The development of Thelohanellus hovorkai, a myxosporean parasite of the connective tissues, and Thelohanellus nikolskii, a fin and scale parasite of common carp (Cyprinus carpio) was studied in experimentally infected oligochaetes Branchiura sowerbyi and Tubifex tubifex, respectively. After infection with mature spores of T. hovorkai, the development of actinosporean stages was first observed light microscopically in the gut of Branchiura sowerbyi 93 days after infection. Free actinospores of T. hovorkai were found in the lumen of the oligochaete's gut 101 days after infection. They were floating in water and showed a typical aurantiactinomyxon form. At 18-22°C, aurantiactinomyxon spores of T. hovorkai emerged from the worms 104 days after infection. The development of T. nikolskii was examined in Tubifex tubifex, from which aurantiactinomyxon spores were released 60 days after infection at 22-24°C. The diameter of spore body was 18.6μm and the lenght of caudal processes 29 μm for T. hovorkai, while 21.1 μm and 13.4 μm for T. nikolskii, respectively. The prevalence of aurantiactinomyxon infection in B. sowerbyi for T. hovorkai proved to be 16.7%, while in T. tubifex for T. nikolskii it was 12.5%.
Penaeid rod-shaped DNA virus (PRDV) is the causative agent of penaeid acute viremia (PAV) in kuruma prawn (Penaeus japonicus) in Japan. To detect the virus, a PCR method using ISOGEN (Nippongene) as a nucleic acid extraction reagent has been used. In order to improve that method, an alternative extraction method named RNase-PEG precipitation (RNPP) method which includes phenol-chloroform extraction, RNase A treatment and polyethylene glycol precipitation was employed in this study. It was found that there was no significant difference in the amounts of extracted PRDV genome between the two methods. However, DNA preparation obtained by the ISOGEN method included contaminants in larger quantities than that by the present method. Comparison of the 2 PCR methods revealed that the RNPP method had a higher sensitivity than the ISOGEN method in detecting PRDV.
Eggs of coho salmon (Oncorhynchus kisutch), rainbow trout (O. mykiss), and masu salmon (O. masou) were artificially inseminated and subsequently immersed in a modified Ordal and Anacker's broth culture of Cytophaga psychrophila (107-108CFU/ml) for 30min. The bacterium was recovered from the eggs at the eyed stage with viable cell counts ranging from 105 to 107CFU/g. The fertilized eggs were disinfected with 50ppm povidoneiodine for 15min immediately after experimental infection. However, C. psychrophila was detected by a broth culture method from 60-80 percent of the surface-disinfected eggs. At the eyed stage, the eggs were disinfected again with povidone-iodine at concentrations of 50-1, 000ppm for 15-120min. C. psychrophila was isolated from all of them at densities of 104-107CFU/g. The uninfected control eggs were all negative for the bacterium. It is concluded that the povidone-iodine treatment is ineffective to eliminate C. psychrophila from salmonid eggs.
Mortalities of yellowtail Seriola quinqueradiata artificially infected with Lactococcus garvieae and of rainbow trout Oncorhynchus mykiss artificially infected with Vibrio anguillarum were compared with the levels of plasma components measured prior to challenge. The levels of plasma total cholesterol, free cholesterol and phospholipid of fish surviving infection were significantly higher in both yellowtail and rainbow trout than those of fish which died during the challenge test. Mortality of yellowtail with plasma total cholesterol levels lower than 250 mg/100 ml was significantly higher than that of fish which had cholesterol levels higher than 275 mg/100 ml (p<0.05). Rainbow trout whose cholesterol was lower than 520 mg/100 ml suffered a significantly higher mortality due to vibriosis than fish having cholesterol levels higher than 560 mg/100 ml (p<0.005). These results indicate that low levels of plasma lipid components may be an indicator of lowered disease resistance in cultured fish.
Cultivation of cells from carp (Cyprinus carpio) kidney tissues including hematopoietic tissues resulted in the formation of a feeder-like cell layer. The carp hematopoietic cells were found to grow in contact with the cell layer. Numerous non-attaching cells were observed in the supernatant of the culture medium. These nonattaching cells were constantly produced in the culture medium, half of which was renewed at 1-4 days intervals for about 1 month. To characterize the proliferating cells, we observed the non-attaching cells with a scanning electron microscope. At a higher magnification, the cells had many short microvilli on their cell surface which is a common feature of carp lymphocytes. Immunofluorescent staining by monoclonal antibodies against carp IgM demonstrated that 1.6-5.6% of the cells were surface immunoglobulin (sIg) positive and 0.1-0.5% of the cells were cytoplasmic immunoglobulin (cIg) positive. Moreover, the percentage of sIg or cIg-positive cells was maintained during the culture, even after the total number of non-attaching cells rapidly increased (more than 20 population doublings) between 10-30 days. These results suggest that immature B-lymphocytes (sIg-negative cells) differentiated into a more matured type of B lymphocytes (sIg or cIg positive cells) during the culture.
Effects of ozonated seawater on disinfection of equipments for aquaculture, fertilized eggs (barfin flounder) and live feeds were investigated by monitoring the viable bacterial counts. Scoopnet, bolting cloth, canvas, hose, beaker, bucket, rubber boot, fertilized eggs, rotifer, Artemia and Thalassiosira were treated with total residual oxidants (0.5mg/l). Treatment for 30 minutes reduced viable bacterial counts of equipments by over 99.9 %. A similar result was obtained on fertilized eggs by treatment for 10 minutes. However, its treatment was not effective for live feeds.
Indirect immunoperoxidase technique (IIPT) was developed for the diagnosis of cold-water disease in cultured ayu (Plecoglossus altivelis). Cytophaga psychrophila cells were clearly distinguished from Pseudomonas sp. cells, the causative bacterium of bacterial hemorrhagic ascites of cultured ayu, in the cross reaction test. The gills, spleens and skins from naturally infected fish were examined for C. psychrophila by IIPT and cultivation with agar plates. The results suggested that IIPT was a quick and reliable method for the diagnosis of cold-water disease.