Detection of Infections Pancreatic Necrosis Virus (IPNV) from leukocytes of carrier rainbow trout Oncorhynchus mykiss

Blood and kidney leukocytes were purified from two-year-old rainbow trout carrying infectious pancreatic necrosis virus (IPNV) and the cells were processed to detect the virus by two methods: 1) indirect immunofluorescence stain followed by flow cytometry analysis, and 2) RT-PCR or nested-PCR amplification. These methods were compared with separate homogenization of visceral samples, followed by inoculation on cell cultures and seroneutralization, a procedure routinely employed by most disease diagnostic laboratories. IPNV was isolated by homogenization of samples in all seven specimens examined. The assay took between 7 and 28 days required for the appearance of cytopathic effects, which is usual in subclinical samples. From purified leukocytes, IPNV was detected by RT-PCR in five out of the seven specimens. The nested-PCR improved the sensitivity of the assay and gave positive results for all the fish. In addition, flow cytometry demonstrated the presence of IPNV in all the fish in 8-24 h. Examination of leukocytes is a practical way of detecting IPNV and much less time-consuming than the homogenization technique.This study was supported by grants from the Comision Interministerial de Ciencia y Tecnologla, project AGF 98-0837 and Comunidad Autonoma de Madrid, project 079/23/98