Moribund Fish Research Articles

Susceptibility of hatchery-reared snubnose pompano Trachinotus blochii to natural betanodavirus infection and their immune responses to the inactivated causative virus

Mass mortality of snubnose pompano Trachinotus blochii fry exhibiting dark coloration, anorexia, and abnormal swimming behavior was recently documented at the hatchery of the Aquaculture Department of the Southeast Asian Fisheries Development Center, Philippines. Samples of brain tissues were collected from affected fish and processed for RT-PCR amplification and virus isolation in cell culture. Infected E-11 cells exhibited cytopathic effect characteristic of betanodavirus. Histopathology of moribund fish showed pronounced vacuolations in the brain, spinal cord, and retina. An RT-PCR product of approximately 430bp was amplified from the culture supernatant of betanodavirus-infected E-11 cells and sequenced. Sequencing of the T4 region of the coat protein gene (RNA 2) revealed clustering of the isolated virus within the red-spotted grouper nervous necrosis virus type. The pathogenicity of the isolated betanodavirus in healthy pompano juveniles and fry was determined via intramuscular injection and immersion challenges, respectively. Higher mortality rates were obtained in challenged fish compared with the controls. An inactivated vaccine was subsequently prepared by treating the clarified betanodavirus with formalin. Pompano juveniles intraperitoneally injected with the inactivated vaccine exhibited neutralizing antibodies from days 15 (mean titer 1:240) to 125 (1:560) with the highest titer noted at day 64 (1:2240) post-vaccination. Additionally, pompano fry bath-vaccinated and consequently bath-challenged with betanodavirus at day 35 post-vaccination showed higher survival rate compared with the control, indicating the potential of the inactivated betanodavirus vaccine against VNN in pompano fry and juveniles.

Pancreas disease (PD) in sea‐reared Atlantic salmon, Salmo salar L., in Norway; a prospective, longitudinal study of disease development and agreement between diagnostic test results

A prospective longitudinal study was performed on three cages at each of three Norwegian Atlantic salmon seawater sites that experienced outbreaks of pancreas disease (PD). Once salmonid alphavirus (SAV) ribonucleic acid (RNA) was detected by real-time RT-PCR (Rt RT-PCR) at a site, it became detected in all studied cages and was persistently found until the end of the study period up to 19 months after first detection. SAV-specific antibodies were detected at all sites until the end of the study period and were also found at a high prevalence in broodfish at the time of stripping. No evidence of increased viral activity was detected in these broodfish. One site tested negative over several months prior to the first detection of SAV by Rt RT-PCR and SAV-specific antibody, which occurred 1 month prior to clinical manifestations of PD. Moribund fish or thin fish/runts that were sampled after the first PD diagnosis had almost twice the risk of testing positive by one or more diagnostic tests compared to that of randomly selected apparently healthy individuals. This paper describes the first detailed investigation of the disease development of PD at site and cage level in Norway, as well as an assessment of the performance and agreement of the commonly used diagnostic tests.

Isolation and identification of Betanodavirus causing mass mortalities in golden grey mullet (Liza auratus) in the Caspian Sea

Outbreaks causing mass mortalities have been observed in the golden grey mullet (Liza auratus) population in southern coastal area of the Caspian Sea since 2002. The clinical signs include neurological abnormalities, such as erratic swimming behavior, spiral swimming, belly-up at rest and over inflation of the swim bladder. Three hundred and twelve samples of moribund fish with sizes that ranged from 15-26 cm showing abdominal enlargement were collected from 2006-2008. No pathogenic bacteria and parasite were found. The observed histopathological changes in 29 fish were necrosis and vacuolation of the brain, optic nerve and retina. Cytopathological effects of nodavirus characterized by vacuolation were observed in the SSN-1 cell line at 25?C, 5 d after inoculation with the filtered supernatant of the brain and eye of four affected fish. The recovered virus from cell line exhibited 104 TCID50 per mL when titrated. Indirect immunoflorescent antibody test showed nodavirus antigens in the retina and positive-CPE SSN-1 cells. Also, a RT-PCR product of approximately 289 bp was amplified from the brain and retina of the 23 samples, including the SSN-1 positive samples. This is the first report of the isolation and characterization of nodavirus from golden grey mullet from the Caspian Sea and, therefore, possible infections in other valuable species of the Caspian Sea warrant further studies.

Pseudobranchial X-cell pseudotumors in young wild and farmed Atlantic cod Gadus morhua in Iceland

This study investigated the prevalence of pseudobranchial X-cell pseudotumors in 2 year classes of wild juvenile cod and to a lesser extent in older cod in NW Iceland. The disease was also monitored in farmed wild-caught juvenile cod. The youngest wild cod detected that had X-cell infections were 6.5 to 13.0 cm in length, with prevalence reaching a maximum level of 7%. The highest prevalence (23%) was recorded in 18.5 to 27.0 cm fish. The mean prevalence in older cod (25.0 to 76.0 cm in length) was 7% but decreased with increasing age. The mean prevalence of X-cell pseudotumors in farmed juvenile cod was < or =1% during winter rearing in land-based tanks. Higher prevalence (2 to 15%) was noted in dead and moribund fish. Diseased farmed fish were usually emaciated, and mortality due to the disease was noticeable.

Dissemination of streptococcal pyrogenic exotoxin G (spegg) with an IS-like element in fish isolates of Streptococcus dysgalactiae

The Lancefield group C alpha-hemolytic Streptococcus dysgalactiae ssp. dysgalactiae (GCSD) causes systemic granulomatous inflammatory disease and high mortality rates in infected fish. Superantigen and streptolysin S genes are the most important virulence factors contributing to an invasive streptococcal infection. PCR amplification revealed that all strains isolated from moribund fish harbored the streptolysin S structural gene (sagA). GCSD fish isolates were PCR negative for emm, speA, speB, speC, speM, smeZ, and ssa. However, the size of the streptococcal pyrogenic exotoxin G (spegg) locus, a superantigen, in positive S. dysgalactiae fish and pig strains was variable. The ORF of the spegg locus of 26 GCSD fish strains and one GCSD pig strain was inserted with IS981SC. Interestingly, the ORF of the spegg locus of two fish strains of GCSD collected in Malaysia was inserted with an IS981SC-IS1161 hybrid IS element. The hybrid IS element was found in all of the GCSD fish isolates and one GCSD pig through PCR screening. Although no insertion sequence (IS) was detected in the spegg locus of S. dysgalactiae ssp. equisimilis (GCSE) strains, a five-nucleotide deletion mutation was detected in the ORF of the spegg locus of one GCSE strain at the supposed site of IS981SC insertion, resulting in a frameshift mutation.

The Laurentian Great Lakes strain (MI03) of the viral haemorrhagic septicaemia virus is highly pathogenic for juvenile muskellunge, Esox masquinongy (Mitchill)

The Great Lakes strain of viral haemorrhagic septicaemia virus (VHSV) isolated from adult subclinical muskellunge, Esox masquinongy (Mitchill), in Lake St. Clair, MI, USA was shown to be highly pathogenic in juvenile muskellunge through intraperitoneal (i.p.) injection and waterborne challenge. Mortality began as early as 3 days after exposure in waterborne challenged fish, whereas fish infected by the i.p. route experienced the first mortality by 5 days post-infection (p.i.). The median lethal intraperitoneal injection dose (IP-LD(50)) was approximately 2.21 plaque forming units (PFU) as opposed to the median lethal immersion challenge dose (IM-LD(50)) of 1.7 x 10(4) PFU mL(-1). A high, medium and low dose of infection caused acute, subacute and chronic progression of the disease, respectively, as was evident by the cumulative mortality data. Clinical signs of disease observed in dead and moribund fish were very pale gills, dermal petechial haemorrhages along the flanks, severe nuchal haemorrhages, intramuscular haemorrhages at the fin-muscle junction and focal haemorrhaging on the caudal peduncle. Internal lesions included livers that were pale, discoloured and friable, and kidneys that were either congested or degenerative in appearance, and petechial to ecchymotic haemorrhages on the swim bladder wall. Histopathologic examination demonstrated massive haemorrhages in the swimbladder wall and muscle, severe vacuolation and multifocal necrosis of the liver, multifocal necrosis of the gills and depletion of lymphoid tissues within the spleen. Kidney tissues also exhibited a mixed pattern of degeneration that included tubular necrosis, interstitial oedema and congestion. Virus was recovered from kidney and spleen tissues through tissue culture and reverse transcriptase-polymerase chain reaction (RT-PCR).

Close Encounters with a Fish Screen III: Behavior, Performance, Physiological Stress Responses, and Recovery of Adult Delta Smelt Exposed to Two‐Vector Flows near a Fish Screen

AbstractThe threatened delta smelt Hypomesus transpacificus is vulnerable to more than 2,000 water diversions distributed throughout the Sacramento–San Joaquin Delta system of California. Although fish screen installation should decrease direct losses, the physiological stress associated with exposure to two‐vector flows near a fish screen may increase delta smelt mortality. We measured the swimming performance, screen contact rate, and stress responses of adult delta smelt to handling and simulated screen exposure at 12°C. Results from handling stress and flow stress experiments showed that fish collection, crowding, and transport elicited significant stress responses in delta smelt, as did exposure to the two‐vector flows and a fish screen. However, delta smelt recovered from the cumulative handling stress within 2 h. Combinations of high approach velocity (10 or 15 cm/s) and high sweeping velocity (31 or 62 cm/s) increased the primary stress response (plasma cortisol concentrations) and the number of moribund fish. Fish screens designed to protect the delta smelt population could minimize physiological stress by decreasing approach velocities in areas with high sweeping velocities.

Identification and immunoprotective analysis of a Streptococcus iniae subunit vaccine candidate

Streptococcus iniae is a Gram-positive bacterium and a severe aquaculture pathogen that can infect a wide range of farmed fish species. In the summer of 2006, an epidemic broke out in a fish farm in north China, and examination of moribund fish (Japanese flounder) identified the possible etiological agent of the outbreak as a strain named SF1, which exhibited apparent virulence in a Japanese flounder infection model and conforms to the description of S. iniae by 16S rRNA sequence analysis and API 20 Strep test. Biochemical and random amplified polymorphic DNA analyses indicated that SF1 is of the serotype I. A putative iron-binding protein, Sip11, was identified from SF1 using a previously established molecular trap that selects exported proteins. Recombinant Sip11 was purified from Escherichia coli and found to be protective against SF1 infection when used as an injection vaccine administered intraperitoneally into Japanese flounder. To improve the vaccine potential of Sip11, an E. coli strain was constructed, which expresses and secrets recombinant Sip11 covalently linked to a carrier protein in the form of a chimera. Vaccination of Japanese flounder with live Sip11-secreting E. coli afforded complete protection upon the fish following lethal SF1 challenge. These results indicate that Sip11, especially when delivered by a live bacterial carrier, is an effective vaccine candidate against SF1 infection.

Pathological Study on the Scuticociliatosis Affecting Farmed Japanese Flounder (Paralichthys olivaceus) in Japan

Pathological findings associated with scuticociliatosis in farmed Japanese flounder in Japan are described. Ten moribund fishes, farmed in Tottori Prefectural Fisheries Experimental Station, showed cutaneous ulcers, darkened skin, fin and tail rot, exophthalmia and alterations in swimming behaviour. Histopathologically, severe epidermal degeneration and necrosis, hyperplasia of branchial epithelium, myositis, myelitis, encephalitis associated with heavy accumulation of scuticociliates in the periorbital cavity and optic nerve fiber were observed. Moreover, masses of ciliates were found to feed on the host tissues such as skeletal muscles, gills and brain, causing severe degenerative changes associated with abundant neutrophilic and lymphocytic infiltration. These findings suggest that the present scuticociliate, Miamiensis avidus, is a highly invasive and destructive pathogen infecting Japanese flounder and capable of developing systemic fatal infection.

PATHOGENECITY OF GROUPER SLEEPY DISEASE IRIDOVIRUS (GSDIV: Megalocytivirus, FAMILY Iridoviridae) TO CORAL TROUT GROUPER Plectrophomus leopardus

Grouper sleepy disease iridovirus (GSDIV), a member of the genus Megalocytivirus in the family Iridoviridae, has been known to cause large scale mortalities resulting in severe economic losses in grouper industries in south-east Asia including Indonesia. In this study, experimental infection of coral trout grouper Plectrophomus indicus with GSDIV was performed to evaluate the viral pathogenecity to this fish species. After virus exposure, the mortalities of coral trout grouper injected with primary and 10-1 dilution of spleen homogenates derived from tiger grouper Epinephelus fuscoguttatus were 100% and 90%, respectively. Histopathology revealed that moribund fish receiving GSDIV inoculum displayed massive formation of enlarged cells in the spleen and hematopoitic tissues. Under electron microscopy, the enlarged cells were observed as inclusion body bearing cells (IBCs) and necrotic cells allowing virus propagation within an intracytoplasmic virus assembly site (VAS). GSDIV virions were 167-200 nm in size. These findings confirmed that GSDIV has severe pathogenicity to coral trout grouper and IBCs as well as necrotic cells were determined to be the pathognomonic sign of megalocytivirus-infected coral trout grouper.

Limnotrachelobdella sinensis, a leech associated with mortality in a wild population of Japanese crucian carp Carassius cuvieri in Korea

Mortality occurred in Japanese crucian carp Carassius cuvieri in a reservoir in Chungnam Province, Korea, in March 2002. Moribund fish showed severe anemia in the gills and piscicolid leeches that were identified as Trachelobdella sp. were reported as the cause of the mortality. Re-examination of the preserved leech specimens reveals that they are not Trachelobdella sp. but Limnotrachelobdella sinensis. The external morphology of the specimens is described. This finding represents the second record of L. sinensis in Korea. The impact of infestation by L. sinensis on a host is discussed in relation to fish mortality.

Challenge studies of European stocks of redfin perch, Perca fluviatilis L., and rainbow trout, Oncorhynchus mykiss (Walbaum), with epizootic haematopoietic necrosis virus

A challenge model for comparison of the virulence of epizootic haematopoietic necrosis virus (EHNV) to European stocks of redfin perch, Perca fluviatilis L., and rainbow trout, Oncorhynchus mykiss (Walbaum), was tested. The model investigated intraperitoneal (IP), bath and cohabitation routes at 10, 15 and 20 degrees C for 5-6 g fish and 15 degrees C for 20 g perch. In the IP challenges of perch, significant mortality occurred at 15 degrees C and 20 degrees C. In challenge trials for rainbow trout, significant mortalities were observed in IP and bath challenges at 20 degrees C. The mortality observed in IP challenged 20 g perch was not significantly different from that recorded for 6 g fish challenged IP. No significant mortality was observed in any other treatment groups. Re-isolation of ranavirus was confirmed by IFAT and was consistently associated with dead or moribund fish in the trial groups challenged with EHNV. The findings indicate that EHNV does not pose a high risk for wild perch and trout populations in Europe by natural exposure. Mortality appears to be primarily a function of environmental factors, with temperature playing an important role, and not just the presence of the virus in the fish.

Isolation and characterization of Pseudomonas sp. KUMS3 from Asian sea bass (Lates calcarifer) with fin rot

Bacteria were isolated from Asian Sea bass, Lates calcarifer kept in a farm, on the South-east coast of India. During an outbreak of fin rot, the affected fish had hemorrhages at the base of fins, mouth and skin muscles and faded pigments. Pure colonies were isolated on NA and ZMA from internal organs of the fish and the bacterial morphology was identified as gram-negative rod-shaped bacteria. Based on different biochemical tests and sequence of 16S rDNA, the causative bacteria were identified as Pseudomonas sp. KUMS3. Bacterial cells were isolated from liver and kidney of all artificially infected moribund fish and confirmed as Pseudomonas sp. KUMS3 by morphological and biochemical characteristics. During the experimental infection, the first incidence of dead fish was observed on 2nd day after exposure to Pseudomonas sp. KUMS3 and no fish died after 12 days post exposure and the cumulative percent of mortality was 70. Histological lesions were observed in the spleen, liver and kidney of the infected fish. Pseudomonas sp. KUMS3 could be considered as an opportunistic pathogen, which can survive on the fish surface or in water or in the gut and may cause disease when unfavorable conditions develop.

Experimental Pseudomonas anguilliseptica infection in turbot Psetta maxima (L.): a histopathological and immunohistochemical study.

Experimental infection with Pseudomonas anguilliseptica was performed both by intraperitoneal (i.p.) and bath route on juvenile turbot (Psetta maxima) in order to evaluate the pathology induced. Turbot was found to be sensitive to i.p. challenge (1.7×106 CFU/fish) but no to bath exposure. The i.p. challenge induced septicaemic infection and mortality. Externally, moribund fish showed distended abdomen and pale areas at day 9. The gross pathological internal signs present were abundant ascitic fluid in the peritoneal cavity, pale and enlarged spleen, pale and friable liver, and congestive and dilated gut with yellowish exudates. On histopathological examination, bacterial invasion was common in all the tissues studied but the most prominent pathological changes were observed in gut, spleen and kidney after 7 day with features of necrosis. The immunohistochemical findings support the widespread localization of the bacteria after the i.p. injection since the P. anguilliseptica was detected in spleen from day 1 post injection, in liver, kidney and gut from day 4, in muscle from day 7 and in brain from day 9. The difficulties in infecting healthy fish by bath challenge can be explained by the opportunistic nature of this pathogen.

THE EFFECTS OF LARGEMOUTH BASS VIRUS ON A QUALITY LARGEMOUTH BASS POPULATION IN ARKANSAS

A 22.4-ha impoundment experienced an outbreak of Largemouth bass (Micropterus salmoides) virus (LMBV) disease in the summer of 2006. All dead or dying largemouth bass observed throughout the entire event were recorded and removed. In this study, we estimated mortality and examined size distribution, condition, and biomass following the outbreak. Boat-mounted electrofishing was used to collect largemouth bass for a mark-recapture population estimate and other population metrics. Fish samples were examined for evidence of LMBV, other infectious diseases, and physical abnormalities. Cell cultures inoculated with samples from moribund fish developed cytopathic effects typical of LMBV, and polymerase chain reaction (PCR) confirmed the presence of LMBV. The total number (N+/-95% confidence interval) of stock-size largemouth bass remaining was estimated to be 2,301+/-528 fish (103 bass/ha). The total observed mortality, including dead and dying individuals, during the LMBV outbreak was 176 largemouth bass (7% of the initial population). The total biomass remaining was estimated at 1,592 kg of stock-size bass and a relative biomass of 71.5 kg of stock-size largemouth bass per hectare. Largemouth bass size structure was dominated by quality and preferred (300-510 mm) size classes, with very few memorable-size or larger (>510 mm) fish, and the relative weight of largemouth bass was unusually variable. These results demonstrate that largemouth bass abundance and biomass in the reservoir remained very high despite mortalities attributed to a LMBV outbreak.

Aeromonas piscicola sp. nov., isolated from diseased fish

Four Aeromonas strains (S1.2 T, EO-0505, TC1 and TI 1.1) isolated from moribund fish in Spain showed a restriction fragment length polymorphism (RFLP) pattern related to strains of Aeromonas salmonicida and Aeromonas bestiarum but their specific taxonomic position was unclear. Multilocus sequence analysis (MLSA) of housekeeping genes rpoD, gyrB, recA and dnaJ confirmed the allocation of these isolates to an unknown genetic lineage within the genus Aeromonas with A. salmonicida, A. bestiarum and Aeromonas popoffii as the phylogenetically nearest neighbours. Furthermore, a strain biochemically labelled as Aeromonas hydrophila (AH-3), showing a pattern of A. bestiarum based on 16S rDNA-RFLP, also clustered with the unknown genetic lineage. The genes rpoD and gyrB proved to be the best phylogenetic markers for differentiating these isolates from their neighbouring species. Useful phenotypic features for differentiating the novel species from other known Aeromonas species included their ability to hydrolyze elastin, produce acid from l-arabinose and salicin, and their inability to produce acid from lactose and use l-lactate as a sole carbon source. A polyphasic approach using phenotypic characterization, phylogenetic analysis of the 16S rRNA gene and of four housekeeping genes, as well as DNA–DNA hybridization studies and an analysis of the protein profiles by MALDI-TOF-MS, showed that these strains represented a novel species for which the name Aeromonas piscicola sp. nov. is proposed with isolate S1.2 T (=CECT 7443 T, =LMG 24783 T) as the type strain.

Experimental Pseudomonas anguilliseptica infection in turbot Psetta maxima (L.): a histopathological and immunohistochemical study

Experimental infection with Pseudomonas anguilliseptica was performed both by intraperitoneal (i.p.) and bath route on juvenile turbot (Psetta maxima) in order to evaluate the pathology induced. Turbot was found to be sensitive to i.p. challenge (1.7x10(6) CFU/fish) but no to bath exposure. The i.p. challenge induced septicaemic infection and mortality. Externally, moribund fish showed distended abdomen and pale areas at day 9. The gross pathological internal signs present were abundant ascitic fluid in the peritoneal cavity, pale and enlarged spleen, pale and friable liver, and congestive and dilated gut with yellowish exudates. On histopathological examination, bacterial invasion was common in all the tissues studied but the most prominent pathological changes were observed in gut, spleen and kidney after 7 day with features of necrosis. The immunohistochemical findings support the widespread localization of the bacteria after the i.p. injection since the P. anguilliseptica was detected in spleen from day 1 post injection, in liver, kidney and gut from day 4, in muscle from day 7 and in brain from day 9. The difficulties in infecting healthy fish by bath challenge can be explained by the opportunistic nature of this pathogen.

Herpesviral haematopoietic necrosis virus (CyHV‐2) infection: case studies from commercial goldfish farms

Herpesviral haematopoietic necrosis is a disease of goldfish, Carassius auratus, caused by Cyprinid herpesvirus-2 (CyHV-2) infection. Quantitative PCR was carried out on tissue homogenates from healthy goldfish fingerlings, broodfish, eggs and fry directly sampled from commercial farms, from moribund fish submitted to our laboratory for disease diagnosis, and on naturally-infected CyHV-2 carriers subjected to experimental stress treatments. Healthy fish from 14 of 18 farms were positive with copy numbers ranging from tens to 10(7) copies microg(-1) DNA extracted from infected fish. Of 118 pools of broodfish tested, 42 were positive. The CyHV-2 was detected in one lot of fry produced from disinfected eggs. Testing of moribund goldfish, in which we could not detect any other pathogens, produced 12 of 30 cases with 10(6)-10(8) copies of CyHV-2 microg(-1) DNA extracted. Subjecting healthy CyHV-2 carriers to cold shock (22-10 degrees C) but not heat, ammonia or high pH, increased viral copy numbers from mean copy number (+/-SE) of 7.3 +/- 11 to 394 +/- 55 microg(-1) DNA extracted after 24 h. CyHV-2 is widespread on commercial goldfish farms and outbreaks apparently occur when healthy carriers are subjected to a sharp temperature drop followed by holding at the permissive temperature for the disease.

Hemorrhage Disease of Cultured Tra Catfish (Pangasianodon hypophthalmus) in Mekong Delta (Vietnam)

This study investigated hemorrhage disease in cultured tra catfish (Pangasianodon hypophthalmus) from An Giang, Ben Tre, Can Tho, and Vinh Long provinces in the Mekong Delta (Vietnam). The disease is characterized by internal organ necrosis, white (spot) nodules in the liver, kidney, and spleen, and petecchial hemorrhages on the tail, fins, and abdomen. Some fish have exophthalmus (pop eye), a reddish and swollen anus, and yellowish fluid in the peritoneal cavity. Moribund fish lose their appetite and swim at the sur- face. Bacteria isolated from the diseased fish consisted of Aeromonas hydrophila (38.8%), A. sobria (4.1%), A. caviae (2.0%), Edwardsiella ictaluri (4.1%), and a gram positive, anaerobic bacteria, Clostridium sp. (40.8%). Histological analyses showed necrotic cells and intranuclear, randomly- arranged, straight rod cells (1.0-1.5 x 3.0-4.0 μm) concentrated in the ulcers. Challenge test with A. hydrophila induced external signs of hemorrhagic dis- ease. Challenge test with Clostridium sp. confirmed the presence of the bac- teria in infected tissues with development of white nodules similar to those in naturally-infected fish. Fish challenged with E. ictaluri exhibited gas bubbles in the stomach and gut with a foul smell. Reovirus-like particles were seen by transmission electron microscopy. Further study is needed to determine the role of each pathogen alone and together with others in the pathogenesis of hemorrhage disease of tra catfish.

The First Isolation of Photobacterium damselae subsp. damselae from Asian Seabass Lates calcarifer

After transporting Asian seabass Lates calcarifer from a fish farm to the Burapha University facilities, they began to die. The affected or moribund fish exhibited abdominal swelling, yellowish decoloration around the anus, darkening of the gills and exophthalmic eyes. The internal signs of the disease included abdominal cavities filled with a cloudy yellow and gelatinous fluid together with liver hemorrhage. Isolated bacteria from several organs were biochemically characterized as Photobacterium damselae subsp. damselae. It was further confirmed by PCR. An infectivity experiment by intraperitoneal injection with an isolate LCA 24907 showed its virulence to Asian seabass with LD50 of 8.1 × 105 CFU/g fish body weight. This is the first report of isolating P. damselae subsp. damselae from Asian seabass.