Introduction
Viral pathogens remain a leading cause of mortality in aquaculture industry, posing major challenges to fish health management and biosecurity. Among these, Salmon gill poxvirus (SGPV) represents a significant threat to the global Atlantic salmon aquaculture industry. SGPV, the causative agent of salmon gill poxvirus disease (SGPVD), can cause severe disease characterized by hyperplasia and apoptosis, with respiratory distress and lethargy being typical clinical signs (1). SGPVD outbreaks have been reported in Norway, Scotland and the Faroe Islands (2-5), leading to high mortality rates during the acute phase of infection (3). In Norway, SGPVD have a great economic impact on the salmon farming industry. Unlike other fish viruses, which has been well studied thanks to the development of cell cultures, SGPV cannot be cultured and vaccination is not yet feasible at the early life stages of fish, when these diseases may cause high mortality. Avoiding contact between the fish and the pathogens may thus be the only available control measure. However, methods of inactivation and disinfection of the virus are not available. Here, we investigated potential effect of UV and chlorine on the inactivation of viable SGPV by conducting standardized challenge experimental infection in Atlantic salmon yolk sac larvae.
Material and Method
SGPV isolated from PCR positive Atlantic salmon gill tissue were treated with10, 25, 90, 250 UVD (mJ/cm2) and 35mg/L, 60 mg/L, 80 mg/L chlorine concentration. Each UV or chlorine treated SGPV was used to bath-challenge 100 salmon yolk sacs for 7hr at 6oC in a total volume of 500mL. After 7 hours, salmon yolk sacs were infected with each UVD (10, 25, 90, 250 mJ/cm2) and chlorine concentration (10mg/L, 25 mg/L, 35 mg/L, 60 mg/L) were transferred to 5 L tanks, providing separate yolk sac groups. After transfer, the yolk sac was monitored for signs of distress for one hour. The challenge experiment lasted for 30 days, with daily recording of yolk sac mortality in all tanks. Ten yolk sacs were sampled from each treated and the uninfected control group at 7, 14, 21 and 30 DPC, in addition to ten yolk sacs sampled before challenge (0-samples). Fish were anesthetized by immersion in benzocaine (40 mg/L), yolk sac head samples were aseptically collected in RNA-Later (Qiagen, Hilden, Germany) for qPCR, and whole yolk sac were fixed in 10% phosphate buffered formalin for histopathology and immunohistochemistry (IHC). Four methods, RT-PCR, histopathology, immunohistochemistry and in situ hybridization were compared in order to determine the viability of SGPV following inactivation with different doses of UV and chlorine.
Results
All yolk sacs samples (at 7, 14, 21 and 30 DPC) from each UV Irradiation and chlorination treated group, were all negative by PCR. Salmon yolk sacs samples collected from the positive control group at 7, 14, 21 and 30 DPC were positive by PCR. However, there were pronounced differences in the percentage of positive individuals. Results from histology, immunohistochemistry and in situ hybridization, are currently being analyzed and will be updated.
Conclusion
A UV dose of 25 mJ/cm2, which is the standard requirement for inlet water treatment in aquaculture facilities, was sufficient to efficiently inactivate poxvirus, comparable to the inactivation observed for VHSV, IHNV, and ISAV. For chlorinated water treatment, the current requirement—an initial residual chlorine concentration of ≥ 50 mg/L and a residual concentration of ≥ 2 mg/L after 25 minutes of treatment was sufficient to inactivate poxvirus.
Acknowledgment
This work was funded by Norwegian Research Council project, number 326585.
References
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