qPCR screening for Yersinia ruckeri clonal complex 1 against a background of putatively avirulent strains in Norwegian aquaculture.

Although a number of genetically diverse Yersinia ruckeri strains are present in Norwegian aquaculture environments, most if not all outbreaks of yersiniosis in Atlantic salmon in Norway are associated with a single specific genetic lineage of serotype O1, termed clonal complex 1. To investigate the presence and spread of virulent and putatively avirulent strains in Norwegian salmon farms, PCR assays specific for Y. ruckeri (species level) and Y. ruckeri clonal complex 1 were developed. Following extensive screening of water and biofilm, the widespread prevalence of putatively avirulent Y. ruckeri strains was confirmed in freshwater salmon hatcheries, while Y. ruckeri clonal complex 1 was found in fewer farms. The formalin-killed bacterin yersiniosis vaccine was detected in environmental samples by both PCR assays for several weeks post-vaccination. It is thus important to interpret results from recently vaccinated fish with great care. Moreover, field studies and laboratory trials confirmed that stressful management procedures may result in increased shedding of Y. ruckeri by sub-clinically infected fish. Analysis of sea water sampled throughout thermal delousing procedures proved effective for detection of Y. ruckeri in sub-clinically infected populations.

Substitution of dietary fish oil with plant oils is associated with shortened mid intestinal folds in Atlantic salmon (Salmo salar).

BACKGROUND: Fish meal and fish oil are increasingly replaced by ingredients from terrestrial sources in the feeds for farmed salmonids due to expanding production and reduced availability of marine feed raw material. Fish oil that is rich in n-3 polyunsaturated fatty acids is considered beneficial to human health in general and to prevent intestinal inflammation and carcinogenesis in particular. In contrast, n-6 fatty acids that are present in many vegetable oils have been associated with increased risk of colitis and colon cancer in rodents and humans, as well as lowered transcription levels of certain stress and antioxidant-related genes in Atlantic salmon.The aim of the present study was to investigate the intestinal health in Atlantic salmon fed with different vegetable oils as partial substitutes of fish oil in the diet. A feed trial lasting for 28 weeks included one reference diet containing fish oil as the sole lipid source and three diets where 80% of the fish oil was replaced by a plant oil blend with either olive oil, rapeseed oil or soybean oil as the main lipid source. These plant oils have intermediate or low n-3/n-6-ratios compared to fish oil having a high n-3/n-6-ratio. The protein and carbohydrate fractions were identical in all the feeds. RESULTS: Morphometric measurements showed significantly shorter folds in the mid intestine in all groups fed vegetable oils compared to the group fed fish oil. In the distal intestine, the complex folds were significantly shorter in the fish fed soybean oil compared to the fish fed rapeseed oil. Histological and immunohistochemical examination did not show clear difference in the degree of inflammation or proliferation of epithelial cells related to dietary groups, which was further confirmed by real-time RT-PCR which revealed only moderate alterations in the mRNA transcript levels of selected immune-related genes. CONCLUSIONS: Shortened intestinal folds might be associated with reduced intestinal surface and impaired nutrient absorption and growth, but our results suggest that partial substitution of dietary fish oil with vegetable oils does not have any major negative impact on the intestinal health of Atlantic salmon.

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.

A novel totivirus and piscine reovirus (PRV) in Atlantic salmon (Salmo salar) with cardiomyopathy syndrome (CMS).

BACKGROUND: Cardiomyopathy syndrome (CMS) is a severe disease affecting large farmed Atlantic salmon. Mortality often appears without prior clinical signs, typically shortly prior to slaughter. We recently reported the finding and the complete genomic sequence of a novel piscine reovirus (PRV), which is associated with another cardiac disease in Atlantic salmon; heart and skeletal muscle inflammation (HSMI). In the present work we have studied whether PRV or other infectious agents may be involved in the etiology of CMS. RESULTS: Using high throughput sequencing on heart samples from natural outbreaks of CMS and from fish experimentally challenged with material from fish diagnosed with CMS a high number of sequence reads identical to the PRV genome were identified. In addition, a sequence contig from a novel totivirus could also be constructed. Using RT-qPCR, levels of PRV in tissue samples were quantified and the totivirus was detected in all samples tested from CMS fish but not in controls. In situ hybridization supported this pattern indicating a possible association between CMS and the novel piscine totivirus. CONCLUSIONS: Although causality for CMS in Atlantic salmon could not be proven for either of the two viruses, our results are compatible with a hypothesis where, in the experimental challenge studied, PRV behaves as an opportunist whereas the totivirus might be more directly linked with the development of CMS.

Agar culture of Piscirickettsia salmonis, a serious pathogen of farmed salmonid and marine fish.

Piscirickettsia salmonis, a serious bacterial pathogen of farmed marine fish, previously considered culturable only in eukaryotic cell-culture systems, was grown for the first time on agar and broth containing enhanced levels of cysteine, thus greatly increasing the potential for isolation, in vitro culture and study of this organism. Virulence towards Atlantic salmon following passage on agar media was retained in a controlled laboratory trial. Of the studied temperatures, optimal growth on agar was observed at 22 degrees C.

The mycobiota of the production environments of traditional Norwegian salted and dried mutton (pinnekjøtt).

In 2013, mould growth on Norwegian ready-to-sell pinnekjøtt (dried and cured lamb ribs) lead to the withdrawal of 200 tons of the product. The aim of this study was to identify the mycobiota at two Norwegian production sites and determine which species present the highest risk for reduced product quality and safety. A total of 485 samples from 2014, 2015 and 2016 were analysed for Penicillium and Aspergillus species. Both production sites showed a persistent mycobiota that remained stable over three seasons. Samples from site A were dominated by P. solitum while samples from site B were equally dominated by P. solitum, P. brevicompactum/bialowiezense and P. nordicum. The presence of P. nordicum was concentrated in one area of the production site where long-time stored hams were produced, but P. nordicum was also found sporadically in other parts of the site. Product samples taken from products with visible mould growth were at both sites dominated by P. solitum, highlighting its importance for product quality. P. nordicum was found frequently in the long-time stored hams, indicating a food safety risk of these products. However, P. nordicum was rarely isolated from pinnekjøtt. Aspergillus spp. were isolated from both sites at all samplings; however, there were no Aspergillus isolated from products, and no sites were repeatedly tested positive for identical species, indicating that Aspergillus is not a part of the persistent mycobiota, but enters the site sporadically. In conclusion, the study showed that a stable mycobiota consisting of few Penicillium species dominated the products and production environments of pinnekjøtt.

Effects of cnidarian biofouling on salmon gill health and development of amoebic gill disease.

This study examines the potential implications of biofouling management on the development of an infectious disease in Norwegian farmed salmon. The hydroid Ectopleura larynx frequently colonises cage nets at high densities (thousands of colonies per m2) and is released into the water during regular in-situ net cleaning. Contact with the hydroids' nematocysts has the potential to cause irritation and pathological damage to salmon gills. Amoebic gill disease (AGD), caused by the amoeba Paramoeba perurans, is an increasingly international health challenge in Atlantic salmon farming. AGD often occurs concomitantly with other agents of gill disease. This study used laboratory challenge trials to: (1) characterise the gill pathology resulting from the exposure of salmon to hydroids, and (2) investigate if such exposure can predispose the fish to secondary infections-using P. perurans as an example. Salmon in tanks were exposed either to freshly 'shredded' hydroids resembling waste material from net cleaning, or to authentic concentrations of free-living P. perurans, or first to 'shredded' hydroids and then to P. perurans. Gill health (AGD gill scores, non-specific gill scores, lamellar thrombi, epithelial hyperplasia) was monitored over 5 weeks and compared to an untreated control group. Nematocysts of E. larynx contained in cleaning waste remained active following high-pressure cleaning, resulting in higher non-specific gill scores in salmon up to 1 day after exposure to hydroids. Higher average numbers of gill lamellar thrombi occurred in fish up to 7 days after exposure to hydroids. However, gill lesions caused by hydroids did not affect the infection rates of P. perurans or the disease progression of AGD. This study discusses the negative impacts hydroids and current net cleaning practices can have on gill health and welfare of farmed salmon, highlights existing knowledge gaps and reiterates the need for alternative approaches to net cleaning.

Mitochondrial dysfunction is involved in the toxic activity of boric acid against Saprolegnia.

There has been a significant increase in the incidence of Saprolegnia infections over the past decades, especially after the banning of malachite green. Very often these infections are associated with high economic losses in salmonid farms and hatcheries. The use of boric acid to control the disease has been investigated recently both under in vitro and in vivo conditions, however its possible mode of action against fish pathogenic Saprolegnia is not known. In this study, we have explored the transformation in Saprolegnia spores/hyphae after exposure to boric acid (1 g/L) over a period 4-24 h post treatment. Using transmission electron microscopy (TEM), early changes in Saprolegnia spores were detected. Mitochondrial degeneration was the most obvious sign observed following 4 h treatment in about 20% of randomly selected spores. We also investigated the effect of the treatment on nuclear division, mitochondrial activity and function using confocal laser scanning microscopy (CLSM). Fluorescence microscopy was also used to test the effect of treatment on mitochondrial membrane potential and formation of reactive oxygen species. Additionally, the viability and proliferation of treated spores that correlated to mitochondrial enzymatic activity were tested using an MTS assay. All obtained data pointed towards changes in the mitochondrial structure, membrane potential and enzymatic activity following treatment. We have found that boric acid has no effect on the integrity of membranes of Saprolegnia spores at concentrations tested. It is therefore likely that mitochondrial dysfunction is involved in the toxic activity of boric acid against Saprolegnia spp.