Six bacteriophages, which are specific to Vibrio harveyi as host, were detected from 67 samples of hatchery water and grow-out pond soil of black tiger shrimp (Penaeus monodon). The bacteriophages were screened for their effects on cell lysis of 35 pathogenic strains of V. harveyi. The drop method showed that two bacteriophages (Vh-Pg₇ and Vh-Pg₁₀) are the most effective in cell lysis activity. However, there were also unclear results on the growth inhibition of V. harveyi when the co-culturing method was employed. The results of virulence tests revealed that both phages have no negative impact on the intestinal bacterial flora and do not cause any mortality in the shrimp samples. When one of the phages was added to shrimp tanks to control V. harveyi infection during the rearing period from naupliar to postlarval shrimps, there was no significant difference (P≥0.05) in the survival rate of control shrimps, those treated with phage, and those treated with oxytetracycline groups.

The development of an “indigenous probiotic” for the grow-out culture of black tiger shrimp (Penaeus monodon) was pursued in this study. Five of nine bacterial isolates with demonstrated anti-luminous Vibrio metabolites showed non-virulence towards exposed P. monodon postlarvae (PLs) compared to the Vibrio harveyi control and the uninoculated negative control after 7 days exposure. Growth of these selected test bacteria at different salinities were optimum with total bacterial counts (TBC) of 10⁷ to 10⁹ cfu/mL at salinities of 15, 20 and 30 ppt. Bacterial population reduction was observed in most bacterial isolates including V. harveyi at 5 ppt. Co-cultivation of the bacterial isolates in pairs with luminous Vibrio demonstrated that most combinations resulted in the loss of their anti-luminous Vibrio properties. Only the co-cultivation of isolates 1213 and 1725 demonstrated consistent maximum inhibition of luminous Vibrio for 24 to 48 hr. Subsequent multiple combinations of four bacterial isolates 508 + 1213 + 1725 + 1896 or 508 + 1213 + 1637 + 1725 were shown to exert maximum anti-luminous Vibrio inhibition in 24 to 48 hr. The application of the “indigenous probiotic” and or Chaetoceros calcitrans in pond waters to shrimp pond soil showed that either or both treatments effectively reduced the luminous Vibrio population in 1-5 days post-exposure causing one to two logs reduction of luminous Vibrio in 5-7 days post-exposure. Penaeus monodon PLs treated with the “indigenous probiotic” before stocking in tanks yielded higher mean survival rates of 62-92% in comparison with the 50% survival rate of untreated control shrimps. The biomass was also higher in treatments where PLs were exposed to the “indigenous probiotic” upon packing at the hatchery source (treatments B, C and D) with weight ranges of 87.04-101.18 g while shrimps in the control tank without treatment (treatment A) or of PLs exposed to the “indigenous probiotic” after transport or upon stocking in tanks (treatment E) had average weights of 79.82 g and 79.43 g, respectively. Overall, both survival rates and biomass were highest in cultured P. monodon exposed to the “indigenous probiotic during transport and with bi-monthly addition (treatment D), yielding a mean weight of 101.18 g and survival rate of 92%. Viability assays of the selected bacteria after inoculation in sterile seawater medium indicated that these remained viable and stable for >45 days. The “indigenous probiotic” when cultivated in varying concentrations of nutrient broth, fish meal and rice bran suspensions, showed a more stable growth in 0.1% nutrient broth and 0.5% fish meal media yielding bacterial populations of 10⁷-10⁸ cfu/mL. Growth depression was observed with rice bran solutions attaining bacterial population levels of only 10¹-10⁵ cfu/mL. By and large, the development of an “indigenous probiotic” in this study is a significant step towards the biocontrol of luminous vibriosis in the grow-out culture of black tiger shrimp.

The ability of the “green water” grow-out culture of black tiger shrimp (Penaeus monodon) to prevent outbreaks of luminous vibriosis was investigated by screening associated isolates of bacteria, fungi, phytoplankton and fish skin mucus for anti-luminous Vibrio metabolites. Among the 87 bacterial isolates tested, 62 (71%) caused inhibition (+~+++) of the pathogen after 24-48 hr co-cultivation. The variation in growth inhibition rates of +, ++, and +++ were demonstrated by 14 (16%), 13 (15%), and 28 (32%) isolates, respectively, 24 hr after treatment. Eight bacterial isolates showed consistently sustained maximum inhibition (+++/+++) of luminous Vibrio after 24-48 hr exposure. Majority of these luminous Vibrio inhibiting bacterial isolates were obtained from tilapia mucus and gut. In tests with fungi, 4 of 20 (20%) yeast isolates showed intracellular metabolites inhibitory to luminous Vibrio. Among filamentous fungi, 5 of 45 (11%) isolates yielded intracellular metabolites while 3 of 41 (7%) isolates had extracellular metabolites inhibitory to luminous Vibrio. These fungal isolates were identified as Rhodotorula sp., Saccharomyces sp., Candida sp., Penicillium sp., mycelia sterilia and two unidentified species. The microalgae, Chaetoceros calcitrans and Nitzchia sp. consistently demonstrated complete inhibition of luminous Vibrio from 24 and 48 hr post-exposure, respectively, and during the 7 day experiment. Leptolyngbia sp. caused a 94-100% reduction of the luminous Vibrio population from 10⁴ to 10¹ cfu/mL 24 hr post-exposure which was sustained throughout the 10-day observation period. In contrast, the inhibitory effects of Skeletonema costatum on luminous Vibrio was bacteriostatic throughout the 7 days exposure while Nannochlorum sp. did not significantly inhibit luminous Vibrio. The skin mucus of jewel tilapia Tilapia hornorum had no resident luminous bacteria and inhibited this bacterial pathogen in 6-48 hr proportionate to the 10³ and 10⁵ cfu/mL test concentrations of luminous Vibrio. This study provides scientific explanation that the effectivity of the “green water” culture of tiger shrimp in preventing outbreaks of luminous vibriosis among P. monodon juveniles in grow-out ponds can be attributed to the presence of anti-luminous Vibrio factors in the bacterial, fungal, phytoplankton flora and the skin mucus of tilapia associated with this novel technique of shrimp culture.

Strain C1 is a bacterial isolate, which can reduce luminous bacteria in seawater, and thus a potential probiotic in crab larval rearing system. A delivery method through colonized substrates like plastic beads, window pane (“bay-ad”) shells of Placuna sella, and corrugated plastic sheets (CPS) was tested, especially for their ability to maintain the stability of C1 population in water where they were introduced. Beads, shells and CPS measuring were washed, and sterilized and soaked in 10⁶ cfu/mL C1 suspension for 24 hr. These substrates were transferred to the following water conditions: sterile seawater (SSW), sterile seawater with luminous bacteria (SSW+LB), and unfiltered seawater (USW). Bacterial load of water was monitored by spread plate method using nutrient agar (NA) and thiosulfate citrate bile salt sucrose (TCBS) agar. Results showed, successful colonization of C1 on the beads, shells and CPS as shown by high strain C1 counts in all treatments at 1 hr after introduction of substrates. With colonized beads, shells, and CPS, the maximum number of C1 bacteria obtained in water in all treatments was 10⁵ cfu/mL. On day 7, strain C1 population remained at 10⁵ cfu/mL in SSW and SSW+LB treatments, but leveled down to 10³ cfu/mL in the USW treatment. With CPS, the maximum strain C1 count obtained was 10⁴ cfu/mL. On day 7, strain C1 population leveled down to 10² cfu/mL in treatments SSW and SSW+LB treatments and 10¹ cfu/mL in USW treatment. The study shows that both windows pane shells and CPS can be used as substrates for colonization of C1 bacteria, but colonized shells can sustain C1 population in their new environment more effectively than the colonized CPS.

A study aimed at obtaining a biological control agent against bacterial diseases of hatchery-reared shrimps and crabs was conducted. Bacteria were isolated from crustacean rearing environments where luminescent vibriosis was not observed, from natural food, and from various feed ingredients. From hundreds of purified strains, 80 bacterial isolates were tested in one-on-one mixed cultures in seawater for their ability to suppress the growth of luminescent Vibrio harveyi. Of the 10 isolates exhibiting that capability, two strains were further studied: C1 from Chlorella culture and P9 from a commercial probiotic preparation. However, due to the indigenous nature of C1 strain from the unicellular alga Chlorella sp. more studies were done on it. Its biochemical characteristics and pathogenicity against crab larvae was determined. Several static bath tests were simultaneously conducted to compare the pathogenicity of luminescent Vibrio harveyi and the benign effect of potential probiotic bacteria on various stages of crab zoeae and shrimp postlarvae. Mortality in all treatments with probiotic bacteria was lower than those challenged with luminescent V. harveyi and the control indicating its benign effect. Initial bacterial inocula of 102 and 103 colony-forming-units (cfu)/mL in ultraviolet-sterilized seawater resulted in a hundred-fold increase in bacterial numbers after 24 hr, proving that in static bath bacterial bioassays, growth and replication of bacteria occurs in the test chambers. However, when the bacterial inocula were 105 to 106 cfu/mL, no significant change in bacterial numbers occurred. Furthermore, control chambers with no bacteria inoculated, were found to harbor between 10⁴ to 10⁵ cfu/mL of mixed bacterial population after 24 hr, part of which was luminescent Vibrio. Since the tests were conducted without feeding, results proved that bacteria associated with the test larvae also proliferated in significant numbers during the experimental period. Thus, monitoring of bacteria should not be done only on the initial hour of inoculation, but the following day as well to show microbial interactions during the experiment.

Prevalences of white spot syndrome virus (WSSV) and monodon baculovirus (MBV) were determined using polymerase chain reaction (PCR) as the detection method in wild black tiger shrimp (Penaeus monodon) collected from seven sampling sites in the Philippines. These seven sampling sites are the primary sources of spawners and broodstocks for hatchery use. Samples for PCR comprised DNA extracts from gills for WSSV detection and from hepatopancreas for MBV. As a result, WSSV was detected in shrimp from all sites except Bohol during dry season, and were negative from all sites except Palawan during wet season. However, all the positive shrimp showed no white spots in the carapace. MBV was also detected from all sites except Palawan, Negros Occidental and Bohol during dry and wet seasons, respectively. The prevalences of WSSV and MBV showed seasonal and sex variations, i.e. the prevalence in dry season (April-May) was higher than in wet season (August-October), and the prevalence in female shrimps was higher than in males. A very low prevalence of dual infection with WSSV and MBV was also observed. These results suggest that the viruses have already established in the local marine environment and in wild populations of P. monodon, and spawners collected during dry season could serve as the main source of contamination for vertical transmission of the virus when they spawn.

A virological survey was conducted in brown-spotted grouper (Epinephelus malabaricus) cultured in cages, earthen ponds and pens from April 2001 to January 2002 on the east and south coasts of Thailand. The adult grouper samples exhibited a wide range of clinical signs beginning from darkening of body color with focal distension of the skin to red boil skin, and to appearance of red spot ulcers on the body and head. Diseased fry to juvenile grouper showed darkening of body color and swirling movement. Thirty-five isolates of viruses were obtained from using freshwater fish cell lines, SSN-1 and EPC. These isolates could be grouped into two different groups of fish viruses. One viral group has been identified as a betanodavirus, which belongs to genotype RGNNV (redspotted grouper nervous necrosis virus). RT-PCR tests in this betanodavirus using specific primers to SJNNV (striped jack nervous necrosis virus) were negative, while specific primers to RGNNV showed positive RT-PCR product. The second viral group could be identified as an iridovirus. This grouper iridovirus had some level of similarities to RSIV (red sea bream iridovirus). One set of specific primers for RSIV could give positive PCR product, while the second set of specific primers gave negative PCR result. The grouper iridovirus was found different from Ranavirus. Betanodavirus could infect both adult and fry to juvenile stages of the grouper and seemed to have more significance during November to January or during the dry and cold season in Thailand. The grouper iridovirus seems to cause disease in adult size of the fish with low level of mortality and seems to infect the fish predominantly in August and September or during rainy season in Thailand. These findings indicate that two different viruses are associated with grouper and identified each as Betanodavirus and grouper iridovirus. The new isolate of grouper iridovirus has some level of variations in nucleotide sequences when compared with RSIV gene. There is a need to do more molecular study of this new isolate of grouper iridovirus as well as its pathogenesis.

This is a summary of investigations conducted on 91 disease/mortality cases observed among marine fish: groupers (Epinephelus coioides, E. malabaricus), sea bass (Lates calcarifer), rabbitfish (Siganus spp.), red snapper (Lutjanus argentimaculatus), milkfish (Chanos chanos) and tilapia (Oreochromis niloticus) from 2000 to 2003 at SEAFDEC/AQD and other sources in the Philippines. Eleven established cell lines were maintained namely: GF-1, SBK-2, SSN-1, E-11, BF-2, CFS, EPC, FHM, SHS, WSS2Cl, and WSSK. Among the 52 cases of grouper larvae, fingerlings, juveniles, and broodstocks sampled, viable viral nervous necrosis (VNN) virus was isolated in E-11 and GF-1 cells from 2/8 (25%), 15/26 (57.7%), 7/14 (50%) and 3/4 (75%) cases in 2000, 2001, 2002 and 2003, respectively. This comes to approximately 51.9% (27/52) being positive for viable VNN virus with a virus titer of TCID₅₀ 10^5.0-5.5. Among the 14 rabbitfish case samples examined, 1/5 and 2/3 of the 2000 and 2002 filtrates, respectively, as well as 5/11, 1/2, 3/10 and 1/2 case samples of diseased red snapper, sea bass, milkfish and tilapia samples, respectively, also yielded viable VNN. The isolations of the VNN virus from naturally-infected rabbitfish, red snapper, milkfish, and tilapia are first reports. Cytopathic effects induced by VNN were characterized by vacuolations followed by complete detachment of the inoculated cells after 3-7 days. The presence of vacuolations in the histological analyses of the brain and retina of naturally-infected fish, plus the positive results of RT-PCR tests (one-step and/or nested) for VNN of all viral isolates, further confirmed the identity of the nodavirus. One viral isolate from rabbitfish remains unidentified. These results indicate that VNN caused the mortalities of majority of cases of marine fish epizootics observed in 2000 to 2003 in the Philippines.

Experiments were undertaken to determine the efficacy of chlorine as a disinfectant in inhibiting HPV infection among P. monodon postlarvae (PLs). Based on histopathological examination, 50 ppm and 200 ppm active chlorine at two different exposure durations (12 and 24 hr), failed to inhibit the transmission of HPV in the experimental PLs. Higher chlorine concentrations, 500 ppm and 1,000 ppm for 24 hr exposure, decreased percentage of HPV infection, but failed to completely inhibit the transmission of the virus in PLs. It is recommended that other disinfecting agents be tested as HPV appears to be resistant to chlorine. Also, the experimental design should be modified in order to actually simulate existence of HPV as an infectious agent in hatchery conditions.

Antibody-based detection methods are relatively simple and rapid, aside from being specific and sensitive. Thus, there is a necessity to develop such techniques for early and rapid diagnosis of viral pathogens such as MBV and HPV. In this study, the development of monoclonal antibodies (MAbs) against MBV and HPV employing the hybridoma technology was initiated. Spleen cells from Balb/C mice, immunized with either semi-purified HPV suspension or purified MBV virions were fused with Sp2/0 myeloma cells to produce hybridoma cells. Supernatants from cells which showed good viability and growth were harvested and screened for the presence of antibodies against the two viruses. An indirect immunofluorescence antibody test (IFAT) was optimized to screen these supernatants. Results showed that 84 of the supernatants raised against HPV showed intense immunofluorescent reaction on either hepatopancreas impression smears or paraffin-embedded tissue. Of the 9 MBV supernatants, only 2 showed intense reaction and one showed very weak fluorescence. These results suggest that antibodies to detect these two pathogens could be derived from the hybridoma cells produced in this study.