Detection of koi herpesvirus (KHV) a er re-activation in persistently infected common carp (Cyprinus carpio L.) using non-lethal sampling methods

Surviving carp which had recovered from KHVD were kept for approximately eleven additional weeks at 20°C water temperature. To induce KHV re-activation, carp were subjected to ne ing stress on day 81 a er infection and gill swabs and dropping samples were collected daily for investigation by quantitative real-time PCR. An increase of KHV concentration of up to 1000 KHV genomic equivalents was detected over a three day period post-ne ing from these non-lethally collected samples. A considerable decrease in KHV genomic concentration was observed a er day four post-ne ing. KHV DNA was not detected in samples from persistently infected carp on day 10 a er stress induction. The results of this study suggest that KHV DNA is more readily detected, in gill swabs, one to three days a er stress induced by capture and ne ing. * Corresponding author’s email: [email protected] Introduction Koi herpesvirus (syn. Cyprinid herpesvirus 3, CyHV-3) has been responsible for worldwide KHV disease (KHVD) outbreaks, o en resulting in high mortality rates (40-100%) and has been reported in both aquacultured and wild common carp in addition to ornamental koi (Cyprinus carpio) (Bretzinger et al., 1997; Hedrick et al. 2000). Typical external signs of KHVD in affected carp include gill necrosis, abundant mucus production, distinct circular skin lesions or necrotic areas and enophthalmus. Internally, severe interstitial nephritis and swollen spleen and / or liver may be observed (Gilad et al., 2003). Due to the nature of the pathogen, an aquatic herpesvirus, taxonomically grouped into the family Alloherpesviridae (Waltzek et al., 2009), an infection with KHV results in latency or persistence (Gilad et al., 2003; St-Hilaire et al., 2005). In disease recovered and healthy appearing carp the virus is difficult to detect, due to these fish being able to suppress the KHV concentration to between 5 and 10 particles per 25 – 30 μg tissue material (Bergmann et al., 2010), as quantified by real-time PCR according to Gilad et al. (2004). Other molecular based methods for genomic detection such as Bull. Eur. Ass. Fish Pathol., 31(3) 2011, 93 PCR, nested-PCR or loop-mediated isothermal amplification (Bergmann et al., 2006; Gilad et al., 2002; Gunimaladevi et al., 2004; Bercovier et al., 2005; Bergmann et al., 2010) o en fail to detect these low viral concentrations. The actual target tissues for KHV persistence / latency are not known but it has been shown that white blood cells, especially polymorphic granulocytes and enlarged mononuclear cells in interstitial tissue of the kidney can contain the virus (Bergmann et al., 2009). It is known that KHV can be re-activated by different stressors, such as transportation, catching, ne ing, hormonal maturation or changes in water temperature in the spring or autumn (St-Hilaire et al., 2005; Meyer, 2007). Based on this knowledge, the aim of this study was to create a simple, safe and reproducible procedure for the detection of re-activated KHV by non-lethal sampling that could subsequently be applied as a diagnostic tool. As ne ing is a procedure that is frequently undertaken during the carp production cycle, this method was utilised as a stress inducer and the KHV genomic concentration from gill and faeces samples determined by real-time PCR. Materials and methods