The electron microscopic examination of the abnormally dark regions of the gill from hard clam, Meretrix lusoria, revealed that there were virus-like particles present in the cytoplasm of necrotic cells. With TO-2 cell line, the virus was isolated. Electron microscopic examinations showed that there were no detactable differences in morphology and location of virions in the gill cells of clam and TO-2 cells except that virus crystals were infrequently observed in clam gills. The subsequent serological and biochemical studies indicated that all the virus isolates from clam were similar to AB IPNV (infectious pancreatic necrosis virus). These studies represent the first complete characterizations of virus isolated from clam in Taiwan.
The pejerrey, Odonthestes bonariensis, given intramuscular injections of Mycobacterium sp., isolated from pond-cultured pejerrey, developed a condition similar to the natural infection. The fish showed 100% mortality at 14 and 30 days after 10 and 1 mg/ml of bacterial suspension was injected, respectively. The bacterium could be easily re-isolated from the kidney of dead fish. In all injected fish, many typical focal granulomas were found in the site of injection and in the examined internal organs. These data proved that the bacterium was certainly a pathogen of pejerrey.
Actual net charges of live cells (RTG-2 cells) were changeable in response to nutrients. Intermetabolites of glycolytic pathway and tricarboxylic acid (TCA) cycle were responsible for net positive and negative charges of RTG-2 cells, respectively. The actual net charges were determined by electrophoresis at pH 7.0 in an appropriate device at 15°C. Infectious pancreatic necrosis (IPN) virus infection shifted the actual net charges of RTG-2 cells from neutral to positive during electrophoresis with minimum essential medium (MEM). Uninfectecd RTG-2 cells used both glucose and amino acids of MEM for nicotinamide adenine dinucleotide+ (NAD+)- and nicotinamide adenine dinucleotide phosphate+ (NADP+)-dependent dehydrogenation. IPN virus infected RTG-2 cells used glucose for the dehydrogenation, and did not used amino acids for it. The lactate dehydrogenase (LDH) activity of the virus infected cells was more enhanced than that of uninfected cells. Instead, isocitrate dehydrogenase (IDH) and malate dehydrogenase (MDH) activities were lower in the virus infected cells. The uninfected cells produced lactic acid and adenosine triphosphate (ATP) from both glucose and amino acids. The virus infected cells produced lactic acid and ATP from glucose. These results indicate that IPN virus infection induces the enhancement of LDH activity and the reduction of IDH and MDH activities to the host cells. The altered dehydrogenases produces further resultant modulation of energy metabolism through glycolysis and TCA cycle.
To increase the fluorescence intensity in the fluorescent antibody test(FAT), fluorescein labeled avidin-biotinylated antibody complex(ABC)was tested for the detection of Renibacterium salmoninarum antigen in the carrier fish of BKD. In comparison with indirect FAT, fluorescence intensity observed by ABC method was increased about 50% by multiscan fluorescent method using 96 well microtiter plate. The detection rate of R. salmoninarum antigens in kidney smears of masu salmon was also increased about 50% in comparison with indirect FAT. The procedure of the ABC method is as follows:A tissue smear was incubated for 30 minutes with antibody against R. salmoninarum. After several washings with phosphate buffered saline, goat anti-rabbit IgG covalently linked to biotin was added and allowed to react for 30 minutes. The preparation was washed again and a solution containing fluorescein-labeled avidin was added andallowed to react for 30 minutes. After final rinsing, the smear is mounted in glycerol buffered to pH 9.0 and observed by fluorescence microscope.
From August to September in 1977, an epizootic occurred among cultured freshwater shrimp Palaemon paucidence in Okayama Prefecture. As the typical symptom of the disease, reddish brown spot was observed on the abdominal segment of shrimp. A vibrio (Vibrio sp.) was purely isolated from diseased specimens, and besides, most of the diseased shrimp were infested with parasitic isopod Tachea chinensis. The pathogenicity of this vibrio was demonstrated by stab-inoculation to the muscle of shrimp or addition to rearing water of isopod-infested shrimp. However, isopod-free shrimp were not infected with the vibrio by the addition method. From these results, this isopod seemed to play an important role in this vibriosis of shrimp.
Sequential changes of leucocytes infiltration were studied quantitatively in orbital inflammation of carp caused by injection of 30% gum arabic solution (0.25ml/each orbit). Inflammatory exudates together with orbital tissues were collected and treated with 5 mg of collagenase (125-200 U/mg) to prepare leucocytes suspension. The volume of the suspension was made up for 1ml by adding Hanks solution, then the total number of leucocytes was counted with a hemocytometer. Number of each type of leucocyte was calculated as the product of percentage of the leucocyte type on a smear and the total number. Differential leucocytes counts in the peripheral blood were estimated on the smear preparations. From stamp preparation of head-kidney, ratio of three maturational stage of neutrophils i.e. mature, immature type I and type II was also estimated. The latter two may be comparable with myelocytes and promyelocytes. Infiltration of neutrophils into the orbital tissue occurred rapidly within one day after the injection of the gum arabic. In advance of the migration, neutrophils showed acute increase in the peripheral blood and decrease in the head kidney. These results indicate that neutrophils stored generally in the head kidney are released to the peripheral blood before the migration to the inflamed lesion. Migrations of monocytes, basophils and lymphocytes were not so active as neutrophils and reached a maximum in about 4 to 6 days. Together with them, blood monocytes and basophils showed some increase, although blood lymphocytes decreased markedly in 6 hours.
Efforts were made to serotype the Vibrio anguillarum strains isolated from fish in Taiwan from 1976 to 1986. Included were 100 strains from milkfish (Chanos chanos), 10 from ayu (Plecoglossus altivelis), 1 from tiger shrimp (Penaeus monodon) and 8 from culture environments. Seven distinct serotypes (T-O-I through T-O-VII) were established from these 119 strains of V. anguillarum examined on the basis of agglutination test with thermostable (O) antigens. Eighty-five strains isolated from milkfish and 5 strains from ayu belonged to serotype T-O-I which appeared to be dominant. However, serotypes II, III, IV, V, VI and VII only occurred sporadically. Besides these, 9 strains isolated from milkfish and 1 from pond water were found to be nontypable. V. anguillarum strains PT213, LS174 and ITAL72 belong to our serotype T-O-I, ATCC19264 (NCMB6), PT24. and SG7701 to our serotype T-O-V and strain PT493 to our serotype T-O-VI.
Comparison on the efficacy of ribosomal and other antigens prepared from Pasteurella piscicida was performed. Yellowtail, Seriola quinqueradiata, was immunized twice by intraperitoneal injection using these antigens to determine the efficacy against artificial challenge by virulent P. piscicida. The antigen preparations used were ribosomal antigen P and S (RBP and RBS), outer membrane fraction (OMF), lipopolysaccharide (LPS), precipitated antigens (PCA), extracellular products (ECP) and formalin killed cells (FKC) as control. After three weeks from the last immunization, RBP gave the highest phagocytic activity although lowest in specific antibody against P. piscicida LPS and serum opsonization in various immunized groups. The RBP preparation was the most effective antigen against as compared to the other preparations.
Twenty-six isolates of gliding bacteria associated with columnaris disease were collected from different species of fish from a wide geographic range. Although there were three phenotypic variations in colony morphology among the strains observed, the growth and biochemical characteristics, as well as, GC content revealed no differences among these isolates. However, when compared by DNA hybridization, there appeared to be three distinct groups based on the DNA homology. Eighteen of the twenty rhizoid strains represented the first group. They were 81-98% homologous with Pacific Northwest strain DD3 of Flexibacter columnaris and included isolates from Canada, Chile, Japan, Korea, ROC and the USA (Atlantic and Pacific Coasts). The mucoid strain K4m was 95% homologous with strain DD3. Taiwanese strains 4G and 5F belonged to the second group and could be classified into new species of gliding bacteria as a result of low DNA homology (<29%) to the Pacific Northwest strain of F. columnaris. A strain with a honeycomb-like colony morphology which came from the southern portion of the USA represented a third group, was less homologous (73%) with strain DD3 of F. columnaris.