Row cytometric analysis of infectious pancreatic necrosis virus attachment to fish sperm

Abstract

Male populations of rainbow trout (Oncorhynchus mykiss W.) from Spanish farms with repetitive outbreaks of infectious pancreatic necrosis (IPN) were investigated for the presence of the virus in milt samples. The analysis was done in parallel by CO-cultivation of sperm cells and seminal fluid on susceptible cell cultures and by flow cytometry, using an indirect immunofluorescence assay (IFA). Pools of sperm cells, stained at the moment the milt sample arrived at the laboratory, approximately 14 h after collection, gave positive results for the presence of IPN virus by flow cytometry. The stained cells could be stored at 4 ' C for 48 h without loss of fluorescence. Allquots of the milt samples inoculated onto cell cultures produced cytopathic effects after 3 consecutive blind passages, usually 15 to 31 d postinoculation.Our results confirm the attachment of IPN virus to the sperm cells and emphasize the importance of new methods, such as flow cytometer analysis, to obtain rapid and quantitative information in experimental studies of vertical virus transmission.