Britt Bang Jensen

Quantification of piscine reovirus (PRV) at different stages of Atlantic salmon Salmo salar production

The newly described piscine reovirus (PRV) appears to be associated with the development of heart and skeletal muscle inflammation (HSMI) in farmed Atlantic salmon Salmo salar L. PRV seems to be ubiquitous among fish in Norwegian salmon farms, but high viral loads and tissue distribution support a causal relationship between virus and disease. In order to improve understanding of the distribution of PRV in the salmon production line, we quantified PRV by using real-time PCR on heart samples collected at different points in the life cycle from pre-smolts to fish ready for slaughter. PRV positive pre-smolts were found in about 36% of the freshwater cohorts and a general increase in viral load was observed after their transfer to seawater. A reduction in viral loads was recorded when fish approached slaughter (18 mo in sea cages). Sequencing of positive samples did not support the hypothesis that outbreaks are caused by the spreading of a particular (virulent) strain of PRV.

Susceptibility of pike Esox lucius to a panel of Ranavirus isolates.

In order to study the pathogenicity of ranaviruses to a wild European freshwater fish species, pike Esox lucius fry were challenged with the following Ranavirus isolates: epizootic haematopoietic necrosis virus (EHNV), European sheatfish virus (ESV), European catfish virus (ECV), pike-perch iridovirus (PPIV), New Zealand eel virus (NZeelV) and frog virus 3 (FV3). The fry were infected using bath challenge at 12 and 22 degrees C. Significant mortalities were observed at 12 degrees C for EHNV, ESV, PPIV and NZeelV. Background mortality was too high in the experiments performed at 22 degrees C for any conclusions about viral pathogenicity at this temperature to be drawn. Viruses could be re-isolated from samples from all challenged groups, and their presence in infected tissue was demonstrated using immunohistochemistry. The findings suggest that pike fry are susceptible to EHNV, ESV, PPIV and NZeelV and can be a vector for ECV and FV3. Statistical analysis of the factors associated with positive virus re-isolation showed that the number of fish in the sample influenced the outcome of virus re-isolation. Moreover, the likelihood of positive virus re-isolation significantly differed among the 6 viral isolates. The temperature from where the sample was taken and the number of days after infection were not associated with the probability of a positive virus re-isolation.

Ranavirus in wild edible frogs Pelophylax kl. esculentus in Denmark

A survey for the amphibian pathogens ranavirus and Batrachochytrium dendrobatidis (Bd) was conducted in Denmark during August and September 2008. The public was encouraged via the media to register unusual mortalities in a web-based survey. All members of the public that registered cases were interviewed by phone and 10 cases were examined on suspicion of disease-induced mortality. All samples were negative for Bd. Ranavirus was isolated from 2 samples of recently dead frogs collected during a mass mortality event in an artificial pond near Slagelse, Denmark. The identity of the virus was confirmed by immunofluorescent antibody test. Sequencing of the major capsid protein gene showed the isolate had more than 97.3% nucleotide homology to 6 other ranaviruses.

Risk-based methods for fish and terrestrial animal disease surveillance.

Over recent years there have been considerable methodological developments in the field of animal disease surveillance. The principles of risk analysis were conceptually applied to surveillance in order to further develop approaches and tools (scenario tree modelling) to design risk-based surveillance (RBS) programmes. In the terrestrial animal context, examples of risk-based surveillance have demonstrated the substantial potential for cost saving, and a similar benefit is expected also for aquatic animals. RBS approaches are currently largely absent for aquatic animal diseases. A major constraint in developing RBS designs in the aquatic context is the lack of published data to assist in the design of RBS: this applies to data on (i) the relative risk of farm sites becoming infected due to the presence or absence of a given risk factor; (ii) the sensitivity of diagnostic tests (specificity is often addressed by follow-up investigation and re-testing and therefore less of a concern); (iii) data on the variability of prevalence of infection for fish within a holding unit, between holding units and at farm level. Another constraint is that some of the most basic data for planning surveillance are missing, e.g. data on farm location and animal movements. In Europe, registration or authorisation of fish farms has only recently become a requirement under EU Directive 2006/88. Additionally, the definition of the epidemiological unit (at site or area level) in the context of aquaculture is a challenge due to the often high level of connectedness (mainly via water) of aquaculture facilities with the aquatic environment. This paper provides a review of the principles, methods and examples of RBS in terrestrial, farmed and wild animals. It discusses the special challenges associated with surveillance for aquatic animal diseases (e.g. accessibility of animals for inspection and sampling, complexity of rearing systems) and provides an overview of current developments relevant for the design of RBS for fish diseases. Suggestions are provided on how the current constraints to applying RBS to fish diseases can be overcome.

Cohort study of effect of vaccination on pancreas disease in Norwegian salmon aquaculture

Pancreas disease (PD) is an economically important viral disease in Norwegian aquaculture, with 75 to 89 annual outbreaks from 2009 to 2011. To hinder further spread of disease from an initial endemic area on the west coast of Norway, measures for surveillance and control are in place, and the disease is notifiable on a national level. Since 2008, the Norwegian coastline has been divided into 2 administrative zones separated by a production-free area of 10 nautical miles at approximately 63°N. At the same time, a vaccination program involving most marine salmonid farms was initiated by the industry, using a vaccine against PD that was made commercially available in 2007. The effects of the vaccine in the field have been questioned, since the annual number of PD outbreaks has not decreased as expected. However, other production parameters can be used for evaluation of vaccine effect, and in this study the effects of vaccination on cumulative mortality, growth rate, feed conversion factor and number of discarded fish were analyzed using data collected from fish cohorts with and without PD put to sea between spring 2007 and spring 2009. The results show that vaccination against PD has a positive effect in reducing the number of outbreaks, and decreasing cumulative mortality and the number of fish discarded at slaughter.

First detection of piscine reovirus (PRV) in marine fish species

Heart and skeletal muscle inflammation (HSMI) is a disease that affects farmed Atlantic salmon Salmo salar L. several months after the fish have been transferred to seawater. Recently, a new virus called piscine reovirus (PRV) was identified in Atlantic salmon from an outbreak of HSMI and in experimentally challenged fish. PRV is associated with the development of HSMI, and has until now only been detected in Atlantic salmon. This study investigates whether the virus is also present in wild fish populations that may serve as vectors for the virus. The virus was found in few of the analyzed samples so there is probably a more complex relationship that involves several carriers and virus -reservoirs.

Risk mapping of heart and skeletal muscle inflammation in salmon farming

Heart and skeletal muscle inflammation (HSMI) is an infectious disease causing losses to the Norwegian salmon farming industry due to increased mortality and high morbidity in infected salmon. The disease is listed as a notifiable disease on list 3 (national list) by the Norwegian Food Safety Authority. HSMI is believed to be a viral disease, but the association to the recently discovered Piscine reovirus (PRV) remains unclear. Undoubtedly, other factors interact to determine whether PRV-infected fish develop disease or not. In this study, logistic regression was used to model the risk of an outbreak of HSMI at the cohort level, by including spatio-temporal risk factors. The data consisted of fish cohorts grown on geo-referenced farms from 2002 to 2010. The risk factors included were: infection pressure, cohort size (maximum number of fish), cohort index (smolt characteristics), cohort lifespan (months in sea) and a geo-index calculated as the position along a local polynomial regression line based on the longitude and latitude of each farm included in the study. The results showed that the risk of developing HSMI increased with increasing cohort lifespan, increasing infection pressure and increasing cohort size, and was mostly low for cohorts grown on farms in Southern-Norway, high for farms in Mid-Norway and variable for farms in Northern-Norway (based on the geo-index). The final model was used to explore three different scenarios with regards to the risk of developing HSMI, and to calculate the probability for each cohort of developing HSMI, independent of their actual disease-status. The model suggested that the probability of developing HSMI was much higher in Mid-Norway than in the rest of the country. Even though PRV seems to be widely distributed in the environment, the finding that infection pressure has a large influence on the probability of developing HSMI, suggests that it might be possible to reduce the number of clinical outbreaks, if measures are taken to reduce infection pressure. However, the prospects of controlling the spread of HSMI and reducing clinical outbreaks might be difficult because of indications of large distance spread of the disease.

Quantitation of ranaviruses in cell culture and tissue samples

A quantitative real-time PCR (qPCR) based on a standard curve was developed for detection and quantitation of ranaviruses. The target gene for the qPCR was viral DNA polymerase (DNApol). All ten ranavirus isolates studied (Epizootic haematopoietic necrosis virus, EHNV; European catfish virus, ECV; European sheatfish virus, ESV; Frog virus 3, FV3; Bohle iridovirus, BIV; Doctor fish virus, DFV; Guppy virus 6, GV6; Pike-perch iridovirus, PPIV; Rana esculenta virus Italy 282/I02, REV282/I02 and Short-finned eel ranavirus, SERV) were detected with the qPCR assay. In addition, two fish cell lines - epithelioma papulosum cyprini (EPC) and bluegill fry (BF-2) - were infected with four of the isolates (EHNV, ECV, FV3 and DFV), and the viral quantity was determined from seven time points during the first three days after infection. The qPCR was also used to determine the viral load in tissue samples from pike (Esox lucius) fry challenged experimentally with EHNV.

Spatio-temporal risk factors for viral haemorrhagic septicaemia (VHS) in Danish aquaculture

Viral haemorrhagic septicaemia (VHS) is an economically very important fish disease in the northern hemisphere. When the VHS virus was first isolated in Denmark 50 yr ago, more than 80% of the 800 Danish fish farms were considered to be infected, but vigilant surveillance and eradication programmes led to a drastic reduction in prevalence, and finally, to complete eradication of VHS. Denmark thus obtained official status as an approved VHS-free member state within the European Union in November 2013. Data on outbreaks within the country have been collected since 1970, and here we combined these data with the geographical coordinates of fish farms to identify clusters of high disease prevalence and other risk factors. Our analyses revealed a statistically significant cluster in the southwestern part of the country, which persisted throughout the study period. Being situated within such a cluster was a significant risk factor for VHS. For freshwater rainbow trout farms situated inland, the number of upstream farms was a determining risk factor for VHS, as was distance to the nearest VHS-infected farm and year. Whether the farm used fresh or marine water in production did not have any influence on the risk of VHS, when accounting for whether the farm was situated inside a cluster of high risk. This information can be used when implementing risk-based surveillance programmes.

Risk factors for cardiomyopathy syndrome (CMS) in Norwegian salmon farming.

Cardiomyopathy syndrome (CMS) has been an economically important disease in Norwegian aquaculture since the 1990s. In this study, data on monthly production characteristics and case registrations were combined in a cohort study and supplemented with a questionnaire-based case-control survey on management factors in order to identify risk factors for CMS. The cohort study included cases and controls from 2005 to 2012. From this dataset differences between all cases and controls were analyzed by a mixed effect multivariate logistic regression. From this we found that the probability of CMS increased with increasing time in the sea, infection pressure, and cohort size, and that cohorts which had previously been diagnosed with heart and skeletal muscle inflammation or which were in farms with a history of CMS in previous cohorts had double the odds of developing CMS. The model was then used to calculate the predicted value for each cohort from which additional data were obtained via the questionnaire-based survey and used as offset for calculating the probability of CMS in a semi-univariate analysis of additional risk factors. Finally, the model was used to calculate the probability of developing CMS in 100 different scenarios in which the cohorts were subject to increasingly worse conditions with regards to the risk factors from the dataset. We believe that this exercise is a good way of communicating the findings to farmers, so they can make informed decisions when trying to avoid CMS in their fish cohorts.