In September 1996, a disease outbreak was reported in one turtle farm in the New Territories, Hong Kong. Soft-shelled turtle (Trionyx sinensis) cultured in tanks showed white spots on their body surface and carapace resulting in gradual, but high levels of mortalities. Microscopic examination of the affected tissues revealed the involvement of thick, non-septate fungal mycelia. A fastgrowing straminipilous oomycete was isolated from the infected animals and identified as Achlya sp. Physiological studies on vegetative growth and sporulation were undertaken and results showed that the isolate could grow from pH 4 to 10 with maximum growth at pH 5 to 9. Growth was also observed at 0-20 salinities, at temperatures of 10-35°C, with an optimum at 0 salinity (distilled water) at 30-35°C. Abundant zoosporangia were produced and primary zoospores released at 0-5 , but secondary zoospores were only released in freshwater (0 ). A wide temperature range was observed for sporulation (10-40°C) with optimum at 15-35°C. This is the first report of a fungal infection in tank-cultured soft-shelled turtle in Hong Kong.

A new frog disease occurred in frog culture farms located in Central Thailand since early 1998. The disease affects 20-100% of the frog population in affected farms. Diseased adult frogs usually exhibit ulcerative lesions on the dorsal part of the body and legs with moderate mortality (20-50%). Some frogs had red lips, ulcerated mouths and rostrums. Diseased tadpoles and small frogs appeared weak with systemic inflammation. Mortality ranged from 50-100%. Histopathological changes observed include cutaneous ulceration and systemic inflammation with exuberant hematopoiesis. No bacteria could be isolated from the kidney, spleen and liver of frogs at the early stage of the disease. Viral investigation was, therefore, conducted. Seventy virus isolates were obtained from 107 diseased frogs collected from 8 provinces using the Epithelioma papulosum cyprini (EPC) cell line at 25°C. One virus isolate (AV9803) was partially characterized. The virions were enveloped, possessed genomic DNA and hexagonal nucleocapsid morphology, and were ~128 nm in diameter. The virus completely lost infectivity when incubated at 56°C for 30 min, in organic solvent or buffer pH 3. These findings indicate that this frog virus belongs to the family Iridoviridae. DNAs of 8 virus isolates from different provinces were extracted and compared using polymerase chain reaction or PCR. Similar sized PCR products were obtained using primers that were specific to different parts of a major capsid protein gene of Ranavirus type genus FV3. Over 99% nucleotide homology was observed between one sequenced PCR product of AV9803 and the sequence of FV3. These findings suggest that a single virus species was isolated which is most likely a strain of Ranavirus. This virus strain is temporally designated as Rana tigrina ranavirus (RTRV) . The RTRV seems to be associated with cutaneous ulceration. Further infection experiments and electron micrograph examinations in the diseased frog need to be done to confirm the causative agent.

An interview-based questionnaire survey of one fish farmer and one fisherman randomly selected in each of the 64 districts of Bangladesh was carried out to study prevalence of EUS, and risk factors associated with EUS outbreaks. The survey was carried out during the EUS season, November 1998 to March 1999. At each site, 100 fish were examined for lesions, and one fish of each species with lesions was sampled for histological diagnosis. A fish farm or wild fishery was classified as affected with EUS if one or more fish of any species had a positive diagnosis based on the presence of characteristic mycotic granulomas in histological sections. Univariate analyses were used to examine the association between EUS occurrence and putative risk factors using crude relative risk (RR) as the measure. Out of 64 districts, fish with lesions were recorded from fish farms in 32 districts and 30 were confirmed EUS positive, and from wild fisheries 52 districts demonstrated lesions and 49 were confirmed as EUS-positive. In total, 6434 wild fish and 6401-farmed fish were examined, and average prevalence of lesions was calculated as 16.0% and 15.5% respectively. Although disease was more widespread in the wild fisheries, the percentage fish with lesions was generally higher at farm sites (8-45%) than wild fisheries (3-32%). A total of 471 fish with lesions was sampled for histology from the 84 affected sites and 80% of these were diagnosed as EUS-positive. Thirty-one species of fish were confirmed as being EUS-positive out of 47 recorded with lesions. Combining data on affected species from both farmed and wild fisheries sites, the highest prevalences of EUS were recorded in Channa marulius (30%); Glossogobius sp. (25%); Cirrhinus mrigala (24%); Channa striata (22%); Channa punctata (21%); and Anabas testudineus (20%); and the lowest prevalence was in Lepidocephalus guntea (3%). Data showed that there is a significantly higher relative risk of EUS occurring in farmed fish when wild fish are present in the pond; pond embankments are not high enough to prevent in-coming flood water; ponds are connected to natural waters; ponds are not dried or limed prior to stocking; ponds are not limed post-stocking; and pond water colour is black or clear (as opposed to red or green). Of the wild-caught fish, those sampled from haors had a significantly higher relative risk of getting EUS. Fish from rivers and flood plains were at a lower risk of EUS infection.

A new aquabirnavirus (BSNV, blotched snakehead virus) was isolated from a persistently infected warmwater fish cell line (BSN) developed from blotched snakehead (Channa lucius). Four polypeptides of molecular mass 112 (polyprotein, PP), 91 (VP1), 44 (VP2) and 37 (VP3) kDa have been identified in BSNV. The reciprocal cross-neutralisation studies involving BSNV and four classical strains of infectious pancreatic necrosis virus (IPNV), strains WB, Sp, Ab and TV-1, revealed that BSNV antiserum was capable of neutralising only homologous virus while no cross reaction was evident against the other viruses. In order to further study and compare the antigenic properties of the virion structural proteins of BSNV and IPNV strain Sp, Western blot analysis of separated viral proteins was conducted using antisera against BSNV and IPNV Sp. The analysis indicated that VP2 protein (44 kDa) of BSNV alone was recognised by homologous antiserum while antiserum against IPNV Sp did not recognise any BSNV polypeptides. This, combined with weak recognition of the structural protein VP2 of IPNV Sp and SGV using BSNV antiserum showed that VP2 is probably the only protein expressed on the virion surface and this protein carries neutralisation epitopes in BSNV.

A Japanese flounder Paralichthys olivaceus cDNA library was constructed from leukocytes that had been previously infected with hirame rhabdovirus (HRV). Eight hundred ninety six independent clones were isolated from which 1,237 sequence reads were obtained. These sequences consist of a total of 925,169 base pairs with an average sequence length of 758 base pairs. Six hundred three (67.3%) of the clones showed significant similarity to previously reported genes. These identified clones were derived from 350 distinct genes, in which 52 clones were considered as putative biodefense and immune-related genes. Forty-one of 52 putative biodefense and immune-related genes, including several clusters of differentiation (CDs), interleukin receptor, TNF receptor, and transcription factors, have not been previously reported in cDNA copies of fish genes.

Hybrid catfish (Clarias macrocephalus x Clarias gariepinus) farming has developed very successfully in Thailand over the last 10 years. Farmers, however, are still encountering disease problems and, as a result, face economic losses. Some disease problems are associated with inadequate farm management practices. This study was carried out to investigate the presence of bacteria in macrophage cells isolated from hybrid catfish cultured in different environmental conditions. Results from farm investigations showed that the percentage of macrophages containing intracellular bacteria was related to water quality parameters. Experimental trials were conducted using high stocking density as stressor and bath challenge with pathogenic Aeromonas hydrophila. The results showed that fish kept in high density and bath challenged with bacteria had higher percentage of macrophages containing bacteria than those kept under normal conditions. The presence of bacterial loading in macrophage cells is, therefore, considered to be an indication of the health of the fish.

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.