Detection and antigenic characterization of salmonid alphavirus isolates from sera obtained from farmed Atlantic salmon, <i>Salmo salar</i> L., and farmed rainbow trout, <i>Oncorhynchus mykiss</i> (Walbaum)

Jewhurst, Victoria; Todd, D.; Rowley, H M; Walker, Ian; Weston, Jonathan; McLoughlin, M F; Graham, D. A.

doi:10.1111/j.1365-2761.2004.00530.x

Cited by 40 publications

(47 citation statements)

References 12 publications

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Section: Methodsmentioning

confidence: 99%

“…Serum samples (without added virus) were run in parallel with each VN test to identify viraemic serum. Immunostaining of these wells was used to identify the presence of virus in collected sera (Jewhurst et al 2004).Statistical analysis. Due to the limited ordinal nature of the recorded lesion scores (scored using a 0 to 4 scale), non-parametric analyses were used on this data.…”

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Virological, serological and histopathological evaluation of fish strain susceptibility to experimental infection with salmonid alphavirus

McLoughlin¹,

Graham²,

Norris³

et al. 2006

Dis. Aquat. Org.

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Pancreas disease (PD) of farmed Atlantic salmon Salmo salar L., which is caused by an alphavirus known as salmon pancreas disease virus (SPDV), can have serious economic consequences. An epidemiological survey carried out in Ireland in 2003 indicated that within individual farms there were significant differences in the susceptibility of different strains of farmed Atlantic salmon to infection with SPDV, as measured by levels of clinical disease and mortality. The aim of this preliminary study was to investigate this field observation by comparing lesion development, viraemia and serological responses of 3 commercial strains of Atlantic salmon (A, B and C) experimentally infected with SPDV. Highly significant differences in the severity of lesions in the pancreas at Day 21 post-infection (pi) were detected (p < 0.01), with Group B being more severely affected. There were also significant differences in the prevalence and severity of lesions in heart and skeletal muscle at Day 21 and 35 pi respectively, with Group B results again significantly higher than those from both Groups A and C (p < 0.05). There was no overlap between viraemia and the presence of specific SPDV antibody. Some fish in all groups had no viraemia, lesions or evidence of seroconversion. There were no significant differences seen between the challenged groups in relation to the percentage of viraemic fish at each time point. Viral loads were not determined. Differences between the number of antibody-positive fish in each challenge group were found at Days 28 and 35 pi (p < 0.1). Highly significant differences (p < 0.01) in the geometric mean titres of seropositive fish were detected at Day 28. These results, obtained using a challenge model, confirm that there are strain differences in the susceptibility to experimental SPDV infection in commercial farmed Atlantic salmon. KEY WORDS: Salmon Pancreas Disease Virus · SPDV · Atlantic salmon · Disease susceptibility · Experimental challenge Resale or republication not permitted without written consent of the publisherDis Aquat Org 72: [125][126][127][128][129][130][131][132][133] 2006 (SPDV), the aetiological agent of PD, was isolated in 1993 (Nelson et al. 1995) and was subsequently classified as a salmonid alphavirus (SAV) (Weston et al. 1999(Weston et al. , 2002.National surveys in Ireland from 1989 to 1994 indicated that mortality owing to PD could be up to 48%, with 94% of Irish marine sites affected, and that PD was the major cause of disease losses at that time (Menzies et al. 1996). From 1996 to 2001 there appeared to be a lower incidence and severity of PD in Ireland (McLoughlin et al. 1998). However, in 2002 there was an increase in both the severity and incidence of PD recorded in Ireland ).An epidemiological survey undertaken in 2003 revealed that 13 of 21 sites (61%) had experienced PD, with mortality reaching 40% in some cages . There have also been reports from Scotland and Norway that PD has re-emerged as a significant problem in specific regions of both countries, resu...

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Section: Methodsmentioning

confidence: 99%

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confidence: 99%

Virological, serological and histopathological evaluation of fish strain susceptibility to experimental infection with salmonid alphavirus

McLoughlin¹,

Graham²,

Norris³

et al. 2006

Dis. Aquat. Org.

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“…With the exception of the last 2 sample sets, the remainder came from sites where SAV-induced disease was present or suspected. These sera had been tested for the presence of SAV viraemia and for virus-neutralizing antibodies as previously described (Jewhurst et al 2004). In some submissions, only antibody negative sera were tested by RRT-PCR.…”

Section: Methodsmentioning

confidence: 99%

“…Recently, a practical and rapid serological assay detecting the presence of virus neutralizing antibodies has been described (Graham et al 2003). This assay, coupled with the recognition that SAV-infected fish undergo a viraemic phase that may be conveniently and rapidly detected in cell cultures by immunostaining (Jewhurst et al 2004) provided a novel diagnostic approach to detect and follow the progress of SAV infections in farm and experimental situations . RT-PCR represents an alternative rapid diagnostic approach that has not been widely used for SAV detection.…”

Section: Introductionmentioning

confidence: 99%

Development and evaluation of a one-step real-time reverse transcription polymerase chain reaction assay for the detection of salmonid alphaviruses in serum and tissues

Graham

Taylor²,

Rodgers³

et al. 2006

Dis. Aquat. Org.

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We designed 4 primer pairs to amplify conserved regions of the E1 or nsP4 genes of salmonid alphavirus (SAV) and evaluated their performance in optimized 1-step SYBR green realtime RT-PCR (RRT-PCR) assays. A single primer pair, amplifying a 227 bp segment of E1 was then chosen for further study. This RRT-PCR was shown to be highly repeatable and reproducible over a wide range of RNA dilutions, with a linear relationship between cycle threshold (Ct) value and RNA concentration over a 10 7 dilution range. The limit of detection was calculated to be ≤1.5 TCID 50 ml -1 . When applied to sera previously screened by virus isolation for SAV viraemia, the RRT-PCR correctly identified all 13 culture-positive samples, as well as finding an additional 28 sera positive. Relative semi-quantitation of sera showed a very highly significant relationship between copy number and TCID 50 (p < 0.001, R 2 = 0.9563). Following experimental infection of salmon, heart samples were consistently positive until 21 d post infection (dpi), with (weak) positive signals still detectable in 50% of fish 70 dpi.

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“…Detection of antibodies or virus isolation in fish cells may also be used to verify the aetiology of the disease (Graham et al, 2003a;Jewhurst et al, 2004). However, the presence of virus-specific antibodies does not provide any information about the viraemic status of an infected fish, and considering the high percentage similarity of the structural proteins among the salmonid alphaviruses, the potential for cross-reactivity of serological assays is high.…”

Section: Introductionmentioning

confidence: 99%

Sensitive and specific detection of Salmonid alphavirus using real-time PCR (TaqMan®)

Hodneland

Endresen

2006

Journal of Virological Methods

136

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Pancreas disease is responsible for major economic losses in the European salmonid farming industry. It was previously believed that a single subtype (salmon pancreas disease virus) of the virus species Salmonid alphavirus (SAV) was responsible for all outbreaks of pancreas disease in the UK and Norway. However, the recent discovery that pancreas disease in Norway is caused by a new and distinct subtype of salmonid alphavirus, exclusively found in Norway, has advocated the need for better diagnostic tools. In the present paper, three real-time PCR assays for all known subtypes of salmonid alphavirus have been developed; the Q nsP1 assay is a broad-spectrum one that detects RNA from all subtypes, the Q SPDV assay specifically detects the salmon pancreas disease virus subtype, and the Q NSAV assay only detects the new Norwegian salmonid alphavirus subtype.The results demonstrated the assays to be highly sensitive and specific, detecting <0.1 TCID 50 of virus stocks. Regression analysis and standard curves calculated from the C t -values from 10-fold serial dilutions of virus stocks showed that the assays were highly reproducible over a wide range of RNA input. Thirty-nine field samples were tested in triplicate and compared with traditional RT-PCR. Overall, the real-time assays detected 35 positive compared to 29 positives in standard RT-PCR, and were thus shown to be more sensitive for detecting salmonid alphaviruses in field samples.

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Detection and antigenic characterization of salmonid alphavirus isolates from sera obtained from farmed Atlantic salmon, Salmo salar L., and farmed rainbow trout, Oncorhynchus mykiss (Walbaum)

Cited by 40 publications

References 12 publications

Virological, serological and histopathological evaluation of fish strain susceptibility to experimental infection with salmonid alphavirus

Virological, serological and histopathological evaluation of fish strain susceptibility to experimental infection with salmonid alphavirus

Development and evaluation of a one-step real-time reverse transcription polymerase chain reaction assay for the detection of salmonid alphaviruses in serum and tissues

Sensitive and specific detection of Salmonid alphavirus using real-time PCR (TaqMan®)

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