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.     

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.     

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.     

Nationwide seroprevalence of Neospora caninum among dairy cattle in Japan

Serum samples from 2420 clinically healthy dairy cattle, randomly selected from stored sera in 18 districts of Japan, were tested for the presence of Neospora caninum antibodies using an indirect fluorescent antibody test (titer > or =1:200). Nationwide seroprevalence is estimated at 5.7% (139/2420). Seropositive cattle were detected in all surveyed districts despite the evidence of confirmed case reports of bovine neosporosis, showing that N. caninum is widely distributed throughout Japan. Age-specific seroprevalence did not increase with cattle age, suggesting that Neospora infection is likely to be transmitted vertically rather than horizontally in Japan. Considering that N. caninum seropositive cows are thought to be more likely to abort, substantial fetal losses may be induced by N. caninum infection in Japan. Devising strategies are needed to reduce the economic impact on the Japanese dairy industry. This is the first study to investigate the nationwide seroprevalence of N. caninum in cattle in Asia.     

Hemolytic compound 3,7,11,15-tetramethyl-hexadecan-1,19-disulfate found in the invasive European sea squirt Ascidiella aspersa

Fisheries Science - The solitary European sea squirt Ascidiella aspersa was introduced to northern Japan, causing dense coverage of the aquacultured Japanese scallop Mizuhopecten yessoensis. There...     

Using landscape metrics and topographic analysis to examine forest management in a mixed forest,Hokkaido, Japan: Guidelines for management interventions and evaluation of cover changes

In this study, we identified the distribution characteristics of a mixed forest of coniferous and broad-leaved trees (a typical forest type in Hokkaido, Japan) using landscape metrics and topographic factors, and attempted to apply this knowledge to examine forest management. This approach provides a new perspective (i.e., the landscape structure) on forest management, which traditionally has been determined on the basis of individual forest stands. We first created a cover type map of the study area by means of aerial photo interpretation. The characteristics of each cover type identified from the photographs were determined using landscape metrics for each cover class. We digitized a forest administrative map (1:20,000 scale) using 20-m contours, and imported this into GIS software to produce a terrain model; on this model, we overlaid the cover types. Our examination of landscape metrics showed that most of the natural forest could be managed similarly. However, our examination of topographic characteristics revealed exceptions (e.g., areas that are difficult to regenerate) that will require particular attention when managing the natural forest. Based on the information we obtained, we proposed a guideline for sustainable forest management. From the land cover map, we proposed an “improved” cover type map to illustrate the development of a high growing stock of forest based on forest management. We compared the current cover map with the “improved” cover map and demonstrated that the improved form would have more significant effects on fauna that do not recognize differences in the proportion of the dominant species types than on those that can recognize these differences. Our results show how the information obtained using landscape metrics and terrain models is an essential tool for various stages of forest management planning.