Cyprinid herpesvirus-2 causing mass mortality in goldfish: applying electron microscopy to histological samples for...

In June 2013, a major fish kill of adult goldfish Carassius auratus occurred in Runnemede Lake, New Jersey, USA: an estimated 3000 to 5000 fish died within ~5 d. Necropsy of 4 moribund fish revealed severely pale gills, and histopathology showed type I and II fusion of the gills, diffuse necrosis of hematopoietic tissue in anterior and posterior kidney, and multifocal necrosis of the spleen. Within necrotic areas, pyknosis and enlarged nuclei with marginalized chromatin were observed. Cyprinid herpesvirus-2, the etiological agent for herpesviral hematopoietic necrosis disease, was confirmed in all 4 fish using PCR. We assessed the efficacy of identifying herpesviral infections (viral morphogenesis and cellular ultrastructure) using transmission electron microscopy (TEM) when applied to tissues fixed in 10% neutral buffered formalin (NBF) and tissue that was removed from paraffin blocks. Both sample types could be used to detect the virus within cells at similar concentrations. Tissues reprocessed from 10% NBF contained all the known stages of viral morphogenesis including empty capsids, capsids with an inner linear concentric density, capsids with an electron-dense core, and in the cytoplasm, mature capsids containing an envelope. Paraffin-embedded tissues showed similar stages, but viral capsids with an inner linear concentric density were rare and mature enveloped virions were not observed. In previously paraffin-embedded tissues, cellular membranes were not preserved, making identification of cell types and organelles difficult, whereas membrane preservation was good in tissues processed from 10% NBF. The results demonstrated that routinely fixed and paraffin-embedded samples can be successfully utilized to diagnose herpesviruses, and formalin-fixed tissue could be used to describe viral morphogenesis by TEM, making this a useful and reliable method for diagnostic virology when other samples are not available.