Inactivation of Piscine orthoreovirus

Piscine orthoreovirus infects various salmonid fish species, and the infection is associated with diseases such as heart and skeletal muscle inflammation (HSMI) in farmed Atlantic salmon (Salmo salar). There are no vaccines available or genetically selected resistant hosts that can efficiently control piscine orthoreovirus (PRV) infection. Currently, the only prophylactic measure against PRV is general biosecurity measures aiming to break the transmission cycle. Methods to eradicate infectious virus from contaminated facilities are desirable, but the knowledge on how to inactivate PRV is lacking. A major bottleneck for inactivation studies is the lack of ability to propagate PRV in cell culture. Therefore, in this study we developed an in vivo model for detection of infectious PRV particles after treatment of the virus with inactivation tools such as heat, pH, iodine, UV and commercially available disinfectants. The results show that standard iodine treatment is efficient in inactivation of the virus, and similarly are high and low pH extremes and treatment with Virocid, a commercially available disinfectant. A UV dose of at least 50 mJ/cm² is required for inactivation, and the virus has high resistance against heat treatment.     

Selenoneine suppresses melanin synthesis by inhibiting tyrosinase in murine B16 melanoma cells and 3D-cultured human melanocytes

Fisheries Science - The novel organic selenium compound, selenoneine, is found in the blood of tuna and has metal-binding activity. In this report, selenoneine displays tyrosinase inhibitory...     

Development of piscirickettsiosis in Atlantic salmon (Salmo salar L.) smolts after intraperitoneal and cohabitant challenge using an EM90‐like isolate: A comparative study

Piscirickettsiosis, caused by the intracellular Gram‐negative bacteria Piscirickettsia salmonis, is at present the most devastating disease in the Chilean salmon industry. The aim of this study was to analyse disease development after challenge with a P. salmonis strain (EM90‐like) under a controlled environment by comparing intraperitoneal challenge with cohabitation challenge. The P. salmonis EM90‐like isolate was cultured in a liquid medium for the challenge of 400 Atlantic salmon (Salmo salar) smolts. Cumulative mortality was registered, necropsy was performed, and bacterial distribution in the tissues and histopathological changes were analysed. The results revealed a similar progression of the disease for the two different challenge models. Pathological and histopathological changes became more visible during the development of the clinical phase of the disease. Bacterial DNA was identified in all the analysed tissues indicating a systemic infection. Bacterial tropism to visceral organs was demonstrated by real‐time quantitative PCR and immunohistochemistry. Better knowledge of disease development during P. salmonis infection may contribute to further development of challenge models that mimic the field situation during piscirickettsiosis outbreaks. The models can be used to develop and test future preventive measures against the disease.     

Levels of Avenanthramides and Activity of Hydroxycinnamoyl‐CoA:Hydroxyanthranilate N‐Hydroxycinnamoyl Transferase (HHT) in Steeped or Germinated Oat Samples

Concentrations of avenanthramides and activity of the biosynthetic enzyme hydroxycinnamoyl‐Co A:hydroxyanthranilate N‐hydroxycinnamoyl transferase (HHT) were analyzed in dry or, steeped nonmilled or milled, non‐heat‐treated (raw) or heat‐treated oat samples (Avena sativa L.). Increased avenanthramide concentrations were found when intact raw groats were steeped. The increase was time‐ and temperaturedependent and maximal after 10 hr of steeping at 20°C. Continuous germination in air, after steeping, only contributed to a further increase in avenanthramides when steeping times were shorter than 10 hr. Concentrations of avenanthramides and HHT activity were positively correlated during steeping of intact groats at 8 and 20°C. The increase in avenanthramides was suggested to be due to de novo synthesis and a whole grain structure seemed to be required as no increase was found when groats were milled before steeping. Avenanthramide levels also increased when heat‐treated samples, lacking HHT activity, were steeped. This increase may be due to release of bound forms, possibly formed during the preceding heat treatment.     

Liquid-chromatographic hybrid triple–quadrupole linear-ion-trap MS/MS analysis of fatty-acid esters of dinophysistoxin-1 in bivalves and toxic dinoflagellates in Japan

Fatty-acid esters of dinophysistoxin-1 (DTX1) in scallops Patinopecten yessoensis, mussels Mytilus coruscus, and toxic dinoflagellate Dinophysis species, collected from Japanese seawater, were analyzed by liquid-chromatography mass spectrometry (LC–MS). Precursor ion monitoring, multiple reaction monitoring for 18 fatty-acid esters of DTX1, and full-scan MS/MS spectra obtained with a hybrid triple–quadrupole linear-ion-trap mass spectrometer showed that 14:0, 16:0, and 16:1 esters were the most abundant 7-O-acyl-DTX1 analogues in bivalves. Fatty-acids esters formed by conjugations at hydroxyl positions other than the 7-position of DTX1 were not detected in the bivalves. DTX1 and okadaic acid-16:0 fatty-acid esters have been reported as the most abundant ester in bivalves in several previous studies; however, we found that 7-O-16:1-DTX1 was the most abundant ester in some mussels in which 16:1 was more dominant than 16:0 in the free fatty-acid profile. Comparison between 7-O-acyl-DTX1 and free fatty-acid profiles in the same bivalve samples suggests that polyunsaturated fatty acids are selectively excluded in enzymatic acylation of DTX1. No 7-O-acyl-DTX1 was detected in any single-cell isolates of D. fortii, D. acuminata, D. mitra, D. norvegica, D. tripos, D. infundibulus, and D. rotundata.     

Effects of olive leaf powder supplemented to fish feed on muscle protein of red sea bream

Olive leaf is known to have the high polyphenol content of 6–9% in dry weight. We investigated the effects of olive leaf powder (OLP) supplemented to fish feed on muscle protein of red sea bream (Pagrus major). Fish reared with feed containing 8% OLP for 40 days had 1.4 times higher myofibril content and 2.2 times higher acid-soluble collagen content than fish reared with control feed for the same period. On the other hand, sarcoplasmic protein content and collagenase activity of the muscle were almost the same between the control fish and OLP-diet fish. Microstructure observation of fish muscle showed that OLP-diet fish has more rigid endomysium structure than that of the control-diet fish. Since collagen fiber in endomysium is responsible for the texture of the muscle, feeding OLP to aquaculture fish will lead to a harder muscle texture. The present study suggests that OLP is a useful feed additive to enhance the texture of aquaculture red sea bream muscle through strengthening of the collagen structure in the muscle.     

Preparation of Photocrosslinked Fish Elastin Polypeptide/Microfibrillated Cellulose Composite Gels with Elastic Properties for Biomaterial Applications

Photocrosslinked hydrogels reinforced by microfibrillated cellulose (MFC) were prepared from a methacrylate-functionalized fish elastin polypeptide and MFC dispersed in dimethylsulfoxide (DMSO). First, a water-soluble elastin peptide with a molecular weight of ca. 500 g/mol from the fish bulbus arteriosus was polymerized by N,N′-dicyclohexylcarbodiimide (DCC), a condensation reagent, and then modified with 2-isocyanatoethyl methacrylate (MOI) to yield a photocrosslinkable fish elastin polypeptide. The product was dissolved in DMSO and irradiated with UV light in the presence of a radical photoinitiator. We obtained hydrogels successfully by substitution of DMSO with water. The composite gel with MFC was prepared by UV irradiation of the photocrosslinkable elastin polypeptide mixed with dispersed MFC in DMSO, followed by substitution of DMSO with water. The tensile test of the composite gels revealed that the addition of MFC improved the tensile properties, and the shape of the stress–strain curve of the composite gel became more similar to the typical shape of an elastic material with an increase of MFC content. The rheology measurement showed that the elastic modulus of the composite gel increased with an increase of MFC content. The cell proliferation test on the composite gel showed no toxicity.     

Structure-Activity Relationship Study of the Neuritogenic Potential of the Glycan of Starfish Ganglioside LLG-3

LLG-3 is a ganglioside isolated from the starfish Linchia laevigata. To clarify the structure-activity relationship of the glycan of LLG-3 toward rat pheochromocytoma PC12 cells in the presence of nerve growth factor, a series of mono- to tetrasaccharide glycan derivatives were chemically synthesized and evaluated in vitro. The methyl group at C8 of the terminal sialic acid residue was crucial for neuritogenic activity, and the terminal trisaccharide moiety was the minimum active motif. Furthermore, the trisaccharide also stimulated neuritogenesis in human neuroblastoma SH-SY5Y cells via mitogen-activated protein kinase (MAPK) signaling. Phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 was rapidly induced by adding 1 or 10 nM of the trisaccharide. The ratio of phosphorylated ERK to ERK reached a maximum 5 min after stimulation, and then decreased gradually. However, the trisaccharide did not induce significant Akt phosphorylation. These effects were abolished by pretreatment with the MAPK inhibitor U0126, which inhibits enzymes MEK1 and MEK2. In addition, U0126 inhibited the phosphorylation of ERK 1/2 in response to the trisaccharide dose-dependently. Therefore, we concluded that the trisaccharide promotes neurite extension in SH-SY5Y cells via MAPK/ERK signaling, not Akt signaling.     

Entry Inhibition of Influenza Viruses with High Mannose Binding Lectin ESA-2 from the Red Alga Eucheuma serra through the Recognition of Viral Hemagglutinin

Lectin sensitivity of the recent pandemic influenza A virus (H1N1-2009) was screened for 12 lectins with various carbohydrate specificity by a neutral red dye uptake assay with MDCK cells. Among them, a high mannose (HM)-binding anti-HIV lectin, ESA-2 from the red alga Eucheuma serra, showed the highest inhibition against infection with an EC₅₀ of 12.4 nM. Moreover, ESA-2 exhibited a wide range of antiviral spectrum against various influenza strains with EC₅₀s of pico molar to low nanomolar levels. Besides ESA-2, HM-binding plant lectin ConA, fucose-binding lectins such as fungal AOL from Aspergillus oryzae and AAL from Aleuria aurantia were active against H1N1-2009, but the potency of inhibition was of less magnitude compared with ESA-2. Direct interaction between ESA-2 and a viral envelope glycoprotein, hemagglutinin (HA), was demonstrated by ELISA assay. This interaction was effectively suppressed by glycoproteins bearing HM-glycans, indicating that ESA-2 binds to the HA of influenza virus through HM-glycans. Upon treatment with ESA-2, no viral antigens were detected in the host cells, indicating that ESA-2 inhibited the initial steps of virus entry into the cells. ESA-2 would thus be useful as a novel microbicide to prevent penetration of viruses such as HIV and influenza viruses to the host cells.     

T4-type bacteriophage communities estimated from the major capsid genes (g23) in manganese nodules in Japanese paddy fields

The present study compared the capsid gene ( g23 ) of T4-type bacteriophages (phages) in Mn nodules with those in the plow layer soil and subsoils of two Japanese paddy fields by applying the primers MZIA1bis and MZIA6 to DNA extracts from the nodules and soils. The deduced amino acid sequences of the g23 genes in the Mn nodules were similar to those in the plow layer soil and in the subsoils. This result indicated that similar T4-type phage communities developed at these sites and that the diversity of T4-type phage communities was wide enough to cover those in the plow layer soil and in the subsoils. The majority of g23 clones formed several clusters with the clones and phages obtained from far-apart paddy fields, and the sequences of two clones were completely identical to a phage and a clone from other paddy fields at the nucleotide or amino acid level, indicating horizontal transfer of g23 genes between those paddy fields. A clone with a long nucleotide residue (686 bp) and a distribution remote from the other clones in the phylogenetic tree indicated that there were many uncharacterized, novel g23 genes in the paddy fields.