Koi Herpesvirus (KHV), a species-specific DNA virus of the family Herpesviridae, is responsible for mass mortalities of common carp (Cyprinus carpio carpio L.) throughout the world. KHV’s broad geographical distribution and relatively high mortality rate among infected fish, creates significant disposal issues when die-offs occur, especially taking into account the body burden of contaminants in the fish. In locales where adequate disposal facilities are unavailable, or are unable to accommodate additional loadings of contaminated fish carcasses, concerns regarding human and environmental health are raised. During the summer of 2008, residents of the Lake Simcoe Region of southern Ontario, Canada, were faced with a massive die-off of carp, infected with KHV. Carp within the Great Lakes and much of the world are known to bioaccumulate (and biomagnify) contaminants, such as, polychlorinated biphenyls (PCBs), pesticides (e.g., dichlorodiphenyltrichloroethane, DDT, and toxic metals (e.g., mercury). These contaminants have been associated with numerous adverse effects on both human and environmental health, and are thus of important considerations when planning for large-scale carcass disposal, following fish die-offs. Although suites of microbiological tests and water quality assessments are frequently conducted to identify causative factors during extensive fish-kills - assessments of relative contaminant burdens in the carcasses, which should dictate the most appropriate method of carcass disposal - are rarely performed. A case study on Snake Island, Lake Simcoe, Ontario was conducted to further examine the implications of this policy. Soil samples from two known disposal sites and three presumed control locations were sampled on Snake Island and sent to the Analytical Services Unit of Queen’s University for chemical analysis. Although none of the soil samples exceeded any legal guidelines in the present study, there is still concern as future die-offs of other fish species or piscivorous birds and the disposal of large numbers of carcasses may be an issue.

Koi Herpesvirus (KHV), a species-specific DNA virus of the family Herpesviridae, is responsible for mass mortalities of common carp (Cyprinus carpio carpio L.) throughout the world. KHV’s broad geographical distribution and relatively high mortality rate among infected fish, creates significant disposal issues when die-offs occur, especially taking into account the body burden of contaminants in the fish. In locales where adequate disposal facilities are unavailable, or are unable to accommodate additional loadings of contaminated fish carcasses, concerns regarding human and environmental health are raised. During the summer of 2008, residents of the Lake Simcoe Region of southern Ontario, Canada, were faced with a massive die-off of carp, infected with KHV. Carp within the Great Lakes and much of the world are known to bioaccumulate (and biomagnify) contaminants, such as, polychlorinated biphenyls (PCBs), pesticides (e.g., dichlorodiphenyltrichloroethane, DDT, and toxic metals (e.g., mercury). These contaminants have been associated with numerous adverse effects on both human and environmental health, and are thus of important considerations when planning for large-scale carcass disposal, following fish die-offs. Although suites of microbiological tests and water quality assessments are frequently conducted to identify causative factors during extensive fish-kills - assessments of relative contaminant burdens in the carcasses, which should dictate the most appropriate method of carcass disposal - are rarely performed. A case study on Snake Island, Lake Simcoe, Ontario was conducted to further examine the implications of this policy. Soil samples from two known disposal sites and three presumed control locations were sampled on Snake Island and sent to the Analytical Services Unit of Queen’s University for chemical analysis. Although none of the soil samples exceeded any legal guidelines in the present study, there is still concern as future die-offs of other fish species or piscivorous birds and the disposal of large numbers of carcasses may be an issue.

Cyprinid herpesvirus 3 (CyHV-3) also known as koi herpesvirus (KHV) 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 may be asymptomatically susceptible to this virus and might play roles as potential carriers or can contribute to biological conservation leading to persistence of this virus in environment. Therefore, it seems to be important to verify not only the susceptibility of other cyprinid and non-cyprinid species, but also their ability to transmit KHV infection to susceptible species. We investigated the susceptibility of stone loach (Barbatula barbatula) and sterbel - a hybrid between sterlet (Acipenser ruthenus) and beluga (Huso huso) to KHV. The investigation was performed by means of their cohabitation together with na?ve koi and intraperitoneally KHV-infected koi (primary challenge). This part of investigation is followed by secondary challenge, when a portion of the surviving stone loach and sterbel was cohabited with health na?ve koi (testing of ability to carry KHV). All samples of fish both from primary challenge and secondary challenge were tested for the presence of KHV DNA by nested PCR. In the primary challenge, results of PCR revealed the presence of KHV DNA in 95% of cohabited na?ve koi samples. Furthermore, PCR analysis of fish samples surviving primary challenge revealed the presence of viral DNA in 77.8% (7/9) of stone loach and in 22.2% (2/9) of sterbel. In case of samples of fish coming from secondary challenge, nested PCR did not reveal any of them to be positive for KHV DNA. Next investigation was focused on assessment of the susceptibility of topmouth gudgeon (Pseudorasbora parva). In this case, we performed cohabitation based on two different conditions. All experiments consisted of primary and secondary challenges as well as in all previous cases. Firstly, we tested topmouth gudgeon under standard conditions (no-stress experiment). After the primary challenge, nested PCR did not reveal the presence of KHV DNA in any specimen of cohabited topmouth gudgeon, but all specimens of dead koi were KHV DNA positive. Nested PCR of fish tissues subjected to the secondary challenge did not show the transfer of virus to naive fish. After that, we changed the experimental conditions and we applied two stress factors (scaring by net and removal of skin mucus) to imitate the stress most commonly encountered in the wild. In this case, all samples were tested for the presence of KHV DNA using real-time PCR. After exposure to stress (removal of skin mucus), real-time PCR revealed four out of five samples (80%) of topmouth gudgeon to be positive for KHV 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 showed 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. Even though PCRs positive findings of KHV DNA in tissues of fish were relatively low, the presented results of cohabitation assays of cyprinid and non-cyprinid fish species indicate other species showing slight asymptomatic susceptibility to CyHV-3. On the other hand, on the base of our results coming from "virus-carrier" assays, we could not prove that hybrids between sterlet and beluga, stone loach and topmouth gudgeon can transfer this virus to naive koi.

The expanding aquaculture industry continues to encounter major challenges in the form of highly contagious aquatic viruses. Control and eradication measures targeting the most lethal and economically damaging virus-induced diseases, some of which are notifiable, currently involve ‘stamping out’ policies and surveillance strategies. These approaches to disease control are performed through mass-culling followed by restriction in the movement of fish and fish products, resulting in considerable impacts on trade. Although effective, these expensive, ethically complex measures threaten the sustainability and reputation of the aquatic food sector, and could possibly be reduced by emulating innovative vaccination strategies that have proved pivotal in maintaining the success of the terrestrial livestock industry. DIVA ‘differentiating infected from vaccinated animal’ strategies provide a basis to vaccinate and contain disease outbreaks without compromising ‘disease-free’ status, as antibodies induced specifically to infection can be distinguished from those induced in vaccinated animals. Various approaches were carried out in this study to assess the feasibility of marker/DIVA vaccination for two of the most important disease threats to the global Atlantic salmon and common carp/koi industries, i.e. infectious salmon anaemia (ISA) and koi herpesvirus disease (KHVD), respectively. Antibody responses of Atlantic salmon (Salmo salar L.), following immunisation with an ISA vaccine, administered with foreign immunogenic marker antigens (tetanus toxoid (TT), fluorescein isothiocyanate (FITC) and keyhole limpet hemocyanin (KLH)) were assessed by antigen-specific enzyme linked immunosorbent assay (ELISA). Although antibodies were induced to some markers, these were unreliable and may have been affected by temperature and smoltification. Detectable antibodies to ISAV antigen were also largely inconsistent despite low serum dilutions of 1/20 being employed for serological analysis. The poor antibody responses of salmon to the inactivated ISA vaccine suggested that DIVA vaccination is not feasible for ISA. A similar approach for KHV, utilising green fluorescent protein (GFP) as the marker, similarly failed to induce sufficiently detectable antibody responses in vaccinated carp (Cyprinus carpio L.). However, as high anti-KHV antibody titres were obtained with an inactivated KHV vaccine (≥1/3200), alternative approaches were carried out to assess the feasibility of DIVA vaccination for carp. Investigations of early KHV pathogenesis in vivo and antigen expression kinetics in vitro (0-10 days post infection (dpi)) provided valuable data for the diagnostics necessary for DIVA surveillance strategies. Following viral infection, molecular methods were shown to be the most effective approach for early detection of KHV infected fish prior to sero-conversion, during which time antibodies are not detectable. An experimental immersion challenge with KHV, however, revealed complications in molecular detection during early infection. The KHV DNA was detected in external biopsies of skin and gills, but also internally in gut and peripheral blood leukocytes ≤ 6 hours post infection (hpi), suggesting rapid virus uptake by the host. The gills and gut appeared to be possible portals of entry, supported by detection of DNA in cells by in situ hybridisation (ISH). However, many false negative results using organ biopsies occurred during the first 4 dpi. The gills were the most reliable lethal biopsy for KHV detection by various polymerase chain reaction (PCR) assays, with a PCR targeting a glycoprotein-gene (ORF56) and a real-time PCR assay being the most sensitive of the 7 methods investigated. Importantly, non-lethal mucus samples reduced the number of false negative results obtained by all KHV PCR assays during the earliest infection stages with large levels of viral DNA being detected in mucus (up to 80,000 KHV DNA genomic equivalents 200 μL-1). KHV DNA was consistently detected in the mucus as a consequence of virus being shed from the skin. Determining the expression kinetics of different viral structural proteins can be useful for DIVA serological tests. Analysis of KHV antigen expression in tissues by immunohistochemistry and indirect fluorescent antibody test was inconclusive, therefore 2 novel semi-quantitative immunofluorescence techniques were developed for determining KHV antigen expression kinetics in susceptible cell lines. During the course of KHV infection in vitro, a greater abundance of capsid antigen was produced in infected cells compared to a glycoprotein antigen (ORF56), as determined by detection with antigen-specific monoclonal antibodies (MAbs). The capsid antigen was characterised as a ~100 kDa protein by SDS-PAGE and identified as a product of KHV ORF84 by matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF/TOF MS). This antigen was subsequently detected in the serum of >25% of KHV infected/exposed carp (6/17), as well as in carp vaccinated with a live attenuated vaccine (3/4), but not with an inactivated vaccine (0/7), by Western blot making it a potential DIVA target for an inactivated vaccine. Attempts were made to improve the sensitivity of KHV serological testing by taking advantage of recombinant proteins specific for KHV (CyHV-3), rORF62 and rORF68 and eliminating any interference by cross-reacting antibodies to carp pox (CyHV-1). These proteins successfully reacted with anti-KHV antibodies. The feasibility of DIVA strategies for KHVD was determined using these recombinant antigens to coat ELISA plates. Differential antibody responses were detected from carp sera to an internal virus tegument protein (rORF62) and external region of a transmembrane protein (rORF68). Fish vaccinated with an inactivated vaccine produced significantly lower antibody responses to rORF62 than to rORF68, whereas infected, exposed and live attenuated vaccinated fish recognised both proteins allowing differentiation between vaccinated and infected carp. However, the sensitivity of the assay was limited, possibly by high levels of natural antibodies detected at the relatively low serum dilutions (1/200) used. As the capsid antigen (ORF84) and tegument protein (ORF62) are derived from internal KHV structural proteins, they induce non-neutralising antibodies, which may be useful for DIVA strategies. Such antibodies are longer lasting than neutralising antibodies and often comprise the majority of fish anti-viral antibodies. This was noted in a fish surviving experimental challenge, which had an antibody titre of 1/10,000, but neutralising titre of 1/45. Such antigens may therefore hold potential for developing effective serological diagnostic tests for KHV and provide the potential for DIVA strategies against KHVD. Natural antibodies will, however, continue to present a challenge to the development of sensitive and reliable KHV serological tests, and hence the application of DIVA strategies.

Eine effektive Strategie zur Verhinderung der Ausbreitung von Infektionskrankheiten in der Aquakultur ist die Anwendung geeigneter Präventionsmaßnahmen, insbesondere die Impfung von Fischen. Das effektivste Impfverfahren stellt die individuelle Injektion des Impfstoffes dar. Bei Milliarden von Fischen jährlich ist dies jedoch sehr zeit- und kostenintensiv. Bei Säugetieren gilt Niederfrequenz-Sonophorese (LFS) als eine der fortschrittlichsten Technologien zur transdermalen Verabreichung von Wirkstoffen. So entstand die Idee, bei Fischen die Aufnahme von Wirkstoffen während einer Tauchbadbehandlung mit Hilfe von LFS zu verbessern. Die vorliegende Arbeit zeigt, dass mit einer Beschallungsintensität von etwa 60 mW/cm^2 die Aufnahme eines Bakterienimpfstoffs in das Kiemengewebe von Regenbogenforellen um den Faktor 240 erhöht werden konnte. Bei dieser Intensität traten geringe oder keine Nebenwirkungen auf. Bei höheren Beschallungsintensitäten wurde eine noch höhere Aufnahme des Bakterienimpfstoffes, aber auch schädliche Nebenwirkungen beobachtet. Darüber hinaus zeigte LFS eine durch eine lokale Entzündungsreaktion und Aktivierung von T-Helferzellen in den Kiemen charakterisierte, Adjuvans-ähnliche Wirkung. Ein Impfversuch mit Koi Karpfen und einem inaktivierten Impfstoff gegen das Koi-Herpesvirus zeigte, dass LFS das Potential hat, den mit einer Tauchbadimpfung erzielbaren Immunschutz zu verbessern. LFS konnte auch für die Verabreichung anderer Substanzen wie Antibiotika verwendet und die Aufnahme von Oxytetracyclin um den Faktor fünf erhöht werden. Zusammenfassend lässt sich sagen, dass die Effektivität einer Tauchbadimpfung von Fischen mit LFS durch die gesteigerte Impfstoffaufnahme und dessen Adjuvans-ähnlichen Wirkung verbessert werden kann. Darüber hinaus könnte mithilfe LFS die therapeutische Dosis von Antibiotika bei Badbehandlungen verringert werden. Es erfordert jedoch noch weitere Studien, um diese Technologie aus dem Labor in die Praxis zu übertragen. / In aquaculture, the use of prevention methods, such as vaccination of fish, is an effective strategy to avoid infectious diseases. However, the most effective route of vaccination for fish is the one-by-one intraperitoneal injection, which that is very laborious and expensive to apply for billions of fish every year. Low Frequency Sonophoresis (LFS) has been recognized as one of the most advanced technologies in transdermal delivery of substances in mammals. Thus, it has been suggested to use LFS to enhance the uptake of substances in fish during bath treatments. The present study shows that a low sonication intensity of ca. 60 mW/cm^2 at 37 kHz increased the uptake of a bacterin into the gill tissue of rainbow trout by up to a factor of 240. At this intensity, no or only minimal side effects occurred. At higher sonication intensities, an even higher bacterin uptake but also deleterious side effects were observed. In addition, LFS showed an adjuvant-like effect characterized by a local inflammatory response and T-helper cell activation in the gills. A vaccination trial with koi carp and an inactivated vaccine against the Koi Herpes Virus (KHV) showed that LFS has the potential to enhance the immune protection achieved by immersion vaccination. In addition LFS can also be used for the administration of other substances, such as antibiotics, here we showed that the uptake of Oxytetracycline could be increased by factor five. In summary, the efficacy of the immersion vaccination of fish could be improved by low-frequency ultrasound due, to the increased vaccine uptake along with its adjuvant-like effect. Furthermore, LFS could also reduce the required therapeutic dose of antibiotics in bath treatments, making them more effective, cheaper and environmentally friendly. However, further practical studies will be required to transfer this technology from the lab to the field.