Cyprinid herpesvirus 3 also known as koi herpesvirus is a causative agent of highly contagious disease (koi herpesvirus disease) and can cause significant losses in fish stocks. The disease is restricted to koi and common carp, but recent investigations have shown that other cyprinids as well as non-cyprinid species are asymptomatically susceptible to this virus and can play either a role as a potential carrier or can contribute to biological conservation of this virus. The susceptibility of two non-target species, stone loach (Barbatula barbatula) and sterbel -a hybrid between sterlet (Acipenser ruthenus) and beluga (Huso huso) to cyprinid herpesvirus 3 was tested by means of their co-habitation together with naïve koi and intraperitoneally KHV-infected koi (primary challenge). On the 15 th day post-infection (dpi), a secondary challenge was started (a portion of the surviving stone loach and sterbel were transferred to tanks with other naïve koi). All dead as well as surviving fish were investigated for the presence of KHV DNA in pooled samples of tissue from individual fish by nested PCR. Sampling for PCR from surviving fish was performed on the 15 th dpi and on the 30 th dpi of the primary challenge, and on the 30 th dpi of the secondary challenge. During the primary challenge (up to the 30 th dpi), average cumulative mortality in duplicated experimental groups was as follows: koi 100%, sterbel and stone loach both 5%. In the primary challenge, no surviving stone loach or sterbel sampled on the 15 th dpi or those that died previously were found to be positive for viral DNA. Results of PCR revealed the presence of KHV DNA in 95% of co-habited naïve koi samples. PCR analysis of tissues taken from surviving fish on the 30 th dpi revealed the presence of viral DNA in 77.8% (7/9) of stone loach and in 22.2% (2/9) of sterbel. Cumulative mortality of fish in the secondary challenge was 100% for stone loach and for koi co-habitating with them, and 50% for koi co-habitating with sterbel, which all survived. Despite the high mortality of koi and stone loach in the secondary challenge (probably caused by malfunction of biofilters or bacterial infection), none of them, nor any of the sturgeon hybrids were considered to be positive for KHV DNA. In summary, the hybrid between sterlet and beluga and the stone loach seemed to be susceptible to cyprinid herpesvirus 3, but we could not prove that they can transfer this virus to naïve koi.Keywords: CyHV-3; KHV; transmission; cohabitation List of abbreviationsCyHV-3 = cyprinid herpesvirus 3, CEFAS = Center for Environment, Fisheries and Aquaculture Science, dpi = days post infection, KHV = koi herpesvirus, PCR = polymerase chain reaction

Cyprinid herpesvirus 3 (CyHV-3), also known as koi herpesvirus, is the causative agent of the highly contagious koi herpesvirus disease, which is restricted to koi and common carp and causes significant losses in both fish stock. Some experimental investigations have shown that other cyprinid or non-cyprinid species may be asymptomatically susceptible to this virus and might play roles as potential carriers of CyHV-3 or might contribute to persistence of this virus in environment. Therefore, it seems important to verify not only the susceptibility of other cyprinid or non-cyprinid species, but also their ability to transmit CyHV-3 infection to susceptible species. Our previous investigation of the susceptibility of the topmouth gudgeon (Pseudorasbora parva) did not reveal the presence of CyHV-3 DNA in the tissues of this species after cohabitation with infected koi. Consequently, we changed the experimental conditions and applied two stress factors (removal of skin mucus and scaring) which would presumably mimic the stress most commonly encountered in the wild. Both experiments (without and with stress factors) consisted of primary and secondary challenges. In both the no-stress and stress experiments, the first challenge was focused only on testing the susceptibility of the topmouth gudgeon to the virus. With the secondary challenge, we investigated potential viral transmission from the topmouth gudgeon to healthy naive koi after exposure to stress factors. All fish (dead, surviving and sacrificed) were tested for the presence of CyHV-3 DNA using nested PCR (no-stress experiment) and real-time PCR (stress experiment). After the primary challenge of the no-stress experiment, PCR did not reveal the presence of CyHV-3 DNA in any specimen of cohabitated topmouth gudgeon, but all specimens of dead koi were CyHV-3 DNA-positive. PCR of fish tissues subjected to the secondary challenge did not show the transfer of virus to naive fish. After exposure to stress (removal of skin mucus), qPCR revealed four out of five samples (80%) of topmouth gudgeon to be positive for CyHV-3 DNA. Two out of five samples (40%) of topmouth gudgeon treated by scaring were found to be positive for the presence of viral DNA. Real-time PCR after the secondary challenge did not reveal any viral DNA positivity in specimens of topmouth gudgeon from groups previously exposed to stress. The stress experiments show that removal of skin mucus might potentially lead to susceptibility of topmouth gudgeon to CyHV-3 infection, but the transmission of the virus to koi carp was not observed.