In the last decade betanodavirus infections have emerged as major constraints on the culture of marine fish in all parts of the world with the exception of the African continent. The occurrence of these infections appears to be a function of the number of species cultured and the intensity of culture. This has been further complicated by the promiscuous translocation of stock within and between countries. Great strides have been made in defining these agents and producing diagnostic techniques but much more remains to be done. Lack of knowledge of the epidemiology of the diseases caused by nodaviruses, except for vertical transmission of the pathogen in some species, has impeded the development of control measures but, even so, the measures identified to date have not been adequately implemented by producers with the result that catastrophic losses still occur on a regular basis.

Betanodavirus infections of teleost fish: a review

The food industry is interested considerably in using ozone to enhance the shelf-life and safety of food products and in exploring new applications of the sanitizer. This interest was recently accompanied by a US governmental approval of ozone for the safe use, in gaseous and aqueous phases, as an antimicrobial agent on food, including meat and poultry. Ozone has a strong microbicidal action against bacteria, fungi, parasites and viruses when these microorganisms are present in low ozone-demand media. Readily available organic constituents in food, however, compete with microorganisms for applied ozone and thus efficacy of the treatment is minimized. Ozone is suitable for washing and sanitizing solid food with intact and smooth surfaces (e.g., fruits and vegetables) and ozone-sanitized fresh produce has recently been introduced in the US market. Use of ozone to sanitize equipment, packaging materials, and processing environment is currently investigated. Efforts to decontaminate bean sprouts and remove biofilm with ozone have not been successful. The antimicrobial efficacy can be enhanced considerably when ozonation is combined with other chemical (e.g., hydrogen peroxide) or physical (e.g., ultraviolet radiation) treatments. Mechanical action is also needed as a means to dislodge microorganisms from the surface of food and expose them to the action of the sanitizer. The food industry also is interested in using ozone to decontaminate processing water and decrease its chemical and biological oxygen demand. This application improves the reusability of processing water and allows for environment-friendly processing operations.

Ozone and its current and future application in the food industry.

Effects of probiotics on growth, stress tolerance and non-specific immune response in Japanese flounder Paralichthys olivaceus were evaluated in a closed recirculating system. Survival and growth of flounder treated by supplying commercial probiotics either in the diet (the probiotic diet group), or into the rearing water (the water supply group), were higher compared to the untreated group (the control group). Water quality parameters, pH, NH4−N, NO2−N and PO4−P showed lower concentration in the probiotic diet group compared with the control group and the supply group. Plasma lysozyme activity in the probiotic diet group and the water supply group was significantly higher (P<0.05) than that in the control group. In heat shock stress tests, flounder in the probiotics-treated groups showed greater heat tolerance (measured by 50% lethal time, LT50) than the control group. Pathogen challenge tests with Vibrio anguillarum (2×107 c.f.u./mL) resulted in significantly higher survival in the probiotics-treated groups than the control group. Results indicated that probiotics supplied in the rearing water and the diet of fish enhanced the stress tolerance and the non-specific immune system of Japanese flounder, providing them a higher resistance against stress conditions and pathogens.

Growth, stress tolerance and non-specific immune response of Japanese flounder Paralichthys olivaceus to probiotics in a closed recirculating system

Striped jack nervous necrosis virus (SJNNV), a nodavirus, is the causative agent of viral nervous necrosis (VNN) in larval striped jack fish. In the present study, the SJNNV coat protein gene was sequenced and compared with that of four known insect nodaviruses and with four other fish nodaviruses causing VNN. The SJNNV coat protein gene was 1410 bases in length and contained a single ORF of 1023 bases encoding a protein of 340 amino acids. The sequence similarities between the coat protein gene of SJNNV and four insect nodaviruses were 28.6% or less at the nucleotide level and 10.6% or less at the amino acid level. A portion of the coat protein gene from four additional fish VNN viruses was amplified by PCR using primers designed for SJNNV and the amplified fragments (870–876 bases) were sequenced. The sequence similarities among SJNNV and the four VNN viruses were 75.8% or greater at the nucleotide level and 80.9% or greater at the amino acid level. In the fish nodaviruses a highly conserved region of 134 amino acids with sequence similarity of 92.5% or greater was detected. This conserved sequence was not found in the coat protein of insect nodaviruses. These results indicate that the fish nodaviruses that cause VNN are closely related to each other but are quite different from insect nodaviruses.

Comparison of the coat protein genes of five fish nodaviruses, the causative agents of viral nervous necrosis in marine fish.

Nodaviruses have emerged as major pathogens of a wide range of larval and juvenile marine finfish in aquaculture worldwide. The causative agents are non-enveloped, icosahedral, RNA viruses with diameters in the range of 25–34nm. They display considerable serological and molecular homology, although the present evidence suggests that there is more than one agent causing disease in a range of species. The diseases produced by these nodaviruses invariably involve the central nervous system and the retina where they usually produce vacuolation and cell necrosis. Virus particles are numerous within the cytoplasm of affected cells and extracellularly. As a result of the lesions, affected larvae/juveniles exhibit a range of neurological signs usually culminating in high mortality rates (not uncommonly 100%). One virus, that of the European sea bass, has recently been cultured in a fish cell line, but to date techniques such as the fluorescent antibody test, enzyme-linked immunosorbent assay and polymerase chain reaction have relied upon the harvest of purified viral antigen from infected tissues rather than obtaining these reagents from viruses grown in cell cultures. The epidemiology of these diseases is only partly understood. All appear to transmit readily by cohabitation of infected fish with naive larvae or juveniles, but vertical transmission has only been recognized with striped jack nervous necrosis and sea bass nervous necrosis viruses. Consequently, some aspects of disease control are based on first principles, rather than application of a full understanding of epidemiological factors.

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Nodaviruses as pathogens in larval and juvenile marine finfish

Effect of chemical and physical treatments on the inactivation of striped jack nervous necrosis virus (SJNNV)

シマアジのウイルス性神経壊死症（VNN）は孵化後2～20日目の仔魚に発生し, 病魚は摂餌をせず, 鰾の膨ら張, 脊索の湾曲, 水面に浮上して斃死するなどの症状が認められた. 孵化後10日目以前に発病した仔魚は数日以内に全滅したが, 11日目以降で発病した場合は, 一部が生き残る例もあった. ELISA 法で測定したウイルス抗原量（SJNNV）の上昇は, 孵化後6日目以前の仔魚では極めて早かったが, 11日目以降の仔魚では比較的遅かった. 同一の親魚群では産卵回数が増す程 VNN の発生率が高くなる傾向が認められた.

Epizootiology of Viral Nervous Necrosis (VNN) in Striped Jack

Intact ovarian follicles, obtained from untreated and human chorionic gonadotropin (HCG) treated Japanese yellowtail Seriola quinqueradiata during different maturational stages, were incubated with radioactive [ 3 H]pregnenolone, [ 3 H]17-hydroxyprogesterone or [ 14 C] androstenedione and steroid metabolites identified by thin layer chromatography (TLC) followed by recrystallization to constant specific activity. In untreated late vitellogenic (0 h) follicles, androstenedione was the major product with smaller amounts of testosterone and oestradiol-17β. In post-vitellogenic (12 h post-injection) intact follicles, androstenedione predominated, and although testosterone and oestradiol-17β were not produced, there were small amounts of 17,20β-dihydroxy-4-pregnen-3-one (17,20β-P) and 17,21-dihydroxy-4-pregnene-3,20-dione (11-deoxycortisol). In HCG-treated fish, a steroidogenic shift resulted in the disappearance of testosterone and oestradiol-17β coinciding with the appearance of 17,20β-P. During early and late final oocyte maturation FOM (24 and 36 h post-injection), there was a five- to seven-fold increase in the production of 17,20β-P, whereas production of 11-deoxycortisol remained almost the same. During FOM, in addition to 17,20β-P, its 5β-reduced metabolite, 17,20β-dihydroxy-5β-pregnan-3-one (5β-17,20β-P) was synthesized, suggesting a decrease in maturation-inducing 17,20β-P activity. 17,20β,21-Trihydroxy-4-pregnen-3-one (20β-S) was not synthesized by ovarian fragments in Japanese yellowtail at any maturational stage. The metabolites identified on TLC during FOM were tested to evaluate their maturation-inducing activity in an in vitro bioassay. Of the steroids tested, 17,20β-P was the most effective inducer of germinal vesicle breakdown (GVBD), followed by 5β-17,20β-P. Timely synthesis of 17,20β-P immediately prior to and during FOM as well as its great potency in inducing GVBD in vitro supports the evidence for a physiological role of 17,20β-P as a maturation-inducing hormone in Japanese yellowtail.

Gonadotropin-induced steroidogenic shift towards maturation-inducing hormone in Japanese yellowtail during final oocyte maturation

The aerobic bacterial flora of larval swimming crabs (Zoea-I to Crab-I) cultured in three hatcheries were investigated in relation to the microflora of rearing pond water and diets. The external and internal bacterial counts of the crab on ZoBell's 2216 e agar ranged from 102 to 104 and 101 to 104 CFU/animal, respectively. The number of the bacterial counts increased as the developmental stages progressed. In respect of constitution, Vibrio and Pseudomonas predominated both in external and internal bacterial flora.The total bacterial counts for rearing pond water, live diets (rotifer and brine shrimp) and minced/artificial foods ranged 104105, 106108, and 103105 CFU/ml or g, respectively. The bacterial flora of rearing pond water and live diets consisted of mainly Vibrio and Pseudomonas, while from the minced food and artificial food only Pseudomonas was predominantly isolated.

Keigo Maruyama

Papers

Effect of chemical and physical treatments on the inactivation of striped jack nervous necrosis virus (SJNNV)

Epizootiology of Viral Nervous Necrosis (VNN) in Striped Jack

Gonadotropin-induced steroidogenic shift towards maturation-inducing hormone in Japanese yellowtail during final oocyte maturation

Bacterial Flora of Cultured Swimming Crab (Portunus trituberculatus) Larvae

A Bacteriological Investigation on the Mass Mortalities of Red Seabream Pagrus major Larvae with Intestinal Swelling