Concentration of Infectious Aquatic Rhabdoviruses from Freshwater and Seawater Using Ultrafiltration

Abstract

Infectious hematopoietic necrosis virus (IHNV), viral hemorrhagic septicemia virus, and spring viremia of carp virus were concentrated and detected from freshwater and seawater samples by using hollow-fiber ultrafiltration. Within 60 min, virus in a 50-L freshwater or saltwater sample was concentrated more than 70-fold, and virus retention efficiencies were consistently greater than 88%. Retention efficiency was highly dependent upon concentrations of column blocking and sample stabilization solutions. A large column with a surface area of 1.15 m² and a filtration capacity of 5–200 L exhibited optimal viral retention when blocked with 2% fetal bovine serum (FBS) and when the samples were supplemented with 0.1% FBS. Conversely, a small column with 100-fold less surface area and a filtering capacity of 0.5–2.0 L was optimized when blocked with 1% FBS and when the samples were supplemented with 0.1% FBS. The optimized ultrafiltration procedure was further validated with water from a tank that contained IHNV-exposed juvenile sockeye salmon Oncorhynchus nerka, resulting in an average virus retention efficiency of 91.6 ± 4.1% (mean ± SE). Virus quantification of concentrated samples demonstrated that IHNV shedding in sockeye salmon preceded mortality; shedding of the virus was observed to increase significantly as early as 7 d postchallenge and peaked at day 14, when virus levels reached 4.87 × 10³ plaque-forming units/mL. We conclude that ultrafiltration is a reliable and effective method for concentrating viable aquatic rhabdoviruses from large volumes of water and has application for the analysis of environmental water samples.

Received April 22, 2011; accepted August 4, 2011

ACKNOWLEDGMENTS

We thank C. McClean and S. Barnetson for providing the sockeye salmon, and we are grateful to C. Baynes for fish rearing assistance. Financial support was provided by Mainstream Canada, Marine Harvest Canada, Grieg Seafoods, and the Aquaculture Coordinated Research and Development Program of Fisheries and Oceans Canada (Project Number P-09-03-006).