Diagnosis of KHVD in Japan

Motohiko Sano and Takaji Iida

National Research Institute of Aquaculture, Fisheries Research Agency, Mie 519-0423, Japan

ABSTRACT

As a certain disease is designated in the Japanese law as the "Specific Disease," which is a term of law for exotic disease threatening to invade into our country and devastate aquatic industry, a guideline was prepared, which provides etiological information, symptom, diagnostic procedures and other important characteristics of the disease. Laboratory diagnosis of the disease should be conducted in accordance with this guideline.

Koi herpesvirus (KHV) infection was specified in the "Specific Disease" on June 30, 2003 and an inspection chart was established in the guideline. According to this chart, prefectural fisheries experimental stations conduct epizootic and routine clinical examination on diseased fish at first and then polymerase chain reaction (PCR) test for detecting viral DNA from the tissues of fish. PCR method described by Gray et al. (2002) is adopted in this step. In case there is some doubt of the KHV infection with inspection, the sample should be sent to the National Research Institution of Aquaculture (NRIA) in order to re-examine by PCR methods of both Gilad et al. (2002) and Gray et al. Virus isolation on KF-1 cell line is also tried. Because of the difficulty of virus isolation, result of the isolation trial is dealt with supplementary data, and therefore confirmatory diagnosis is based on the results of PCR examinations.

The Ministry of Agriculture, Forestry and Fisheries (MAFF) officially announced the first occurrence of KHV infection in Japan at the beginning of November 2003. At that time, each prefectural fisheries experimental station did not have enough equipments and skills in doing PCR test for KHV detection. Therefore, most PCR tests were carried out in NRIA until PCR system in the experimental stations was prepared, and confirmatory diagnosis was carried out on the result of PCR in NRIA. A seminar in NRIA to learn PCR procedure for KHV detection was held for the research staff of each prefectural fisheries experimental station in mid-November. After that, PCR inspection for KHV infection was established to do in accordance with the chart in the guideline.

NRIA accepted 529 fish of 87 cases to be diagnosed, KHV-infected carp were found in 23 out of 47 prefectures. The sequences of the PCR products from the samples were identical and there was no nucleotide mismatch in the 9/5 primer regions reported by Gilad et al. KHV was isolated from tissue samples in a few cases, and their characteristics including pathogenicity are now under investigation.