engleski

Molecular epidemiology of Koi Herpes Virus outbreak in Croatia

Introduction: Koi herpes virus disease (KHVD) is a highly contagious viral disease of worldwide importance, particularly in regions and countries with intensive cultivation and trade of koi carp and common carp (Cyprinus carpio). The causative agent is a double stranded DNA virus CyHV-3 (koi herpes virus (KHV)) of the genus Cyprinivirus within the family Alloherpesviridae (order Herpesvirales) (Gotesman et al., 2013). Generally there are considered to be two main genetic lineages of CyHV-3 ; the European lineage comprising several European isolates along with the representative US (KHV-U) and Israeli strain (KHV-I) and an Asian lineage circulating in different Asian countries (representative KHV-J strain from Japan) (Aoki et al., 2007 ; Kurita et al., 2009). However, strains similar to the European lineage have been detected in China (Dong et al., 2013). Apart from the TK gene as the main target for phylogeny, several other targets have been identified, namely ORF25, 65 and 116 (Han et al., 2013) or ORF29/31 and region near ORF133 (marker I and marker II, respectively) (Bigarre et al., 2009) that enable more in-deep analysis. The first descriptions of KHVD presence in Europe date back in 1998 (Bretzinger et al., 1999) and since then it has been reported in numerous European countries and in our neighborhood (Slovenia, Hungary) (Gotesman et al., 2013 ; Láng et al., 2014). However, Croatia was declared free of KHVD by the EU Commission in 2015 after 10 years of surveillance program. But in early spring of 2016, the suspicion has been notified after the advent of mass mortalities in the common carp farm. Herein we report a case description of the first outbreak of KHVD in Croatia and we describe the genetic background of detected strains and their epidemiological tracking. Materials and Methods: Clinical appearance and sampling: In April 2016 a common carp farm in eastern Croatia reported high mortalities (water temperature of 14°C) which continued for a month later (water temperature of 16°C). Affected fish showed typical KHVD symptoms ; light pigmentation of the skin, focal loss of scales, excess mucous secretion, pale gills with extensive necrotic areas and enophthalmia. Upon necropsy, tissue samples of anterior kidney, gills and spleen were taken for molecular detection of KHV (five pools of two fish). Post-outbreak surveillance included sampling and molecular testing of 29 cyprinid fish farms and 11 open water locations (including put-and-take fisheries) which resulted in total of 940 samples (470 pools of two fish). Molecular diagnostics: Tissue samples were homogenized in transport medium and supernatant was taken for viral DNA extraction using iPrep PureLink Virus kit (Invitrogen, USA). A real-time PCR (Gilad et al., 2004) was conducted to detect a KHV DNA fragment using QuantiFast Pathogen PCR + IC kit (Qiagen, Germany). The conventional PCR (Bercovier et al., 2005) was performed in order to confirm the KHV positives. All positive samples were confirmed once more by the EURL for fish diseases. Molecular characterization: Selected KHV positive samples from all four positive locations were sequenced for TK gene (Kurita et al., 2009) which was followed by phylogenetic analysis of generated sequences in MEGA6 (Tamura et al., 2013). Results and Discussion The fish with KHVD clinical appearance tested positive by real-time PCR for the presence of KHV after second sampling when water temperature was at 16°C. Risk mitigation measures were implemented by Veterinary Directorate in order to control the outbreak, which also included the epidemiological survey. After the primary outbreak in eastern Croatia, the post-outbreak surveillance resulted in three additional KHV positive locations. The second positive common carp farm was only 50km apart from the primary outbreak and these two farms were from the same enterprise. The last two positive locations were put-and-take fisheries that had bought the fish from the primary outbreak farm. However, the initial route of infection in the primary outbreak is still unknown. The possibility of unreported trade with farms in neighboring countries (Hedrick, 1996) that have the KHVD or the entry by wild species of fish (Fabian et al., 2013) cannot be excluded. In total we have detected 20 positive pooled samples out of 475 tested at four locations which were confirmed by conventional PCR and EURL for fish diseases. The sequencing results confirm that CyHV-3 was the causative agent, and more thorough molecular characterization will be presented in details. To conclude, this is the first occurrence of KHVD in Croatia which emphasize the need of implementation of strict monitoring and biosecurity measures at common carp farms in order to protect the production and to keep the country’s KHVD free status.

KHV ; outbreak ; molecular epidemiology ; Croatia

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