Aquaculture Extension Manual No

Experience on Common Carp Mass Mortality in Japan

Motohiko Sano¹, Takafumi Ito¹, Jun Kurita¹, Kei Yuasa¹
Satoshi Miwa¹ and Takaji Iida²

¹Inland Station, National Research Institute of Aquaculture, Fisheries Research
Agency, Tamaki, Mie 519-0423, Japan
²National Research Institute of Aquaculture, Fisheries Research Agency,
Nansei, Mie 516-0193, Japan

Abstract
The mortality rate among common carp for food reared in net pens in Lake Kasumigaura, the second largest lake in Japan, in Ibaraki Prefecture, increased from early October 2003 and koi herpesvirus (KHV) was detected in the affected fish by the National Research Institute of Aquaculture (NRIA) in late October using PCR methods of Gilad et al. (2002) and Gray et al. (2002). The Ministry of Agriculture, Forestry and Fisheries of Japan officially announced the first occurrence of KHV disease in Japan. In late October 2003, the water temperature of Lake Kasumigaura was 16-18⁰C and the fish losses were severe, particularly in market-sized carp. The apparent symptoms of affected fish were presence of mucus-like substance on the body surface, sunken eyes, and pale and necrotic gills, which were similar to those reported by Hedrick et al. (2000). Approximately 1,200 metric tons of common carp cultured in the lake were lost by mid-November. Prior to this, however, infected carp cultured in Lake Kasumigaura had already been transferred to farms,
wholesalers, restaurants and game fishing facilities. Consequently, the infection spread to other areas in Japan. Independent of the outbreak in Lake Kasumigaura, a massive carp loss of over 10 thousand fish, the cause of which was initially diagnosed as columnaris disease, occurred in some rivers and a lake in Okayama Prefecture from late May to mid-July 2003. In November, the NRIA detected KHV DNA by PCR from samples of the diseased fish stored in a freezer. This demonstrated that KHV was present in Japan before late May 2003. By the end of 2003, KHV was detected in carp from 23 out of 47 prefectures in Japan. No occurrence of the disease was observed during the winter period. However, as the water temperature increased in spring of 2004, KHV reappeared in the area where the disease had been previously recorded, and also in new places. In many of the facilities that experienced KHV outbreak in 2003, the disease was not observed by June 2004 because all carp had been removed together with other fish species and the facilities were disinfected thoroughly after the outbreaks. From January to the end of May 2004, KHV infections were reported in 24 of 47 prefectures in Japan.

Diagnostic System for Exotic Diseases and Koi Herpesvirus Disease

Some diseases are designated as “Specific Diseases” in the Japanese law. These are principally exotic diseases such as spring viremia of carp (SVC) and viral hemorrhagic septicemia (VHS) of salmonids that have the potential to devastate the aquaculture industry in Japan. For such diseases, protective guidelines have been established in Japan. The guidelines provide etiological information, diagnostic procedures, description of the symptoms and other important characteristics of the diseases. Laboratory diagnosis of the diseases must be conducted in accordance with these guidelines. A newly isolated herpesvirus, designated as koi herpesvirus (KHV), was first reported as a causative pathogen of mass mortality that occurred among common and ornamental (koi) carp Cyprinus carpio cultured in Israel and the USA in 1998 (Hedrick et al., 2000). A similar virus was also isolated after massive mortality of carp in Germany (Neukirch and Kunz, 2001) and Israel (Perelberg et al., 2003). The virus isolated in Israel was identified as carp nephritis and gill necrosis virus (CNGV) based on the histopathological results (Ronen et al., 2003). Subsequently, this viral infection has been observed in western Europe since 2000, Indonesia in the spring of 2002 and Taiwan in the fall of 2002 (Tu et al., 2004), revealing that this disease is rapidly spreading worldwide in carp-trading countries. In Japan, there was no such mass mortality of carp before 2003 and KHV was not detected by a survey conducted in the Niigata Prefecture in 2001 (Amita et al., 2002). As KHV is highly contagious and virulent in juvenile and adult carp (Hedrick et al., 2000; Perelberg et al., 2003), KHV infection was designated as a “Specific Disease” by the Japanese law amended on 30 June 2003, and an inspection procedure was established as part of the guidelines (Fig. 1). According to the procedure, Prefectural Fisheries Experimental Stations (PFESs), which

belong to the local government, first conduct an epizootic and routine clinical examination of diseased fish. The most important epizootiological aspect of KHV disease is that it affects only carp Cyprinus carpio and occurs apparently only in a limited range of water temperature from 18-28⁰C (Hedrick et al., 2000; Gilad et al., 2003). Therefore, the water temperature and susceptible fish species should be determined during a field examination. Few distinguishable external signs are usually visible, but pale and necrotic gills are frequently found. Flexibacter columnaris infection and some
protozoan parasites, such as Chilodonella and Trichodina, are sometimes found on necrotic gill lesions, which easily lead to misdiagnosis of KHV disease. In case any doubt remains as to the presence of KHV, a polymerase chain reaction (PCR) test can be used to detect KHV DNA in the tissues of fish. The PCR method described by Gray et al. (2002) is adopted in the inspection procedure as the primary examination conducted by PFESs. When the PCR test is positive for KHV, the sample is sent to the National Research Institute of Aquaculture (NRIA) for further examination by PCR methods of both Gilad et al. (2002) and Gray et al. (2002) for confirmation. Virus isolation on the KF-1 cell line is also attempted using the KF-1 cell line (Hedrick et al., 2000). Because of difficult isolation of KHV using the cell line, results of the isolation trial is treated as supplementary data and confirmation of KHV is solely based on the results of the PCR tests.

Occurrence of KHV Disease in Japan and Practical Diagnosis of the Disease

In Lake Kasumigaura, central Japan, the mortality among common carp cultured in net pens increased from early October 2003, when the water temperature of the lake was 16-18⁰C. The fish were lethargic and swam near the water surface. There were no marked external signs in most of the affected fish, but the appearance of whitish mucous-like substance on the body surface, redness of the fin and body, fin rot, and discoloration of the gill with some necrosis were sometimes observed. Mortality was over 60% in the most severe cases, especially in larger carp over 2 years old. The losses of cultured carp were estimated at 660 metric tons (MT) in early November and this reached approximately 1,200 MT by mid-November. This represents approximately one fourth of the lake’s annual production.External parasites, such as Chilodonella, Trichodina, and Gyrodactylus, were sometimes seen on the necrotic gill of affected fish. Marked histopathological changes were observed in the gill of diseased carp (Fig. 2). The secondary lamellae were often fused with the hyperplastic branchial epithelium where cell necrosis or infiltration of lymphocytes were often observed. Congestion and hemorrhage were sometimes observed. In some cases, the branchial tissues were severely degraded and numerous bacteria were seen in the lesions. These histopathological changes are similar to those previously reported (Hedrick et al., 2000; Tu et al., 2004). Unlike a previous report (Hedrick et al., 2000), however, nuclear changes characterized by hypertrophy and margination of chromatin were rarely observed. No bacteria were isolated from the kidney of affected fish using trypticase soy agar. The PCR test for KHV revealed specific bands amplified by the methods of Gray et al. (2002) and Gilad et al. (2002)(Fig. 3). The sequence of the amplicon by the primer set of Gray et al. (2002) was identical to the sequence deposited in the GenBank with accession no. AY568951, and that with the primer set of Gilad et al. (2002) showed 99% matching to AF411803.

The Ministry of Agriculture, Forestry and Fisheries of Japan officially announced the first occurrence of KHV disease in Japan on 2 November 2003. It was also reported to the Office International des Epizooties (OIE). According to the law, the Ibaraki Prefectural Governor prohibited any shipment or removal of cultured carp from the lake and ultimately ordered that all carp cultured in the lake would be destroyed by the end of March 2004.

Evidence of the Presence of KHV before the Outbreak in Lake Kasumigaura

Independent of the outbreak in Lake Kasumigaura, a massive loss exceeding 10,000 pieces of carp occurred in some rivers and a lake in Okayama Prefecture in late May to mid-July 2003. In November 2003, the NRIA detected KHV DNA by PCR in samples of diseased fish stored in a freezer. This demonstrates that KHV had been introduced into Japan before May 2003, much earlier than the Lake Kasumigaura outbreak.

The Spread of KHV in Japan

KHV-infected common carp cultured in Lake Kasumigaura were transferred to other areas in Japan before the first detection of KHV resulting in the spread of the virus. Mortalities of carp with KHV were reported in some facilities, but there were many facilities where KHV was detected in carp without mortality. This could be attributed to the fact that the water temperature was gradually decreasing at the time of investigation. By the end of 2003, the NRIA examined 529 carp in 87 cases, and KHV was found in 23 out of 47 prefectures in Japan. Half of the KHV positive cases had no obvious relations with the Lake Kasumigaura. There was no occurrence of KHV disease during the winter period. However, as the water temperature increased in the spring of 2004, KHV reappeared in those areas where the disease was recorded in 2003, and also in new places. However, in many of the facilities that experienced KHV outbreak in 2003, the disease was not observed by June 2004. This is because in these places, all carp were removed together with other fish species, and the facilities were thoroughly disinfected after the outbreaks. There has been no occurrence of KHV disease in ornamental (koi) carp farms to date. From January to May 2004, KHV infection was reported in 24 of 47 prefectures in Japan.

Research Activity for KHV Infection at the NRIA

The NRIA and other research groups, including some universities and the Southeast Asian Fisheries Development Center (SEAFDEC), began to conduct a research project funded by the Ministry of Agriculture, Forestry and Fisheries of Japan to control KHV infection. This project will last for 3 years and consists of three major research aspects: 1) molecular virology and histopathology of KHV infection, including viral behavior in infected fish at different temperatures and at a carrier state, 2) development and evaluation of diagnostic tools such as the loop-mediated isothermal amplification (LAMP, Eiken Chemical Co.) method or immunofluorescence technique, and 3) control measures of the infection, including efficacy of disinfectants, vaccination and elevation of the rearing water temperature. The results could contribute to the control of KHV infection in both wild and cultured carp populations.

Acknowledgements

We are grateful to Prof. Dr. Ronald P. Hedrick of the University of California, Davis, USA, for his invaluable suggestions and for providing KF-1 cell line and a KHV isolate. We thank the staff of the Ibaraki Prefectural Experimental Station and Okayama Prefectural Experimental Station for providing the sample fish, and also the staff of the Aquatic Animal Health Division, National Research Institute of Aquaculture (NRIA), for running the diagnosis of KHV. This study was partially supported by a grant from the Ministry of Agriculture, Forestry and Fisheries of Japan.

References

Amita K, Oe M, Matoyama H, Yamaguchi N, Fukuda H. 2002. A survey of koi herpesvirus and carp edema virus in colorcarp cultured in Niigata Prefecture, Japan. Fish Pathol. 37:197-198.

Gilad O, Yun S, Andree KB, Adkison MA, Zlotkin A, Bercovier H, Eldar A, Hedrick RP. 2002. Initial characteristics of koi herpesvirus and development of a polymerase chain reaction assay to detect the virus in koi, Cyprinus carpio koi. Dis. Aquat. Org. 48: 101-108.

Gilad O, Yun S, Andree KB, Adkison MA, Way K, Willits NH, Bercovier H, Hedrick RP. 2003. Molecular comparison of isolates of an emerging fish pathogen, koi herpesvirus, and the effect of water temperature on mortality of infected koi. J. Gen. Viol. 84: 2661-2668.

Gray WL, Mullis L, LaPatra SE, Groff JM, Goodwin A. 2002. Detection of koi herpesvirus DNA in tissues of infected fish. J. Fish Dis. 25: 171-178.

Hedrick RP, Gilad O, Yun S, Spangenberg JV, Marty GD, Nordhausen RW, Kebus MJ, Bercovier H, Eldar A. 2000. A herpesvirus associated with mass mortality of juvenile and adult koi, a strain of common carp. J. Aquat. Anim. Health 12: 44-57.

Le Deuff R-M, Way K, Ecclestone L, Dixon PF, Betts AM, Stone DM, Gilad O, Hedrick RP. 2001. Development and comparison of techniques for the diagnosis of koi herpesvirus (KHV). Abstract of 10th International Conference of the European Association of Fish Pathologists. p. 257.

Neukirch M, Kunz U. 2001. Isolation and preliminary characterization of several viruses from koi (Cyprinus carpio) suffering gill necrosis and mortality. Bull. Eur. Ass. Fish Pathol. 21: 125-135.

Perelberg A, Smirnov M, Hutoran M, Diamant A, Bejerano Y, Kotler M. 2003. Epidemiological description of a new viral disease afflicting cultured Cyprinus carpio in Israel. Israeli J. Aquat. - Bamidgeh 55: 5-12.

Ronen A, Perelberg A, Abramowitz J, Hutoran M, Tinman S, Bejerano I, Steinitz M, Kotler M. 2003. Efficient vaccine against the virus causing a lethal disease in cultured Cyprinus carpio. Vaccine 21: 4677-4684.

Sunarto A, Taukhid, Rukyani A, Koesharyani I, Supriyadi H, Huminto H, Agungpriyono DR, Pasaribu FH, Widodo, Herdikiawan D, Rukmono D. 2002. Field investigations on a serious disease outbreak among koi and common carp (Cyprinus carpio) in Indonesia. Paper presented in the 5th Symposium on Diseases in Asian Aquaculture, 24-28 November 2002, Gold Coast, Australia. p. 106.

Tu C, Weng M-C, Shiau J-R, Lin S-Y. 2004. Detection of koi herpesvirus in koi Cyprinus carpio in Taiwan. Fish Pathol. 39: 109-110.

Current Status of Koi Herpesvirus Disease in Taiwan

Chien Tu¹, Shih-Yuh Lin¹ and Hwa-Tsung Sung²

¹National Animal Health Research Institute, Council of Agriculture
376 Chung-Cheng Rd., Tamsui 251, Taiwan ROC
²Bureau of Animal and Plant Health Inspection and Quarantine, Council of
Agriculture, 51 Sec. 2 Chung-Ching South Rd., Taipei 100, Taiwan ROC

Abstract

The first reported case of koi herpesvirus disease (KHVD) occurred in northern Taiwan in December 2002. Later, there were three more cases in 2003 and one outbreak of KHVD in 2004. Externally, the affected fish did not show any prominent lesions except swollen gills sometimes accompanied by bleeding. Consistent histopathological findings were in the gill tissues, where hyperplasic epithelia and eosinophilic granular cells were observed within fused secondary lamellae. Electron microscopy revealed negativelystained icosahedral viral nucleocapsids measuring 112±1 nm in diameter. Also, the koi herpesvirus was detected in the homogenate of diseased fish by PCR assay using specific primers for koi herpesvirus (KHV). The amplicon was cloned, sequenced and compared with previously published data. The sequenced data showed 99% identity with the American KHV sequence in the GenBank. The above evidence suggests that KHVD have already invaded carp culture systems in Taiwan.

The First Case of KHVD in 2002

The first outbreak of KHVD occurred in a private carp pond in Taipei County in northern Taiwan. On December 6, 2002, two 2-year-old colored carp were submitted for examination by a private hobbyist to our research facility. The owner had about 30 koi, averaging in age between 2 to 3 years, which were reared in two different ponds. He had bought several of the koi from farmers at Taoyuan County in northern Taiwan about one month before the occurrence. The water temperature in both ponds was approximately 20-22⁰C when the disease outbreak occurred. At first, the affected fish appeared lethargic and lacked appetite for several days before death. The owner observed a light-reddish discoloration of the pond water so that he decided to submit his fish for examination. Upon necropsy, congestion was observed at the base of the fins and tail, and the gills were swollen. Bleeding from the gill was observed in one fish. There were no other lesions found in the internal organs. For histological examination, tissues were fixed in 10% neutral-buffered formalin for 24 hours, embedded in paraffin, sectioned and stained with hematoxylin and eosin using all routine techniques. Histopathology of the gills showed hyperplasia and fusion of the secondary lamellae in the diseased fish. Necrotic epithelial cells accompanied by some eosinophilic granular cells were noted among the hyperplasic gill epithelia. Samples for bacterial examination of the liver, spleen and kidney were obtained by stabbing the organs with a sterile loop, inoculating samples onto blood agar (5% sheep red blood cell), then incubating them at 25⁰C for 48 hours. No bacterial growth was obtained from these samples. For viral isolation, the tissues (spleen, kidney and gill) of two diseased fish were homogenized with 10´PBS and centrifuged at 1500´g for 15 min. The supernatants were filtered through a 0.45µ pore-size filter and inoculated onto a monolayer of FHM, EPC, and BF-2 cell lines at 25⁰C; CHSE-24 and RTG-2 cell lines at 20⁰C, and observed for 14 days. There was no CPE in the inoculated cell lines after one blind passage. For electron microscopy, a herpeslike viral particle was found in the negatively-stained samples. For PCR assay, specific amplicons were produced using specific primers designed by Gilad et al. (2002) and Gray et al. (2002). The amplicons were cloned, sequenced and compared with all the data submitted to GenBank. Our sequenced result had 99% identity with that of American KHV in Genbank. KHV was diagnosed as the etiological agent of this outbreak. As soon as KHVD was confirmed in the National Animal Research Institute, the Taipei County Livestock Disease Control Center and the Bureau of Animal and Plant Health Inspection and Quarantine (BAPHIQ) were notified of the results. These facilities are responsible for controlling the spread of newly emerging exotic animal diseases. Upon receiving our notice BAPHIQ sent an official document to all local County Livestock Disease Control Centers requesting the centers to investigate the current status of carp cultures in their respective counties. The public veterinarians began an investigation and discovered no new disease outbreaks among cultured carp in Taiwan. In addition, upon advise of the owner of the diseased fish, we also visited the suspected farm at Taoyuan County as the possible source of infection. Our investigation was unsuccessful because the owner of the fish farm had already closed his farm and fled the premises. Therefore, the source of the KHVD introduction to Taiwan is still unknown.

The First Case of KHVD in 2003

The first occurrence of KHVD in 2003 was at a private koi pond in Taipei. The case was transferred from the Taipei Animal Health Inspection Center to our laboratory. The total number of cultured koi in this incident was 20. Clinically, the affected fish were observed to be very sluggish and after 7-10 days, death occurred. Upon necropsy, only swelling of the gills was observed. Similar histopathological examinations and PCR assays were performed in this case, and results obtained were the same with findings in the 2002 case. Therefore, this case was also diagnosed as KHVD. All fish were eradicated and buried. As in the previous case, the origin of infection remains unknown.

The Second Case of KHVD in 2003

The second case of KHVD also occurred in Taipei in 2003. The koi were reared in an artificial lake together with tilapia in a public park. The lake had approximately 300 koi with no recent introduction of new fish into the park ponds. The tilapia remained unaffected and had no deaths throughout the koi herpesvirus outbreak. The affected koi (2-3 years old) were lethargic and were floating near the water surface before death. Both dead and moribund fish were sent to our laboratory by the Taipei City Animal Health Inspection Center. The gross lesions were similar to those in the previous two outbreaks. The series of examinations were performed at our laboratory as previously described. Similar histopathological changes and PCR results were also obtained in this case. Since the park is open to the public for 24 hours, it is very easy to gain access into the artificial lake. Because park officials have not introduced new koi, it was speculated that the outbreak of KHVD might have resulted from unauthorized release of diseased fish into the lake by unknown persons. All the fish in this case were destroyed.

The Third Case of KHVD in 2003

The third outbreak of KHVD in 2003 also occurred in Taipei. Seven hundred 2-3 year old koi that were reared in a lake located at a public memorial hall became infected. Similar pathological changes and PCR results were found during examinations. It was suggested that all the fish be culled to prevent further spread of the disease. Since the public has access to the pond for 24 hours, the origin of this outbreak of KHVD is probably caused by the unintentional (unexpected) introduction of diseased fish by unknown park visitors.

The First Case of KHVD in 2004

The first outbreak of KHVD in 2004 occurred in a suburban area in Taipei County. The owner is a farmer who hatches, raises and sells the koi. The farmer’s koi hatchery is located in southern Taiwan. After hatching, the fries are grown to juvenile, moved to the grow-out farm, and later sold in northern Taiwan. Water for the grow-out ponds comes from a spring. The water temperature was 22-23⁰C when the disease outbreak occurred. The diseased fish did not show any prominent clinical signs or gross lesions during the visual examinations. The histopathological and PCR results were identical with the findings in all previous cases. All the affected fish (about 1000 pieces) were destroyed. After disinfecting the rearing water with chlorine, it was drained and the ponds were left empty for 2 months before being re-stocked with new fish. This case may have been caused by the owner’s acceptance of unhealthy koi returned by his customers.

Diagnosis and Control System of Exotic Aquatic Animal Diseases in Taiwan

The diagnostic system for aquatic animal diseases is a collaboration of the central government and the local county. In the central government, the Animal Health Research Institute (AHRI) of the Council of Agriculture is responsible for the final diagnosis of exotic aquatic animal diseases. In the AHRI, routine monitoring program for animal diseases is in place and it also receives suspected specimens submitted from all over Taiwan. In the local county, the aquatic health section of the county Livestock Disease Control Center (LDCC) is responsible for routine aquatic animal disease diagnosis and control. If a disease is suspected to be caused by a new and emerging disease agent, it will be referred to the AHRI for final confirmation. Furthermore, aside from receiving suspected specimens from local LDCC, AHRI can also accept specimens submitted directly by private individual for diagnosis. Regarding the control system for exotic aquatic animal diseases, the BAPHIQ in the central government is in charge of the control plan, including confinement, eradication and compensation related to the exotic aquatic animal disease outbreaks. In the local county, the LDCC executes the control plan
determined by BAPHIQ.

Spread of KHV in Taiwan

According to our official data, the outbreaks of KHV are found in Taipei and Taoyuan County in northern Taiwan only.

Research on KHV in Taiwan

There are on-going studies in the development of cell lines for viral isolation, development of rapid diagnostic tool, research on viral pathogenesis in the molecular level, and the development of a vaccine against KHVD at AHRI and other universities in Taiwan.

References

Gray WL, Mullis L, LaPatra SE, Groff JM, Goodwin A. 2002. Detection of koi herpesvirus DNA in tissues of infected fish. J. Fish Dis. 25: 171-178.

The Role of Quarantine in Preventing the Spread of Serious Pathogens of Aquatic Animals in Southeast Asia

J. Richard Arthur

P.O. Box 1216
Barriere, British Columbia
Canada V0E 1E0

Abstract

Quarantine, in the strict sense, is the confinement of aquatic animals of unknown or questionable health status in secure facilities such that neither they nor any pathogens they may be carrying can escape into the external environment. During the period of quarantine, the animals are observed, tested, and treatment may be applied, and a decision will be made as to whether or not they should be released to the external environment. While the concept of quarantine for aquatic animals has existed for many years, within the current framework of “national biosecurity”, quarantine is seen as one of a number of risk mitigation options that governments can apply to reduce the likelihood of serious pathogens being introduced with the importation of live aquatic animals and their products. Although the concept of quarantine is relatively simple, its effective implementation may be complex, due to the need for specialized infrastructure, capability and expertise. Several Southeast Asian countries have considered or attempted to implement border quarantine for live aquatic animals; however, these efforts have met with little success. This has been due to a number of reasons, including failure to carefully define the scope and purpose of quarantine within a national aquatic animal health program, the diversity of forms in which trade occurs, the sheer volume of commodity traded, the lack of simple and accurate diagnostics tests for some pathogens, and the limited capital and human resources that governments are able to commit to this effort. To improve this situation, risk analysis can be used to determine whether or not the importation of a given commodity (living aquatic animal or its product) poses an unacceptable disease risk to national biosecurity. In those cases where an unacceptably high level of risk exists, possible risk mitigation measures can then be examined to determine what actions, if any, can be applied to reduce the risk to within the country’s appropriate level of protection (ALOP). In this way, quarantine, as one of a suite of possible risk reduction measures, can be applied effectively on a case-by-case basis to reduce the risk of introduction, establishment and spread of serious aquatic animal pathogens into new areas.

Introduction

What is Quarantine?

Quarantine has been defined in a number of ways. The International Aquatic Animal Health Code of the Office International des Épizooties (OIE, the World Animal Health Organization) defines the term “quarantine” as: “Maintaining a group of aquatic animals in isolation with no direct or indirect contact with other aquatic animals, in order to undergo observation for a specified length of time and, if appropriate, testing and treatment, including proper treatment
of effluent waters.” (OIE 2003). A similar but slightly different definition was used by the Food and Agriculture Organization of the United Nations (FAO) and the Network of Aquaculture Centres in Asia-Pacific (NACA) during the recent regional Technical Cooperation Project “Assistance for the responsible movement of live aquatic animals” (FAO/NACA TCP RAS 6714(A) and 9605(A) (FAO/NACA 2000): “Holding or rearing of aquatic animals under conditions which prevent their escape, and the escape of any pathogens they may be carrying, into the surrounding environment. This usually involves sterilisation/disinfection of all effluent and quarantine materials.” In contrast, in Australia, the legal basis for import biosecurity, the Quarantine Act (1908), defines “quarantine” with a wide scope, to include pre-border (e.g., health certification), border (e.g., quarantine sensu stricto) and post-border (e.g., monitoring and surveillance) activities. Thus the operational agency, the Australian Quarantine and Inspection Service (AQIS), uses the term “quarantine” in a very wide sense (see Bernoth 1998). Biosecurity Australia, however, generally considers the terms “biosecurity” and “quarantine” to be equivalent when quarantine is used in the sense that it has in the Quarantine Act (i.e., in the broadest sense). Thus in legal situations, Biosecurity Australia uses the word “quarantine”, while in other situations “quarantine” is avoided because it is confusing to people from outside Australia, who generally consider that it means a period of mandatory detention (Peter Beers, pers. comm.). In recent Australian risk analyses for aquatic animals, the term “quarantine measures” is used in the sense that other countries use the term “quarantine.” In this paper, “quarantine” will be discussed using the concept of mandatory detention as applied by OIE and FAO/NACA.

The Purpose of Quarantine

The primary purpose of quarantine is to minimize the risk of introducing infectious agents (pathogens) into the national territory of the importing country and their escape and spread to susceptible species. The secondary purpose is to prevent the entry of aquatic organisms that have not been approved for introduction.

Attempts to Establish National Quarantine Programs in Southeast Asia

The international spread of serious pathogens of aquatic animals has been a concern to Southeast Asian countries for several decades (see Davy and Graham 1979, Davy and Chouinard 1983, Shariff 1987, Arthur and Shariff 1991, Arthur 1995). With the support of donor agencies such as the International Development Research Centre (Canada), the United States Agency for International Development (USAID), and the British Overseas Development Agency (ODA, now the Department for International Development (DFID), several Southeast Asian countries began to establish quarantine and/or health certification procedures for aquatic animals in the late 1970s, and at least two (Indonesia and Malaysia) have devoted considerable national resources and effort to training quarantine officers and establishing quarantine holding facilities and supporting diagnostic laboratories. As the current status of these national efforts will be reviewed during the individual country presentations, I will not discuss these national efforts in more detail. However, I would like to explore briefly why past quarantine efforts have not been effective in preventing the international spread of serious pathogens of aquatic animals, and how the concept of “risk” and the use of “risk analysis” can lead to the application of quarantine in more effective and cost efficient ways.

Why Have Southeast Asian Countries Had Difficulties in Implementing Quarantine?

Although the concept of quarantine is relatively simple, its effective implementation can be complex, due to the need for specialized infrastructure, capability and expertise. The efforts of countries such as Indonesia in Malaysia in attempting to implement quarantine for aquatic animals are laudable, and have certainly increased national capacity to diagnose diseases of aquatic animals and provided much basic infrastructure and expertise. However, it must be admitted that these efforts have not been as effective in preventing the entry of serious exotic diseases of fish, shellfish and molluscs as hoped. There is ample documentation of the inability of national governments of Southeast Asian countries to prevent the spread of exotic pathogens, such as epizootic ulcerative syndrome (EUS) of freshwater fish, white spot syndrome virus (WSSV) of penaeid shrimp, and more recently, koi herpes virus (KHV) of koi and common carp and Taura syndrome virus of penaeid shrimp, which are discussed elsewhere in this volume. The inability to prevent the entry and spread of exotic diseases has been due to a number of reasons, including: most importantly, the lag time between when a new disease emerges, when it is first recognized as a serious pathogen of international importance, and when accurate and reliable diagnostics tests are developed and become generally available;

the diversity of forms in which trade occurs;
the sheer volume of commodity traded;
the lack of simple and accurate diagnostics tests for some pathogens (e.g., white tail disease of Macrobrachium); and
the limited capital and human resources that governments are able to commit to this effort.

It must also be admitted that while various multinational and bilateral donor agencies have promoted the value of establishing quarantine programs to national governments in Southeast Asia, there has, until quite recently, been little technical guidance to assist governments in designing effective policy and approaches to aquatic animal disease control. Thus in the past, national governments have had difficulty defining the scope and purpose of quarantine within national aquatic animal health programs.

The Role of Quarantine in National and Regional Biosecurity

In the past, quarantine has often been seen as a separate activity, and as a procedure that should be applied to all imports of living aquatic animals, often with the unrealistic goal of “zero risk” of disease entry to the importing country. This thinking has changed considerably in the past 10 years, so that national governments are increasingly viewing quarantine as one component of a national aquatic animal health strategy. In Southeast Asia, the components of such a national program have been defined through a regional FAO/NACA TCP project that has the support of 21 countries in the Asia-Pacific and a number of international agencies. One of the major outputs of this program was the “Asia Regional Technical Guidelines on Health Management for the Responsible Movement of Live Aquatic Animals and the Beijing Consensus and Implementation strategy” (FAO/NACA 2000). These guidelines, which outline an agreed-upon general approach and framework that countries in the Asia-Pacific should use in developing and implementing national programs to reduce the risk of pathogen transfers with live aquatic animals and their products, has been officially adopted as a policy document by the Association of South East Asian Nations (ASEAN). The guidelines act as a platform for greater cooperation and implementation of aquatic animal health management measures within the region and will be utilized in a wider context to support the development of sustainable aquaculture in ASEAN (see http:// www.aseansec.org/13553.htm).
The components of a national strategy for aquatic animal health are shown in Box 1. It can be noted that health certification and quarantine measures are key components that countries should consider when developing a national aquatic animal health strategy. In cases where a risk assessment has determined that the level of risk associated with trade in a commodity exceeds the appropriate level of protection (ALOP) of the importing country, the importing country can then consider ways to reduce the risk to an acceptable level. The possible options for risk management will vary depending on the nature of the commodity and the individual hazard. Quarantine is one of the options that may be applied (Box 2). Note that during the risk analysis, the management options for each hazard (pathogen) must be carefully evaluated as to their likely effectiveness, and the risk presented by the hazard reassessed based on the expected results. Figure 1 shows a summary of possible risk management steps recommended by the risk assessment for hypothetical movement of live cultured juvenile fish between two countries. In this scheme initial screening for viruses, external lesions and parasites is conducted in the exporting country. Fish that pass this initial inspection are then exported and upon arrival in the importing country, they are placed in quarantine, where they are held for further observation and tested for disease. Only batches of fish that have shown no evidence of disease are released from quarantine. It is important to note that this is a working procedure for routine importation of juvenile fish, not a procedure for the introduction of an exotic species. Previous experiences with the supplier, and a good knowledge of the history of the stock and of the capabilities of the Competent Authority in the exporting country will also increase confidence in the health status of the imported animals.

The Basic Requirements of Effective Quarantine

The basic requirements for effective quarantine include:

Adequate physical infrastructure appropriate to the level of containment required (secure facilities, secure intake water source, etc.);
Established operating protocols (including chain of custody); and
Well-trained staff.

Detailed information on the requirements for setting up and operating quarantine facilities for exotic species and for routine ornamental fish trade are given by MAFF (2001), AQIS (2003) and Arthur (2003).

The necessary supporting services for quarantine include:

Adequate legislation;
Effective enforcement (e.g., border customs and inspection, postborder follow up);
Knowledgeable and supportive aquaculture industry;
Sufficient political will;
Competent and readily available diagnostics support;
Existence of reliable diagnostics tests for major pathogens;
Good working relationships between importing and exporting country
Competent Authorities;
Good knowledge base of pathogens present in importing country (surveillance and monitoring, disease surveys); and
Good information base on pathogen biology, prevention, treatment, etc.

Conclusions

Consideration of quarantine is a fundamental activity when setting up a National Aquatic Animal Health Strategy. Quarantine may be highly important to some countries having significant aquaculture, capture fisheries and/or natural biodiversity. In other cases, national situations may make quarantine a low priority or an unnecessary activity. For most country situations, quarantine need not be applied generally. Whether or not to require quarantine should be determined based on the results of a risk analysis for each commodity or situation. In some cases, risk analysis may show that quarantine of a given commodity is not required to achieve the national ALOP, while in others, less costly and/or less restrictive measures may be equally effective. The responsibility for establishing a quarantine facility (e.g., whether private sector or government), the place of quarantine (preborder or border) and the stringency of quarantine (level of security, duration, testing, etc.) should also be decided on a case by case basis based on the nature of the importation. Importations of exotic species for aquaculture development, because of the high likelihood of introducing serious pathogens and the extensive economic, biological and social damage such pathogens may cause, will require more stringent quarantine measures than, for example, routine importations of strictly ornamental species.

References

Alimon SB, Vijiarungam AF, Ming TT, Yahya MTBM, Shariff M. 1983. Country Report. Malaysia. p. 31-34. In: Davy FB, Chouinard A. (eds) Fish Quarantine and Fish Diseases in Southeast Asia. Report of a workshop held in Jakarta, Indonesia, 7-10 December 1982. Intern. Developm. Res. Centre Publ. IDRC-210e, Ottawa.

AQIS. 2003. Quarantine premises criteria. 7.1 Fresh water and marine ornamental fin fish. Quarantine Premises Register, Class Criteria. Class 7.1, 22/06/2003, 6 p.

Arthur JR (ed). 1987. Fish quarantine and fish diseases in South and Southeast Asia: 1986 update. Report of the Asian Fish Health Network Workshop held in Manila, The Philippines, 30 May 1986. Asian Fish. Soc. Spec. Publ. No. 1, 86 p.

Arthur JR. 1995. Efforts to prevent the international spread of diseases of aquatic organisms, with emphasis on the Southeast Asian region. p. 9-25. In: Shariff M, Arthur JR and Subasinghe RP (eds) Diseases in Asian Aquaculture II. Asian Fish. Soc., Fish Health Sect., Manila.

Arthur JR. 2003. Draft Aquaculture (Import and Export) Regulations and Annexures. Project TCP/NAM/0168(A) Assistance in Establishing a Legal Framework for Responsible Aquaculture Development, Food and Agriculture Organization of the United Nations, Rome, 99 p.

Arthur JR, Bondad-Reantaso M, Baldock FC, Rodgers CJ, Edgerton BF. 2004. Manual on Risk Analysis for the Safe Movement of Aquatic Animals (FWG/01/2002). APEC/DoF/NACA/FAO, 59 p. APEC Publ. No. APEC #203-FS-03.1.

Arthur JR, Shariff M. 1991. Towards international fish disease control in Southeast Asia. Infofish Intern. 3/91: 45-48.

Bernoth E-M. 1998. Australia. p. 10-11. In: First Training Workshop of the FAO/NACA/OIE. Regional Programme for the Development of Technical Guidelines on Quarantine and Health Certification, and Establishment of Information Systems, for the Responsible Movement of Live Aquatic Animals in Asia, Bangkok, Thailand, 16-20 January 1998. Food and Agriculture Organization of the United Nations, Network of Aquaculture Centres in Asia-Pacific, and Office International des Epizooties, Bangkok, TCP/RAS/6714, Field Doc. No. 1.

Davy FB, Graham M (eds). 1979. Diseases of fish cultured for food in Southeast Asia. Report of a workshop held in Cisarua, Bogor, Indonesia, 28 November - 1 December 1978, Intern. Develop. Res. Centre Publ. IDRC-139e, 32 p.

FAO/NACA. 2000. Asia regional technical guidelines on health management for the responsible movement of live aquatic animals and the Beijing consensus and implementation strategy. FAO Fisheries Technical Paper 402, 53 p.

FAO/NACA. 2001. Manual of procedures for the implementation of the Asia Regional Technical Guidelines on Health Management for the Responsible Movement of Live Aquatic Animals. FAO Fisheries Technical Paper 402/1,106 p.

AFF. 2001. Transitional facilities for ornamental fish and marine invertebrates. MAF Biosecurity Authority, Animal Biodiversity, Standard 154.02.06, Ministry of Agriculture and Forestry, Wellington, 23 March 2001, 30 p.

OIE. 2003. International Aquatic Animal Health Code. 6th edn. Office International des Épizooties, Paris. (http://www.oie.int/eng/normes/fcode/a_summry.htm)

Shariff M. 1987. Current status of programs for fish health certification and quarantine systems. p. 48-52. In: Arthur JR (ed) Fish quarantine and fish diseases in South and Southeast Asia: 1986 update. Report of the Asian Fish Health Network Workshop held in Manila, The Philippines, 30 May 1986. Asian Fish. Soc. Spec. Publ. No. 1.

The Role of the Office International des Epizooties (OIE) in Health Improvement of Aquatic Animals

Yoshiyuki Oketani
OIE Regional Representation for Asia and the Pacific
Japan

History of OIE in Aquatic Animal Health

The World Organisation for Animal Health (OIE) is an inter-governmental organization that was established in 1924 in order to promote world animal health. OIE missions that have become increasingly important and its mandate that has been expanded to meet requirements from the world are strongly supported by the Member Countries now reaching 167. OIE Regional Offices have been established in Tokyo, Buenos Aires, Beirut, Sofia and Bamako covering Asia and the Pacific, the Americas, the Middle East, Eastern Europe and Africa.

The main objectives of the OIE are:
1) To ensure transparency in the global animal disease situation;
2) To collect, analysis and disseminate scientific veterinary information;
3) To contribute expertise and encourage international solidarity in the control of animal diseases;
4) Within its mandate under the WTO SPS Agreement, to safeguard world trade by publishing health standards for international trade in animals and animal products;
5) To improve the legal framework and resources of Veterinary Services; and
6) To provide a better guarantee of the safety of food of animal origin and to promote animal welfare through a science-based approach.

Previously, aquatic animals were included in the category of terrestrial animals. The initial body of the current OIE Aquatic Animal Standards Commission was established in 1960 under the name of “Commission for the Study of Diseases of Fishes” and since then the Commission has organized scientific symposia regularly (in Turin in 1962, in Munich in 1965, in Stockholm in 1968, and in Paris in 1991, 1995 and 2000). The symposia have been held under the auspices of the OIE on the most important aspects of aquatic animal health, notably on fish diseases and their control. In 2000, OIE published for the first time the Aquatic Animal Health Code and the Manual separately from those for terrestrial animals. Diagnostic procedures for some aquatic animal diseases used to be included in the OIE International Animal Health Code starting from the 1986 edition, but it became clear that separate publications specific to Aquatic Animal Health would be needed for reasons that the conditions, problems and requirements in this field are different from those encountered in other animals, and that international trade in aquatic animals and their products has become increasingly important.

Aquatic Animal Health Standards Commission

Presently, the OIE Aquatic Animal Health Standards Commission is in operation in Paris as one of the Specialist Commissions. This Commission, which consists of five elected members (presently from Australia, the United Kingdom, Chili, France and Uganda) experienced in methods for surveillance, diagnosis, control and prevention of infectious aquatic animal diseases, meets twice yearly to address its work program. The Aquatic Animal Health Standards Commission also collaborates closely with the OIE Terrestrial Animal Health Standards Commission on issues requiring harmonized approach, and with the Biological Standards Commission and the Scientific Commission to ensure that the Aquatic Animal Health Standards Commission is using the latest scientific information in its work.

Disease Notification
Once an infectious animal disease occurs in a country, many people including producers, government officials, policy makers, exporters, importers and consumers require accurate information as quickly as possible. The OIE has worked to secure transparency in the global animal disease situation, and collection, analysis and dissemination of scientific information. To accomplish this purpose, OIE Member Countries have obligations for disease notification, by fax or e-mail, within 24 hours, of the suspected or confirmed first occurrence or re-occurrence of the OIE list A diseases. Every
Member Country of the OIE recognizes the right of the OIE to communicate directly with the Veterinary Administration of its territory. In 2003, 61 emergency alert messages from 46 Member Countries were sent by e-mail to the Delegates of Member Countries, OIE Regional Representatives and other interested international organizations, or by fax to Member Countries without an e-mail address. This information was also disseminated via the OIE public access Web-site, the messages being systematically published in the “Alert Messages” section, and through the open access “OIE Info” mailing list.
When a serious infectious disease occurs, sometimes various rumors circulate and unconfirmed reports are accepted as facts. Therefore, people ask for official information provided by responsible source. OIE receives official disease notification with the signature of the OIE Delegate directly from the relevant Member Country. Furthermore, OIE collects animal health information based on the active search policy, which is enhanced by exchanges of relevant information with OIE Reference Laboratories, Collaborating Centres and other international organizations. The information thus collected, including nonofficial information, is systematically evaluated before deciding whether to verify it with the Delegate of the relevant Member Country. OIE information is recognized and used as official information in the world. Therefore, during the Avian Influenza crisis, more than 40,000 people visited the OIE Web-site in only one week. The OIE collaborates with the Food and Agriculture Organization of the United Nations (FAO), and the World Health Organization (WHO) with the design of a joint global early warning system. The OIE world animal health situation, lists of disease free countries, etc. are commonly used in Web-sites of these organizations as official information.

Disease Free Status

Official reporting is made by a Member Country not only when an infectious disease breaks out, but also when the disease is eradicated. If the country or a zone in the country was previously considered to be free from the particular disease, the Delegate will, with necessary documents including results of monitoring and surveillance, apply to the OIE to declare freedom from the disease. So far, to meet the demand of the Member Countries, the OIE has been
publishing the “Disease Free Country List” for Foot and Mouth Disease, Rinderpest, Bovine Pleuropneumonia, and now for Bovine Spongiform Encephalopathy (BSE). In recent years, many Member Countries submitted formal applications for BSE free status to the Director General of the OIE. Those applications were evaluated by an ad hoc group of experts on BSE that has been set up, and also by the Scientific Commission. During the 72nd OIE General Session held in May 2004, the Director General established a list of BSE provisionally-free countries or zones in accordance with the chapters of the Terrestrial Code. The first countries listed as provisionally-free countries included Argentina, Uruguay, Iceland and Singapore.

Surveillance and Monitoring

So far, only OIE’s procedures about “disease notification” and “recognition of disease free status” using examples of some terrestrial animal diseases have been introduced. In both procedures, the official information as the result of monitoring and surveillance in accordance with the OIE Code and Manual are indispensable. Then, what kind of surveillance and monitoring are needed? According to the OIE definitions: “Surveillance” means the continuous investigation of a given population to detect the occurrence of disease for control purposes, which may involve testing of a part of population; and “Monitoring” constitutes on-going program directed at the detection of changes in the prevalence of disease in a given population and in its environment. The ability of the animal health authorities to substantiate elements of the reports on the animal health situation in their country by surveillance data, results of monitoring program and details of disease history is highly relevant to the procedures of risk analysis. The science of epidemiology provides the foundation for surveillance and monitoring. A national epidemiological system should incorporate agent surveillance and monitoring, description of host population characteristics, and environmental assessment. An effective infrastructure is necessary to support this epidemiological system. Agent surveillance and monitoring may involve the clinical pathological examination of animals, the identification of pathogens, and the detection of immunological or other evidence of previous exposure of animals to pathogen. The first step is early investigation of clinical diseases. Investigating the suspicious cases of animal diseases is one of the most important means of agent surveillance. Investigation may focus on exotic or new and emerging diseases within the country. The next step is detection of agent and confirmation of disease prevalence. A complete epidemiological system may also require the screening of animals for OIE listed diseases having the major economic impact on trade in animals and animal products, as appropriate to the animal health situation of the country. Now, the Asia-Pacific region is producing approximately 79% in value and 88% in volume of aquaculture worldwide. Nevertheless, the aquatic animal sector in the region is not as well provided with professional health services. It appears that, while aquaculture has been growing rapidly in many countries, there has been no matching expansion of the supporting aquatic animal health infrastructure. However, there is a relatively good coverage of aquatic animal health at international conferences, hands-on trainings, seminars and timely symposia on particular diseases organized by SEAFDEC, OIE , FAO and other organizations.
Recently, the following Conferences and Training course were held:

OIE International Conference on Risk Analysis in Aquatic Animal Health, held in Paris in 2000;
SEAFDEC Hands-on Training for Important Viral Diseases of Shrimps and Marine Fish, held in Iloilo, 2002 and 2003; and
SEAFDEC/OIE/FRA/MAFF Japan International Symposium on Koi Herpesvirus Disease, held in Yokohama in 2004.

Reference Laboratory

With the aim to diagnose each OIE listed diseases, 15 fish disease institutes, 4 mollusc disease institutes and 2 crustacean disease institutes have been given the qualification as OIE Reference Laboratories in the world. The list includes 3 fish disease institutes located in the Asian region.

OIE Aquatic Code

The International Aquatic Animal Health Code contains health recommendations relative to international trade in aquatic animals and aquatic animal products. The practical application of OIE recommendations relating to international trade requires, in particular, the importing country to conduct risk analysis, preferably in liaison with the exporting country. The aim of the Aquatic Animal Health Code is to assure the sanitary safety of international trade in aquatic animals (fish, molluscs and crustaceans) and their products. This is achieved through the detailing of health measures to be used by the Veterinary Administrations and Competent Authorities in the importing and exporting countries to avoid the transfer of agents pathogenic for animals or humans, while avoiding unjustified sanitary barriers. The health measures in the Aquatic Code (in the form of standards, guidelines and recommendations) have been formally adopted by the OIE International Committee and the General Assembly of all Delegates of OIE Member Countries, which constitute the organization’s highest decision-making body. The development of these standards, guidelines and recommendations is the result of the continuous work of the OIE Aquatic Animal Health Standard Commission. This Commission draws upon the expertise of internationally renowned specialists to prepare draft texts for new chapters of the Aquatic Animal Health Code or revise existing chapters in light of advances in veterinary science. The views of the Delegates of Member Countries are systematically sought through the circulation of draft and revised texts. The value of the Aquatic Animal Health Code lies in the fact that measures published in it are the result of consensus among the OIE Member Countries.

OIE Aquatic Manual

The purpose of the Manual of Diagnostic Tests for Aquatic Animals is to provide a uniform approach to diagnosis of diseases listed in the OIE Aquatic Animal Health Code so that the requirements for health certification, in connection with trade in aquatic animals and their products, can be met. Also many publications exist on the diagnosis and control of aquatic animal diseases. The Aquatic Manual is a key document describing the methods that can be applied to the OIE listed diseases in aquatic animal health laboratories all over the world, thus increasing efficiency and promoting improvements in aquatic animal health worldwide.

OIE as an International Standard Setting Organization

The World Trade Organization (WTO) Agreement on the Application of Sanitary and Phytosanitary Measures (SPS Agreement) conferred on the OIE new responsibilities under international law by specifying “the standards, guidelines and recommendations developed under the auspices of the OIE” as the international standards for animal health and zoonoses. The SPS Agreement is aimed at establishing a multilateral framework of rules and disciplines to guide development, adoption and enforcement of sanitary measures in order to minimize their negative effects on international trade. Guidelines for conducting risk analyses are described in the Aquatic Animal Health Code. The Code thus forms an integral part of the regulatory reference system established by the WTO.

Conclusion

Effective realization of disease control needs to be based on accurate disease information resulting from well-designed surveillance and monitoring schemes. Especially with reference to trans-boundary animal diseases, a single country could not necessarily accomplish to prevent and control of such diseases. In order to carefully plan surveillance and monitoring schemes which would be acceptable worldwide, the OIE Aquatic Code and Manual should be referred to as the global standards. The OIE Regional Representation based in Tokyo will work with the Member Countries, SEAFDEC and relevant organizations for aquatic animal disease control.

Research and Training on Fish Diseases at the SEAFDEC Aquaculture Department in 2000-2004: A Review

Kazuya Nagasawa
Southeast Asian Fisheries Development Center, Aquaculture Department
Tigbauan 5021, Iloilo, Philippines

Abstract

This paper reviews various research and training activities on fish diseases at the Aquaculture Department of the Southeast Asian Fisheries Development Center (SEAFDEC) in Iloilo, Philippines. The activities were implemented through the “Regional Fish Disease Project” of the Government of Japan Trust Fund starting in March 2000. A total of 29 research studies were conducted from 2000-2004 in the following aspects: (1) establishment and standardization of diagnostic methods; (2) biology and pathogenesis of disease pathogens; (3) disease prevention and control; (4) establishment of evaluation methods for residual chemicals in aquaculture products; and (5) epizootiology and prevention of koi herpesvirus disease. Some of these studies were conducted by scientists from the Department of Fisheries in Thailand, and from the Marine Fisheries Research Department (MFRD) of SEAFDEC in Singapore. Two sessions of hands-on training on “Important Viral Diseases of Shrimp and Marine Fish” was implemented in 2002 and 2003. Participants from the SEAFDEC member countries were funded by the project to attend the training course. The course consisted of both lecture and practical hands-on sessions. The latter focused on the use of molecular tools and other important techniques in the diagnosis of viral diseases of shrimp and marine fish. This review also provides information on publications such as proceedings, manuals, review articles, scientific papers, terminal report, annual reports, flyers, pamphlets and others as the outputs of research activities and international meetings that were organized with financial support from the project.

Introduction

Fish disease is a major constraint and threat to aquaculture production in Southeast Asia. Numerous infections diseases have been reported from fish and shrimp cultured in this region. Currently, several new diseases have emerged in the region. These diseases cause mass mortality of cultured species, resulting in devastating losses to the regional aquaculture production. Various chemicals including antibiotics, pesticides, disinfectants and others are often used to control fish diseases in the region. There is, however, a need to ensure that aquaculture products are safe for humans since the presence of chemical residues can negatively affect international trade of the products. Since the year 2000, the “Regional Fish Disease Project” has been implemented at the Aquaculture Department of the Southeast Asian Fisheries Development Center (SEAFDEC) in Tigbauan, Iloilo, Philippines to address various regional fish disease problems and food safety issues through the Government of Japan (GOJ) Trust Fund. The first phase of the project entitled “Development of Fish Disease Inspection Methodologies for Artificially- Bred Seeds” started in 2000 and will end in 2004. It was initially planned to end in 2003, but was extended to 2004 because of the urgent need to study an emerging viral disease of common carp and koi (Cyprinus carpio)(= koi herpesvirus [KHV] disease), which was reported in Indonesia and Taiwan in 2002 and Japan in 2003. After this 5-year project, the second phase of the Regional Fish Disease Project entitled “Development of Fish Disease Surveillance System” has been proposed for another 5 years duration (2004 to 2008). The project is being conducted as one of the collaborative projects of the ASEAN-SEAFDEC Fisheries Consultative Group (FCG). This paper reviews the activities under the Regional Fish Disease Project from 2000 to 2004, focusing on research and training conducted at the SEAFDEC Aquaculture Department.

Objectives and Activities of the Regional Fish Disease Project

The Regional Fish Disease Project supported by the GOJ Trust Fund aims to: (1) assist the health development in aquaculture in Southeast Asia; (2) promote the healthy and wholesome trading of aquaculture products in the region; and (3) develop a fish disease surveillance network in the region. To achieve these objectives, the project conducted the following activities from 2000-2004:

1. Research. The specific objectives of research were to (1) develop standardized diagnostic methods for major diseases affecting economically important aquaculture species in the region; (2) develop effective prevention and control measures against microbial and parasitic diseases; (3) assess the pathogenesis of newly emerging diseases; and (4) develop monitoring methods for residual chemicals in aquaculture products.

2. Hands-on training. This activity was specifically aimed at developing and enhancing capability in aquatic animal health diagnosis and management of technical staff working at research centers and institutions in the region.

3. International meetings. These were conducted to (1) discuss the status of fish disease problems in the region, the available diagnostic methods, and prevention and control measures; (2) discuss the results of research studies conducted under the project and those generated in other regions; (3) identify and discuss aquatic animal disease issues to be solved further for sustainable aquaculture growth; and (4) discuss collaboration with other international organizations such as the Office International des Épizooties (OIE).

4. Extension. This activity was done to disseminate research results and technology generated by the project through (1) training courses on fish diagnosis and health management; (2) production of manuals; (3) publication of primary results in international scientific journals; (4) international meetings; and (5) sampling and field visits.

To coordinate and promote the project, two Japanese fish disease experts were dispatched to the SEAFDEC Aquaculture Department as long-term experts by the Japan International Cooperation Agency (JICA). Dr. Yasuo Inui worked as the first expert from March 2000 to March 2003, and the second expert, Dr. Kazuya Nagasawa acted as the project leader from April 2003 to date.

Research during the First Phase of the Project (2000 - 2004)
Research is the main activity component of the Regional Fish Disease Project. When the project started in 2000, it was undertaken only by scientists of the SEAFDEC Aquaculture Department. Subsequently, scientists from three research institutions under the Department of Fisheries in Thailand and those of the SEAFDEC Marine Fisheries Research Department (MFRD) in Singapore joined the project in 2001 and 2002, respectively. During the period from 2000 to 2004, a total of 29 research studies were conducted in the following five categories:

A. Establishment and Standardization of Diagnostic Methods

In Southeast Asia, various viral diseases have been reported from cultured shrimp and fish, causing devastating losses in aquaculture production. White spot syndrome (WSS) of black tiger shrimp (Penaeus monodon) and viral nervous necrosis (VNN) of marine fish are well known examples of such viral diseases affecting aquaculture in the region. The research in this category was undertaken to establish and standardize diagnostic techniques, such as PCR (polymerase chain reaction) methods for viral diseases, which are applicable and practical in the region. Some research studies surveyed the distribution, occurrence and prevalence of important viral diseases. There was also a study to prevent and control VNN infection in the marine finfish hatchery.

1. Standardization of diagnostic methods for viral diseases of shrimps (SEAFDEC Aquaculture Department [AQD], 2000)

2. Standardization of PCR technique as the detection method for WSSV infection in Penaeus monodon (SEAFDEC/AQD, 2001)

3. Development of shrimp cell culture in vitro (Marine Shrimp Research and Development Center [MSRDC], Thailand, 2001-2002)

4. Standardization of diagnostic methods for monodon baculovirus (MBV) and hepatopancreatic parvovirus (HPV): Establishment of monoclonal antibodies (MAbs) against MBV and HPV (SEAFDEC/AQD, 2001-2003)

5. Epizootiology of economically important viral diseases of wild Penaeus monodon (SEAFDEC/AQD, 2001-2003)

6. Viral diseases of cultured marine fishes in Southeast Asia

6-1. Detection and identification of viral pathogens in cultured marine finfishes in the Philippines (SEAFDEC/AQD, 2000-2003)

6-2. A viral survey in diseased grouper in Thailand using virus isolation and polymerase chain reaction (PCR) technique (Aquatic Animal Health Research Institute [AAHRI], Thailand, 2001-2002)

6-3. Survey of iridoviral disease in freshwater fishes in Thailand (AAHRI, Thailand, 2003)

7. Establishment of preventive measures against viral nervous necrosis (VNN) in fish broodstocks: (1) grouper, (2) milkfish, (3) red snapper, and (4) sea bass (SEAFDEC/AQD, 2001-2003)

B. Biology and Pathogenesis of Disease Pathogens

Diseases caused by protozoan and metazoan parasites often cause mass mortality of cultured fish, and, like microbial agents, the parasites are important pathogens. However, there remains limited information available on fish parasites in Southeast Asia. For example, less than 10 % of more than 2,030 species of marine and freshwater fish in the Philippines have been examined for the parasites. There are only a few studies on the parasites of cultured fish
in the region. The research in this category aimed to screen economically important fish for the presence of parasites, determine diagnosis and pathology of infections, and establish prevention and control methods.

1. Parasitosis in marine and freshwater fishes: diagnosis, pathology, prevention and control of infection
1-1. Screening of important parasites in economically important aquaculture fish (SEAFDEC/AQD, 2000-2003)
1-2. Biology and pathology of the gill monogenean parasitic to grouper (SEAFDEC/AQD, 2000-2003)
1-3. Leech infestation and its associated blood parasitic protozoans (SEAFDEC/AQD, 2000-2003)
1-4. Establishment/application of prevention and control methods against parasites (SEAFDEC/AQD, 2000-2003)

2. Study on parasites of groupers in Thailand (AAHRI, Thailand, 2001-2002)

3. Screening of important parasites of freshwater fish in Thailand and neighboring countries (AAHRI, Thailand, 2003)

C. Disease Prevention and Control

Luminous vibriosis caused by Vibrio spp., especially V. harveyi, is a major bacterial disease of black tiger shrimp cultured in Southeast Asia. The research studies were intended to develop husbandry techniques, such as the use of live bacteria (probiotics) and “green water” culture system, as alternatives for chemotherapy to control vibriosis. The “green water” culture system is the finfish-integrated shrimp culture system, utilizing finfish rearing water for shrimp culture. The mechanisms on how the system works to control vibriosis were analyzed.

1. Use of bacteria as biological control agent against microbial diseases in shrimp (Penaeus monodon) and crab (Scylla serrata) hatcheries (SEAFDEC/AQD, 2000-2003)
2. Screening of probiotics as biocontrol/bioremediation in the rearing of P. monodon. I. Tank experiment (SEAFDEC/AQD, 2001-2003)
3. Antibacterial metabolites in the microbial and phytoplankton flora of the “green water” cultured Penaeus monodon (SEAFDEC/AQD, 2000-2003)
4. Investigation on the mechanism of the effect of tilapia culture water on luminous bacteria (SEAFDEC/AQD, 2001-2003)
5. Screening of Vibrio harveyi bacteriophage for controlling luminous disease in marine shrimp hatchery (Samutsakhon Coastal Aquaculture Development Center [SCADC], Thailand, 2001-2003)
6. Development of immunological indices for monitoring health status in P. monodon (SEAFDEC/AQD, 2001-2003)

D. Establishment of Evaluation Methods for Residual Chemicals in Aquaculture Products

The presence of chemical residues in aquaculture products threatens human health. To ensure safe and healthy aquaculture products, a research activity addressed the development and standardization of detection methods of residual chemicals, especially pesticides and antibiotics, in aquaculture products. The usage of antibiotics in shrimp culture was also monitored.

1. Establishment and monitoring on antimicrobial usage in shrimp aquaculture (SCADC, Thailand, 2001-2003)
2. Detection of pesticide residues in aquaculture products (SEAFDEC/AQD, 2000-2003)
3. Detection of antibiotic residues in aquaculture products (SEAFDEC/MFRD, 2002-2003)

E. Epizootiology and Prevention of Koi Herpesvirus Disease

Koi herpesvirus (KHV) disease was found in common carp and koi cultured in Indonesia and Taiwan in 2002 and Japan in 2003. The disease caused mass mortality of the fish and became a new threat to freshwater aquaculture in Southeast Asia. The research studies were conducted to elucidate various aspects of KHV infection.

1. Transmission and control of koi herpesvirus (SEAFDEC/AQD, 2004)
2. Development of PCR-based detection method and phylogenetic analysis of koi herpesvirus isolated from Asian countries (SEAFDEC/AQD, 2004)
3. Histopathology of koi herpesvirus disease (SEAFDEC/AQD, 2004)
4. Hematology of carp infected with koi herpesvirus (SEAFDEC/AQD,2004)
5. Determination of the virucidal effects of various disinfectants on koi herpesvirus (SEAFDEC/AQD, 2004)

Hands-on Training during the First Phase of the Project (2000 - 2004)

The Seminar/Workshop on “Disease Control in Fish and Shrimp Aquaculture in Southeast Asia-Diagnosis and Husbandry Techniques” was convened by SEAFDEC and OIE in Iloilo City, Philippines on 4-6 December 2001. One of the major recommendations of the Seminar/Workshop was to conduct the international training course on diagnosis of viral diseases, which became the basis for the “SEAFDEC Hands-on Training for Important Viral Diseases of Shrimp and Marine Fish.” The implementation of the said training course was done at the SEAFDEC Aquaculture Department in collaboration with OIE and the Network of Aquaculture Centres in Asia-Pacific (NACA). The objective of the training course was to provide executive training on the diagnosis of viral diseases to core persons from the SEAFDEC member countries and other interested participants. These persons were expected to serve as national trainers in their respective countries. The training course consisted of the first and second phases, which were held on 6-19 November 2002 and 5-21 November 2003, respectively, at the SEAFDEC Aquaculture Department. The same set of trainees was invited to attend the two phases, but there were new participants who replaced those who were unable to come for some reasons. A total of 12 and 11 participants attended the first and second phases of the training course, respectively. The participants came from the SEAFDEC member countries (one from each country: Brunei Darussalam, Cambodia, Indonesia, Lao PDR, Malaysia, Myanmar, Philippines, Singapore, Thailand, Vietnam) and other countries (China and India) although there was no participation from Brunei Darussalam in the second phase. All participants from the SEAFDEC member countries were funded by the Regional Fish Disease Project. The first phase of the training course focused on the use of molecular tools and other important techniques in the diagnosis of viral diseases of shrimp and marine fish, while the second phase was a continuation of the first phase, which then completed the two-year plan.
The 14-day course of the first phase consisted of lectures (11 hours or 15%) and practical activities (64 hours or 85%). Similarly, the 17-day course of the second phase was composed of 17 hours of lecture sessions (17.3%) and 81 hours practical activities (82.7%). Most of the lectures were done at the Research Division (RD) conference room. The practical activities were undertaken either at the Fish Health laboratory or in the Biotech laboratory. For the second phase, the participants brought shrimp and fish tissue samples from their respective countries for the laboratory activities. The first phase of hands-on sessions included dissection and preservation of fish and shrimp samples, rapid detection methods for monodon baculovirus (MBV) and hepatopancreatic parvovirus (HPV) using hepatopancreas impression smears, histopathological analysis of viral diseases of shrimp and marine fish, extraction of nucleic acids (DNA and RNA) and detection of white spot syndrome virus (WSSV) and MBV in shrimp, and viral nervous necrosis virus (VNN) and iridovirus in grouper by one-step and nested polymerase chain reactions (PCR). The diagnostic techniques used were consistent with the standards set in the OIE’s “Diagnostic Manual for Aquatic Animal Diseases” and the FAO’s “Asia Diagnostic Guide to Aquatic Animal Diseases.” Demonstrations on cell culture- and antibody-based detection methods for viruses were undertaken. The second phase of the hands-on training consisted of preparation and preservation of tissue filtrates, cell culture passage, immune system parameters, histopathology, observation of virus using electron microscopy, detection of viruses by cell culture, antibody (Ab)-based detection of HPV and MBV, detection of MBV, WSSV, VNN and iridovirus using PCR. Demonstrations on hitopathological slide preparation were also done. In addition to these activities, all participants presented country reports describing the status of aquatic animal diseases and diagnostic capability in their respective countries and institutions.

Outputs

As the outputs of research and hands-on training activities and international meetings that were organized with financial support from the Regional Fish Disease Project, there are already publications available and many are in preparation for publication. These are listed under various categories below.

Proceedings

1. Disease Control in Fish and Shrimp Aquaculture in Southeast Asia-Diagnosis and Husbandry Techniques (ed by Inui Y, Cruz-Lacierda ER), 2002, SEAFDEC Aquaculture Department, Iloilo. 215 p.

2. Transboundary Fish Diseases in Southeast Asia: Occurrence, Surveillance, Research and Training (ed by Lavilla-Pitogo CR, Nagasawa K), 2004, SEAFDEC Aquaculture Department, Iloilo. 254 p.

Manuals

1. Borlongan I, Ng Poh Chuan J. 2004. Laboratory Manual of Standardized Methods for the Analysis of Pesticide and Antibiotic Residues in Aquaculture Products. SEAFDEC Aquaculture Department, Tigbauan, Iloilo. 46 p.

2. de la Peña LD, Lavilla-Pitogo CR. 2004. Control Measures against Important Viruses in Shrimp Hatchery with Emphasis on White Spot Syndrome Virus (WSSV). SEAFDEC Aquaculture Department, Iloilo. (in press).

3. de la Peña LD. 2004. Control Measures against Viral Nervous Necrosis (VNN) in Finfish Hatchery. SEAFDEC Aquaculture Department, Iloilo. (in press).

4. Lavilla-Pitogo CR, de la Peña LD. 2004. Health Management in Crab Hatchery System. SEAFDEC Aquaculture Department, Iloilo. (in press).

5. Nagasawa K, Cruz-Lacierda ER (eds). 2004. Diseases of Cultured Grouper. SEAFDEC Aquaculture Department, Iloilo. (in press).

6. Ruangpan L, Tendencia EA. 2004. Standardized Methods for Minimal Inhibitory Concentration Test and Determination of Antimicrobial Resistance for Vibrio Bacteria Isolated from Shrimp. SEAFDEC Aquaculture Department, Iloilo. (in press).

Review Articles

1. de la Peña LD. 2004. Transboundary shrimp viral diseases with emphasis on white spot syndrome virus (WSSV) and Taura syndrome virus (TSV). In: Lavilla-Pitogo CR, Nagasawa K (eds), Transboundary Fish Diseases in Southeast Asia: Occurrence, Surveillance, Research and Training. SEAFDEC Aquaculture Department, Iloilo. p. 67-69.

2. Inui Y. 2002a. Fish disease control project of SEAFDEC Aquaculture Department. In: Inui Y, Cruz-Lacierda ER (eds), Disease Control in Fish and Shrimp Aquaculture in Southeast Asia-Diagnosis and Husbandry Techniques. SEAFDEC Aquaculture Department, Iloilo, p. 181-185.

3. Inui Y. 2002b. Activities of the Fish Disease Project of SEAFDEC/AQD with relevance to shrimp disease control. In: Proceedings of the Third National Philippine Shrimp Industry Congress (Shrimp Congress 2002). 1-4 July 2002, Bacolod Convention Plaza Hotel, Bacolod City, Negros Occidental, Philippines. p. 60-64.

4. Lavilla-Pitogo CR, Torres PL. 2004. AquaHealth Online: A new learning environment for building capacity in aquatic animal health. In: Lavilla-Pitogo CR, Nagasawa K (eds), Transboundary Fish Diseases in Southeast Asia: Occurrence, Surveillance, Research and Training. SEAFDEC Aquaculture Department, Iloilo. p. 53-66.

5. Lavilla-Pitogo CR, Catedral DD, Pedrajas SDG, de la Peña L. 2002. Selection of probiotics for shrimp and crab hatcheries. In: Inui Y, Cruz- Lacierda ER (eds), Disease Control in Fish and Shrimp Aquaculture in Southeast Asia-Diagnosis and Husbandry Techniques. SEAFDEC Aquaculture Department, Iloilo, p. 136-150.

6. Lio-Po GD. 2004. Summary brief: International Symposium on Koi Herpesvirus Disease. In: Lavilla-Pitogo CR, Nagasawa K (eds), Transboundary Fish Diseases in Southeast Asia: Occurrence, Surveillance, Research and Training. SEAFDEC Aquaculture Department, Iloilo. p. 71-73.

7. Lio-Po GD, Cruz-Lacierda ER, de la Peña LD, Maeno Y, Inui Y. 2002. Progress and current status of diagnostic techniques for marine fish viral diseases at the SEAFDEC Aquaculture Department. In: Inui Y, Cruz-Lacierda ER (eds), Disease Control in Fish and Shrimp Aquaculture in Southeast Asia-Diagnosis and Husbandry Techniques. SEAFDEC Aquaculture Department, Iloilo. p. 97-106.

8. Lio-Po GD, Leano EM, Usero RC, Guanzon Jr. NG. 2002. Vibrio harveyi and the “green water culture” of Penaeus monodon. In: Inui Y, Cruz-Lacierda ER (eds), Disease Control in Fish and Shrimp Aquaculture in Southeast Asia-Diagnosis and Husbandry Techniques. SEAFDEC Aquaculture Department, Iloilo. p. 172-180.

9. Nagasawa K. 2004. Background and objectives of the Meeting on Current Status of Transboundary Fish Diseases in Southeast Asia: Occurrence, Surveillance, Research and Training. In: Lavilla-Pitogo CR, Nagasawa K (eds), Transboundary Fish Diseases in Southeast Asia: Occurrence, Surveillance, Research and Training. SEAFDEC Aquaculture Department, Iloilo. p. 3-9.

10. Nagasawa K. 2004. Research and training on fish diseases at the SEAFDEC Aquaculture Department in 2000-2004: a review. In: Lavilla-Pitogo CR, Nagasawa K (eds), Transboundary Fish Diseases in Southeast Asia: Occurrence, Surveillance, Research and Training. SEAFDEC Aquaculture Department, Iloilo. p. 41-52.

11. Nagasawa K. 2005. Proposed activities for koi herpesvirus disease at the SEAFDEC Aquaculture Department. Bull. Fish. Res. Agen., Supplement 2. (in press).

Scientific Papers

1. Catap ES, Lavilla-Pitogo CR, Maeno Y, Travina RD. 2003. Occurrence, histopathology and experimental transmission of hepatopancreatic parvovirus (HPV) infections in Penaeus monodon postlarvae. Diseases of Aquatic Organisms, 57: 11-17.

2. Catap ES, Travina RD. 2004. Experimental transmission of hepatopancreatic parvovirus (HPV) infection in Penaeus monodon postlarvae. Proceedings of 5th Symposium on Diseases in Asian Aquaculture, 24-28 November 2002, Queensland, Australia. (in press).

3. Cruz-Lacierda ER, Maeno Y, Pineda AJ, Matey VE. 2004. Mass mortality of hatchery-reared milkfish (Chanos chanos) and mangrove red snapper (Lutjanus argentimaculatus) caused by Amyloodinium oceallatum (Dinoflagellida). Aquaculture, 236: 85-94.

4. de la Peña LD, Lavilla-Pitogo CR, Namikoshi A, Nishizawa T, Inui Y, Muroga K. 2003. Mortality in pond-cultured shrimp Penaeus monodon in the Philippines associated with Vibrio harveyi and white spot syndrome virus. Fish Pathology, 38: 59-61.

5. Ho J-S, Kim I-H, Cruz-Lacierda ER, Nagasawa K. 2004. Sea lice (Copepoda, Caligidae) parasitic on marine fishes cultured in the Philippines. Journal of the Fisheries Society of Taiwan. (in press).

6. Maeno Y, de la Peña LD, Cruz-Lacierda ER. 2002. Nodavirus infection in hatchery reared orange-spotted grouper Epinephelus coioides: first record of viral nervous necrosis in the Philippines. Fish Pathology, 37: 87-89.

7. Maeno Y, de la Peña LD, Cruz-Lacierda ER. 2003. Development of control methods of factors suppressing sustainable production of aquaculture species: isolation of a piscine nodavirus from hatchery-reared sea bass Lates calcarifer in the Philippines. In: Ogawa Y, Ogata GY, Maeno Y, Shimoda T, Fujioka Y, Fukuda Y (eds), Sustainable Production Systems of Aquatic Animals in Brackish Mangrove Areas. JIRCAS Working Paper No. 35. Japan
International Research Center for Agricultural Sciences (JIRCAS), Tsukuba, Japan. p. 81-87.

8. Maeno Y, de la Peña LD, Cruz-Lacierda ER. 2003. Development of control methods of factors suppressing sustainable production of aquaculture species: experimental transmission of piscine nodavirus-induced viral nervous necrosis to the orange-spotted grouper Epinephelus coioides. In: Ogawa Y, Ogata GY, Maeno Y, Shimoda T, Fujioka Y, Fukuda Y (eds), Sustainable Production Systems of Aquatic Animals in Brackish Mangrove Areas. JIRCAS Working Paper No. 35. Japan International Research Center for Agricultural Sciences (JIRCAS), Tsukuba, Japan. p. 89-94.

9. Maeno Y, de la Peña LD, Cruz-Lacierda ER. 2004. Mass mortalities associated with viral nervous necrosis in hatchery-reared sea bass Lates calcarifer in the Philippines. Japan Agricultural Research Quarterly, 38: 69-73.

10. Moravec F, Cruz-Lacierda ER, Nagasawa K. 2004. Two Procamallanus spp. (Nematoda: Camallanidae) from fishes in the Philippines. Acta Parasitologica. (in press).

11. Tendencia EA , de la Peña M. 2003. Investigation of some components of the greenwater system which makes it effective in the initial control of luminous bacteria. Aquaculture, 218: 115-119.

12. Tendencia EA, de la Peña MR, Femin AC, Lio-Po G, Choresca, CH Jr, Inui Y. 2004. Antibacterial activity of tilapia Tilapia hornorum against Vibrio harveyi. Aquaculture, 232: 145-152.

Papers Presented at International and National Meetings

1. Catap ES, Travina R. 2002. Experimental transmission of hepatopancreatic parvovirus (HPV) infection in Penaeus monodon postlarve. Abstracts of 5th Symposium on Diseases in Asian Aquaculture, 24-28 November 2002, Queensland, Australia, p. 63.

2. Catap ES. 2003. Purification of monodon baculovirus (MBV) and hepatopanacreatic parvovirus (HPV) virions from postlarval Penaeus monodon. Paper presented at the Philippine Society for Microbiology – Visayas Chapter 11th Annual Meeting and Regional Scientific Convention held in Cebu City, Philippines on October 23-24, 2003.

3. Cruz-Lacierda ER, Maeno Y, Pineda AJ. 2002. Occurrence and pathology of an Amyloodinium-like parasite on hatchery-reared milkfish (Chanos chanos). Abstracts of 10th International Congress of Parasitology, 4-9 August 2002, Vancouver, Canada, p. 85.

4. Cruz-Lacierda ER, Maeno Y, Pineda AJ, Matey VE. 2003. Mass mortality of hatchery-reared milkfish (Chanos chanos) mangrove red snapper (Lutjanus argentimaculatus) caused by Amyloodinium ocellatum (Dinoflagellida). Paper presented at the Philippine Society for Microbiology-Visayas Chapter 11th Annual Meeting and Regional Scientific Convention held in Cebu City, Philippines on October 23-24, 2003.

5. Erazo-Pagador G. 2002. Biology and pathogenicity of the gill monogenean (Pseudorhabdosynochus sp.) in grouper. Abstracts of 5th Symposium on Diseases in Asian Aquaculture, 24-28 November 2002, Queensland, Australia, p. 113.

6. Lavilla-Pitogo CR, Catedral DD, de la Peña LD, Inui Y. 2002. Evaluation of pathogenicity of bacterial strains in crustacean larvae by static bath: significance of monitoring bacterial counts. Abstracts of 5th Symposium on Diseases in Asian Aquaculture, 24-28 November 2002, Queensland, Australia, p. 162.

7. Lavilla-Pitogo CR, Catedral DD, de la Peña LD. 2003. Delivery of strain C1 probiotic bacteria through colonized Placuna sella shells and corrugated plastic sheets. Paper presented at the Philippine Society for Microbiology- Visayas Chapter 11th Annual Meeting and Regional Scientific Convention held in Cebu City, Philippines on October 23-24, 2003.

8. Lio-Po GL, Peñaranda MD, Duray MN. 2002. Viral infections of hatchery reared marine finfishes in the Philippines. Abstracts of 2nd International Symposium on Stock Enhancement and Sea Ranching, 28 January-1 February 2002, Kobe, Japan, p. 36.

9. Lio-Po G, Franco A. 2003. Preliminary results on the comparative effects of two commercial probiotics on total bacterial counts and on luminous Vibrio. Paper presented at the Philippine Society for Microbology-Visayas Chapter 11th Annual Meeting and Regional Scientific Convention held in Cebu City, Philippines on October 23-24, 2003.

10. Maeno Y, de la Pena L, Cruz-Lacierda ER. 2002. Susceptibility of marine fish species to piscine nodavirus from orange-spotted grouper, Epinephelus coioides in the Philippines. Abstracts of 5th Symposium on Diseases in Asian Aquaculture, 24-28 November 2002, Queensland, Australia, p. 46.

11. Tendencia E. 2002. Effect of Tilapia hornorum on luminous bacteria Vibrio harveyi. Abstracts of 5th Symposium on Diseases in Asian Aquaculture, 24-28 November 2002, Queensland, Australia, p. 172.

Terminal Report

1. Recent Advances in Diagnosis and Prevention of Fish and Shrimp Diseases in Southeast Asia: Terminal Report of the Regional Fish Disease Project on “Development of Fish Disease Inspection Methodologies for Artificially-Bred Seeds” Supported by the Government of Japan Trust Fund (ed by K Nagasawa), 2005, SEAFDEC Aquaculture Department, Iloilo. (in press).

Annual Reports

1. Anonymous. 2001. Annual Report 2000 - Component: Development of Fish Disease Inspection Methodologies for Artificially-Bred Seeds.
SEAFDEC Aquaculture Department, Iloilo. 64 p.

2. Anonymous. 2002. Annual Report 2001 - Component: Development of Fish Disease Inspection Methodologies for Artificially-Bred Seeds.
SEAFDEC Aquaculture Department, Iloilo. 152 p.

2. Anonymous. 2002. Annual Report 2001 - Regional Fish Disease Program: Development of Fish Disease Inspection Methodologies for Artificially-Bred Seeds. SEAFDEC Aquaculture Department, Iloilo. 10 pp. (abstracted version).

4. Anonymous. 2003. Annual Report 2002: Component - Development of Fish Disease Inspection Methodologies for Artificially-Bred Seeds. SEAFDEC Aquaculture Department, Iloilo. 131 pp.

5. Anonymous. 2004. Annual Report 2003: Development of Fish Disease Inspection Methodologies for Artificially-Bred Seeds. SEAFDEC Aquaculture Department, Iloilo. 223 p.

Hands-on Training Reports

1. Cruz-Lacierda E. 2003. Terminal report on SEAFDEC hands-on training for important viral diseases of shrimp and marine fish in collaboration with OIE and NACA, 06-19 November 2002. SEAFDEC Aquaculture Department, Iloilo. 48 p.

2. Gonzales-Corre K. 2004. Terminal report on 2nd SEAFDEC hands-on training for important viral diseases of shrimp and marine fish in collaboration with OIE and NACA, 05-21 November 2003. SEAFDEC Aquaculture Department, Iloilo. 21 p.

Pamphlet

1. Sugiura S (ed). 2001. Trust Fund Highlights 2000. SEAFDEC, Bangkok. 28 pp.

Flyers

1. Anonymous. 2003. Regional Fish Disease Project. SEAFDEC Aquaculture Department, Iloilo.
2. Anonymous. 2003. Living with White Spot Disease in Shrimp Farming. SEAFDEC Aquaculture Department, Iloilo.

AquaHealth OnLine: A New Learning Environment for Capacity Building in Aquatic Animal Health

Celia R. Lavilla-Pitogo and Pastor L. Torres Jr.
Southeast Asian Fisheries Development Center
Aquaculture Department, Tigbauan 5021
Iloilo, Philippines

Abstract

Due to significant requirement of trained personnel in Fish Health Management, the Aquaculture Department of the Southeast Asian Fisheries Development Center (SEAFDEC/AQD) offered 14 sessions of face-to-face (F2F) training (1988–2002) at its station in Iloilo. However, shrinking fellowship and travel funds necessitated a shift in training paradigm. Thus, transformation of teaching materials used in F2F trainings resulted to AquaHealth Online, a team developed and electronically delivered course on Health Management in Aquaculture. The general objective of the course transformation was to train a large pool of geographically dispersed participants at minimum cost, and this paper reports on the experience earned in course development, delivery and outcome.
To enable Fish Health specialists to develop materials and skills to deliver courses for the online environment, SEAFDEC/AQD collaborated with the University of the Philippines Open University to help adapt, enhance, and reformulate materials in the F2F course for online delivery. The specialists
underwent hours of training in “techno-pedagogy”, or ways of transforming teaching activities into formats that could be understood even in our absence.
The primary learning resource is a CD-ROM that provides interactive information with self-assessment questions. The course covers 12 modules in 4 units: I. Introduction to Fish Health Management; II. Infectious Diseases of Fish and Crustaceans; III. Non-Infectious Diseases; and IV. Disease Diagnosis, Prevention and Control. Learning enhancement and discussion occurs through internet-based Discussion Boards (DBs) presided over by module specialists. The DBs serve as media for asynchronous discussions and makes a permanent record of lessons learned. When first offered in 2002, AquaHealth Online had 25 enrollees from 10 countries. In 2003, there were 17 participants from 8 countries. Participants were led to “just-in” relevant information and encouraged to submit assignments from internet resources. This course is an example that a state-of-the-art online course can be as effective as F2F training.

Introduction

Training on specialized subjects in aquaculture such as marine fish hatchery, freshwater aquaculture, nutrition and feed development, aquaculture management course, and many others were offered every year to participants from SEAFDEC member countries with funding for fellowships and travel
from the Government of Japan. Training course delivery was through faceto-face (F2F) lectures, field trips and hands-on laboratory exercises. The Fish
Health Management Training Course (FHMTC) was offered for fourteen sessions and became one of the most sought-after and well-attended international classroom type training courses at the Aquaculture Department of the Southeast Asian Fisheries Development Center (SEAFDEC/AQD). This was due to the realization that no aquaculture venture would ever succeed without due consideration to proper health management practices and the emergence of serious infectious disease outbreaks from the late 1980s. While the demand for trained personnel in fish health management was sustained, SEAFDEC/AQD anticipated the worldwide trend of generalized reductions in the public funding of institutions, diminishing access to private and charitable donations (Abrioux, 2001) and the need to become more self reliant in its course offerings. Thus, transformation of teaching materials used in F2F trainings resulted to a team developed and electronically delivered full course on Principles on Health Management in Aquaculture or AquaHealth Online. Elearning in the Philippines is relatively new and many requirements need to be in place to catch up with developments worldwide (Khanser, 2003).

Objectives of AquaHealth Online

AquaHealth Online is an elearning course targeting full-time working professionals. It enables learning to take place in different places, both physical and virtual. Through elearning, it is convenient and practical for a learner to acquire knowledge and skills in aquaculture health management at his own place and at his own time as long as a computer and an Internet access are made available to him to communicate with highly qualified teachers or with fellow learners. The general objective of the course was to ensure delivery of efficient training to a large pool of geographically dispersed participants at minimum cost. As with the F2F FHMTC, AquaHealth Online’s goals remained the same wherein at the end of the course learners should be able to:

RECOGNIZE diseased shrimps and fish;
IDENTIFY the cause of the disease;
EXPLAIN how a disease develops;
APPLY preventive and control measures to lessen the risks posed by the disease;
USE appropriate techniques for the preparation of samples for disease diagnosis.

The Course Transformation Process

The most effective learning, whether delivered as conventional F2F instruction or an elearning solution, is a result of careful planning and systematic design derived from the needs of the organization and its clients. In conceptualizing SEAFDEC/AQD’s elearning courses, it was recognized that expertise in course transformation to elearning mode was generally lacking. Thus, collaboration with the University of the Philippines Open University (UPOU) and SEAFDEC/AQD was formalized through a Memorandum of Agreement whereby the former provided expertise and guidance needed in online course transformation. The project was spearheaded by the Training and Information Division with the cooperation of specialists and content experts from the Fish Health Section. An editor was assigned to see to it that course materials are expressed in a language suited for online delivery. UPOU provided expertise in instructional and graphic design. The team worked in a collaborative manner to develop the course, whereby specialists received constant support from the instructional designer to develop the course structure, create the course webpages, and package them in a CD-ROM.

A. The Fish Health Team

Twelve senior research staff of SEAFDEC/AQD contributed in the course development (Table 1). Seven have PhD degrees and majority have acted as lecturers in the F2F FHMTC. Put together, their work experiences total 210 man-years of research and teaching in fish health and related disciplines.
During the conceptualization of AquaHealth Online, many specialists were hesitant and got intimidated in delivering courses online completely. Issues
on pedagogy have surfaced, but the team from the UPOU gave the necessary motivation and technical help in transforming the pedagogy of F2F instruction
into online instruction materials. The specialists underwent a restructuring process with the goal of better preparing them to effectively integrate technology into their teaching and developing courses that make extensive use of Web-based technologies. This transformational process was to preserve the constructionist learning environment (Davies and Carbonaro, 2000) of the traditional course while at the same time optimizing the course delivery mode to make it more accessible to a wider audience of students.

B. Source of Course Materials

The transformation from F2F to elearning was not very difficult since basic students’ references had already been transformed from loose handouts

to a well-illustrated textbook (Fig. 1) on Health Management in Aquaculture (Lio-Po, Lavilla and Cruz-Lacierda, 2001). The book has 187 pages, 11 chapters, and over 140 diagrams, tables, drawings and colored photographs to illustrate the principles. Instructional media had also been upgraded from 35
mm slides and transparencies to PowerPoint presentations and short film clips. The Fish Health Section team underwent hours of training in what we
now know as “techno-pedagogy”, or ways of transforming teaching activities into multimedia formats that could be understood even in our absence. Thus,
after “teacher training”, online course design, review of system’s capabilities, and provision of platform for online interaction, development of materials for
AquaHealth online commenced.

Fig. 1. The textbook on Health Management in Aquaculture edited by Lio-Po, Lavilla and Cruz-Lacierda (2001) that became the source of materials for the CD-ROM learning resource.

C. Course Design and Coverage

AquaHealth online covers up-to-date knowledge on fish and crustacean diseases, their causal organisms, and tried and tested methods of disease
prevention and control. The course runs for a minimum of 16 weeks and is presented in 4 units consisting of 12 modules (Table 2). The duration of
modules that deal with highly technical subjects was doubled allowing two weeks of discussion between the learners and the specialists.

The AquaHealth Online Learning Package

The advantage of an elearning package is that it not only provides a marriage of digital technology, Internet, and learning, but it also facilitates

learner-centered learning. The students are at the center of the teachinglearning
process, and teachers act as mentors, navigators, facilitators, or
“guides” to help the learners access, organize, construct, and transfer
information to grasp the principles being imparted to them.

A. The AquaHealth Online CD-ROM

The FHMTC materials that were transformed into interesting and easily learned modules were rendered by the UPOU multimedia specialists and packaged in a CD-ROM “Principles of Health Management in Aquaculture” (Fig. 2). This software provides our learners with basic interactive information. Every module contains several interactive self-assessment-questions (SAQs) that help students gauge their learning progress. Formulation of SAQs took into account design guidelines formulated by Race (1997). Each of the 12 modules was authored by at least one specialist in the field. Recognizing that the key component in an elearning approach is the students’ ability to obtain more information and research materials, online materials with hyperlinks to relevant websites were provided to encourage the learners to actively participate in the search for resources and answers to enhance their research and diagnostic skills.

B. Course Guide

A course guide (Fig. 3) was provided at the start of the course. The document provides the learner with the course basics: introduction, description, goals and objectives, outline, requirements (skills and equipment), manner of assessment (grading system), as well as activities for each chapter. Also in the document is a study schedule, instructions on navigating the CDROM, house rules and important contact numbers and addresses in case the learner needs rechnical support. Annexes are provided like Netiquette Guidelines, an introduction to the discussion platform in the Integrated Virtual Learning Environment (IVLE), starting discussions using the DBs, and submission of assignments and reports through the Workbin. The Course Guide also provides tips on how to become a successful online student and some frequently-asked-questions.

C. The Discussion Platform

The AquaHealth Online website used the IVLE structure hosted at the UPOU server (Fig. 4) and was accessible through links in the SEAFDEC/AQD or UPOU sites. The design of the course forum allowed discussion content to be accessible from any computer anywhere, as long as it was connected to the
Internet with the user assigned account and password providing the gateway. The proposed structure consisted of a homepage with icons for establishing links to the course outline, schedule, content, email, discussion board, and technical support (Fig. 5). Interaction and exchange of ideas in each
module was through the Discussions Boards (Fig. 6), each of which was mentored by at least one specialist. This set-up offered a semi-permanent record of what transpired during the module discussions.While absence in class is too conspicuous to ignore in face-to-face classrooms, an online student

always keeps tracks of discussion as long as the DBs remain posted. Asynchronous discussion and interaction through the DBs provide a permanent record of lessons learned as a result of interaction. Most importantly, the DB allowed for course material contents updates without necessarily revising the CD-ROM. In addition to board postings, email was also used to inform learners about activities, grades, and reminders of upcoming deadlines and submissions. However, learners were not encouraged to use email as a platform for discussion in order not to disperse the sites where exchange of information is located. This is very important since discussion is asynchronous.class is too conspicuous to ignore in face-to-face classrooms, an online student always keeps tracks of discussion as long as the DBs remain posted. Asynchronous discussion and

interaction through the DBs provide a permanent record of lessons learned as a result of interaction. Most importantly, the DB allowed for course material
contents updates without necessarily revising the CD-ROM. Recruitment of Trainees When first offered in 2002, AquaHealth Online had 25 enrollees from 10 countries namely: Cambodia (2), Egypt (1), India (1), Indonesia (2), Malaysia (2), Myanmar (2), Singapore (3), Thailand (2), Vietnam (3), and the Philippines (7). In 2003, there were 17 participants from 8 countries (Table 3). The Government of Japan provided fellowships for two participants each
from 10 SEAFDEC member countries. They were nominated by their respective Directors to the SEAFDEC Council. Interestingly, 30% of

participants in 2002 and 2003 are privately funded. The main requirement was that all learners must have taken a subject in college biology. The maximum number of learners at any time is set at 30. Overall, participants from 12 countries have participated in the course.

Conduct of the Course

Upon enrollment, learners were provided with User Identifications and Passwords that entitled them to enter the virtual classroom. Access to the virtual
classroom was either through the SEAFDEC/AQD website at www.seafdec.org.ph of through UPOU’s website at www.upou.org. Upon access to the course site, students “met” at the Café for Students where they introduced each other. AquaHealth Online runs for 16 weeks where a designated specialist of a particular module encourages discussion and information exchange. A Course Officer moderates the whole process. Learners proceeded with the course as

if they were in a classroom, except they face computer screens instead of instructors. Under the guidance of specialists, learners performed exercises
individually or as a group and submitted reports of their work either through assigned workbins, by email or by posting them in the DBs for everyone’s
perusal. Group work was encouraged among learners from the same country to encourage F2F meetings, where possible. Most people learn better when
computer-mediated lessons are combined with study groups, team exercises, and off-line events. Although computers can make aspects of learning more
convenient, they do not eliminate the need for human intervention. In the first year, learners took examinations administered by proctors near the places
of their work, but during last year’s AquaHealth online, essay type or investigative take-home examinations were given. Of utmost importance was the unlimited interaction among learners, sharing insights and experiences, enhancing further the learning process. Together with learning the principles of health management in aquaculture, AquaHealth learners enhanced their basic computer skills. Learners found the interactive SAQs and tests in the CD engaging. Those who could not hold the mouse prior to the course, learned to access the IVLE website, took active part in the discussion forum, learned how to send and receive emails, type documents, attach files and submitted assignments through workbins. The links taught them how to access online dictionaries, abstracts of journals and interactive sites full of movie clips. Indirectly, the activities opened the gates to a wide array of online resources.
Out-of-“Classroom” Interaction One problem with online learning is the perceived isolation of learners. Knowing that learning is a social experience, an Internet Café for AquaHealth learners was constructed where informal exchanges between them took place. This is where learners and specialists “meet” to learn more about each other, the nature of their work, to exchange pictures, and other personal contact that enhanced their interaction. One learner even sent a drawing depicting his interpretation of the on-going online course. While many learners and specialists keep in touch only during the course, a few remain in contact and arrange F2F meetings at every opportunity. Although “chat” was not used as a means of course delivery and discussion, it was used as a regular means of communication among learners.

Outcome of Courses

To evaluate the learners’ performance, the following assessment criteria for AquaHealth online were adopted: examination and reports (60%), discussion board participation (20%), and learning activities (20%). The total point to be accumulated was 100%, and the passing mark is 70%. All participants of AquaHealth Online were working full time and have tight work schedules, and many would have been attending to their families’ needs after work. Thus, participation and completion of requirements varied. Table 4 summarizes the performance of two batches of participants. Learners who passed the course were awarded a “Certificate of Completion”. Those who failed to get the passing mark of 70% but participated in the discussions were awarded “Certificate of Attendance”. No recognition was given to enrollees who failed to participate significantly in the discussions and they were considered drop-outs.
Successful enrollees were those with high self-motivation. Although elearning course is accessible at learners’ work place, home, cyber café, etc. some learners are unable to cope with the demands of the course concurrently with their normal workload and personal obligations. The required repeated use of resources like computers, floppy discs, printers, Internet connections, email and discussion forums to send, retrieve, and process information actually empowered rather than intimidated learners via the development of their computing skills. Absence of computer skills was less of a deterrent to learning that having no access to it at all.

Discussion

For SEAFDEC/AQD, online delivery of courses offers many benefits because it is cost-saving and course delivery through a CD with discussion through internet-based discussion boards drastically reduced or eliminated travel cost, thus decreasing per-student training expense. Online teaching also provides higher quality of interactive and flexible training using “justin” materials available in the internet. The students were very positive about the elearning format of Health Management in Aquaculture with many of them seeing it as superior to conventional classroom instruction because of the added benefit of honing
computer and internet navigation skills. The CD-ROM also provides readily accessible module contents that can be translated in the learners’ own language at their own pace. This positive impact on student learning is an outcome that most likely could not have been achieved through conventional training as has been observed by Oliver and Lake (1998). Aware that attrition is a phenomenon that occurs at an alarming rate in an online learning environment, AquaHealth Online tried to provide interesting web-based links to capture the enthusiasm and interest of learners. The present state-of-the-art in online courses shows that the F2F teaching can even be surpassed by the online course pedagogy.

Looking Forward

It is a challenge to every good researcher to be able to reach an audience worldwide at a lesser cost. In ASEAN countries where many participants’ command of English may become a deterrent to effective face-to-face learning, online learning is an effective tool since the learner can study the modules through the CDs offline at his own pace. As soon as he finds the need to interact online with his classmates worldwide, the 24x7 DB is there for asynchronous discussion. Specialists from the Fish Health Section of SEAFDEC/AQD are already experiencing the fun and benefits of online interaction in virtual classrooms. Notwithstanding the difficulty in shifting to a new teaching (mentoring) paradigm, online teaching (and learning … yes, we do learn with our learners!) is a necessa