Forty-one strains of Edwardsiella tarda including both virulent and avirulent strains were examined in their iron-acquisition ability. The strains were divided into two groups by growth under an iron-Iimiting condition (LB broth containing casamino acid in the presence of 6.25 μm EDDHA); one group was able to grow more than 102 times the initial bacterial density, but the other was not. The bioassay and the modified CAS assay for detecting bacterial siderophore production showed that the strains of the former group produced the siderophore with a higher ability for ironacquisition than those of the latter group. The siderophore-producing strains include all virulent ones. In addition, a natural mutant that showed lowered siderophore production had a remarkably reduced virulence. All of strains produced two outer membrane proteins, one of which is considered to be a receptor of the siderophore under the iron-limiting condition, indicating that these proteins are hardly related to the virulence of E. tarda. Our data in the present study suggest that the iron-acquisition ability of E. tarda by the siderophore is involved in its virulence.
A group consisting of 20 individuals of Japanese flounder Paralichthys olivaceus was reared for 178 days, during which edwardsiellosis occurred. Monitoring of immunological (phagocytic rate, NBT reduction and potential killing activity of blood phagocytes and lysozyme activity in the plasma) and hematological (protein concentration and glucose concentration in the plasma) parameters were carried out throughout the period, using the blood periodically collected from each fish. Relative standard deviation (SD/average) values of most of the parameters were expanded several weeks before mortality started. The expansion of these values is thought to be caused by increased physiological differences between normal and infected fish in the group. The variance value of these parameters is suggested to be useful to recognize a sign of outbreaks of infectious diseases.
An inoculum of white spot syndrome virus (WSSV), called penaeid rod-shaped DNA virus (PRDV) in Japan, was prepared for challenge tests in kuruma shrimp Penaeus japonicus. The hemolymph was drawn with PBS from moribund shrimp, which were intramuscularly (IM) inoculated three days before with a virus suspension prepared from naturally affected shrimp. The virus concentration in the hemolymph was quantified as 1.5×107 genome copies/μL hemolymph by competitive PCR. The LD50 of the inoculum prepared from the hemolymph stored at -80°C for 40 days was calculated by IM challenges as 10-4.2μL hemolymph /g shrimp or 9.5×102 genome copies/g shrimp. Virulence testes were also conducted through IM injections with the virus preparation stocked for 5 and 16 months. As a result, there was no significant difference in cumulative mortalities (p> 0.05) among the 3 challenge tests made after preservation of the virus in the hemolymph at -80°C for 40 days, 5 or 16 months.
Mortalities of white shrimp Litopenaeus vannamei juveniles exceeding 80% have occurred in Taiwan since late 1998. It has been determined that Taura syndrome virus (TSV) is responsible for the epizootic outbreaks. In order to clarify the origin of these epizootics, a 3288 base pair fragment that encodes a TSV coat protein was amplified by RT-PCR. Analysis showed that the Taiwan isolate had 97% and 98% identity to the sequences in Genebank originated from isolates obtained from Mexico and Hawaii, respectively, both in cDNA and deduced amino acid sequences. These results suggest that TSV that infected the white shrimp in Taiwan in late 1998 and early 1999 is similar to the Western hemisphere isolates. We conclude that the Taiwan isolate originated in the Western hemisphere. Urgent and strict quarantine is advised to prevent Taura syndrome dissemination to other areas in Asia.
A disease characterized by white nodules in the kidney, swollen abdomen and ascitic fluid occurred in cultured ayu Plecoglossus altivelis from April to July, 2001. The bacterial isolate on KDM-2 from the affected ayu was identified to Renibacteirum salmoninarum using IFAT and PCR, and experimental infection resulted in the same clinical signs and re-isolation of the bacterium. From clinical signs, bacteriological examinations and challenge test, this disease in ayu was diagnosed as bacterial kidney disease (BKD). In experimental infection, cumulative mortalities of ayu and masu salmon Oncorhynchus masou were 100% and 60% when injected with 107 cells/fish, and 100% and 0% when injected with 103 cells/fish, respectively. These results indicate that ayu was more susceptible to the isolate than masu salmon, although there is no reported case of BDK in ayu. The affected ayu were originally introduced from another farm in April 2001, where salmonid fishes were reared in neighboring ponds, and it was revealed that masu salmon cultured in the latter farm was infected with R. salmoninarum. This result indicates that R. salmoninarum was horizontally transmitted from masu salmon to ayu.
A population of juvenile (28 cm, 180 g) summer flounder Paralichthys dentatus developed severe coelomic distention, anorexia and lethargy over a period of 4 to 6 months. Gross internal lesions included a large coelomic mass that replaced the posterior kidney and yellow foci on the heart, spleen and liver. Histopathologically, the posterior kidney was obliterated by extensive granulomatous inflammation and rare calcified granulomas. Special stains revealed extracellular acid-fast bacilli in the areas of inflammation. An isolated bacterium belonging to the genus Mycobacterium was the presumptive causative agent of these lesions.
Mass mortality occurred in 34-day old larval orange-spotted grouper Epinephelus coioides reared at a hatchery in the Philippines with clinical signs such as anorexia and abnormal swimming behavior. Histopathology of moribund fish demonstrated marked vacuolation of the brain, spinal cord and retina. Cytopathic effects were observed in SSN-1 cells inoculated with the tissue filtrate of affected grouper. Electron microscopy revealed non-enveloped virus particles measuring 20 to 25 nm in diameter in the cytoplasm of degenerated SSN-1 cells. Piscine nodavirus (betanodavirus), the causative agent of viral nervous necrosis (VNN), was detected in the affected tissues and SSN-1 cells inoculated with the tissue filtrate of affected fish by RT-PCR. This is the first record of VNN in the Philippines.
The effectiveness of a vaccine made of the cell culture supernatant of RSIV (red sea bream iridovirus) infected cells inactivated with formalin (0.1% v/v) in yellowtail Seriola quinqueradiata, amberjack S. dumerili, kelp grouper Epinephlelus moara, striped jack Pseudocaranx dentex and spotted parrot fish Oplegnathus punctatus was evaluated under experimental conditions. Juvenile or young fish were intraperitoneally injected with the vaccine, and challenged with RSIV by intraperitoneal injection at 10th day post-vaccination. Statistical analysis showed significantly higher survival rates in the vaccinated groups of all tested fish species.
Age-dependent humoral immune response of barfin flounder Verasper moseri was investigated. One hundredμL of recombinant coat protein of barfin flounder nervous necrosis virus (BFNNV) adjusted to 250 μg/mL in Tris-EDTA buffer (pH 8.0) was injected intravascularly to 4, 5, 6 and 7 month-old fish. One month after injection, an enzyme-linked immunosorbent assay (ELISA) antibody titer was measured. Although no ELISA antibody titers were detected in injected or noninjected fish of 4 and 5 month-old, the antibody titers were remarkably increased (p<0.025) in 6 and 7 month-old fish. High ELISA antibody titers were kept for at least 2 months.
The effect of water temperature on virus propagation, mortality and histopathological changes of Japanese flounder Paralichthys olivaceus intraperitoneally injected with an isolate (KRRV-9601) of VHSV from flounder were examined. Mortalities for 15 days at 10, 15 and 20°C were 100, 60 and 0%, respectively. At 10°C, all of the infected fish showed high virus titers in both the heart and kidney, and histopathologically displayed necrotizing myocarditis and necrotic lesions in the spleen, hematopoietic tissue and liver. At 15°C, all fish showed higher virus titers in the heart than in the kidney, and myocardial necrosis. No histopathological changes were observed and no VHSV was recovered from fish at 20°C.