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We used (1) ultracentrifugation followed by RT-PCR and (2) real-time RT-PCR to detect and quantify nodaviruses in seawater in which Atlantic halibut Hippoglossus hippoglossus larvae/fry had been held at rearing facilities. Evaluated against in vitro propagated viruses, the viral concentration corresponded to 1.6 × 10 4 TCID 50 (50% tissue culture infectious dose) ml-1. Evaluated against in vitro transcribed RNA, the concentration was estimated at 2 × 10 7 virus particles ml-1 seawater.

The effects of filleting method, pre-slaughter stress, storage and season on drip loss, water content, water-holding capacity, rigour contraction and colour in Atlantic cod, Gadus morhua L., were examined. A total of 182 fish were sampled under commercial conditions. To test the effects of filleting method and stress, stressed (S) and unstressed (C) fish were filleted either pre (Pr) or post rigour (Po) at 1 and 48 h post mortem, receptively. The muscle pH significantly decreased from 0 to 144 h of storage in all groups. The onset of the rigour contraction was more pronounced in the Pr-S group as compared with the Pr-C group, but after 144 h of storage, no difference in degree of contraction was observed. Filleting method, stress and storage time influenced the colour of the fillets. Post-rigour filleting caused a significantly increased in measured Lightness (L*) during storage. Stress caused a significant increase in measured redness (a*). No significant difference was found for water-holding capacity. The water content was influenced by filleting method, where the Pr-S group had a significantly lower water content compared with the Pr-C groups; water content changed also according to season. The findings of this study show that stress, filleting method, time of storage and season had a significant effect on the drip loss, where a combination of all factors will determine the total loss.

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Traditionally farmed fish are slaughtered and processed over several steps, which often include live chilling, stunning, exsanguination, chilling, gutting, rinsing, decapitation, filleting before the fillets are packed into polystyrene boxes and shipped with ice. These processes are often time, laboring, space, and energy consuming. A novel processing line for filleting of farmed fish is gutting and filleting the fish directly after decapitation and replacing exsanguination with spray washing the fillets. In addition, all the cooling steps are replaced by superchilling the fillets. This novel process line gives fillets with comparable if not superior quality compared to the traditional process.

We have developed a real-time nucleic acid sequence based amplification (NASBA) procedure for detection of infectious salmon anaemia virus (ISAV). Primers were designed to target a 124 nucleotide region of ISAV genome segment 8. Amplification products were detected in real-time with a molecular beacon (carboxyfluorescin [FAM]-labelled and methyl-red quenched) that recognised an internal region of the target amplicon. Amplification and detection were performed at 41°C for 90 min in a Corbett Research Rotorgene. The real-time NASBA assay was compared to a conventional RT-PCR for ISAV detection. From a panel of 45 clinical samples, both assays detected ISAV in the same 19 samples. Based on the detection of a synthetic RNA target, the real-time NASBA procedure was approximately 100× more sensitive than conventional RT-PCR. These results suggest that real-time NASBA may represent a useful diagnostic procedure for ISAV.KEY WORDS: Infectious salmon anaemia virus · Orthomyxovirus · NASBA · Diagnostics · Fish · Nucleic acid amplification Resale or republication not permitted without written consent of the publisherDis Aquat Org 72: [107][108][109][110][111][112][113] 2006 Sensitive and specific diagnostic procedures are essential for the effective control of ISA. Methods currently used for the detection of ISAV include virus isolation in SHK-1, ASK, or CHSE-214 cells (Cipriano & Miller 2003), in situ hybridization (Gregory 2002), indirect fluorescent antibody testing (Falk & Dannevig 1995), and RT-PCR (Mjaaland et al. 1997, Rimstad et al. 1999, Devold et al. 2000. A real-time RT-PCR method for the diagnosis of ISAV utilising SyBr Green I detection of amplification products has been described by Munir & Kibenge (2004).Nucleic acid sequence based amplification (NASBA) is an isothermal nucleic acid amplification procedure based on the activity of 3 enzymes; reverse transcriptase, RNase H, and T7 RNA polymerase (Compton 1991). Real-time detection in NASBA can be performed using molecular beacon probes (Leone et al. 1998). NASBA detection methods have been described for several viruses including human immunodeficiency virus type 1 (de Baar et al. 1999 (Starkey et al. 2004) and SARS-associated coronavirus (Keightley et al. 2005).In the present study we report on the development of a real-time NASBA procedure for detection of ISAV. The real-time NASBA assay was compared to a conventional RT-PCR assay for ISAV using previously described primers (Mjaaland et al. 1997). MATERIALS AND METHODSClinical samples. A panel of 45 clinical samples (Atlantic salmon kidney) obtained from outbreaks of ISA in Scotland, the Faroe Islands and Norway was used in this study (see Table 1). Samples 1 to 20 were archival, and following collection were stored at -70°C. Samples 21 to 45 were obtained from Atlantic salmon during an outbreak of ISA, and were stored in RNAlater (Ambion) at -20°C.RNA isolation. Isolation of RNA from tissue samples for use in both RT-PCR and NASBA was performed using the Nucleospin procedure (Machery Nagel)...