Detection of koi herpesvirus DNA in river water in Japan

Koi herpesvirus (KHV) has a diameter of 170– 230 nm and possesses a double-stranded DNA genome (Pokorova, Vesely, Piackova, Reschova & Hulova 2005). KHV was first discovered in the USA in 1998, followed by outbreaks in koi, Cyprinus carpio koi and common carp, C. carpio carpio, in Israel and the USA (Hedrick, Gilad, Yun & Spangenberg 2000). The virus subsequently spread to numerous countries worldwide (Haenen, Way, Bergmann & Ariel 2004). In Japan, the first large outbreak of KHV occurred in October 2003 in Kasumigaura Lake, where more than half of Japan’s farmed carp are produced (Sano, Ito, Kurita, Yanai, Watanabe, Miwa & Iida 2004). Subsequently, many outbreaks were reported in almost all of the prefectures in Japan, probably due to transport of infected carp from Kasumigaura Lake to other lakes, ponds or rivers (Kimiya 2004). Koi herpesvirus is suspected to be transmitted via water as the virus is excreted with faeces of the infected carp (Dishon, Perelberg, Bishara-Shieban, Ilouze, Davidovich, Werker & Kotler 2005). The virus can grow in carp at water temperatures from 15 to 25 C (Gilad, Yun, Adkison, Way, Willits, Bercovier & Hedrick 2003), thus KHV infection is suppressed during winter months. However, outbreaks of KHV can reoccur the following spring, partly because the virus remains infective in water for a long period (Perelberg, Smirnov, Hutoran, Diamant, Bejerano & Kotler 2003). Before resuming koi farming after an outbreak, it is important to confirm the absence of KHV in the water source. In our previous studies, we succeeded in detecting various kinds of human enteric viruses, such as noroviruses, adenoviruses or enteroviruses, in aquatic environments (Katayama, Shimasaki & Ohgaki 2002; Haramoto, Katayama, Oguma & Ohgaki 2005). A key step in the procedure is concentration of virus particles (<1 mL concentrated virus sample from 100 to 1000 mL of water sample). Viruses were then detected by polymerase chain reaction (PCR) amplification of the concentrated sample. This method may be more broadly applicable to virus detection in aquatic environments and was used in this study to detect KHV. The diagnosis of KHV infection is usually based on virus isolation using KF-1 cells, followed by amplification of viral DNA using the PCR technique (Pokorova et al. 2005). However, due to the limited susceptibility of KF-1 cells, it is sometimes difficult to isolate KHV even from tissues with high titres of KHV, such as the gill, kidney and spleen of carp (Pokorova et al. 2005). Recently, a real-time quantitative TaqMan PCR system was developed for rapid, sensitive and specific detection of KHV (Gilad, Yun, Zagmutt-Vergara, Leutenegger, Bercovier & Hedrick 2004); this system could be a powerful tool to detect KHV at low levels in water. In this study, the occurrence of KHV in river water in Japan was investigated using a virus concentration method developed in our previous study (Haramoto, Katayama & Ohgaki 2004) in concert with a real-time PCR system (Gilad et al. 2004). Journal of Fish Diseases 2007, 30, 59–61