Innovative research to guarantee food safety and sustainable production

Molecular diagnosis and prevention of economically-important viruses in fish and shrimp - The plasmid positive control for WSSV was initially developed.

- Optimization of q-PCR protocols for WSSV, RSIV, KHV, VNN, IMNV and IHHNV were already completed.

- LAMP assay for WSSV showed that the optimum incubation is at 61°C for 58 min.

Establishment of immunization regimen for the prevention of viral nervous necrosis in high value marine broodfish

- Pompano broodstocks were intraperitoneally (IP) booster vaccinated with inactivated Philippine strain of NNV, while control fish were IP injected with L-15 medium. Determination of antibody titers in the sera of vaccinated/control fish collected is ongoing.

Establishment of novel prophylactic and therapeutic methods for the prevention of viral infections in commercially important maricultured fish

- U. pertusa extracts showed potent antibacterial activity against Aeromonas hydrophila and A. sobria at 100 and 50 mg.

- Determination of the minimum inhibitory concentration and minimum bactericidal concentration of the U.

pertusa aqueous extract on Vibrio alginolyticus, V. parahemolyticus, Edwardsiella tarda, and Streptococcus spp. is ongoing.

- Mortality for A. hydrophila-challenged fish fed U. pertusa supplemented diet (100 g U. pertusa leaves/kg feed, 5% BW) was not significantly different (40%) from those for A. hydrophila-challenged fish fed with SEAFDEC/AQD formulated diet (35%).

Evaluation of carriers for practical delivery of vaccines to shrimp and other crustaceans

- The carriers+vaccine were administered orally to the shrimp via the feed. The survival rates against WSSV challenge were 65.3%, 61.9%, 50% and 25.9% in inclusion bodies (IB)+chitosan, IB+alginate, naked IB, and inactivated recombinant bacteria groups, respectively, and 0% in the unvaccinated group. There were no apparent differences in survival among IB+chitosan, IB+alginate, and naked IB, but were significantly higher compared to inactivated recombinant bacteria and unvaccinated group.

- To determine appropriate dosages and vaccine: carrier ratios, a third trial is ongoing.

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Parasitic and shell diseases of abalone (Haliotis asinina) in Philippines

- Abalones are routinely infested by shell-infesting polychaete worm belonging to the family Dorvilleidae (prevalence, 20%).

- The condition indices of infested abalone consistently yielded significantly lower condition indices than uninfested abalone.

- In the presence of infested abalone, uninfested abalone becomes infested within 48-72 hours with an average of 5 worms (crawling larvae) per abalone.

(3) Outcomes/Outputs during the project period and expected achievement rate till the end of 2013 (tentative)

Expected outcomes/outputs Achievement rate (%) - To accelerate awareness on fish health management in resource-

deprived countries through industry-wide capacity building 80%

- To guarantee food safety and sustainable production through innovative

research 80%

- To disseminate output of the project 80%

3.2.6 Evaluation of Project Activities in 2013

The study plans are considered to be appropriately implemented as a whole in this project.

4. PROPOSED ACTIVITIES FOR THE YEAR 2014 4.1 Planning of the Project Activities

Activity/inputs Duration Remarks

Activity 1: Accelerating awareness about fish health management in resource- deprived countries through industry-wide capacity building Sub-activity 1.1 Survey on the status and needs of primary aquatic animal health

care in small-scale aquaculture

Follow-up training and information activities will be implemented either in Lao PDR or Cambodia.

Sub-activity 1.2 Surveillance and training of fish-borne zoonotic parasites of commercially important freshwater fish in some Southeast Asian countries

1) Parallel sampling for zoonotic parasites will be continued in some regions in the Philippines, where freshwater fish farming activities are widely practiced; 2) Another re-visit to the Member Countries (Lao PDR or Cambodia) that still need intensive capacity building of fish health diagnosis through expanded training will be conducted; In addition, 3) guidelines and recommendations of prophylactic and control methods of these zoonoses will be formulated.

Activity 2: Innovative Research to Guarantee Food Safety and Sustainable Production

Sub-activity 2.1 Molecular diagnosis and prevention of economically-important viruses in fish and shrimp

1) Susceptibility experiments will continue for Penaeus monodon on the first quarter, Litopenaeus vannamei on the second quarter, P. indicus on the third quarter and P. mergueinsis on the last quarter of 2014; and 2) Determination of infection /threshold level of TSV will be conducted on the first quarter, VNN on the second quarter, RSIV on the third quarter and KHV on the last quarter of 2014.

Sub-activity 2.2 Establishment of immunization regimen for the prevention of viral nervous necrosis in high value marine fish

1) Monitor the kinetics of NNV-neutralizing antibodies in the sera of vaccinated and unvaccinated pompano Trachinotus blochii broodfish at scheduled intervals post-vaccination; 2) Conduct booster vaccination and L-15 injection in vaccinated and unvaccinated pompano broodfish, respectively; 3) Conduct induce spawning of sexually matured vaccinated and unvaccinated pompano broodfish; 4) Detect NNV

Jan – Dec

Jan – Dec

Jan – Dec

Jan – Dec

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Activity/inputs Duration Remarks

in milt and eggs collected from both vaccinated and unvaccinated pompano broodfish by RT-PCR and cell culture isolation; 5) Detect NNV in spawned eggs of vaccinated and unvaccinated pompano broodfish by RT-PCR and cell culture isolation; and, 6) Quantify the levels of NNV-neutralizing antibodies in spawned eggs of vaccinated and unvaccinated pompano broodfish.

Sub-activity 2.3 Establishment of novel prophylactic and therapeutic methods for the prevention of viral infections in commercially important cultured fish

1) Determine the in vitro susceptibility of newly acquired fish pathogens such as Vibrio alginolyticus, V. parahemolyticus, Edwardsiella tarda, and Streptococcus spp. to Ulva pertusa aqueous extract; 2) Determine the minimum inhibitory concentration (MIC) and minimum bacteriacidal concentration (MBC) of the U.

pertusa aqueous extract against the above fish pathogens; 3) Examine the prophylactic and therapeutic efficacy of U. pertusa aqueous extract against the above fish pathogenic bacteria in vivo through experimental infection; 4) Determine the anti-nervous necrosis virus activity of U. pertusa aqueous extract in vitro; and 5) Test the prophylactic and therapeutic efficacy of U. pertusa aqueous extract against nervous necrosis virus in vivo through experimental infection.

Sub-activity 2.4 Evaluation of carriers for practical delivery of vaccines to shrimp and other crustaceans

Challenge trials with different vaccine-carrier ratios and dosages will be conducted in shrimp/ crustacean species other than P. monodon.

Sub-activity 2.5 Parasitic and shell diseases of abalone (Haliotis asinina) in Philippines

1) Reproductive characteristics of shell-boring polychaetes using scanning electron microscope (SEM) will be done; 2) Continued parasitological screening of hatchery-reared abalone from different sites; 3) Confirmatory runs on the mode of infestation (cohabitation) will be continued; and 4) Guideline of prevention and control method for shell-boring polychaetes infections will be formulated.

Activity 3: Dissemination of Output of the Project

Sub-activity 3.1 Publication of new information into popular materials (No activity scheduled in 2014)

Sub-activity 3.2 Maintenance and updating of website contents (No activity scheduled in 2014)

Activity 4: Annual progress meeting and international workshop Sub-activity 4.1 Annual progress meeting

Annual meeting will be held to review the project achievements. Evaluators will be invited to join the meeting to review/evaluate the project achievements.

Sub-activity 4.2 International workshop (No activity scheduled in 2014)

Activity 5: Coordination by the project leader

The project leader will coordinate and encourage the research, training and dissemination, and also facilitate information exchange not only between activities but also among Member Countries so that awareness and capacity-building in fish health management will be effectively promoted in Southeast Asia. Semi-annual meeting will be held in July or August to confirm the progress of respective activities and sub-activities. Project achievements will be summarized at the end of year. Annual progress report will be prepared.

Jan – Dec

Jan – Dec

Jan – Dec

December

March

Jan – Dec

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The envisaged outcomes for the fifth year are : 1) increased awareness on primary aquatic animal health care in small-scale aquaculture; 2) prophylactic and control methods of zoonotic parasite of freshwater fishes formulated; 3) plasmid positive controls for IHHNV, TSV, IMNV and VNN developed, 4) level of neutralizing antibodies in the sera of booster vaccinated and unvaccinated sea bass broodstocks determined; 5) antibacterial activity in vitro and in vivo evaluated; 6) practical delivery system examined and the administration method optimized; 7) reproductive characteristics and infection mechanisms of shell-boring polychaetes clarified; and 8) guidelines on prevention methods for parasite infections formulated.

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Appendix 10 of Annex 4 PROJECT DOCUMENT

Project id: 021004

Program Categories:

Programs under the Fisheries Consultative Group of the ASEAN-SEAFDEC Strategic Partnership (FCG/ASSP) Mechanism

Project Title: Food Safety of Aquaculture Products in Southeast Asia

Program Thrust: II Total Duration: 5 years (2010-2014) Lead Department: Aquaculture Department Lead Country: Philippines

Project Sponsor: Japanese Trust Fund Project Partner:

Proposed Budget: This year budget:

Prepared by: Teruo Azuma, Deputy Chief

Project Leader: Teruo Azuma, Deputy Chief 1. INTRODUCTION/BACKGROUND

The use of antibiotics and other chemicals in aquaculture is widely practiced to help meet the increasing demand for aquaculture food. These antibiotics and chemicals detected in aquaculture products appear to derive from material inputs during rearing, mostly from contaminated feed ingredients and therapeutants for prevention or treatment of diseases. On the other hand, with the ever-growing and worldwide concern for food safety, fish farmers are faced with the challenge of producing safe food from farm to fork.

Uncontrolled use of chemicals should be prevented to secure human health and wholesome aquatic ecosystem and to keep from loading of harmful chemicals. Certificates guaranteeing safety as foods would be increasingly important, particularly for trading.

The mechanisms of accumulation and withdrawal of some antibiotics and chemicals have already been studied in developed countries. However, these data were generated using their species and under environmental conditions that are different from the conditions prevalent in the Southeast Asian region.

There are still very limited data available on the withdrawal period of antibiotics and the presence of chemical residues in aquaculture products from the region. Considering the growing-awareness on issues of food safety of aquaculture products, it is an urgent matter that SEAFDEC should take the lead in establishing regional guidelines on the right usage of antibiotics and other chemical inputs.

At almost the same time when this project started, ASEAN also initiated preparation of guidelines for the use of chemicals in aquaculture and measures to eliminate the use of harmful chemicals, with the Department of Fisheries Malaysia as the lead country. This is based on the declaration on the ASEAN Economic Community Blueprint to realize the ASEAN Economic Community (AEC) during the 13^th ASEAN Summit held in Singapore in November 2007. Since the purposes are common between the activities under the present project funded by Government of Japan and the guidelines led by ASEAN, SEAFDEC/AQD and ASEAN agreed to collaborate to make a better guideline so that more practical, detailed information could be disseminated and utilized effectively in the Southeast Asian region.

The results of this project will also be useful for the formulation of policy recommendations for a concerted action by governments of the ASEAN Member Countries.

2. PROJECT

2.1 Goal/Overall Objectives and Performance Indicators:

The project goal is to attain food safety of aquaculture products in Southeast Asia. The project objectives are to:

1) Contribute in the establishment of guidelines on the production of safe aquaculture products from Southeast Asia;

2) Determine the presence and levels of commonly used chemicals in aquaculture in aquaculture products such as fish and shrimps;

3) Compile and disseminate SEAFDEC guidelines on the use of antibiotics and chemicals in aquaculture

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4) Implement training course/workshop to promote food safety awareness in the ASEAN region.

The performance indicators are as follows.

1) Detection of withdrawal period of antibiotics in important cultured fishes;

2) Surveillance and monitoring of the chemical contaminants in aquaculture products and feeds especially in developing ASEAN Member Countries;

3) Compiling guidelines on appropriate administration and regulation of antibiotics/other chemicals; and 4) Implementation of training course/workshop

2.2 Expected Outcomes and Outputs:

Expected outcomes are:

1) Determination of the withdrawal periods of antibiotics in a high value fin fish;

2) Screening the present status of the usage of chemical contaminants in aquaculture products and feeds;

3) Establishment of a new method of determining antibiotics in aquaculture product samples;

4) Publishing guidelines for the SEAFDEC Member Countries; and

5) Implementation of International Workshop on “Food Safety of Aquaculture Products in Southeast Asia”.

Expected outputs are:

1) Information on withdrawal period of antibiotics in important culture species;

2) Baseline data on chemical contaminants in aquaculture products and feeds;

3) New techniques for detection of antibiotics in aquaculture products and feeds;

4) Guidelines on appropriate administration and regulation of antibiotics/other chemicals; and 5) Practical information to promote food safety in the Southeast Asian region.

2.3 Project Description/Framework

Activity 1: Withdrawal period of antibiotics in important species cultured in the tropics

Developments in aquaculture discouraged the use of antibiotics because of the risk of developing antibiotic resistance to human pathogens, effect on non-target organisms, safety of workers, and effect on the environment, among others. However, the use of these drugs is sometimes inevitable, especially in an outbreak of a disease.

Milkfish is the most common cultured species in the Philippines and is now becoming a fast export aquaculture commodity. Most often, monoculture technique is employed. However, polyculture system is also being practiced and the milkfish-shrimp polyculture is quite common. Although outbreaks of bacterial infection during milkfish culture have seldom occurred, risk of exposure to antibiotics in a polyculture system increases if its co-culture species is at risk of bacterial infection. Likewise, freshwater fish species like tilapia could be at risk to exposure to antibiotics during culture.

Withdrawal periods, bioavailability and pharmacokinetic studies of some antibiotics, have been conducted in temperate countries in several species. However, data are very limited, if there is any, for fishes raised in the tropics.

In the present activity, therefore, withdrawal periods of antibiotics from marine fish such as milkfish and freshwater fish such as tilapia are revealed to contribute in the establishment of guidelines on the production of safe aquaculture products from Southeast Asia.

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Activity 2: Surveillance of chemical contaminants in aquaculture products and feeds: Levels and classification of contaminants in aquaculture products, aqua feeds and aqua feed ingredients in the Philippines and other Asian countries

A wide range of chemicals are used in the culture of popular aquaculture species. There are also chemicals applied or used in the processing of feed ingredients and in the manufacture of aquafeeds. The indiscriminate use of these chemicals; however, poses dangers to human health since some of these chemicals have been detected in aquaculture products. The increased usage and the number of these drugs and other chemicals throughout the years are predictable since world aquaculture production and the numbers of species for culture have been increasing. The trend towards high density culture systems is also a contributing factor in the increased use of chemicals in aquaculture. For health reasons of consumers and the safety regulations imposed by importing countries on aquaculture products, there is a pressing need to survey at this time the chemicals used in aquaculture.

The fast growing aquaculture sector exerted a great demand for aqua feed and feed ingredients. This has translated into adulteration and indiscriminate use of chemicals in these commodities. A survey of these chemicals is important in the sustainability of the aquaculture industry which is much related to the environment.

This Activity 2 will determine the presence and levels of commonly used chemicals such as antibiotics, fungicides, feed additives, toxin binders etc. in aqua feed and feed ingredients as well as aquaculture products through the conventional method using HPLC. In addition, the Activity 2 also tries to develop a new method to detect antibiotics, which is reliable, inexpensive, mass-determination-possible, and simple so that the Member Countries can easily adopt the method for screening of chemicals.

Activity 3: Establishment of guidelines on appropriate administration and regulation of antibiotics/other chemicals

Comprehensive information on the use of chemicals in aquaculture in Asia with emphasis on the various aquaculture systems and species to which chemicals are applied as well as the various country regulations regarding their distribution and use was presented during the Expert Meeting on the Use of Chemicals in Aquaculture in Asia at the SEAFDEC AQD in 1996. Concerns for the safe, effective and minimal use of chemicals to protect human health and the environment are also reflected in the FAO Code of Conduct for Responsible Fisheries. A wide range of chemicals are being used in aquaculture worldwide in different aquaculture systems and for various reasons. Sustained efforts are needed to update the general information base on chemical usage in aquaculture in Asia and understand the realities and uncertainties in the regulatory frameworks governing the use of chemicals to ensure food safety and minimal impacts on public health and the environment. Many countries are now imposing strict food safety requirements (maximum residue levels and monitoring banned chemicals) on imported aquaculture products, which will likely pose significant difficulties to countries exporting aquaculture products in the future. Therefore, the Activity 3 will establish guidelines on appropriate administration and regulation of antibiotics and other chemicals used in aquaculture in Southeast Asian countries.

Activity 4: Dissemination of food safety awareness and manual publication

Dissemination of information on food safety is inevitable to responsible approach to aquaculture. Manuals, posters, pamphlets and fliers describing information on food safety will be published and distributed.

Activity 5: Annual Progress Meeting and International Workshop

Annual progress meeting and International Workshop will be organized to review the progresses and check the appropriateness of the project, and to disseminate new information to each member country.

Sub-activity 5.1: Annual progress meeting

Annual progress meeting will be held to review the project achievement. Evaluators will be invited to join the meeting to review/evaluate the project achievements.

Sub-activity 5.2: International Workshop

New information on food safety will be presented and discussed by participants from ASEAN Member Countries, resource persons as well as study leaders in this project. Emerging problems that require urgent

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solutions will also be discussed to pave the way to promote and to assure the food safety practices in the Southeast Asian region.

Activity 6: Coordination by the Project Leader

The project leader will coordinate and assist the research, training and dissemination, and also facilitate information exchange not only domestically but also among Member Countries so that food safety will be effectively promoted in Southeast Asia. Semi-annual meeting will be held on July or August to confirm the progress of respective activities and sub-activities. Project achievements will be summarized at the end of year. Annual progress report will be prepared.

3. PROGRESS/ACHIEVEMENTS OF ACTIVITIES IN THE YEAR 2013 3.1 Activities Achievements in the Year 2013

Achievements based on Activities Duration Remarks 1. Withdrawal period of antibiotics in important species cultured in the

tropics

- Determine the withdrawal periods of commonly used and generally approved antibacterial agents, specifically oxytetracycline (OTC) and oxolinic acid (OXA)

2. Surveillance of chemical contaminants in aquaculture products and

In document THE THIRTY-SIXTH MEETING OF THE PROGRAM COMMITTEE OF THE SOUTHEAST ASIAN FISHERIES DEVELOPMENT CENTER (pahina 119-126)