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This study was funded by the Department of Science and Technology-National Research Council of the Philippines (DOST-NRCP; NRCP project no. E-225) and partly by SEAFDEC AQD (study code: FH02-F2013-T). We would like to thank Dr. Evelyn Grace de Jesus-Ayson for the critical review of the manuscript, and Mr. Eric Ledesma and the laboratory staff of NPPMCI for the invaluable assistance during our sampling.

The increasing demand for slipper oyster ( Crassostrea iredalei ) has propelled farmers to expand oyster cultivation areas in the Philippines, chiefly for local consumption and feasibly for export overseas. Being filter feeders, oysters can accumulate pathogens from their surrounding waters, which can cause foodborne diseases once consumed. Monitoring oyster farming areas for microbiological quality and levels of heavy metals is therefore crucial. In the current study, the microbiological quality of oysters and culture waters of the major oyster farming areas in Cogon and Palina rivers and Cabugao bay, located in Roxas City and municipality of Ivisan, Capiz Province, Western Visayas, Philippines, respectively, were examined monthly during the wet (May to October) and dry (November to April) seasons over a period of 12 months. Regardless of the sampling period, high levels of fecal coliforms in the water and Escherichia coli in oysters were noted, clearly illustrating that these oyster growing areas would comply with the lower Class B standard and 'Prohibited' areas under the European Union and United States classification systems, respectively. Moreover, while Salmonella was erratically detected in oysters, V. cholerae and V. parahaemolyticus count were not detected and within acceptable limit, respectively. The levels of heavy metals in oyster's meat were also determined twice, i.e. during the wet (July) and dry (March) seasons. Zn and Cu were the most abundant metals detected while the levels of Pb, Cd, Hg, and Cr were below the regulatory limits set by the European Union and United States Food and Drug Administration, respectively. Taken together, these oyster culture areas studied should be urgently rehabilitated to improve oysters' poor microbiological quality. Additionally, depuration or relaying of oysters harvested from these sites is imperative to ensure quality and safety.

Changes in innate immunity parameters and epinecidin mRNA transcript levels were examined to characterize the non-specific immune response of E. coioides to pathogenic V. harveyi JML1 isolated from affected cage-cultured fish. After fish had been injected with bacteria at a dose causing 30% mortality, blood and tissue samples were collected at 0, 6, 12, 24, 48, 72, 96, 120, and 240 h post-infection (hpi) for assessment of indices such as the oxidative burst (OB) and phagocytic index (PI) of head kidney cells, and lysozyme activity (LYS) and total immunoglobulin (Total Ig) levels of the plasma. The epinecidin mRNA transcript levels (EGE) from skin, gills, liver, kidney, and spleen tissues were also determined by gel-based RT-PCR. Lastly, daily mortality (DM), liver total bacterial load (TBC), and presumptive Vibrio count (TVC) were monitored up to 240 hpi. The results revealed that bacteria proliferated rapidly in fish tissue, reaching peak densities at 24 hpi for both TBC and TVC but was on a downward trend thereafter. The pattern in fish mortality closely correlated with TBC and TVC. Total Ig, OB, and PI in E. coioides were suppressed in the early part of infection when V. harveyi load was high but recovered and later increased as bacterial density declined. LYS and EGE were consistently high and their activities were not hampered by bacterial infection. The study demonstrated that V. harveyi JML1 interacts with E. coioides by transiently inhibiting some immune parameters resulting in mortalities. However, consistently high LYS, upregulated EGE, and resurgent PI, OB and Total Ig conferred resistance and subsequent recovery in the fish. The study provides new insights on the interaction between E. coioides and V. harveyi JML1 that can aid in formulating health management strategies for groupers. Further studies on prophylactic interventions to enhance the innate immune response in grouper during infection with V. harveyi JML1 are suggested.

Milkfish (Chanos chanos) is a commercially important species widely cultured and consumed in the Philippines. It is a hardy fish but due to culture intensification, occurrence of bacterial disease is inevitable. The causative agent of fin rot disease in milkfish fingerlings (total length [TL] = 2.8–4.5 cm) reared in intensive nursery earthen ponds in the Philippines was investigated in the current study. Following biochemical characterization tests and 16S rRNA sequencing, seven isolates recovered from affected fish were identified as Vibrio harveyi-like bacteria. Immersion challenge of milkfish (TL = 5.64 ± 0.76 cm) using a representative strain (CCL-01) at inoculum dose of 108 CFU/mL resulted in fin rot as early as 1-day post-infection (dpi) and concomitant mortalities of 57% ± 0.58 at 7 dpi. Moreover, when milkfish (TL = 6.18 ± 0.66 cm) stocked at 5, 10, and 20 fish/5 L were exposed to the computed 168-h lethal dose (LD50) at 6.63 × 104 CFU/mL, significant mean mortality of 45% ± 1.0 coupled with typical signs of fin rot were particularly obtained in fish stocked at 20 fish/5 L (biomass = 4 g/L) while low mortality of 0% and 7% ± 0.58 were recorded in fish stocked at 5 fish/5 L and 10 fish/5 L, respectively, at 7 dpi. Vibrio harveyi-like bacteria was reisolated in lesions and kidney of all challenged fish while none in any of the control fish. The current data clearly indicate that the isolated V. harveyi-like bacteria is an opportunistic pathogen capable of instigating disease epizootics in milkfish fingerlings stocked at higher densities.

This study is the first attempt to assess parasitic infestations in grow-out cage culture and land-based grow-out tanks of the donkey's ear abalone Haliotis asinina in the Philippines. This histopathological study of the parasites was conducted from 2010 to 2013. Using 30 samples of abalones collected monthly from two sites, samples were measured and fixed in 10% formaldehyde in seawater, and routine histological techniques were used to identify the parasites. Water temperatures of the grow-out floating sea cages and the abalone hatchery facilities whose abalones were collected measured 27.5°C–32.5°C and 26°C–29°C, respectively; salinity in the area of the sea cages was 32–34 ppt, whereas salinity of the abalone hatchery was 32–35 ppt. Examination of the shell from cage-cultured abalones revealed the presence of burrowing polychaetes belonging to the families Serpulidae (prevalence, 32.5%) and Dorvilleidae (prevalence, 30%); on the other hand, only Dorvilleidae (prevalence of 28.5%) was detected in abalones from the hatchery. Histological examination revealed the presence of unknown ciliates (prevalence, 37.5%), Nematopsis sp. (prevalence, 17%), and metacestode Tylocephalum sp. (prevalence, 8.5%) from the grow-out cage-cultured abalones, whereas unidentified ciliates (prevalence, 32.5%) and Nematopsis sp. (prevalence, 10.5%) were detected from hatchery stock. The data collected showed that there was low-intensity infection and prevalence of parasites in abalones from grow-out cages and hatchery. These records of parasites constitute baseline information for future research work on abalones.

Outbreaks of viral nervous necrosis (VNN) in Asian sea bass (Lates calcarifer) at the larval stages via vertical transmission of nervous necrosis virus (NNV) from asymptomatic broodfish remain as a major deterrent during seed production. A five-year study was conducted to produce NNV-specific-free sea bass broodfish reared in land-based tanks through an annual immunization regimen with the formalin-inactivated NNV. We primarily immunized (intraperitoneal injection) sea bass juveniles (5 g) and monitored the neutralizing antibody (Nab) titers in the sera of these fish at scheduled intervals post-immunization. Nab titers in the sera of immunized fish peaked at Month 2 (titer: 1:4480 ± 1185) but thereafter gradually declined and significantly dropped (1:260 ± 83) at Month 12 post-primary immunization. Booster immunization of these fish at Month 12 post-immunization led to abrupt increases in Nab titers in booster immunized (B-Im) fish at Month 1 (1:12800 ± 6704) but thereafter declined and dropped at Month 12 (1:480 ± 165) post-booster immunization. The annual booster injections with the inactivated vaccine or L-15 (Unimmunized [U-Im]) were consecutively conducted for 4 years until the fish became sexually mature. Mature fish from both groups were successively induced to spawn twice (1-month interval) via intramuscular injection with luteinizing hormone-releasing hormone analogue (LHRH-a; 100 µg/kg BW). NNV was not detected by RT-PCR in oocytes and milts, and spawned eggs of B-Im fish. In contrast, oocytes and milts, and spawned eggs of U-Im fish were NNV positive. Spawned eggs of B-Im broodfish exhibited Nab titers ranging from 1:192 ± 34 to 1:240 while such was not detected (

Unwarranted accumulation of halogenated compounds in the rivers and streams has in recent years emerged due to the widespread use agricultural pesticides. The presence of these halogenated compounds in the water does not only suppress the immune system of fish but adversely induce serious morbidity and mortality among cultured stocks. Importantly, gradual accumulation of these compounds in the system of cultured and wild freshwater fish species cultured in ponds and floating net-cages in dams and rivers, respectively, poses some risks to humans, the end users. In this study, we attempted to isolate bacteria from the gut of pond-reared rohu (Labeo rohita) in Myanmar, screened the isolated bacteria for dehalogenase gene using molecular technique and tested the ability of these bacteria to degrade halogenated compounds in vitro. The eight bacterial strains studied were identified as Enterobacter mori strain MK- 121001, Enterobacter cloacae strains MK121003, MK-121004, MK121010, Ralstonia solanacearum strain 121002, Acinetobacter baumannii strain MK121007, Chromobacterium violaceum strain MK121009 and Pantoea vagans strain 121011. Only three bacterial strains (MK121002, MK121007 and MK121009) were capable of degrading 2,2-dichloropropionic acid (2,2-DCP) as the sole carbon source up to a final substrate concentration of 20 mM. Their mean growth doubling time ranging from 6-23 hours with the maximum of chloride ion released of 85%. PCR amplifica- tion with oligonucleotide primers designed from group I dehalogenase revealed the presence of deha- logenase genes in all isolates suggesting dehalogenase gene in strains 121001, 121003, 121004, 121010 and 121011 were silenced. In contrast, group II dehalogenase primers did not show any PCR amplification. These results suggest that MK121002, MK121007 and MK121009 only encode a group I dehalogenase and its non-stereoselectivity is in agreement with previoulsly described group I haloacid dehalogenase. The partial gene sequences were blasted but no significant sequence identity was observed. Therefore, it suggest the 2-haloacid dehalogenase of MK121002, MK12-1007 and MK121009 might be a novel group I 2-haloacid dehalogenase. The results indicated a broad distribution of dehalogenation genes in many micro- bial genomes that harbor dehalogenase(s) due to the exposure of the microorganisms to the naturally occurring or man-made halogenated compounds in the environmental systems. So far, microorganisms capable of producing dehalogenases were mainly isolated from soil and scarcely from aquatic animals and their environments. To the authors’ knowledge, this is the first report on the isolation of dehalogenase-producing bacteria from the gut of pond-reared freshwater fish, Labeo rohita, in Myanmar.

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 430 bp 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.

Pseudomonas sp. strain S3 was isolated from Paddy (rice) field agricultural area. This organism, which can utilize a halogenated compound of D,L-2-Chloropropionic acid as sole carbon and energy source, catalyses the hydrolytic dehalogenation of both D- and L- isomers of 2-Chloropropionic acid. Identification of Pseudomonas sp. S3 is still ambiguous due to the lack of basic studies, especially their molecular genetic information. In this study, the amplified 16S rRNA gene sequence of Pseudomonas sp. S3 (Accession No. FJ968758) was compared to other nine selected gene sequences from the same group of Pseudomonas sp. and/or dehalogenase producing bacteria using in silico method. Their phylogenetic relationships were then determined. The results were analysed using MEGA4 software to ascertain its evolutionary distance by reconstructing a phylogenetic tree of these organisms. The evolutionary history and bootstrap consensus tree were inferred using the Neighbour-Joining method from 500 replicates. The tree is drawn to scale, with branch lengths (next to the branches) in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the p-distance method and were in the units of the number of base substitutions per site. Based on this analysis, Pseudomonas sp. S3 16S rRNA gene was closely related to the Pseudomonas chlororaphis with genetic distance 0.170 base substitutions per site. S3 gene was also compared among known dehalogenase producing bacteria 16S rRNA genes. Results suggested that S3 was closely related to the Pseudomonas sp. R1 with a genetic distance 0.040 base substitutions per site. From present study, evolutionary relationships of 16S rRNA gene of Pseudomonas sp. S3 were elegantly illustrated by phylograms, comparable to a pedigree showing which microorganisms are most closely related.

A bacterial strain tentatively identified as Pseudomonas sp. R1 was isolated from a paddy (rice) field that could degrade monochloroacetic acid (MCA) for concentrations ranging from 5 to 40 mM. Quantitative agreement between the amount of MCA introduced and chloride released was also found. MCA dehalogenase activity in this strain was found to be inducible. Cell-free extracts displayed dehalogenating activity with specific halogenated organic compound with no activity on dichloropropionic acid or monochloropropionic acid. The estimated Km values for MCA was 0.14 mM. The optimal pH range for MCA dehalogenase activity (between pH 6.5 and 8.0), whereas the thermal stability profile stable up to 50 °C. The results of our current study demonstrated the potential use of Pseudomonas sp. R1 as suitable biological agent for biodegradation of MCA in contaminated agricultural area.

非病原性アクアビルナウイルス(ABV)を予め接種され、その 7 日後にベータノダウイルス(NNV)で攻撃されたマハタは NNV 感染に対して強い抵抗性を示した。他方、ヒラメではNNVに対する感受性が低いため死亡率からABVの効果をみることはできなかったが、脳および腎臓におけるNNVの消長をみると、両魚種とも ABV 処理魚において明らかにウイルス感染力価の低下が認められた。この防御は、ABVにより誘導されるインターフェロン関連タンパク質に因ると考えられた。, Experimental dual-infections with a non-lethal aquabirnavirus (ABV) and a lethal betanodavirus (redspotted grouper nervous necrosis virus: RGNNV) were carried out in Japanese flounder Paralichthys olivaceus and sevenband grouper Epinephelus septemfasciatus. In the dual-infection group, ABV was intramuscularly (IM) injected into fish seven days before the IM-injection with RGNNV. In the experiments with flounder, a high expression of an Mx gene, a molecular marker for type I interferon(s) (IFM) production, occurred in the head kidneys and brains at Day 7 post-ABV injection. Although-no mortality was found not only in the dual-infected group but also in the single infection group with RGNNV (control group), the infective titers of RGNNV in the tissues of the dual-infected group were significantly lower at any sampling times than those in the control group. In the experiments with grouper, the preceding ABV infection resulted in complete protection against RGNNV infection. The infective titers of RGNNV in the tissues were also lower in the dual-infected group than in the control group throughout the experiments, and finally the virus disappeared from the head kidneys and brains of the dual-infected group at Day 14 and Day 56 postinjections, respectively. These results suggest that an ABV-induced IFN(s) effectively suppresses the progression of secondary betanodavirus infection.

The resistance of Japanese flounder (Paralichthys olivaceus Temminck et Schlegel) against a viral haemorrhagic septicaemia virus (VHSV) challenge induced by a preceding non-lethal aquabirnavirus (ABV) challenge was investigated through experimental dual-infections with different intervals between the two challenges. The non-specific protection conferred by the primary ABV infection against the secondary VHSV infection commenced at Day 3 and persisted up to Day 14 but vanished at Day 21 post-ABV challenge. The in vitro assay using HINAE (hirame natural embryo) cells demonstrated anti-VHSV activity in the serum of ABV-challenged flounder from Day 1 to Day 14 but not at Day 21 post-ABV challenge. A high expression of a Mx gene, a molecular marker of type I interferon(s) (IFN) occurred in the head kidneys of ABV-challenged flounder from Day 1 to Day 7. These results suggest that the non-specific protection against the secondary VHSV infection in flounder was due to IFN(s) induced by the primary ABV infection.

Japanese flounder Paralichthys olivaceus, farmed or wild, are ofoen infected with aquabirnavirus (ABV) but the virus does not usually exhibit pathogenicity to the fish under usual experimental conditions. The present study investigated the experimental coinfection in flounder with ABV and three flounder pathogens, i.e. viral hemorrhagic septicemia virus (VHSV), Edwardsiella tarda and Streptococcus iniae. When young flounder were injected with ABV (106.5 TCID50/fish) and subsequently (1 week later) injected with VHSV (106.7 or 104.7 TCID50/fish), higher survival rates were obtained in these flounder compared with those in fish infected with VHSV alone. In contrast, higher mortalities occurred in the flounder dually injected with ABV (106.8 TCID50/fish) and E. tarda (1.4 × 101 CFU/fish) or S. iniae (1.7 × 102 CFU/fish) at a 1 week-interval than those in fish infected with bacteria alone. These results indicate that the primary ABV infection in flounder suppresses the secondary viral infection but facilitates the secondary bacterial infections.

A survey of virus isolation was conducted on 160 samples of wild Japanese flounder Paralichthys olivaceus and 366 samples of other 9 wild fish species collected in 8 coastal areas of Japan in 2001. Aquabirnavirus (ABV) was isolated from flounder (15%), Japanese horse mackerel Trachurus japonicus (23%), and dark banded rockfish Sebastes inermis (4%). Viral hemorrhagic septicemia virus (VHSV) was isolated from flounder (10%) and sand lance Ammodytes personatus (2%) while the other 6 fish species were virus-negative. Concurrently, the distribution of viruses was also examined in 200 flounder collected from different hatcheries and grow-out farms. Only 1.5% of the farmed flounder were ABV-positive and none was VHSV-positive.

Viral nervous necrosis (VNN) caused by betanodaviruses has been recently implicated in serious mortalities of groupers in the grow-out culture system. A safe and effective vaccine against this disease is urgently needed. This study demonstrates that a single intramuscular vaccination with formalin-inactivated Philippine strain of piscine betanodavirus (genotype: redspotted grouper nervous necrosis virus; RGNNV) induces potent immune responses and substantial protective immunity against an intramuscular challenge with the homologous virus in brown-marbled grouper, Epinephelus fuscogutattus, a highly susceptible marine fish species to VNN. Seroneutralization assay conducted on sera of vaccinated fish revealed the occurrence of substantial neutralizing-antibody titers from Days 15 (mean titer 1:800) to 190 (1:400) with the highest titer observed at Day 60 post-vaccination (1:5120). When vaccinated fish were challenged with the homologous virus at Days 15, 30 and 75 post-vaccination, significantly higher survival rates were obtained in these fish compared with their corresponding controls (L-15 injected fish). Abrogation of virus multiplication in all vaccinated survivors was indicated by undetectable virus titers in the brains and kidneys paralleled by significantly high levels of neutralizing antibodies in the sera of these fish. Consecutively, replicates of vaccinated fish that survived betanodavirus challenge at Days 15 and 75 post-vaccination were maintained in flow-through aquaria and rechallenged with the homologous virus 3 and 5 months later, respectively. A significant drop in neutralizing-antibody titers of 3 and 8 folds, respectively, were observed in the sera of Days 15 and 75 post-vaccinated fish assayed before the virus rechallenge. Interestingly, reversion in the levels of neutralizing antibodies to significantly high levels (8-15 folds) were noted in these fish after the virus rechallenge. Taken together, our current data clearly demonstrate that a single administration of the inactivated Philippine strain of betanodavirus vaccine can effectively mount a specific anamnestic response and concomitant long-term protection against VNN in grouper at the grow-out culture system.

Asian sea bass, Lates calcarifer (Bloch), exhibited strong immune responses against a single injection of the formalin-inactivated red-spotted grouper nervous necrosis virus (RGNNV), a betanodavirus originally isolated in Japan. Fish produced neutralizing antibodies at high titre levels from days 10 (mean titre 1:480) to 116 (1:1280), with the highest titre at day 60 post-vaccination (1:4480). When fish were challenged with the homologous RGNNV at day 54 post-vaccination, there were no mortalities in both the vaccinated and unvaccinated control fish. However, a rapid clearance of the virus was observed in the brains and kidneys of vaccinated fish, followed by a significant increase in neutralizing-antibody titres. Furthermore, the vaccine-induced antibodies potently neutralized Philippine betanodavirus isolates (RGNNV) in a cross-neutralization assay. The present results indicate the potential of the formalin-inactivated RGNNV vaccine against viral nervous necrosis (VNN) of Asian seabass.