First clinically apparent koi herpesvirus infection in the Czech Republic

Koi herpesvirus disease (KHVD) is a highly contagious viral illness causing significant morbidity and mortality in common carp (Cyprinus carpio). KHVD has been confirmed in almost all countries around the world except Australia. KHV has been previously detected in the Czech Republic based on PCR screening but with no clinical signs of KHVD. An outbreak of KHVD with clinical signs, and severe mortality was confirmed in July 2009 by histopathology and virological testing of hepatopancreas, anterior kidney, spleen and gill samples. Introduction Koi herpesvirus (KHV) is a highly contagious viral illness that can cause significant morbidity and mortality in common carp (Cyprinus carpio) (Hedrick et al., 2000). The aetiological agent is a DNA virus belonging to the virus family Herpesviridae cyprinid herpesvirus-3 (CyHV-3) commonly known as KHV (Ronen et al., 2003; Waltzek et al., 2005). This virus is closely related to carp pox virus (CyHV-1) and hematopoietic necrosis herpesvirus in goldfish (CyHV-2). The first recognized case of KHV occurred in England in 1996, with subsequent confirmation in almost all countries that culture koi and/or common carp, with the exception of Australia (Hedrick et al., 2000). Other related cyprinid species such as the common goldfish (Carassius auratus) and grass carp (Ctenopharyngodon idella) do not show clinical signs of KHV although DNA of this virus has been identified in tissue of these species using polymerase chain reaction (PCR) (Bergmann et al., 2010). Hybrid goldfish (male goldfish C. auratus x female common carp (C. carpio) was moderately resistant to mortality following experimental infection with KHV (Hedrick et al., 2005). Presence of KHV was also suspected in the Czech Republic based on the screening of PCR results from carp that did not manifest with any clinical signs (Pokorova et al., 2005; Pokorova et al., 2007). No clinical cases of KHVD were reported in the Czech Republic until the occurrence in July 2009. Bull. Eur. Ass. Fish Pathol., 30(3) 2010, 86 Materials and Methods Origin of the fish The koi carp originated from a private garden pond (vol. 12 000 l-1) in Lazne Bohdanec district (Loc: 50°4’32.172”N, 15°40’47.224”E). Fish stock within the pond consisted of koi carp (n = 48) ranging from 10 to 60 cm body length, goldfish (n = 26), ornamental tench (n = 3) and Caspian sturgeon (n = 2). The water temperature was 22°C. The water purity was maintained by a multi-segment (mechanical and biological) filters. The fish were fed a commercial diet for koi carp. All species in the pond did not show any signs of infection until the described case. Diagnostic methods Pathology Five individuals of koi carp suspected of having KHV infection based on clinical signs, were euthanased with overdosing of tricaine methanesulphonate (MS – 222). Fresh mounts and histopathology samples were taken from gills, hepatopancreas, anterior and posterior kidney, spleen and gut. Samples for histopathology were processed with 10% buffered formalin and fixed for 24 h. Samples were embedded in paraffin, sections cut at 5 μm thickness and stained with haematoxylin and eosin (H&E) or as per the periodic acidSchiff (PAS) method. Virology Sample collection for virological testing Individual samples of hepatopancreas, anterior kidney, spleen and gills were collected from two fish. A er dilution with culture medium (1:5 v/v), organ homogenates from each fish were centrifuged and a part of the supernatant was used for nucleic acid extraction for virus detection by PCR. The other part of supernatant was used for virus culture isolation in the cell line Cyprinus carpio brain (CCB) (Neukirch et al., 1999).