The impact of pooling samples on surveillance sensitivity for the megalocytivirus Infectious spleen and kidney necrosis virus.

Movements of large volumes and species varieties make the ornamental fish industry a high-risk pathway for the transfer of aquatic pathogens to new geographical regions and naïve hosts, potentially resulting in emergency disease events. Infectious spleen and kidney necrosis virus (genus Megalocytivirus) is considered exotic to Australia despite documented incursions since 2003. There are current import controls requiring freedom from infection for entry to Australia. The objective was to evaluate the effect of tissue pooling strategies for qPCR testing using a SYBR® assay for freedom from ISKNV at 2% expected prevalence with 95% confidence. Tissue homogenates from apparently healthy imported ornamental fish were tested as individuals and in pools of 5 and 10. Analytical sensitivity of the qPCR assay was reduced by two orders of magnitude when the nucleic acid extraction process was accounted for by spiking the plasmid in fish tissues and compared with molecular grade water. Diagnostic sensitivity of the assay was substantially reduced when testing tissues in pools compared with individual testing. For Population 1 (66% positive for ISKNV with moderate viral loads), surveillance sensitivity was only achieved using individual testing. For Population 2 (100% positive ISKNV with high viral loads), surveillance sensitivity was achieved using 260 fish in pools of 10 for a total of 26 tests or 200 fish in pools of 5 for 40 tests. Surveillance sensitivity could be maximized even when there was a reduction in pooled diagnostic sensitivity compared with diagnostic sensitivity for individual fish by increasing the sample size. Pooled sensitivity was influenced by the prevalence and variable virus load among fish with subclinical infections. Pooled testing is highly effective when the prevalence is >10% which should be informed by prior knowledge or pooling can be used for a screening test to rapidly identify populations with high prevalence.

Detection of dwarf gourami iridovirus (Infectious spleen and kidney necrosis virus) in populations of ornamental fish prior to and after importation into Australia, with the first evidence of infection in domestically farmed Platy (Xiphophorus maculatus).

The movement of ornamental fish through international trade is a major factor for the transboundary spread of pathogens. In Australia, ornamental fish which may carry dwarf gourami iridovirus (DGIV), a strain of Infectious spleen and kidney necrosis virus (ISKNV), have been identified as a biosecurity risk despite relatively stringent import quarantine measures being applied. In order to gain knowledge of the potential for DGIV to enter Australia, imported ornamental fish were sampled prior to entering quarantine, during quarantine, and post quarantine from wholesalers and aquatic retail outlets in Australia. Samples were tested by quantitative polymerase chain reaction (qPCR) for the presence of megalocytivirus. Farmed and wild ornamental fish were also tested. Megalocytivirus was detected in ten of fourteen species or varieties of ornamental fish. Out of the 2086 imported gourami tested prior to entering quarantine, megalocytivirus was detected in 18.7% of fish and out of the 51 moribund/dead ornamental fish tested during the quarantine period, 68.6% were positive for megalocytivirus. Of fish from Australian wholesalers and aquatic retail outlets 14.5% and 21.9%, respectively, were positive. Out of 365 farmed ornamental fish, ISKNV-like megalocytivirus was detected in 1.1%; these were Platy (Xiphophorus maculatus). Megalocytivirus was not detected in free-living breeding populations of Blue gourami (Trichopodus trichopterus) caught in Queensland. This study showed that imported ornamental fish are vectors for DGIV and it was used to support an import risk analysis completed by the Australian Department of Agriculture. Subsequently, the national biosecurity policy was revised and from 1 March 2016, a health certification is required for susceptible families of fish to be free of this virus prior to importation.