Reinforcement and optimization of fish health management and effective dissemination in the Southeast Asian Region Accelerating

Awareness on Fish Health Management in Southeast Asia

Viral and bacterial diseases have caused major constraints in shrimp farming in most Asian countries and in the world. Early detection of these devastating pathogens is the most efficient response to be able to implement immediate and appropriate interventions for the control of the spread of infection. AQD determined the threshold infection levels (viral DNA/RNA copies in an organism that can result to an infection) for WSSV and Vibrio parahaemolyticus (VP_AHPND). Knowing these will enable the farmers to strictly monitor the health status of shrimp so that early and effective intervention strategies can be implemented. Also, the known threshold levels for the pathogens will serve as a reference in the regular monitoring and diagnostic schemes in the farm level, if it is still safe or dangerous.

Standard curve has been established using WSSV plasmid. Preliminary infection experiments to determine the concentration of viral inoculum that killed 50% of injected shrimp (LD₅₀) were conducted for the 3 weight ranges (3.81, 7.42 and 16.83 g) which showed that LD₅₀ at viral dilution of 10^-6 were achieved faster in smaller weight range (9 days for ABW 3.81 g) compared to bigger weight range (10 days for ABW 7.42 g). LD₅₀ for ABW 16.83 g was achieved with lower viral dilution of 10^-5 at 9 days. All the mortalities were found to be WSSV one-step positive.

Time-course experiment was subsequently conducted. Mortality for ABW 3.81 g has started at the range of 1.6x10⁹ to 3.3x10⁹ copies/g, 195 h post infection (pi). The range for the survivors was from 3.3x10⁶ to 4.1x10⁶ copies/g, 231 h pi. Mortality for ABW 7.42 g has started at the range of 3.7x10⁹ to 5.1x10⁹ copies/g, 231 h pi. The range for the survivors was from 6.3x10⁶ to 8.9x10⁶ copies/g, 264 h pi. Mortality for ABW 16.83 g has started at the range of 1.2x10⁹ to 4.6x10⁹ copies/g, 162 h pi. The range for the survivors was from 6.8x10⁶ to 8.8x10⁶ copies/g, 219 h pi.

Enhancement of efficacy of vaccine treatment in tropical cultured species

Viral nervous necrosis (VNN) caused by nervous necrosis virus (NNV), a piscine betanodavirus, is a destructive disease that induces neuropathological abnormalities in maricultured fishes generally at the larval and juvenile stages. A study was carried out to develop and adopt methods that would enhance the efficacy of the present NNV vaccines through the use of immunoadjuvants and other substances that promote the activation of antiviral responses in marine fish. A practical method to deliver the vaccine to fish would be developed to prevent unwarranted outbreaks of VNN in hatcheries and grow-out culture systems.

AQD investigated the field efficacy of the inactivated NNV vaccine in orange-spotted grouper (Epinephelus coioides) reared in net-cages in earthen pond. Seroneutralization assay conducted on the sera of vaccinated fish revealed the presence of neutralizing antibody titers from day 30 to day 150 with the highest titer observed at day 60 post-vaccination. Nil and 25% mortality were obtained in both vaccinated and unvaccinated fish, respectively, when challenged with NNV through intramuscular injection. Additionally, NNV-challenge of day 120 vaccinated and control fish likewise resulted in nil mortality, suggesting an age or weight dependent susceptibility to NNV.

Taken together, the data suggests that single vaccination with inactivated NNV vaccine could mount and confer respectively the production of protective antibodies and concomitant protection against VNN in groupers especially during the early phase of grow-out culture in earthen ponds when these fish species are highly susceptible to the disease.

Application of adjuvants, carriers and RNAi technology to enhance the antiviral immune response of shrimp to WSSV

In shrimp aquaculture, a safe, effective, and inexpensive antiviral treatment is required to limit the impact of WSSV and other shrimp viruses. RNAi is a new technology that is based on gene silencing. The antiviral effect of RNAi is based on silencing a viral or host gene that is primarily involved in viral pathogenesis. The main constraint of RNAi as an antiviral agent is production cost and a practical method of delivery. Thus, dsRNA was produced using a low-cost bacterially expressed dsRNA production method. The efficacy of dsRNA was tested in several challenge experiments using various dsRNA doses, different frequency of dsRNA administration, and inclusion of heterologous dsRNA to test the specificity of gene silencing.

The best treatment was determined to be a dose of 20 µg/shrimp administered 4 times over 28 days (2 times before and 2 times after challenge (total = 80 µg/shrimp)). Furthermore, the silencing was found to be specific to VP28 dsRNA. Oral delivery using different ratios of dsRNA to rVP28 entrapped in microparticle carriers will be tested by challenge experiments in tanks.

Cumulative mortalities of unvaccinated ( ) and vaccinated ( ) orange-spotted grouper (Epinephelus coioides) juveniles intramuscularly

injected with nervous necrosis virus

Grouper affected by viral nervous necrosis (VNN )

Establishment of protective measures against persistent and emerging parasitic diseases of tropical fish

Results of the study that aims to develop practical strategies could be adopted by farmers to address the pressing problem on mass mortalities of net-caged and pond- reared fishes attributed to persistent and emerging fish parasites. This study examines the anti-parasitic effect of garlic (Allium sativum) in the form of allicin powder against monogenean parasites (Pseudorhabdosynochus lantauensis) infecting groupers (Epinephelus coioides).

Acute toxicity bioassays to determine the 96 h LC₅₀ value of allicin powder were carried out in static systems.

Results showed that the median lethal concentration (LC₅₀) of allicin powder to grouper for 24, 48, 72 and 96 h of exposure are 172.37, 168.52, 134.90 and 73.63 ppm respectively. Oral treatments using allicin powder- supplemented diet (0.10%, 0.50%, and 1.0%) and a control diet without allicin were tested on groupers infected with gill monogenean P. lantauensis for 14 days.

Results showed that groupers fed with allicin powder

supplemented diets showed reduced prevalence and mean intensity of monogenean parasites as compared to the control. Histological examination showed no pathological changes in the liver, intestine and kidneys of the fish.

Also, hematological changes were compared in both Trichodina sp.-infected and uninfected tilapia (Oreochromis niloticus). Results showed that hematocrit, hemoglobin and red blood cell count were lower in infected fish than in healthy individuals. In contrast, white blood cell count was higher in infected fish compared to non-infected fish.

Acute toxicity bioassays to determine the 96 h LC₅₀ value of allicin powder in tilapia (Oreochromis niloticus) were carried out in static systems. The LC₅₀ values of garlic extract for 24, 48, 72 and 96 hours were 398.1, 360.7, 316.21 and 208.95 ppm respectively.

Epidemiology of the Early Mortality Syndrome (EMS)/ Acute Hepatopancreatic Necrosis Disease (AHPND)

Early Mortality Syndrome (EMS) or Acute Hepatopancreatic Necrosis Disease (AHPND) is a shrimp disease caused by the Vibrio parahaemolyticus affecting most Southeast Asian Countries. This activity aims to develop protective measures, in cooperation with farmers and hatchery operators, based on the causative agents together with identification of risk and protective factors. Results of the pathogenicity tests indicate that the threshold level of VP_AHPND bacteria in the environment that shrimp may overcome is 10⁶ cfu/ml.

Parasites (Pseudorhabdosynochus lantauensis) in the gills of

grouper

Shrimp on top infected with AHPND shows an empty stomach, pale hepatopancreas,

and empty midgut

Furthermore, exposure to 10⁷ cfu/ml VP_AHPND bacteria may cause significant mortality in P. monodon postlarvae. High temperature (35°C), high (28 ppt) and low (10 ppt) salinities are other possible factors that may increase the risk of mortality due to VP_AHPND infection.

Use of greenwater that has been stocked with siganid (Siganus spp.) for not less than 2 weeks might provide some protection against the disease as shown by less V. parahaemolyticus detected in the hepatopancreas of shrimp. Use of siganid water to culture shrimp was further shown to improve shrimp growth and survival. Use of brown mussel (Perna perna) may also improve shrimp survival but needs further investigation.

Technology extension and demonstration

On-site evaluation of the aquaculture practices in selected developing countries was conducted to assess the compounding factors that trigger persistent diseases caused by parasites, bacteria and viruses in freshwater fishes. To boost the capability of fish health personnel, basic on-site training courses on fish bacteriology and parasitology were also organized, e.g. the on-site training course on “Health Management of Parasitic Diseases of Freshwater Fishes” from 6 to 10 December 2017 at the Fish Health Laboratory of the Marine Research and Development Center, Sihanoukville, Cambodia, as requested during the SEAFDEC Council Meeting in 2017. Eleven staff from various aquaculture agencies in Cambodia attended the training. As per request from Lao PDR, on-site training course was also conducted on 20-24 November 2017 at Namxoung Aquaculture and Development Center, Namxoung, Lao PDR. A total of 15 participants from different institutions in Lao PDR participated. The lectures highlighted on the major bacterial and parasitic diseases currently affecting cultured freshwater fishes in the region. Hands-on exercises including fish necropsy, quantitative determination of parasite load in the gills and skin of fish and subsequent identification of the parasite present in the fishes examined were also carried out.

Additionally, an update on tilapia lake virus infection, an emerging and pressing problem currently besetting the tilapia aquaculture industry in Asia was presented. On the last day of the training, a field trip to a farm was conducted to observe the actual pond culture practices.

Demonstration of laboratory procedures on parasitology during the on-site training in Cambodia (left), and practical session during the on-site training on “Health Management of Parasitic Diseases of

Freshwater Fishes” in Lao PDR (right)

THRUST 3. IMPROVING MANAGEMENT CONCEPTS AND APPROACHES FOR