Epizootiology of Koi herpes virus disease in Croatia

Koi herpes virus disease (KHVD) has spread globally, particularly to the regions and countries with intensive cultivation and trade of koi carp and common carp (Cyprinus carpio). Croatia has implemented surveillance programme for the presence of the KHVD for last 10 years and in 2015. the whole country has been declared free by EU Commission decision. The sampling for diagnostics on KHV was scheduled in the warmer period of year with water temperature exceeding 20oC. In the early spring of 2016, high mortalities have been notified from the common carp farm with the water temperature of 14oC. Affected fish showed typical KHVD symptoms ; light pigmentation of the skin, focal loss of scales, excess mucous secretion, and pale gills with extensive necrotic areas, endophthalmia. All samples tested negative for Koi herpes virus (KHV) using real time PCR as well as for Carp Edema Virus (CEV). During the next month mortalities were persistent with the same symptoms and at the end of May another clinical inspection and sampling were undertaken at the water temperature of 16oC. All samples tested positive for KHV by means of real time PCR. The epizootiological survey and sampling was undertaken during June and July in all carp producing farms. Finally, two farms were confirmed as infected. All movements into and from two affected farms were assessed and two put and take fisheries which imported fish from one of the infected farms were also found positive. All positive results were confirmed also by conventional PCR targeting TK gene. All isolated viruses were submitted to partial sequencing of poly gene for DNA polymerase and showed 100% similarity with Cyprinid herpes virus 3. The temperature ranges of mortality onset make a suitability of the surveillance program during period with water temperature above 20oC questionable.