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Antibody response of two populations of common carp, Cyprinus carpio L., exposed to koi herpesvirus.

Common carp, Cyprinus carpio L., exposed to koi herpesvirus (KHV) may become persistently infected and populations containing such virus-infected individuals may transmit the virus to other fish when co-habited. Detection of virus-infected fish in a population is thus critical to surveillance and control programmes for KHV. A study was therefore designed to detect anti-KHV serum antibodies, with an enzyme-linked immunosorbent assay, in common carp following experimental exposures to KHV under varying environmental conditions. The study determined that a proportion of fish within a population experimentally exposed to KHV (at least 10-25%) develop high antibody titres (1/1600 or greater) to the virus, and this immunological response was detectable for several months (observed at the termination of the experiments at 65, 46 and 27 weeks post-exposure). Furthermore, this response was detected in one population of fish that did not succumb to a high level of mortality when maintained at water temperatures that were non-permissive for KHV. Elevating the water temperatures to permissive conditions for KHV resulted in recurrence of disease despite the presence of anti-virus antibodies, suggesting that serum antibodies alone are not protective under the conditions of our trials.

Comparison of the resistance of selected families of common carp, Cyprinus carpio L., to koi herpesvirus: preliminary study.

Koi herpesvirus disease (KHD), caused by koi herpesvirus (KHV), is a highly infectious disease of carp, Cyprinus carpio L., usually causing high mortality (reviewed by Dixon 2008). The disease has a worldwide distribution and affects ornamental carp (koi carp) as well as common carp in both aquaculture and open waters (rivers, lakes, etc.). The disease is economically very important, having an impact on the trade in high value ornamental fish, sport fisheries and food production. Attempts have been made to reduce the economic impact of the disease by vaccination (Ronen, Perelberg, Abramowitz, Hutoran, Tinman, Bejerano, Steinitz & Kotler 2003; Perelberg, Ronen, Hutoran, Smith & Kotler 2005), but as yet no vaccine has been approved for use outside Israel. An alternative method of controlling the disease by use of resistant strains has been investigated (Shapira, Magen, Zak, Kotler, Hulata & Levavi-Sivan 2005; Zak, Perelberg, Magen, Milstein & Joseph 2007). The latter authors bred three hybrid and three pure crosses from two Hungarian and one Israeli strain of fish and reported survival rates after challenge with KHV of between 4% and 20%. Shapira et al. (2005) crossed two strains of domesticated carp with one strain of wild carp and exposed the progeny to KHV both in the laboratory and in the field. The mortalities ranged from 8.9% to 89.7% in the field trial and from 8.6% to 68.5% in the laboratory trial, and with one exception there was good comparability of survival rates of the different crosses in the two trials. To further explore the potential of producing strains of carp resistant to KHV, we have investigated the survival of a large number of carp families following exposure to the virus, and present the results here. This study is part of a larger study, which includes investigating production characteristics, the resistance of the families to Aeromonas hydrophila, post-infection changes at the genomic level (Williams, Li, Hughes, Gonzalez, Vernon, Vidal, Jeney, Jeney, Dixon, McAndrew, Bartfai, Orban, Trudeau, Rogers, Matthews, Fraser, Gracey & Cossins 2008) and proteomic level, heritability characteristics, the genetic correlation between different traits and gene linkage mapping. The production and other characteristics of the fish families will be described in full in a separate communication (Z. Jeney, unpublished data). Briefly, 96 families were produced by diallele crossing of four strains of carp (Duna, Amur, Tata and HAKI 15) maintained in a live gene bank at HAKI, Hungary. Five females and 10 males from each strain were used to produce six families per strain cross. The families were maintained in separate tanks until they were large enough to be injected with a passive integrated transponder (PIT) tag. Twenty fish from each of 94 families (there Journal of Fish Diseases 2009, 32, 1035–1039 doi:10.1111/j.1365-2761.2009.01081.x

Studies on the effect of temperature and pH on the inactivation of fish viral and bacterial pathogens

Disposal of fish by-products in the European Community must comply with Regulation (EC) No 1069/2009 which categorizes animal by-products according to risk, and specifies methods of disposal of by-products according to that risk. There is provision under the regulation for composting or ensiling to be used for by-products from aquatic animals. Biosecurity considerations require knowledge of the parameters of time and temperature, or time and pH, required to inactivate any fish pathogens that may be present. To provide those data, we undertook laboratory studies on the inactivation of a number of fish pathogenic viruses and bacteria at 60 °C, pH 4.0 and pH 12.0 as a preliminary to conducting subsequent trials with the most resistant viruses and bacteria in fish tissues. The most resistant bacterium to 60 °C, pH 4.0 as well as pH 12.0 was Lactococcus garvieae. Its concentration was reduced to the level of sensitivity of the test after 24-48 h exposure to 60 °C, but it survived for at least 7 days at pH 4.0 and 14 days at pH 12.0. The most resistant virus to 60 °C was infectious pancreatic necrosis virus, and to pH 12.0 was infectious salmon anaemia virus. The majority of the viruses tested survived exposure to pH 4.0 for up to 28 days. The results suggest that the process of acid ensiling alone is not an effective method for the inactivation of many viral and bacterial pathogens, and fish by-products would need further treatment by a method approved under the regulation following ensiling, whereas alkaline or heat treatment are likely to provide an increased degree of biosecurity for on-farm processing of mortalities.

Studies on the inactivation of selected viral and bacterial fish pathogens at high pH for waste disposal purposes

This study investigated the use of alkaline hydrolysis at ambient temperature for inactivation of selected fish pathogens in fish tissues under conditions approximating those that are likely to be found in the aquaculture industry. Infectious salmon anaemia virus (ISAV) and Lactococcus garvieae have been determined in a previous study to be the most resistant virus and bacteria to pH 12 from a wide range of viruses and bacteria tested. They were spiked at high titres into fish extracts that were then treated with 1 m sodium hydroxide (NaOH). Viable L. garvieae was not detected in the treated fish extract after 1 h, and ISAV was not detected after 24-h exposure. Field mortalities of Atlantic salmon, Salmo salar L., caused by infectious pancreatic necrosis virus were treated by alkaline hydrolysis at ambient temperature. The macerated fish mortalities contained a high titre of virus (3.38 × 10⁸ TCID₅₀ g⁻¹) that was reduced to approximately 2.2 × 10³ TCID₅₀ g⁻¹ after 24-h exposure to NaOH, and virus was not detected after exposure for 48 h. The results suggest that alkaline hydrolysis at ambient temperature has potential as a biosecure treatment method for fish by-products containing fish pathogens.

Risks associated with commodity trade: transmission of viral haemorrhagic septicaemia virus (VHSV) to rainbow trout fry from VHSV-carrying tissue-homogenates.

Movements of commodity fish present a potential risk of transferring pathogens. Within a study to estimate the risk from imported rainbow trout Oncorhynchus mykiss carcases, fry were exposed to tissue homogenates from market size rainbow trout infected experimentally with viral haemorrhagic septicaemia (VHS) by waterborne exposure to VHS virus (VHSV, isolate of genotype Ia). Tissues were collected from fish that showed clinical signs and from recent mortalities. Homogenates of (i) internal organs, (ii) brain/gills and (iii) muscle tissue were prepared and added to tanks holding the fry. Virus transmission occurred from all tissues tested, causing high mortality of the fry. The results underline the potential risk of introduction of VHSV through the trade of fish products.

Puffy Skin Disease Is an Emerging Transmissible Condition in Rainbow Trout Oncorhynchus mykiss Walbaum.

The transmission of puffy skin disease (PSD) to rainbow trout Oncorhynchus mykiss Walbaum was tested in the laboratory by conducting co-habitation challenges with puffy skin (PS)-affected fish (Trojans) collected from the field. Two separate challenges were conducted using Trojans sourced from two different sites and diploid (first trial) or triploid (second trial) naïve fish. PSD-specific clinical signs were observed in both groups of naïve fish, with 66% of the fish sampled during the challenges showing signs of varying severity. The first clinical features of PSD were presented as white oval skin patches on one or both flanks 15–21 days post-challenge (dpc). The extent of the lesions ranged from 10 to 90% of the body surface, depending on the severity of the lesion. Both the severity and number of affected fish increased during the challenge. Macroscopically, oedema of the skin and multifocal petechial haemorrhaging were observed towards the end of the trials. Abnormal fish behaviour consisting of “flashing” and excessive mucous production was noted from 15 dpc onwards. Fish with severe PSD lesions also displayed inappetence and associated emaciation. Rodlet cells were observed in 41% of the fresh skin scrapes analysed from the second trial. Histologically epidermal oedema was observed in 31% of the naive fish showing gross pathology, with additional 12% displaying epidermal hyperplasia, mostly observed at the end of the challenge. Other concomitant features of the PSD lesions in challenged fish were epithelial erosion and sloughing, and occasionally mild or focal inflammation. No consistent pathology of internal organs was observed. The parasites Ichthyophthirius multifiliis and Ichthyobodo necator were observed in skin samples of a proportion of naïve challenged fish and in Trojans but not in control fish. The presence of these and other known fish pathogens in the skin of PSD-fish was confirmed by high-throughput sequencing analysis. In summary, we have demonstrated that PSD is a transmissible condition. However, even though a number of known fish pathogens were identified in the skin tissues of PSD-fish, the actual causative infectious agent(s) remain(s) unknown.