measured by an ELISA. In summary, this is the first study demonstrating the presence of viral proteins in cells of caudal fin, kidney and spleen of European seabass.

Fig. 1

Immunohistochemistry (IHC) signals in sections of non-nervous tissues. Positive cells contain dark purple signals (arrows). (A) Kidney from non-infected fish (negative control). (B) Kidney sampled 17 days post-infection (PI) showing red-spotted grouper nervous necrosis virus (RGNNV) antigens in tubular epithelial cells. (C) Liver section from non-infected fish (negative control). (D) Liver section from infected fish 3 days PI. Staining was observed in hepatocytes and endothelial cells. (E) Spleen section from non-infected fish (negative control). (F) Positive signal in white pulp of the spleen on day 10 PI. (G) Caudal fin section from non-infected fish (negative control). (H) Positive cartilage cells in the caudal fin 17 days PI. Scale bars = 100 µm.

Fig. 2

Sections of nervous tissues evaluated with IHC. Positive cells contain dark purple signals (arrows). (A) Brain from non-infected fish (negative control). (B) Brain from inoculated fish 24 days PI. Staining is in the area between the tectum opticum and cerebellum. (C) Retina from non-infected fish (negative control). (D) Retina from inoculated fish 17 days PI. Stained cells are in the outer limiting membrane (olm), outer plexiform layer (opl), and ganglion layer (gl). Scale bars = 100 µm.

Fig. 3

Histological damage caused by RGNNV infection. (A) Brain from non-infected fish (negative control). (B) Brain from infected fish. Vacuolation was observed in the cerebellum (arrow). (C) Retina from non-infected fish (negative control). (D) Retina from infected fish. Vacuoles (arrows) were found in the outer nuclear layer (onl), inner nuclear layer (inl), inner plexiform layer (ipl), and ganglion layer (gl). (E) Liver from non-infected fish (negative control). (F) Accumulation of fat vacuoles in hepatocytes from challenged fish. All tissues shown were sampled 17 days PI. H&E stain. Scale bars = 50 µm (B, C, and D), and 25 µm (A, E, and F).

Fig. 4

The mean optical density (OD) values from the ELISA analysis of three replicates of three samples composed of pooled sera from five fish. Titration of anti-RGNNV antibodies in sera obtained from challenged animals 24 days (♦) and 2 months (•) PI. Sera collected from control fish 24 days (◊) and 2 months (○) PI were also tested. (1) and (2) are the thresholds for samples collected 24 days and 2 months PI, respectively.