A birnavirus was isolated from cultured loach (Misgurnus anguillicaudatus) in Taiwan with TO-2 cell line. Evident cytopathic effects of the virus, including karyopycnosis, cytoplasmic inclusion bodies and cell destruction, were observed in TO-2 cells by light microscopy. The naked icosahedral virion layered by a single capsid with a diameter of 69 ± 4 nm was observed by electron microscopy. Sometimes, complete virions were found to be enclosed by lysosomes to form cytolysome. The virus propagated in several fish cell lines and its replication cycle was completed in 24 hours at its optimum multiplication temperature of 25°C. This virus has a two segmented double-stranded RNA genome and 3 structural proteins, showing 1.33 g/cm3 buoyant density in CsCl. The chemico-physical properties of the virus together with the result of cross neutralization test indicated that it is a birnavirus and is closely related to Ab serotype of infectious pancreatic necrosis virus (IPNV).
Attempts were made to classify the antiviral properties of bacteria which has been isolated from various environmental waters and had the anti-infectious effects on infectious hematopoietic necrosis virus (IHNV). The majority of these bacterial strains were considered to produce the substances that cause direct inactivation of virus or inhibition of viral invasion into CHSE-214 cells. While a few of the isolates were considered to produce the substances that cause inhibition of viral replication. Among the four strains selected, two strains of Pseudomonas produced heat-stable and relatively low molecular weight substances; one strain of Alteromonas produced a high molecular weight, heat-labile substance; and one strain of Pseudomonas produced at least two kinds of substances unstable against heating.
In this work, we conducted a phenotypic and serological characterization of a group of Vibrio mimicus and Vibrio cholerae non-01 strains isolated from different fish species in the Chesapeake Bay area (Maryland, USA) in comparison with reference strains. Pathogenicity for fish and mice as well as the biological activities present in their extracellular products (ECP) were also evaluated. The taxonomic analysis revealed that all the strains possessed similar physiological and biochemical profiles with the exception of sucrose fermentation, which is the main differential trait between these two Vibrio species.The isolates showed drug resistance patterns very similar, being all of them sensitive to the majority of the chemotherapeutic agents employed. The serological analysis revealed an antigenic diversity among the strains which was confirmed by the LPS profiles. However, although differences were observed in the profiles of total and outer membrane proteins, the western blot analysis showed that most of these Vibrio isolates shared several immunoreactive bands in the region of 60-70 kDa. Virulence assays demonstrated that although the V. cholerae non-01 and V. mimicus isolates were not pathogenic for the fish tested, all of them proved to be virulent for mice. The extra cellular products (ECP) of all V. cholerae and V.mimicus strains showed high proteolytic and phospholipase activities. Moreover, they exhibited a positive cytotoxic response for all the fish and homoiothermic cell lines tested and produced exotoxins with lethaleffects for fish and mice. The virulence properties for mammals exhibited by all these Vibrio strains is a question of special concern mainly in countries where raw fish or fish products are consumed.
Growth responses to three different media, temperatures, and sodium chloride concentrations in the media were determined for 13 salmonid and 14 non-salmonid fish cell lines. Most cell lines showed better growth in Eagle's MEM than in Medium 199 or in Leivobitz L-15 medium. Nine salmonid cell lines grew well in the normal sodium bicarbonate buffer in Eagle's MEM, while 11 non-salmonid cell lines grew better in Eagle's MEM buffered with either HEPES-bicarbonate or Tris-bicarbonate. Optimum temperature for growth ranged from 15 to 20°C for almost all salmonid cells and 20 to 30°C for non-salmonid cells. Most of the cell lines showed highest growth in commercial medium preparations with the lowest concentration of sodium chloride (0.116M) examined. However, three of the six cell lines derived from rainbow trout, Oncorhynchus mykiss and cell lines from eels, Anguilla japonica, showed optimum growth response at a higher sodium chloride concentration of 0.171M in the medium.
For the purpose of obtaining basic knowledge for screening IHN resistant stocks, infection experiments were performed by using a virulent strain of IHNV, TK8901 from 37 g diseased rainbow trout and several different brood and size groups of rainbow trout juveniles. In the infection experiments, 22 progenies, derived from individual pairing of rainbow trout, were inoculated with TK8901 by immersion method at the stages of 1, 8 and 25 g body weight. The results indicated that evident differences in mortality existed among the progenies. Cumulative mortalities of the virus infected fish ranged from 65%to 100% (87.4%in average) in 1 g groups, 33%to 90% (57.2%) in 8 g groups and 10%to 85% (57.0%) in 25 g groups.However, changes in susceptibility of fish with size differed among the progenies. These results suggest that challenge tests using different developmental stages of fish are necessary to estimate the susceptibility of rainbow trout to IHN for screening IHN-resistant strains.
Extracts of skin mucus of ayu, Plecoglossus altivelis agglutinated rabbit erythrocytes (RRBC), but not sheep, guinia pig or human erythrocytes. Hemagglutinins for RRBC were partially purified through ConA Sepharose affinity column chromatography and Sephacryl S-300 gel filtration. RRBC aggregates formed by partially purified hemagglutinin were dissociated by the addition of D-galactose, L-rhamnose and D-fucose. Eluate with 0.05 M α-methyl-D-mannoside from a ConA Sepharose column to which the skin mucus extract had been applied showed high affinity to the lipopolysaccharide purified from Vibrio anguillarum cell wall.