Detection of cyprinid herpesvirus-3 DNA in lake plankton Toshifumi Minamoto a, * , Mie N. Honjo a , Hiroki Yamanaka a , Nobuyuki Tanaka b,c , Tomoaki Itayama a,b , Zen’ichiro Kawabata a a Research Institute for Humanity and Nature (RIHN), 457-4 Motoyama, Kamigamo, Kita-ku, Kyoto 603-8047, Japan b Research Institute of Environmental Eco-Technology (E-TEC), 1-5-26 Shintera, Wakabayashi-ku, Sendai 984-0051, Japan c Water and Soil Environment Division, Marine Environment Section, National Institute for Environmental Studies (NIES), 16-2 Onogawa, Tsukuba, Ibaraki 305-8506, Japan article info Article history: Received 26 February 2010 Accepted 3 July 2010 Keywords: Cyprinid herpesvirus-3 Koi herpesvirus Lake Plankton abstract The disease caused by cyprinid herpesvirus-3 (CyHV-3) severely impacts the natural freshwater ecosys- tem and damages carp and koi farming, however, the pathway of CyHV-3 transmission remains unclear. It is possible that the virus adheres to plankton, which then facilitate viral movement and transmission, and therefore, it is hypothesised that plankton are involved in the disease dynamics. In this study, plank- ton were collected at eight sites in the Iba-naiko lagoon; we detected and quantified CyHV-3 DNA from plankton samples. The results of the correlation analysis showed a significant positive correlation between CyHV-3 copies and the number of Rotifera, suggesting that CyHV-3 binds to and/or is concen- trated by Rotifera. Our results suggest that plankton affect viral ecology in the natural environment. Ó 2010 Elsevier Ltd. All rights reserved. A newly emerging fatal disease of common carp (Cyprinus car- pio carpio) and koi (Cyprinus carpio koi) is known to be caused by cyprinid herpesvirus-3 (CyHV-3). This disease was formerly known as koi herpesvirus (KHV) or carp interstitial nephritis and gill necrosis virus (CNGV). CyHV-3 was first reported in the late 1990s and has subsequently spread worldwide (Hedrick et al., 2000). It has been thought that CyHV-3 infects only carp, however, recently it was reported that other species are infected with this disease (El-Matbouli et al., 2007; Sadler et al., 2008). In some lab- oratory analyses, more than 80% of infected fish died (Ronen et al., 2003), and this highly contagious and virulent disease severely im- pacts the natural freshwater ecosystem and damages carp and koi farming. CyHV-3 belongs to the family Alloherpesviridae, and its genome consists of 295-kbp double-stranded DNA (Aoki et al., 2007). CyHV-3 DNA has been detected in the droppings and organs (e.g., gills and brain) of infected fish (Dishon et al., 2005). Furthermore, viral DNA has been detected in water from the surrounding habitat (i.e., environmental water) before, during, and after an outbreak of the disease (Haramoto et al., 2007; Minamoto et al., 2009b). Although it is known that the skin is the major access point for CyHV-3 (Costes et al., 2009), the pathway of CyHV-3 transmission remains unclear. It is possible that the virus adheres to plankton, which then facilitate viral movement and transmission. Therefore, it is hypothesised that plankton are involved in the disease dynamics of CyHV-3. In this study, we attempted to detect CyHV-3 DNA in plankton collected from Iba-naiko, a lagoon connected to Lake Biwa in Japan, where mass mortality due to CyHV-3 disease was observed in 2004. Iba-naiko is a muddy-bottomed, shallow lagoon with 49 ha of open water area, which is connected to Lake Biwa by a canal (Fig. 1). The maximum water depth is approximately 3 m and nearly all sections of the lake are shallower than 1.5 m. The lagoon is used by common carp as a feeding and reproductive habitat. A previous study reported that 54% of adult carp (>300 mm in length) caught in this lagoon and the neighbor area show antibody-positiveness, and that infection and transmission of CyHV-3 among natural carp population are still occurring through reproductive behavior of carp (Uchii et al., 2009). Surface water and plankton were collected on 14 November 2008 at eight sites in the lagoon (Fig. 1). Water quality parameters were measured on site using a multi-parameter water quality sonde (6600EDS; YSI, Yellow Springs, OH, USA). Total plankton were collected from surface water using a NXX17 plankton net (average pore size, 72 lm; Rigo, Tokyo, Japan). For identification of zooplankton and phytoplankton, fresh and fixed (2% Lugol’s solution) plankton samples were transported to the laboratory at 4 °C, and plankton were identified under an optical microscope. Prior to further processing, the plankton samples were not washed to prevent dissociation of virus and plankton. For DNA extraction, 25 ml of plankton samples were fixed on site with 50% ethanol and transported to the laboratory. Total DNA was extracted using proteinase K and SDS, and purified using the phenol–chloroform method (Sambrook and Russell, 2001); 500 ll of total DNA was 0034-5288/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2010.07.006 * Corresponding author. Tel.: +81 75 707 2333; fax: +81 75 707 2507. E-mail address: minamoto@chikyu.ac.jp (T. Minamoto). Research in Veterinary Science 90 (2011) 530–532 Contents lists available at ScienceDirect Research in Veterinary Science journal homepage: www.elsevier.com/locate/rvsc