Special issue: International Symposium on Koi Herpesvirus Disease


abstract

Keynote
Title Initial Isolation and Characterization of a Herpes-like Virus (KHV) from Koi and Common Carp
Authers Ronald P. HEDRICK, Oren GILAD, Susan C. YUN, Terry S. MCDOWELL, Thomas B. WALTZEK, Garry O. KELLEY and Mark A. ADKISON
Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California, Davis, California 95616 USA
Keywords koi, common carp, koi herpesvirus, KHV, herpes-like viruses
Citation Bull. Fish. Res. Agen. supplement No.2, 1-7, 2005
Abstract
In September of 1998 our laboratory was asked to investigate epidemics characterized by high mortality occurring in populations of koi (Cyprinus carpio) in both the U.S.A. and Israel. Either live fish or frozen tissues arrived from both locations in September and November of 1998. In addition, tissues fixed for both light and electron microscopy from fish in Israel and the U.S.A. were obtained for examination. The virus observed and then isolated in 1998 from these initial samples has become the focus of a worldwide concern for the health and welfare of both captive and wild populations of common carp and ornamental koi. In this short report we review the initial isolation and characterization of the herpes-like virus referred to as koi herpesvirus or KHV. We also discuss the important role of temperature on in vivo and in vitro infections with the virus, recent comparisons of KHV to other herpeslike viruses from fish and lastly review current methods to detect the virus or evidence that fish have been exposed to the virus.


Country Report
Europe
Title Prevention of a Mortal Disease of Carps Induced by the Carp Interstitial Nephritis and Gill Necrosis Virus (CNGV) in Israel
Authers Ariel RONEN, Ayana PERELBERG, Marina HUTORAN, Yechiam SHAPIRA, Michael STEINITZ, Berta Levavi-SIVAN, Eli PIKARSKY and Moshe KOTLER
Department of Pathology, The Hebrew University-Hadassah Medical School, Jerusalem, ISRAEL
Citation Bull. Fish. Res. Agen. supplement No.2, 9-11, 2005
Abstract
Massive mortality of Koi and Common carp - Cyprinus carpio species - was observed in many farms throughout Israel, resulting in severe financial losses.
 This lethal disease is highly contagious and extremely virulent, but morbidity and mortality are restricted to Koi and Common carp populations .
 We isolated a carp nephritis and Gill necrosis virus (CNGV), which is the etiologic agent of this disease. The virus propagates and induces severe cytopathic effects five days post infection in fresh Koi fin cell cultures (KFC). The virus harvested from KFC cultures induced the same disease with mortality of 75-95% upon inoculation of naive Koi and Common carp (Pikarsky et al., 2004).
 Electron microscopy revealed viral cores with icosahedron morphology of 100-110 nm resembling the herpes virus. Electron micrographs of purified pelleted CNGV sections, together with sensitivity to ether and Triton x 100 suggest that it is an enveloped virus. However, the genome of the isolated virus is a double-stranded DNA molecule of 250-300 Kbp, larger than that of known Herpesviridae members. The viral DNA seems highly divergent and bears only small (16-45 bp) fragments similar to the genomes of several DNA viruses. We suggest, therefore, that the etiologic agent of this disease may represent as yet unclassified virus species endemic to cyprinids (Hutoran et al., 2004).
 Carps, exposed to the virus at 23 ℃ for 3- 5 days and then transferred to the nonpermissive temperature of 30 ℃ , became resistance to a challenged infection and their sera demonstrated a high level of virus specific antibodies .
 We have isolated attenuated nonpathogenic viruses that render virusvaccinated carps resistant to the disease (Ronen et al., 2003). Furthermore, vaccinated fish developed high levels of antibodies against the virus. We suggest, therefore, that this attenuated virus could be used as a live vaccine for the eradication of the mortal disease afflicting common and ornamental carp fisheries in many countries (Perelberg et al., 2005).
 We examined the pathobiology of this disease in carp using immuno histochemistry. We found large amounts of the virus in the kidneys of sick fish, and lesser amounts in liver and brain. A rapid increase in the viral load in the kidneys was detected using both immuno-fluorescence and semi-quantitative PCR. Histological analyses of fish at various times after infection revealed signs of interstitial nephritis as early as 2 days postinfection, which increased in severity up to 10 days post-infection. There was severe gill disease evidenced by loss of villi with accompanying inflammation. Minimal focal inflammation was noted in livers and brains.
 Two diagnostic methods for identifying the CNGV in live fish are in use in our laboratory: Using PCR with authentic primers is applied on blood, kidney or gills DNA samples and immunological diagnostic kit. The immunological kit is designed to be a simple, rapid and low cost diagnostic kit, which will be appropriate for retailers as well as hobbyists to identify the CNGV in presymptomatic fish. We believe that these means will be instrumental in preventing the distribution of sick fish world wide.

Title KHVD, Diagnosis, Control, Research and Future in The Netherlands and Europe
Authers Marc Y. ENGELSMA and Olga L.M. HAENEN
CIDC-Lelystad, Fish and Shellfish Diseases Laboratory, P.O. Box 2004, 8203 AA Lelystad, The NETHERLANDS
Citation Bull. Fish. Res. Agen. supplement No.2, 13-14, 2005
Abstract
In recent years koi herpesvirus disease (KHVD) has quickly spread over Europe. In several European countries the first KHVD cases were observed in 2002 (Italy, Denmark) and 2003 (France, Austria, Switzerland). In contrast to Western Europe, where koi and carp are mainly kept as ornamental fish, in Eastern Europe carp is farmed for consumption and KHVD could have a huge impact on the carp industry. At the moment however, Eastern Europe still seems free from KHVD. However, common carp exported from Poland to Germany were shown to be KHV positive. The highest incidence of KHVD cases in Europe are found in Germany, England and the Netherlands.
 Germany has had over 250 cases since 2002. England had 36 cases in 2002, and more cases followed in 2003.
 In the Netherlands a steady increase was seen from 2 KHV positive cases in 2001 up till 27 cases in 2003 .
 Concern for these countries is that KHVD could possibly not only be restricted to koitrading and private ponds but already be spread into the natural environment.
 At the moment however there is still no evidence for this. As KHVD is not notifiable for the European Union (EU), the control on spread of the disease within the EU is minimal.
 The majority of the KHV cases in the Netherlands were detected by PCR (Gilad et al., 2002), with or without using gill histopathology as a confirmatory method . In a few cases the herpes virus could be cultured successfully on Epithelioma Papilloma Carposum (EPC) or Koi Fin-1 (KF- 1) cells .
 In 2001 we succeeded in isolating the virus for the first time in the Netherlands from a clinical diseased koi. The inoculated sample of gills and pooled internal organs showed cytopathic effect (CPE) at the 2nd passage on EPC cells.
 Unfortunately the sensitivity of viral isolation is inadequate for diagnostics of KHV. Additional methods, as PCR, are required for correct diagnosis of the disease.
 Currently effort is put into developing alternative detection methods as immunofluorescence tests (IFT) to support diagnostics of KHVD .


Asia
Title Indonesian Experience on the Outbreak of Koi Herpesvirus in Koi and Carp (Cyprinus carpio)
Authers Agus SUNARTO1, Akhmad RUKYANI2 and Toshiaki ITAMI3
1: Fish Health Research Laboratory, Jl. Ragunan 20, Jakarta 12540, INDONESIA
2: Directorate of Fish Health and Environment, Jl. Harsono RM 3, Jakarta, INDONESIA
3: Miyazaki University, Gakuen Kibana-dai Nishi 1-1, Miyazaki 889-2192, JAPAN
Keywords Koi herpesvirus (KHV), Outbreak, Control, Indonesia
Citation Bull. Fish. Res. Agen. supplement No.2, 15-21, 2005
Abstract
Koi Herpes Virus (KHV) is a new emerging disease known to cause gill and skin damage in koi and carp (Cyprinus carpio). The disease suspected to have been introduced into Indonesia through importation of koi from Hongkong. It is currently occurring in Indonesia since March 2002 starting in the area of Blitar in East Java. Since then it has been spreading rapidly throughout Java Island, Bali, southern part of Sumatra, East Kalimantan and Central Sulawesi. The disease cause very high mortality (80-95%) to both koi and common carp with estimated losses of more than 150 billion rupiahs (equal to US$15 M) as of December 2003. To prevent the spread of the outbreaks to other islands, the Government of Indonesia issued Ministerial Degrees that declared Java and Bali Islands as an isolated area of the disease and moving koi and carp from Java and Bali Islands to other islands are strictly prohibited or should follow quarantine check for KHV. In addition, importing koi and common carp is permitted only from free KHV countries. A Task Force consisted of international, national and local experts were organized to conduct emergency assessment of the disease situation through epidemiological investigations, field observations and laboratory examinations. Information on KHV was disseminated widely across the country with the use of TV, radio, newspaper, posters, pamphlets and technical guideline. In farm level, biosecurity concept was applied to reduce the risk of KHV outbreak. The implementations of government regulations pertaining the outbreak and key components of biosecurity are discussed in this paper.

Title The Status of Viral Diseases of Carp in Korea: Its Control and Research Development
Authers Mi-Young CHO1, Sang-Kyu SHON2 and. Soo-Il PARK3
1: Pathology Team, National Fisheries Research and Development Institute 408-1, Shirang-ri, Kijang-up, Kijang-gun, Busan 619-902, Republic of KOREA
2: Aquaculture and Environment Institute, NFRDI, 361, Yeongun-ri, Sanyang-up, Tonyeong-si, Kyeongsangnam-do 650-943, Republic of KOREA
3: Department of Aquatic Life Medicine, Pukyoung National University, 559-1, Daeyon-dong, Nam-gu, Busan 608-739, Republic of KOREA
Citation Bull. Fish. Res. Agen. supplement No.2, 23-25, 2005
Abstract
The carp (Cyprinus carpio) is the most common species of cultured fish, in particular, common carp and Israel carp have been cultured as an edible fish for several decades in Korea. Viral diseases of carp have never been reported until 1998 despite its long history of aquaculture in Korea. However, mass mortality occurred in populations of cultured carp from 1998 to 1999 resulting in a drastic reduction of carp production in Korea. The infected fish showed dark coloration, excessive mucus secretion from the surface and gills and branchial necrosis.
 Several cases were reported that dermal ulcers were noticed due to secondary infection by bacteria or parasites.
 In moribund carp, the clinical signs were formation of white patches on body surface or gills due to excessive mucus secretion. Dermal lesions caused by secondary infection were also found.
 So, we called that the viral disease is a “viral systemic necrosis of carp (VSNC)” based on their clinical signs. The mortality was the highest at 20~28℃, and there being virtually no occurrence at below 20℃ or above 30℃. Although the characteristics of VSNC are similar to those of KHV, PCR tests proved that it was not KHV.
 Oh et al. (2001) reported that the virions with 70~80nm diameter were presumed to be a causative virus in the kidney and spleen tissues of infected fish. They also reported that the artificial infection using a viral suspension of 103TCID50/mL obtained from the infected FHM (fathead minnow) cells, gave 80 to 100 percent mortality in carp.
 Kim et al. (2002), as a result of conducting gene cloning of the virus, suggested that the virus being a new species different from the known reported viruses of carps such as grass carp reovirus (GCRV), grass carp hemorrhage virus (GCHV), koi herpesvirus (KHV) and spring viremia of carp virus (SVCV).
 Unlikely such findings, Choi et al. (2004) found a herpes-like virus of 82nm size within the nucleus of spleen cells from infected carps .
 During the past three years, the present study was established to development of prevention technologies for epidemics in order to protect the carps from the viral diseases.
 First, two types of inactivated vaccines were prepared with the viral suspension of VSNC that obtained from the infected FHM cells: formalin-killed vaccine (FKV: 0.4% formalin) and heat-killed vaccine (HKV: 60℃, 1hour).
 The lysozyme activity of serum and chemiluminescent responses of head-kidney leucocytes showed increasing in the vaccinated fish, and as measured by ELISA (enzyme-linked immunosorbent assay), vaccinated groups showed a significant increasing in the virus-specific serum antibodies between 2 week post-first injection and 6weeks post-boost injection.
 Results of the virus challenge showed that the fish vaccinated with FKV have induced protective immunity, while HKV injection hardly provided protection. And secondly, the antiviral activity of carp was examined using the potent inducers of interferons such as poly inosinic: cytidylic acid (Poly I:C) and spring viremia of carp virus (SVCV).
 The results showed that carp injected Poly I:C developed an anti-viral protection which resulted in lower cumulative mortality against SVCV than untreated fish did. Moreover, in vitro study showed that head kidney leucocytes produced an interferon-like cytokine (ILC) after stimulation with Poly I:C.
 Since 1998, many studies have been conducted in diversified aspects to find out the causative virus of the diseases and prevention measures against viral disease of carp in Korea. However, identification of the virus was not clear and effective control measures were not established so far. Therefore, further studies on the causative virus including characterization and infection mechanisms are required.

Title Viral Studies on Carp Disease in China
--- with a Special Reference to Herpesvirus on Common Carp---
Authers Xu PAO and Min KUANHONG
Freshwater Fisheries Research Center Chinese Academy of Fishery Sciences, Wuxi 214081, CHINA
Keywords viral diseases, Chinese carps, herpesvirus on carps, preventive measures
Citation Bull. Fish. Res. Agen. supplement No.2, 27-33, 2005
Abstract
The present paper mainly deals with the general situation of the viral diseases affected on the Chinese carps in a recent decade in China. The viral disease is highly characterized by the fast transmission, great mortality and being difficult to control which has caused a heavy loss in the aquaculture systems. The paper further and specifically introduces the general status of herpesvirus on carps in China and preventive measures against the outbreak of the disease from the ecological point of view and the environmental issues.

Title Quarantine, Surveillance and Monitoring of Koi Herpesvirus in Singapore
Authers Kai Huat LING, Susan Ling Fung KUEH and Yew Kwang POH
Agri-Food & Veterinary Authority of Singapore 5 Maxwell Road SINGAPORE 069110
Keywords Koi Herpes Virus, Accredited Ornamental Fish Exporters Scheme, laboratory examinations, surveillances, Aquatic Animal Disease, consignments, compulsory inspection, Quarantine
Citation Bull. Fish. Res. Agen. supplement No.2, 35-40, 2005
Abstract
The Koi industry in Singapore is a sizable one. Thus the potential introduction of any significant disease such as Koi Hrpesvirus (KHV) or Spring Viremia of Carp Virus (SVCV), will be of concern to Singapore. Singapore’s ornamental fish are imported and exported by traders licensed under the ‘Accredited Ornamental Fish Exporters Scheme’. Under this Scheme, the exporters have to get their premises approved according to guidelines set by the Agri-Food and Veterinary Authority of Singapore (AVA), which include the provision of designated quarantine area, fish disease treatment area, and packing area. These approved premises are inspected monthly by AVA inspectors. As part of routine fish disease surveillances conducted by AVA, regular fish samples are taken from each exporter premise once every six months for laboratory examinations, to ensure the absence of any significant diseases. Additional samples are taken for laboratory examinations at greater frequencies should any significant disease outbreaks occur in these premises. Significant results from these surveillances are reported in the Quarterly Aquatic Animal Disease Report (Asia and Pacific Region), which is submitted to the OIE and NACA. Since January 2003, no positive case has been detected to date from AVA’s surveillance for KHV in Singapore.

Title Thailand’s Current Quarantine Status on Aquatic Animal Diseases
Authers Somkiat KANCHANAKHAN
Inland Aquatic Animal Health Research Institute, Bureau of Inland Fisheries Research and Development Department of Fisheries, Paholyothin Rd., Jatuchak, Bangkok 10900 THAILAND
Keywords aquatic animal quarantine, koi herpesvirus, KHVD, trans-boundary disease, import requirement
Citation Bull. Fish. Res. Agen. supplement No.2, 41-45, 2005
Abstract
An awareness of aquatic animal diseases spread through an international trade has been increasing since the first edition of OIE on Aquatic Animal Health Code in 1995. How to control the diseases through an international trade and a development of national strategy for controlling the diseases had been discussed in great details among representative from 21 Asian governments during a three years (1998-2000) seminar and workshop program organised by NACA. As part of the region, the strategic plans for aquatic animal health management in Thailand have been developed. Presently, Thailand has over 21import/export regulations announced through Emergency Decree, Royal Decree, Ministerial Regulation, Notification or Rule to control the movement of certain aquatic animals both from importation and exportation. The aquatic animal disease quarantine enforcement is based on the Animal Epidemic Act B.E. 2499 (1956) and Fishery Act B.E. 2490 (1947). The Department of Fisheries has developed an importation of live aquatic animals under quarantine enforcement framework, which is based on disease control program and disease surveillance. The quarantine measures include pre-arrival, arrival at the entry port and post-arrival of aquatic animals that will be discussed. Koi herpesvirus disease surveillance system has been in place since August 2002 and Thailand is still free.


Japan
Title Survivability of Fish Pathogenic Viruses in Environmental Water, and Inactivation of Fish Viruses
Authers Mamoru YOSHIMIZU, Toko YOSHINAKA, Shuichi HATORI, and Hisae KASAI
Faculty of Fisheries and Graduate School of Fisheries Sciences, Hokkaido University, Hakodate, Hokkaido 041-8622, JAPAN
Keywords fish virus, survivability, environmental water, inactivation, disinfectant, UV, ozonization, electrolyzation
Citation Bull. Fish. Res. Agen. supplement No.2, 47-54, 2005
Abstract
Survival of three salmonid viruses and two marine fish viruses in fish rearing water or coastal sea water were observed at 0, 5, 10 and 15 ℃ for 7 or 14 days. Interaction between viruses and microorganisms present in the rearing water was observed. Infectious pancreatic necrosis virus (IPNV) and fish nodavirus (BF-NNV) were stable in waters used at every temperature tested for 14 days, but it was observed that, for infectious hematopoietic necrosis virus (IHNV), Oncorhynchus masou virus (OMV), and hirame rhabdovirus (HIRRV), as the temperature increased, the loss of infectivity also increased. When IHNV and OMV were suspended in filtrated and autoclaved rearing water, infectivity was reduced in comparison with the untreated water. Subsequently, adsorption of IHNV to mud or small particles was studied. IHNV adsorbed to several clays (kaolin, bentonite, Japanese acid clay) and diatomaceous earth in sterilized water with a wide range of pH (5-11) at concentrations of 1, 10, and 100 mg/mL. Except for bentonite, infectivity of clay-adsorbed IHNV persisted for at least 9 weeks. The clay-adsorbed IHNV also persisted in infectivity to rainbow trout Oncorhynchus mykiss, causing cumulative mortality rates of more than 73 %. Then, inactivation effects of UV irradiation, ozonization, and electrolyzation of water were studied against six fish rhabdoviruses, three fish herpesviruses, one fish birnaviruses, one fish iridovirus, and one fish nodavirus. Six rhabdoviruses, three herpesviruses, and lymphocystis disease virus were found to be sensitive to UV irradiation, ozonization, and electrolyzation. Susceptibility of IPNV, chum salmon virus (CSV), and BFNNV to UV was found to be low. IPNV and CSV were low sensitive to ozonization and electrolyzation. Virucidal effects of six kinds of disinfectants were examined against OMV, IPNV, IHNV, and HIRRV at 15 and 20℃ for 30 sec and 20 min. At 15℃ for 20 min, minimum concentrations showing 100 % plaque reduction of viruses tested by iodophore, sodium hypochlorite solution, benzalconium chloride solution, saponated cresole solution, formaldehyde solution, and potassium permanganate solution were 40, 50, 100, 100, 3500, and 16 ppm, respectively.

Title The Present State of Carp Fisheries and Aquaculture in Japan
Authers Kazumasa IKUTA and Motoyoshi YAMAGUCHI
Ecosystem Conservation Section, Freshwater Fisheries Research Division, National Research Institute of Fisheries Science, Fisheries Research Agency Ueda, Nagano 386-0031, JAPAN
Keywords carp, nishikigoi (koi), Lake Kasumigaura, KHV infection
Citation Bull. Fish. Res. Agen. supplement No.2, 55-58, 2005
Abstract
Carp (Cyprinus carpio), which is thought to have originated from central Asia, is the world’s oldest aquacultured fish. In Japan, farmers have cultured carp in paddy fields for nearly two thousand years, particularly in inland areas, such as in Nagano, Gunma and Akita Prefectures, which have been traditionally characterized by a lack of animal protein resources. Since the 1960s, carp production, particularly in Lake Kasumigaura in Ibaraki Prefecture, has been accelerated by new developments in artificial feed and net-pen aquaculture technology. Culture levels peaked in 1977, when total annual production was about 30,000 tons. Subsequently, with the diversification of people’s food preferences and product availability, total production decreased gradually in accordance with lower demand and depressed prices. As a consequence, total annual production of cultured carp reached a low of 9,949 tons in 2001.
 While, nishikigoi (koi) have been bred as ornamental fish since the Edo Era in the 18th century. Nowadays, koi shows and contests are important activities inside of Japan, and koi traded is conducted throughout the country. International trading is also being actively promoted, and these fish are well-known and admired in many foreign countries. Nishikigoi farms are located in many areas of Japan; 48% of the farms are found in Niigata Prefecture, followed by Gifu (8%) and Hiroshima (6.5%) Prefectures.

Title Diagnosis of Koi Herpesvirus (KHV) Disease in Japan
Authers Motohiko SANO1, Takafumi ITO1, Jun KURITA1, Satoshi MIWA1 and Takaji IIDA2
1:Inland Station, National Research Institute of Aquaculture, Fisheries Research Agency, Hiruta 224-1, Tamaki, Mie 519-0423, JAPAN
2:National Research Institute of Aquaculture, Fisheries Research Agency, Nakatsuhamaura 422-1, Nansei, Mie 516-0193, JAPAN
Keywords diagnosis, koi herpesvirus, KHV, specific disease, common carp, Cyprinus carpio
Citation Bull. Fish. Res. Agen. supplement No.2, 59-64, 2005
Abstract
The koi herpesvirus (KHV) disease was designated as a “Specific Disease” in a Japanese law amended on June 30, 2003. The Ministry of Agriculture, Forestry and Fisheries officially announced the first case of KHV disease in cultured common carp Cyprinus carpio in Japan in early November 2003. At that time, the Prefectural Fisheries Experimental Stations (PFESs), which belong to local government, lacked sufficient equipment and skill required to conduct a polymerase chain reaction (PCR) tests for KHV detection. Therefore, at first, most PCR tests for KHV were carried out at the National Research Institute of Aquaculture (NRIA), Fisheries Research Agency (FRA). The NRIA gave a short training course of PCR for the research staff of PFESs in mid-November. According to the established guideline for the KHV disease, the PFESs now conduct epizootic and routine clinical examinations on diseased fish followed by PCR test to detect viral DNA in the tissues. When there is a positive reaction with the PCR test, the sample is sent to the NRIA for further confirmation by PCR. Virus isolation on the KF-1 cell line is also tried, but confirmatory diagnosis is based on the results of PCR tests. By the end of 2003, the NRIA has accepted 529 individuals of 87 cases to be diagnosed and KHV-infected carp have been found in 23 out of 47 prefectures.

Title The Status of Koi Herpesvirus Disease Outbreaks in Lake Kasumigaura and Kitaura
Authers Youji TAKASHIMA1, Naoki WATANABE1, Takanori YANAI1 and Takeo NAKAMURA2
1: Ibaraki Prefectural Freshwater Fishery Research Institute, Tamatsukuri, Namekata-gun, Ibaraki 311-3512, JAPAN
2: Ibaraki Prefectural Kasumigaura-Kitaura Fishery Office, Tuchiura, Ibaraki 300-0051, JAPAN
Keywords koi herpesvirus, KHV,common carp, Cyprinus carpio,Lake Kasumigaura and Kitaura
Citation Bull. Fish. Res. Agen. supplement No.2, 65-71, 2005
Abstract
Lake Kasumigaura and Kitaura in Ibaraki Prefecture is the second largest lake in Japan. In the lake, common fisheries such as trawling and set net as well as net cage culture are practiced. Net cage culture produces about 5,000 tons of edible common carp (Cyprinus carpio) per year, which is one half of the total production in Japan. At the beginning of October 2003, deaths of unknown origin of common carp in net cages occurred in Lake Kasumigaura. After inquiry of the cause, PCR testing detected DNA of the Koi herpesvirus (KHV) in the affected fish and natural carp captured in Lake Kitaura, confirming the occurrence of the Koi herpesvirus disease (KHVD). We conducted an interview survey on aquaculturers regarding the date, quantity, and circumstance of death of cultured carp in Lake Kasumigaura and Kitaura, and investigated the circumstances of death in net cage farms. Another interview survey was also conducted on other fishermen regarding the occurrence of abnormality in natural carp, and both cultured and natural carps were sampled for PCR testing. The death of cultured carp in Lake Kasumigaura was generated in two areas dense with net cage farms, and then was likely expanded. The cumulative amount of death according to the interview surveys increased rapidly: 200-300 tons in the middle of October, 660 tons at the end of the same month, and 1,200 tons at the beginning of November. The damage amount reached one quarter that of the annual common carp products. Since the KHVD is one of the designated diseases under the Law to Ensure Sustainable Aquaculture Production, to prevent the spread of this disease according to this law, Ibaraki Prefecture requested aquaculturists of Lake Kasumigaura and Kitaura to exercise restraint on the transfer of cultured carp in net cages on November 2, and issued orders prohibiting transfer on November 12 and implementing incineration and landfill disposal on December 21. The incineration disposal commenced from January 20. This report aims to shed light on the state of mass cultured carp kills in Lake Kasumigaura and Kitaura, caused by this KHV, and the state of outbreaks of abnormal fish among cultured and natural carp.

Title Preventive Measures against Koi Herpesvirus Disease in Fancy Carp in Niigata Prefecture
Authers Kazuo YAMADA, Hideyuki KARAKISAWA and Hiroko WATANABE
Niigata Prefectural Inland-water Fisheries Experimental Station, Ogawara-machi 2650, Nagaoka, Niigata 940-1137, JAPAN
Citation Bull. Fish. Res. Agen. supplement No.2, 73-75, 2005
Objectives
 Following by the outbreaks of koi herpesvirus (KHV) in worldwide including Israel, many areas of Europe, the United States, and Indonesia, the Fisheries Division of the Niigata Prefectural Government started implementing a polymerase chain reaction (PCR) tests on the prefecture’s carp in October of 2001.
 The aim of the test was twofold--to prevent the entry of the KHV into the prefecture and to prevent the spread of the virus within and out of the prefecture, both domestic and abroad, if the virus is detected in carp in Niigata. Although there have been no reports of a KHV outbreak in Niigata’s fancy carp, the prefecture’s fancy carp industry has voluntarily suspended all of its shipments temporarily. This measure has been taken to prevent the possible transfer of the virus following the detection of KHV in common carp in Niigata on November of 2003 (Note: These KHV-affected fish, originally imported from Ibaraki prefecture, were obtained from an entertainment fishing pond and a breeder of common carp in Niigata prefecture.).

Method
 A polymerase chain reaction (PCR) tests were conducted in Niigata prefecture during the following three periods: from October to November of 2001 on fancy carp owned by twenty carp breeders, in December of 2002 on fancy carp owned by thirty carp breeders, and from July to August of 2003 on fancy carp owned by 114 breeders. (Note: Many of the breeders have participated in the PCR tests in multiple years.)
 The procedures of PCR-test were as follows : 1) To avoid a great financial loss, sample fish were selected only for young fancy carps less than one-year old which mean valueless in terms of unable to reproductive activities. 2) These young carps were kept in tank during 3 weeks under maintaining condition of water-temperature from 20℃ to 25 ℃ , together with adult ones that would be shipped for commercial purposes. 3) Sample fish for examined were maintained by designated staff members who conduct PCR tests under the supervision of the Niigata Prefectural Inland-water Fisheries Experimental Station. 4) Afterwards tissues from the gill, spleen, kidney, and heart (sometimes just a sample from the gill tissue) were taken from two to five of each sample fish. 5) These tissues were homogenized to extract DNA, which was obtained by using a DNA extraction kit. 6) Each sample was mixed with an agent that contained types of primers such as 9/5 and SphI-5. 7) After this stage, the samples received an agarose gel electrophoresis treatment to examine the DNA fragments. and 8) A KHV DNA sample, which Niigata had received from Dr. R. P. Hedrick, was used to compare the results of the samples taken in Niigata.

Results
 All the fish that were examined in the PCR tests during the previously specified periods tested negative. During these test periods also, Niigata provided carp business owners in the region with meetings and lectures about KHV to enable the breeders to be able to detect KHV-affected carp at an early stage of the disease and thus minimize the risk of spreading the virus.
 Currently PCR tests are being conducted on the largest scale ever in Niigata with the participation of 150 carp breeders, each of which presented tissue samples from thirty-one fish from their farm. Originally, the voluntary policy to temporary suspend ornamental carp shipments prohibited any shipments of fancy carp until the test results of the fish from all the breeders concerned were confirmed; however, the voluntary policy to temporary suspend fancy carp shipments has been revised to allow overseas shipments of fish upon confirmation of favorable test results of the fish from each individual breeder.
 Upon Niigata Prefectural Government’s request, the carp industry in Niigata is creating a KHV prevention policy that instructs carp breeders how to avoid exposing fish to the virus. Under this policy, any fish that is shipped from another breeder will have to be isolated from other fish in a low temperature water tank for three weeks during which time the breeder will check to see whether or not the fish is infected with the virus. It also advises the breeder to avoid using river water when maintaining the fish and to sanitize all equipment that comes in contact with fish water or the fish themselves, including staff’s boots and gloves. Staff members are also required to wash their hands at their facility and to keep a detailed journal of the conditions of their fish.

Title Further Researches for the Control of KHVD in Japan
Authers Satoshi MIWA
Fish Health Division, National Research Institute of Aquaculture Fisheries Research Agency, Hiruta 224-1, Tamaki, Mie 519-0423, JAPAN
Citation Bull. Fish. Res. Agen. supplement No.2, 77, 2005
In the fall of 2003, the first outbreak of Koi Herpes Viral disease occurred in Lake Kasumigaura in Japan, and the disease spread rapidly over the country. The disease has caused massive mortality in both cultured and wild carp. As the water temperature has decreased in winter months, the disease seems to have been suppressed now, but people fear that the disease will reappear in this coming spring through summer. Prevention of KHV outbreak in cultured carp and control of the disease in wild waters are strongly required.
 To achieve these goals, Fisheries Research Agency is planning a research project in collaboration with both academic and private sectors. There are three major areas of research or development in the project. In the first area, the basic pathogenesis of the disease and molecular genetics of KHV will be studied. Since the mechanisms or steps whereby KHV brings about disease in carp are largely unknown, these will be studied using histopathological techniques.
 Epizootiological study including molecular genetics of the virus will also be conducted, so that the spreading route of the disease in Japan will be clarified, at least partly.
 Second, new diagnosis or detection methods for KHV will be developed. Currently, diagnosis of the disease is exclusively carried out by PCR. However, this method requires a thermal cycler, and there is a concern that the present PCR methods for KHV may not be sensitive enough to detect the virus at the latent stage of the disease. On the other hand, PCR has a possibility of yielding false positive results by contamination of a minute amount of viral genes. Hence, techniques with which we can overcome these problems will be developed, as well as improvement of the present PCR techniques.
 Third, preventive measures for KHV should be developed. Susceptibility of KHV to various chemicals and physical treatments will be tested to develop effective methods of disinfection of fish farming equipment and rearing water. Development of inactivated vaccine and effective vaccination techniques will also be tried. Furthermore, a procedure will be developed to cure the diseased fish by raising water temperature.

Title Proposed Activities for Koi Herpesvirus Disease at the SEAFDEC Aquaculture Department
Authers Kazuya NAGASAWA
Southeast Asian Fisheries Development Center, Aquaculture Department Tigbauan 5021, Iloilo, PHILIPPINES
Keywords koi herpesvirus disease, KHV, common carp, koi, Cyprinus carpio, Southeast Asian Fisheries Development Center, Southeast Asia, Regional Fish Disease Project, proposed activities
Citation Bull. Fish. Res. Agen. supplement No.2, 79-86, 2005
Abstract
The Southeast Asian Fisheries Development Center (SEAFDEC) is a regional treaty organization with 11 member countries. This was established in 1967 to promote fisheries development in Southeast Asia. As one of four SEAFDEC departments, the Aquaculture Department based in Tigbauan, Iloilo, Philippines, has conducted activities for aquaculture research and development in the region. Since 2000, the Regional Fish Disease Project has been implemented at the SEAFDEC Aquaculture Department through the Government of Japan Trust Fund. Under this project, research studies were conducted on various aspects of viral, bacterial and parasitic diseases of fishes and shrimps. In East Asia, koi herpesvirus (KHV) disease initially occurred in Indonesia and Taiwan in 2002. KHV infection was also found in Japan in 2003. This disease had a serious, devastating impact on common carp and koi (Cyprinus carpio) production in Indonesia and common carp production in Japan. Common carp is an important food resource in the rural areas of the region, while koi is internationally traded as ornamental fish among Southeast Asian countries. Under these situations, the Regional Fish Disease Project identified KHV as a serious, transboundary pathogen in the region and decided to work on KHV disease at the SEAFDEC Aquaculture Department in coordination with the SEAFDEC member countries to prevent the spread of KHV in the region. The planned research includes survey of the distribution of KHV in the region, standardization of the PCR (polymerase chain reaction) detection method, characterization of the virus isolated from the region, mode of transmission of KHV, and pathophysiology of KHV-infected fish. To support establishment of the fish disease quarantine and surveillance in Southeast Asia, the Regional Fish Disease Project has, since 2002, annually conducted a hands-on training at the SEAFDEC Aquaculture Department on viral diseases of fishes and shrimps for scientists and technical staff from the SEAFDEC member countries. The trainees are expected to play key roles in the diagnosis, prompt information exchange, and surveillance of fish diseases, including KHV disease, in their respective countries. The Regional Fish Disease Project organized two meetings in March 2004 and will convene another meeting in June 2004: Pre-KHVD Symposium Meeting, International Symposium on Koi Herpesvirus Disease, and Meeting on Current Status of Transboundary Fish Diseases in Southeast Asia: Occurrence, Surveillance, Research and Training.