One of the most studied fish pathogens is Vibrio anguillarum. Development of the genetics and biochemistry of the mechanisms of virulence in this fish pathogen together with clinical and ecologic studies has permitted the intensive development of microbiology in fish diseases. It is the intention of this review to compile the exhaustive knowledge accumulated on this bacterium and its interaction with the host fish by reporting a complete analysis of the V. anguillarum virulence factors and the genetics of their complexity.
We developed a long-term culture system for Japanese flounder Paralichthys olivaceus leukocytes supported by JFF07-1 feeder cells established from Japanese flounder fin tissue. Kidney leukocytes were seeded onto a monolayer of the feeder cells in enriched RDF medium supplemented with 20% fetal bovine serum and 2.5% flounder serum. Several colonies adhered to the feeder cells after 7 days of cultivation, demonstrating leukocyte proliferation. Increasing numbers of floating cells, which signified colony growth, were observed as the length of the culture period increased. The optimum culture conditions consisted of an incubation temperature of 25°C, the addition of 2.5% flounder serum to the medium and the inoculation of kidney leukocytes at a density of 2 × 106 cells/mL. The proliferated cells were grouped into three types based on May-Grünwald Giemsa staining: basophilic cytoplasmic cells (65%), neutrophilic cytoplasmic cells (30%) and large cells containing many vacuoles (5%). The cells showed acid-phosphatase activity (90%), peroxidase activity (31%) and non-specific esterase activity (57%). Electron microscopy revealed that many of the cells contained endoplasmic reticula and mitochondria, but not specific granules with the fibrillar structure that characterizes flounder granulocytes. A monocyte lineage thus appeared to be the dominant population among the proliferated cells in the culture system. The composition of growing cells was also kept after 20 passages.
The gene vah4 encoding Vibrio anguillarum hemolysin was cloned and hetelogously expressed. The purified recombinant VAH4 protein was then injected into Japanese flounder Paralichtys olivaceus as an immunogen. Activities of superoxide dismutase, catalase, alkaline phosphatase and acid phosphatase in liver became higher in the immunized group. However, these improvements vanished in 72 h after the injection. VAH4-specific antibodies were detected at a titer of 1:10,240 by ELISA on the 10th day after the injection. In a separate experiment, fish were immunized with VAH4 twice and challenged with V. anguillarum. The immunization with the VAH4 protected fish from V. anguillarum infection.
A recombinant fimbrial protein of Edwardsiella tarda was designed to detect the specific antibody in Japanese flounder Paralichthys olivaceus. The specific antibody was detected from the fish intraperitoneally injected with E. tarda expressing the fimbria on LB agar containing 3% NaCl, but not from those injected with E. tarda without the fimbria, Streptococcus iniae, S. parauberis or viral hemorrhagic septicemia virus. In a flounder farm suffered with edwardsiellosis, the antibody-positive fish increased in ration with the occurrence of mortality. These results suggested that E. tarda with the fimbria was important for epidemics of edwardsiellosis at flounder farms.
In this study, pathogenicity of Edwardsiella tarda isolates from turbot Scophthalmus maximus as well as other strains from different hosts were tested against turbot, sole Solea senegalensis and sea bass Dicentrarchus labrax. In addition, the influence of challenge route and temperature in the pathogenic potential of E. tarda strains from turbot were also examined. The results obtained showed that E. tarda did not have host specificity in virulence and that the turbot isolates were highly virulent regardless of the inoculation route and temperature. Moreover, inoculation experiments performed in mice suggest that these isolates are virulent for mammals. All these findings suggest that this emergent turbot pathogen constitutes an important risk factor for the marine aquaculture.
Our previous suggestion that the gill was a key organ for the development of pseudotuberculosis at the early phase of infection was evaluated in natural infection. From naturally infected amberjack Seriola dumerili groups at two fish farms, total 15 dead fish and total 18 apparently healthy fish were collected. In the dead fish, bacterial isolation rate from the kidney and the detection rates of the pathogen in the gills, kidney and spleen by fluorescent antibody method were 87, 100, 100 and 100%, respectively. In the apparently healthy fish, those rates were 6, 72, 33 and 33%, respectively. The quite high detection rate of the bacteria at the gills of the apparently healthy fish may confirm the suggestion.
Protective response was investigated in rainbow trout Oncorhynchus mykiss that had recovered from a primary low-dose cohabitation challenge of Loma salmonae and were re-challenged via a high dose oral exposure under experimental conditions. Compared to uninfected control fish, the previously exposed trout had 82.6% and 86.0% fewer xenomas, and many of the recovered fish (47% and 55%) showed no signs of infection whereas 95% and 100% of the control fish developed xenomas on their gills in two experimental tanks. From the results we conclude that rainbow trout develop protective immunity following low-dose exposure to L. salmonae acquired through cohabitation. The relevance of this with respect to unexpected patterns of disease presentation at commercial salmon farming sites is discussed.
We developed a sensitive method for the detection of Nymphonella tapetis (sea spider) larvae in Manila clam Ruditapes philippinarum. The gills were removed from infected clams and lysed in 2N NaOH at 60°C. The lysate was centrifuged and the pellet was stained in 0.2% Uvitex 2B solution and observed under a fluorescent microscope. Using this method, we were easily able to enumerate the larvae. The majority of larvae were found in the gills, which were indicated as the primary site of infection for N. tepetis larvae.