Efficacy of an inactivated vaccine and nutritional additives against white spot syndrome virus (WSSV) in shrimp (Penaeus monodon)

Although the immune system of shrimps is not comparable to that of vertebrates, shrimps can acquire protection against pathogenic challenge by building up immunity. In this study, formalin-inactivated virus (FIV) was administered by injection, bath-immersion, or orally to determine levels of vaccination-mediated protection against the pathogenic white spot syndrome virus (WSSV). Diets supplemented with alfalfa, methyl sulfonyl methane (MSM), or wheat grass were provided with or without FIV. Shrimp injected with FIV and challenged 3, 15, or 30 days after vaccination had cumulative and relative survivals of 83%, 67%, and 33%, respectively. Survival of shrimp challenged by bath-immersion 3-45 days after vaccination by immersion was significantly higher than in the unvaccinated control. Orally vaccinated shrimp challenged by bath-immersion were partially protected up to 45 days after vaccination (cumulative survival 63.7%, relative 61.7%) but not til 60 days after vaccination (cumulative 8%, relative 3.2%). Survival of unvaccinated shrimp challenged by bath-immersion improved when shrimp were fed a diet supplemented with wheat grass or MSM, but not alfalfa. Survival was further enhanced when FIV was provided together with diets supplemented with wheat grass (cumulative 72.7%, relative 94.8%) or MSM (cumulative 73.3%, relative 96.3%)

Induction of immunity and resistance to white spot syndrome virus (WSSV) in shrimp Penaeus monodon (Fabricius) by synthetic oligodeoxynucleotide and bacterial DNA

Shrimps like all invertebrates are believed to lack true adaptive immunity but recent evidence indicate that they can be protected against pathogenic organisms by priming their immune system with immunostimulatory substances. Here, we describe results of investigation aimed to strengthen shrimp immunity as a preventive strategy against white spot syndrome virus (WSSV) infection. Oligodeoxynucleotides (ODNs) with and without Cytosine-Guanine (CpG) motifs, and Vibrio harveyi genomic DNA (VHD) were administered by intramuscular injection and shrimp responses were assessed by ex vivo assays and experimental infection trials. Results showed that CpG ODN significantly increased ex-vivo immunity indices such as total hemocyte count (THC), plasma agglutination titer (PAT) and hemocyte lysate agglutination titer (HLAT). VHD significantly increased immune indices such as THC, plasma total protein (PTP) and HLAT. Reverse (GpC) motifs increased THC only. At a lower viral challenge dose, both CpG and GpC motifs, and VHD, were able to reduce shrimp mortality significantly but only CpG and VHD did so at a higher challenge dose. Strengthening shrimp immunity by the use of immunostimulatory nucleotides and bacterial genomic DNA could be a feasible preventive approach in the management of WSSV infections in shrimp.This study was supported by research funds from SEAFDEC AQD and the Regional Fish Disease Project of the Government of Japan Trust Fund Phase 4 under Study Code 8001-T-FD-FH1204

Field efficacy evaluation of a formalin-inactivated white spot syndrome virus (WSSV) vaccine for the preventive management of WSSV infection in shrimp grow-out ponds

A shrimp grow-out trial was conducted in four 700 m2 earthen ponds in Dumangas Brackishwater Station of SEAFDEC Aquaculture Department, in Iloilo, Philippines to evaluate the efficacy of a combination of immersion- and orally-administered formalin inactivated vaccine (FIV) as an antiviral strategy against WSSV. Purified virus prepared by sucrose gradient ultracentrifugation was inactivated by overnight incubation in 0.5% buffered formalin. PCR-screened Penaeus monodon postlarvae (PL14) obtained from a commercial hatchery were divided into two groups: 1) immersed in FIV solution for 2 h before stocking in ponds (VAC); and 2) unexposed control (CON). Both groups were stocked at a density of 15 m−2 and grown for 115 d following recommended good practices. Pond water was aerated using two 20-HP electric blowers. Shrimp were given commercial feed following the manufacturer's protocol. They were booster-vaccinated by administering FIV-coated feed every 15 d until the termination of the trial. Shrimp samples were regularly collected to obtain estimates of growth, survival, and feed conversion ratio. In addition, shrimp and extraneous crustacean species were analyzed for the presence of WSSV and IHHNV by PCR. Water quality and bacterial load were monitored regularly. After 115 d of culture (DOC), shrimps grew to 12.93 ± 1.26 g in VAC and 8.54 ± 0.78 g in CON. Likewise, survival and yield was higher in VAC (71.2 ± 3.13% and 1311 ± 70.4 kg ha−1, respectively) compared to CON (52.7 ± 5.05% and 640 ± 3.0 kg ha−1, respectively). PCR analysis showed both shrimp and extraneous crustaceans to be lightly infected with WSSV and IHHNV. A few dead shrimp were noted during routine pond inspection as shrimp biomass increased towards the end of the trial. However, no mass mortalities occurred and shrimp were successfully harvested on 115 DOC. VAC and CON shrimp were also collected on day 70, 90, and 105 and experimentally infected with WSSV in tanks. Significant differences in survival rates (p < 0.05) between the groups were obtained 15 d post-challenge. Further studies in tanks and in ponds on prophylactic oral delivery of antiviral treatments with the use of protein vaccines and vectors/carriers are recommended.This work was fully supported by project funds from SEAFDEC/AQD and the Government of Japan Trust Fund under Study Code 8400-T-RD-FH0210. We wish to thank the management of the Dumangas Brackishwater Station (DBS) for use of the facilities during the field trial. The generous assistance of DBS and Fish Health Section staff are gratefully acknowledged

Microbiological quality and heavy metal concentrations in slipper oyster (Crassostrea iredalei) cultured in major growing areas in Capiz Province, Western Visayas, Philippines: Compliance with international shellfish safety and sanitation standards

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. As filter feeders, oysters can accumulate pathogens from the surrounding waters, and these pathogens can cause foodborne diseases in consumers. Therefore, oyster farming areas must be monitored for microbiological quality and heavy metal concentrations. In the present study, the microbiological quality of oysters and their growing waters in the major oyster farming areas of the Cogon and Palina Rivers and Cabugao Bay (in Roxas City and the Municipality of Ivisan, respectively, Capiz Province, Western Visayas, Philippines) were examined monthly during the wet (May to October) and dry (November to April) seasons over 12 months. Regardless of the sampling period, high levels of fecal coliforms in the water and Escherichia coli in oysters were found, clearly illustrating that these oyster growing areas would meet only the class B standard under the European Union classification system and would be considered “prohibited” for growing oysters under the U.S. classification system. Although Salmonella was occasionally detected in oysters, Vibrio cholerae was not detected and Vibrio parahaemolyticus was within acceptable limits. The heavy metal concentrations in oyster meat were also determined during the wet (July) and dry (March) seasons. Zinc and copper were the most abundant metals detected, and concentrations of lead, cadmium, mercury, and chromium were below the regulatory limits set by the European Union and the U.S. Food and Drug Administration. These oyster culture areas should be rehabilitated immediately to improve the microbiological quality of the oysters. Oysters harvested from these sites must be depurated or relayed to ensure quality and safety.We express our heartfelt gratitude to Southeast Asian Fisheries Development Center/Aquaculture Department, Bureau of Fisheries and Aquatic Resources—Region VI, Negros Prawn Producers Cooperative, the local government units of the Municipality of Ivisan and Roxas City, and collaborators from Palina Greenbelt Ecopark, Barangay Cabugao, and Barangay Cogon. This study was funded by the Department of Science and Technology, Philippine Council for Agriculture, Aquatic and Natural Resources Research and Development (study code 6286-T-RD-DOST03)

Growth, plasma cortisol, liver and kidney histology, and resistance to vibriosis in brown-marbled grouper, Epinephelus fuscoguttatus fed onion and ginger

The health-promoting effects of dietary onion and ginger were studied in brown marbled grouper, Epinephelus fuscoguttatus. An eight-week feeding trial was conducted to evaluate the effects of dietary onion and ginger on growth, cortisol levels, histology and disease resistance in the fish. Five experimental diets were formulated to contain either onion (2%), ginger (2%), β-glucan (1%) or vitamin C (3%) and a control diet (without immunostimulants). Each diet was fed to triplicate groups of fish with an average weight of 10.85±0.69 g. Fish supplemented with either of the immunostimulants exhibited a significantly higher growth compared to the control group. Specific growth rate (SGR) and feed conversion efficiency (FCE) were significantly higher in the onion and ginger-supplemented fish than the control. Cortisol level was higher in the control compared to the immunostimulant-fed groups with onion and ginger-fed fish showing significantly lower cortisol levels. When experimentally infected with Vibrio harveyi, fish fed onion or ginger exhibited significantly reduced mortality when compared with the control and β-glucan, but not when compared with the vitamin C-fed group. Liver sections sampled 4 days postinfection showed no remarkable pathology except for the slight reduction in glycogen granules in the supplement-fed fish. The liver of non-supplemented infected fish showed necrosis, fatty globule deposition, vacuolation, and presence of short rod-shaped bacteria. Kidney sections in the supplemented groups, likewise, did not show significant pathology similar to the uninfected control, whereas those of infected control fish showed necrosis of the tubules and glomeruli resulting in severely altered morphology of the tissues and presence of bacteria in the necrotic areas. As a result of circumventing tissue damage, wound healing was faster in fish supplemented with ginger, onion, and vitamin C compared to the β-glucan and the control groups. This study showed the benefits of onion and ginger in promoting growth and alleviating stress and severity of vibriosis in grouper.The University of the Philippines Visayas is gratefully acknowledged for providing the research fund and the Southeast Asian Fisheries Development Center, Aquaculture Department (SEAFDEC/AQD) for the use of their research facilities

Temporal changes in innate immunity parameters, epinecidin gene expression, and mortality in orange-spotted grouper, Epinephelus coioides experimentally infected with a fish pathogen, Vibrio harveyi JML1

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 gelbased 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.This study was supported by SEAFDEC/AQD under Study Code 5203-T-RD-FH0510. The assistance of the Fish Health Section Staff is gratefully acknowledged

Achieving high production of micropropagated seaweed through optimization of the culture protocol

Kappaphycus and Eucheuma are the two carrageenanproducing red algae extensively cultured and farmed in tropical and subtropical waters. Seaweed production accounted for 5.4 percent of the world’s aquaculture production in 2019, which was over USD 15 billion in value. With this value share, the production of seaweed farming was higher than other aquatic commodities, such as tilapia, carp, shrimps, and others (Cai et al., 2021). The red algae production of the Philippines started in the 1960s (Ronquillo & Gabral-Llana, 1989), and by the early 2000s, the country had become the world’s top supplier of seaweed, particularly Kappaphycus sp., until 2007, when Indonesia outperformed it. Problems and challenges in seaweed farming, including climate change, pests and diseases, and biosecurity issues, caused the continuous decline of the seaweed production yield (Faisan, Sollesta-Pitogo, & de la Peña, 2022). Furthermore, the deterioration of seaweed quality caused by the repetitive use of vegetative cutting methods has also become one of the most pressing concerns in seaweed farming. Unfortunately, this procedure of growing plantlets for farming is one of the most common methods many seaweed farms used in the Philippines and other countries. Research in SEAFDEC Aquaculture Department (AQD) looks into breaking from the conventional method of farming seaweed plantlets and optimizing laboratorybased production. With this, it hopes to create a more sustainable source of propagules, better growth and survival in grow-out, and a higher carrageenan quality in cultured Kappaphycus alvarezii

Dietary onion and ginger enhance growth, hemato-immunological responses, and disease resistance in brown-marbled grouper, Epinephelus fuscoguttatus.

A 12-week (September to December 2009) feeding trial was conducted to evaluate theimmunostimulatory effects of different substances administered orally through the diet in the brown-marbled grouper, Epinephelus fuscoguttatus. Five experimental diets containing either onion, ginger, β–glucan, or vitamin C and a control diet (without immunostimulants) were fed to the fish weighing about 44 g for 12 weeks. Onion-fed fish showed significantly increased weight gain, hematocrit, and total Ig compared to the control group; however, leukocyte differential count and ROS production were unaffected. Ginger-fed fish likewise significantly increased total Ig, ROS production and lysozyme activity. However, it did not affect growth and hematocrit value. β-glucan significantly increased growth and total Ig but had no effect on the other parameters. Vitamin C significantly increased hematocrit, total Ig and ROS production but did not increase growth. Upon challenge with a bacterial pathogen Vibrio harveyi, mortality was significantly reduced in the onion, ginger and vitamin C-fed fish but not in the β–glucan-fed fish. This study demonstrated that onion and ginger could positively affect the innate immune responses and protect grouper against Vibrio harveyi infection.This study was supported by the University of the Philippines Visayas in-house funds through the National Institute of Molecular Biology and Biotechnology (NIMBB) and the Institute of Aquaculture, College of Fisheries and Ocean Sciences (IA-CFOS). We also gratefully acknowledge Biotec AHN AS, Norway for supplying the â-glucan (MacroGard)

Strategies to reduce disease incidence in mud crab culture

Abstract only.Mud crab hatchery suffers from low survival due to susceptibility to bacterial infection in the early larval stages among many causes. Despite food safety issues, antibiotics continue to be used in the absence of effective alternatives. In this study, screening of plant extracts was conducted to determine their suitability as antimicrobial agents against pathogens causing low survival in the hatchery. In addition, potential probionts were isolated from the gastrointestinal tract of mud crab, and water and sediments of mud crab grow-out ponds. Crude ethanolic extracts from various terrestrial and mangrove trees were tested for in vitro antimicrobial activity and toxicity to mud crab zoea and megalopa. In addition, the in vivo antimicrobial efficacies of the selected extracts were tested by oral administration followed by experimental challenge with Vibrio harveryi. On the other hand, the putative probionts, were tested for pathogenicity against mud crab zoea and megalopa and quorum sensing inhibition activity against V. harveyi. Finally the extracts and probionts were tested for their efficacy in simulated hatchery and grow-out trials. Results showed that extracts of Terminalia cattapa and the potential probiont Bacillus subtilis G100R11 showed antimicrobial and probiotic activity in in vitro and in vivo tests. In simulated hatchery trials, T. cattapa administration successfully produced crab instar with a survival of 1.3-1.8% in trials 1 and 2 comparable to antibiotic control. B. subtilis produced crab instar with survival of 0.8-1.0% in trials 1 and 2, better than the commercial probiotic with 0-0.13% survival. Using T. cattapa and B. subtilis, survival was above 30% until zoea 5 but suddenly dropped below 5% during metamorphosis to megalopa where high incidence of incomplete molting was observed. If difficulty affecting the molting process is addressed, high survival from zoea to megalopa and crab instar will be achievable

Evidence of WSSV transmission from the rotifer (Brachionus plicatilis) to the black tiger shrimp (Penaeus monodon) postlarvae and means to control rotifer resting eggs using industrial disinfectants

Rotifers are considered possible vectors of the white spot syndrome virus (WSSV) and have been implicated in its recurrence in pond-cultured shrimp. However, direct evidence of the transmission and the pathogenicity of this virus from rotifers to shrimp has been lacking. In the present study, the pathogenicity of WSSV transmitted from infected rotifers (Brachionus plicatilis) to post larval black tiger shrimp (Penaeus monodon) was investigated. Results show that WSSV transmitted from infected rotifers to post-larval P. monodon caused an 82% cumulative mortality as compared to a 9% mortality in the non-infected control group. We also investigated the possibility of industrial disinfectants (sodium hypochlorite, granulated calcium hypochlorite and formalin) as possible means to inhibit the viability of rotifer resting eggs, considered a biological reservoir of WSSV in earthen ponds. Among the disinfectants that were tested, granulated calcium hypochlorite at 5 mg/L was the most effective. The present study provides direct evidence of the high pathogenicity of WSSV transmitted from rotifers to post larval P. monodon and shows the potential use of granulated calcium hypochlorite in pond disinfection. This treatment could be a promising strategy to inhibit the spread and recurrence of WSSV outbreaks in P. monodon culture