Experimental transmission of sleeping disease in one-year-old rainbow trout, Oncorhynchus mykiss (Walbaum), induced by sleeping disease virus.

Sleeping disease (SD) is a serious disease of rainbow trout, Oncorhynchus mykiss, reared in fresh water caused by sleeping disease virus (SDV). In this study a detailed clinical, histological, virological and serological description of the experimental reproduction of SD in 1-year-old rainbow trout exposed to SDV was carried out. Two hundred disease-free fish were intraperitoneally inoculated with a SDV isolate and 100 fish were inoculated with an uninfected cell culture lysate as a negative control. Infected and control fish were randomly removed at days 4, 7, 14, 21, 42 and 70 post-infection. Blood and tissues were collected for virus isolation, histopathological examination and serum neutralization. SDV was detected in serum, kidney and brain of infected fish from 4 to 21 days post-infection (dpi). Characteristic pathological lesions were observed in infected fish as early as 7 dpi. Lesions were first detected in exocrine pancreas and subsequently observed in heart and skeletal muscle. Neutralizing antibodies to SDV were detected in infected fish from 14 to 70 dpi. Infected fish displayed typical signs of SD 1-month pi and the mortality reached 18.7% within 44 days. This study experimentally reproduced all the pathognomonic features of natural outbreaks of SD in 1-year-old rainbow trout.

Pathogenesis and immune response in Atlantic salmon (Salmo salar L.) parr experimentally infected with salmon pancreas disease virus (SPDV).

Atlantic salmon parr were injected intraperitoneally with salmon pancreas disease virus (SPDV) grown on CHSE-214 cells. The viraemia, the histopathological changes in target organs and some immune parameters were taken at intervals up to 30 days post-infection (dpi). The earliest kind of lesion was necrosis of exocrine pancreas, appearing as soon as 2 dpi. It progressed towards complete tissue breakdown at 9 dpi before resolving gradually. Concurrent to this necrosis, a strong inflammatory response was in evidence from 9 dpi in the pancreatic area for a majority of fish. A necrosis of the myocardial cells of the ventricle occurred in infected fish mainly at 16 dpi and it faded thereafter. The monitoring of the plasma viral load showed a rapid haematogenous spreading of SPDV, peaking at 4 dpi, but also the absence of a secondary viraemia. No interferon (IFN) was detected following the infection of parr with SPDV, probably owing to an IFN activity in Atlantic salmon below the detection level of the technique. Neutralising antibodies against SPDV were in evidence from 16 dpi and they showed a time-related increasing titre and prevalence. The phagocytic activity in head-kidney leucocytes was always significantly higher in the infected fish than in the control fish, being particularly high by 9 dpi. Lysozyme and complement levels were both increased and they peaked significantly in the infected fish at 9 and 16 dpi respectively. These results demonstrated that an experimental infection of Atlantic salmon parr with SPDV provoked a stimulation of both specific and non-specific immunity with regards to the viraemia and the histopathology.

An RT-PCR-based method for the diagnosis of the sleeping disease virus in experimentally and naturally infected salmonids.

The sleeping disease (SD) of rainbow trout (Oncorhynchus mykiss) is a worldwide disease for which the causative agent, the sleeping disease virus (SDV), has been recently characterized as an atypical alphavirus (Villoing et al. 2000). Up to now, no diagnostic tools were available and thus no epidemiological studies have been undertaken to evaluate the occurrence of this disease on the field. We present in this paper a sensitive and highly specific 1 working day method, which allows the detection of SDV from experimentally and naturally infected fishes. This method, based on a reverse transcriptase/polymerase chain reaction (RT-PCR) assay on total RNA extracted from SDV-infected fish organs, enables the specific DNA amplification of part of the gene encoding the SDV glycoprotein E2, as early as 2 d post-infection (d.p.i.) and as late as 70 d.p.i., at which time clinical signs of infection are no longer apparent. Moreover, we show that this RT-PCR method can be successfully used for the diagnosis of fish infected by a closely related virus, namely salmon pancreas disease virus (SPDV). This report is the first description of a very powerful diagnostic assay which could provide a more accurate replacement for the classical virological, histological and immunochemistry methods.

Comparative study of viral encephalopathy and retinopathy in juvenile sea bass Dicentrarchus labrax infected in different ways.

The transmission of viral encephalopathy and retinopathy (VER) was investigated in juvenile sea bass (3 g) Dicentrarchus labrax by using cell culture supernatant (SSN-1 cell line) containing nodavirus. Five methods of infection were tested: intramuscular injection (IM), intraperitoneal injection (IP), oral infection, bath exposure and cohabitation of healthy fish with infected fish. Some differences were observed in time of disease onset and severity of symptoms depending on the mode of infection used. Clinical symptoms such as whirling swimming and lethargic or hyperactive behaviour were generally reproduced, except for fish infected via oral and IP infection. First mortalities occurred 3 d after IM and IP infection and 6 d after for the other modes of infection. Cumulative mortalities were also variable: 100% after IM infection, 10% after IP infection, 32% for bath exposure, 43% after cohabitation and 24% via oral infection. Histopathologically, vacuolation was observed in the central nervous tissues and in the retina. The observed lesions were more or less severe depending on the mode of infection, the sampling time and the organs: lesions on the surviving fish (42 days post infection, d p.i.) seemed to be generally more conspicuous in the retina than in the brain of the same fish. In most cases, the presence of nodavirus was confirmed in the same samples of brain and retina by immunohistochemistry and reverse transcriptase-polymerase chain reaction (RT-PCR). The virus was not detected in other organs examined. The present results suggest that 2 forms of VER can be induced: IM injection leads to an acute form (severe nervous disorders with high and fast mortality) whereas oral infection, bath exposure and cohabitation induce a subacute form (less severe disorders and weak daily mortality). This experiment demonstrates experimentally induced horizontal transmission of VER in sea bass for the first time.