LAUSR

Abstract The genus Megalocytivirus (MCV) includes Infectious spleen and kidney necrosis virus (ISKNV) and red seabream iridovirus (RSIV) which are listed by the World Organization for Animal Health (OIE). MCV has a broad host range and can be present as a persistent subclinical infection in some fish. Australia is considered free from MCV and important aquaculture and wild fisheries are at risk. Consequently, biosecurity measures are implemented to ameliorate transmission pathways through imported ornamental fish which include certification of freedom from infection with MCV. Evaluation of practical and cost effective disinfection protocols suitable for recirculating aquaculture facilities is an important aspect of the biosecurity plan to facilitate eradication in the event of early detection of an outbreak. An authentic sample matrix was prepared by in vivo amplification of ISKNV in Murray cod (Maccullochella peelii) and the biological load of a clarified tissue homogenate was standardized by addition of 10% v/v foetal bovine serum. In the absence of a cell culture system for ISKNV, a bioassay was used to assess infectivity after disinfection. A cellulose membrane buffer exchange device was used to remove residual disinfectants before intraperitoneal injection of juvenile Murray cod. Fish were maintained in 100 L aquaria at 23 °C and observed for clinical signs over 14 days. Each bioassay was conducted in duplicate tanks with 6 to 19 fish per tank. A positive assay was defined by an increase in ISKNV DNA quantified by qPCR in any fish from a subsample of challenged fish collected at 7 d or those remaining at 14 d. Negative bioassays were defined by the absence of ISKNV DNA in all fish at the both sampling times. Clinical disease and amplified viral DNA was detected after injection of a dilute positive control, indicating greater analytical sensitivity compared to qPCR (with a detection limit of 100 copies per reaction). Further, the system was used to demonstrate that ISKNV can remain infectious in aquaria without fish for at least 48 h at 25 °C. Effective disinfection measures included: heating to 65 °C for 20 min; pH 3; pH 11; 1% Virkon™; 1000 ppm sodium hypochlorite and benzalkonium chloride at the recommended concentration and contact time. These data can be interpreted to provide effective disinfection protocols for MCV in a wide variety of disease control scenarios.