A monoclonal antibody (MAb), 8D10, was produced against Aeromonas hydrophila strain 94030 isolated from giant gourami (Osphronemus gourami). This was classified as an IgG2a immunoglobulin by ELISA, and recognised a 41 kDa band on whole cell preparations of the A. hydrophila by Western blotting. The reactivity of the MAb against twenty-five different A. hydrophila isolates was examined by an indirect ELISA and Western blotting. Twelve other bacterial species were also tested by the indirect ELISA to examine the specificity of the MAb. One of the A. hydrophila isolate (strain 95014) gave a strong reaction (89.9%) with the MAb in the ELISA and also recognised the 41 kDa band. Sixteen other strains were weak positive in the ELISA, and eleven of these strains were weakly positive by Western blotting. No cross-reaction with the non-A. hydrophila isolates occurred.

The objective of the experiment was to determine the immunogenicity of various concentrations of formalin-killed whole cells of Flavobacterium columnare and lipopolysaccharide (LPS) from the bacterium, with and without Freund s adjuvant. The immunological protective capability of each antigen preparation was then evaluated. Five antigen preparations of F. columnare; the LPS, LPS plus Freund s complete adjuvant (FCA), formalin-killed whole cells (FKWC) at 1 x 10⁷CFU/ml or optical density (OD) of 0.35 at₅₅₀ nm, 0.35 OD₅₅₀ FKWC plus FCA and 6 x 10¹⁰ CFU/ml or 0.9 OD₅₅₀ FKWC, were immunogenic when injected intraperitoneally in 2nd year channel catfish (Ictalurus punctatus). An increase in antigen concentration from 1 x 10⁷CFU/ml to 6 x 10¹⁰ CFU/ml FKWC did not significantly (p>0.05) increase the antibody titer. The antigen FKWC at a concentration of 1 x 10⁷ CFU/ml was also exposed to the fish by the immersion method. An increase in exposure time of immersion in 1 x 10⁷ CFU/ml from 5 to 15 and 30 min resulted in an increase in antibody titers. Higher antibody production was seen in fish held at 27°C and exposed by immersion to antigen twice. Improved protection in channel catfish fingerlings immunized by intraperitoneal injection with LPS+FCA, FKWC (0.35 OD₅₅₀), FKWC (0.35 OD₅₅₀) + FCA, and FKWC (0.9 OD₅₅₀) antigens over challenged saline and formalin-saline control groups was seen. Fish immunized by immersion in FKWC (OD 0.35₅₅₀) for 15 min and those immunized by multiple exposure immersion and kept at 27°C also showed improved protection compared to controls or fish immunized by four other immersion immunization regimes.

The lipopolysaccharide (LPS) profiles of five Vibrio alginolyticus strains (14, 17, 26, 78 and ATCC 17749) were studied by SDS-PAGE. Strains (ST) 14, 17 and 26 were found to possess high molecular weight bands, suggesting that their LPS was highly immunogenic. ST 78 and ATCC possessed only low molecular weight bands, suggesting that their LPS was not highly immunogenic. The LPS and FKC antigens of ST 17, 26 and 78, were injected intraperitoneally in seabass (Lates calcarifer) at 0.2 mg/fish and 0.1 ml/fish at 11.9 X 106 CFU/ml, respectively. Fish were also vaccinated by immersion in formalin-killed cells (11.9 X 106 CFU/ml) for 15 minutes. The mean agglutination titers of fish immunized with ST 17 and 26 were higher compared to strain 78. Fish immunized with antigens from strain 17 showed the highest protection and the protection being significantly different (p<0.05) from that of ST 78. Fish immunized with LPS from the three strains showed significantly (p<0.05) higher protection (survival rate of 80-90%) than that of those injected with FKC. LPS, was thus shown to be a good candidate for a vaccine against vibriosis.

A non-radioactive in situ hybridization (ISH) protocol was used as a diagnostic tool for several fish pathogens including Candidatus Xenohaliotis californiensis, the etiologic agent of withering syndrome, which is a new disease in wild and cultured abalone Haliotis spp., the microsporidian Nucleospora salmonis and the myxozoan parasite Tetracapsula bryosalmonae (previously referred to as PKX) that causes proliferative kidney disease in salmonids. Most applications of the ISH protocol in our -laboratory were used in pathogenesis studies of whirling disease caused by the myxosporean Myxobolus cerebralis.

This paper describes three diseases of seabass (Lates calcarifer) larvae: viral nervous necrosis (VNN), glugeosis microsporidian infection, and a bacterial infection observed in private hatcheries in Indonesia from 1997-1998. VNN of seabass larvae was initially recorded in one hatchery in Situbondo, East Java in August 1997. Occurence in hatcheries in Bali and Banyuwangi, East Java was recorded in the same year. Cumulative mortalities in each case reached 100% within one week. The signs include abnormal swimming, or fish remaining on the tank bottom. Histopathologically, necrosis and vacuolation were observed in the brain and retina of affected larvae. Abundant spherical viral particles, 30 nm in diameter, were found in the cytoplasm of affected nerve cells. Glugeosis and bacterial infection were observed in seabass larvae in Banyuwangi, East Java in 1998. Larvae with glugeosis had numerous whitish cysts (xenoma), 0.5-1.5 mm in diameter, in the abdominal cavity. The cysts were composed of Glugea spores measuring 5-6.5 µm x 2.0-2.5 µm in size. The disease caused mortalities up to 10%. Outbreaks of bacterial disease resulted in 100% mortalities within one week. Diseased larvae showed sluggish swimming near the water surface or weak swimming near the tank bottom. Histological sections of liver tissues showed Grain-negative rods, but isolation of the bacteria was not successful.

Recent advances in fish parasitological studies in Japan focus mainly on myxosporean, microsporean and monogenean infections. Myxosporeans: The two-host life cycle has been elucidated for several freshwater species in Japan. Actinosporean stages were studied, not only their morphology, but also their biology, viz. their viability in water, response to fish mucus, and portals of entry to fish. Myxobolus artus and Thelohanellus hovorkai, both parasites of carp Cyprinus carpio, cause severe damage when spores leave the host. Kudoa amamiensis infection of yellowtail, Seriola quinqueradiata, had a patchy geographical distribution in Okinawa, perhaps reflecting the distribution of its unknown alternate invertebrate host. With the development of PCR probes, the sensitivity of detection of infection in fish has dramatically increased. Microsporeans: Little is known about their mechanism of infection. Macrophages of ayu, Plecoglossus altivelis, recognized glycoproteins on the surface of Glugea plecoglossi spores, and produced much more H2O2 than O2 against phagocytized spores. It may be that G. plecoglossi utilizes this host defense mechanism to establish the infection in ayu. With no chemotherapeutants commercially available, Microsporidium seriolae infection of yellowtail was prevented by using filtered water to maintain fry in tanks, and Glugea-infected ayu was treated by raising the temperature of rearing water. Both PCR and a fluorescent dye, Uvitex 2B, were very sensitive tools to detect infection. Monogeneans: Egg entanglement on the culture net mesh was quantified in Heterobothrium okamotoi infection of tiger puffer, Takifugu rubripes. In vitro attachment of several species of oncomiracidia on cell wells coated with lyophilized extracts of skin epithelia of different fish species suggests a complex nature of their host specificity. Japanese flounder, Paralichthys olivaceus, and tiger puffer infected with Neobenedenia girellae and H. okamotoi, respectively, acquired immunity against reinfection. Tiger puffer develops antibodies against H. okamotoi, but the mechanism of immunity is not yet completely understood. Freshwater or H2O2 bath treatment of B. seriolae-infected yellowtail is effective, but laborious. Experiments showed that, as an alternative method, oral administration of praziquantel was easier and as effective as bath treatment. Most studies introduced here are related to parasite biology. This is because we aim to develop control measures against parasitic diseases with minimal reliance on chemotherapeutic agents.

Heavy mortality of cultured seabass (Lates calcarifer), groupers (Epinephelus spp.) and snappers (Lutjanus spp.) due to bacterial infection is a common problem in Malaysia. All the three species exhibited a wide range of clinical signs in the presence of suspected Vibrio spp. infection. Early stages of infection in the three cultured species ranged from abnormal body color change, poor swimming activity and feeding, and erythema and hemorrhage in and around the mouth and at the base of fins. Advance stages of infections showed petechiae and ulcerative haemorrhagic lesions. This study was carried out to isolate the various pathogenic Vibrio spp. affecting the cultured finfish and to differentiate their strains by morphological, physiological and biochemical characters.

Lymphocystis-like disease was observed in the pond cultured juveniles of tiger grouper (Epinephelus fuscoguttatus) at a farm in Penang Island, Malaysia in May 1999. Clinical signs were abnormally dark colored patches with numerous nodules on the skin. The histopathological study showed that the nodules were composed of many enormously hypertrophied cells commonly 300 - 400 µm in size. Each cell was surrounded by a hyaline capsule and epithelioid structure. The cytoplasm consisted of an enlarged nucleus with prominently stained nucleoli. These histological characteristics of the hypertrophied cells were similar to the lymphocystis cells that were reported in other fish species like bluegill (Lepomis marcochirus) and European flounder (Platichthys flesus). Under electron microscopy, many virus particles were observed in the hypertrophied cell s cytoplasm. These particles showed typical hexagonal profiles with a size of 220-257 nm. The nodules disappeared from the affected parts of the body after fish were maintained for 75 days in an aquarium with clean water. The clinical signs, histopathological findings, and electron microscopical observations showed that the fish were suffering from lymphocystis disease. Lymphocystis is well known to have a wide range of hosts among freshwater and marine fishes. This is the first reported occurrence of the disease in tiger grouper in Malaysia.

Recently, some serious disease problems have occurred in cultured species of gastropods and bivalves in Japan. Among the current problems, amyotrophia of abalones (Nordotis species) and epizootic mortality of pearl oysters (Pinctada fucata martensii), are briefly described in this minireview. Amyotrophia occurs in juveniles of Japanese black abalone (Nordotis discus discus) and two other species of abalones (N. discus hannai, N. madaka) in hatcheries in the late spring, causing muscle atrophy in the mantle and epipodium and abnormal cell masses in the nerve tissues and gills. Although the disease was produced in susceptible juveniles of black abalone by inoculating filtered homogenate of affected abalones, the causative agent has not yet been determined. Epizootic mortalities of pearl oyster have been observed in western Japan since 1996. The adductor muscle of diseased animals often becames reddish and various histopathological changes, such as necrosis and atrophy, occur in both the adductor muscle and heart. Association of the toxic dinoflagellate Heterocapsa circularisquama, the protistan parasite Perkinsus sp. and birnavirus were first suspected, but now a virus or a filterable agent is thought to be the causative agent of the mortality.