In situ hybridization and histopathological observations during ostreid herpesvirus-1-associated mortalities in Pacific oysters Crassostrea gigas

Abstract

In a previous longitudinal study conducted during a mortality investigation associated with ostreid herpesvirus-1 (OsHV-1) microvariant in New Zealand Pacific oysters in 2010-2011, temporality of OsHV-1 nucleic acid detection by real-time PCR assay and onset of Pacific oyster mortality was observed. The present study further elucidated the role of OsHV-1 using an in situ hybridization (ISH) assay on sections of Pacific oysters collected from the same longitudinal study. Hybridization of the labelled probe with the target region of the OsHV-1 genome in infected cells was detected colorimetrically using nitro blue tetrazolium (NBT). OsHV-1 presence and distribution in spat indicated by the ISH signal was then compared with the existence of pathological changes in oyster tissues. Dark blue to purplish black NBT cell labelling was seen predominantly in the stroma of the mantle and gills at Day 5 post introduction to the farm. The distribution and location of ISH signals indicated the extent of OsHV-1-infected cells in multiple tissues. Histopathological abnormalities were mostly non-specific; however, a progressive pattern of increasingly widespread haemocytosis coincided with the appearance of OsHV-1-infected cells in spat collected at different time-points. The visualisation of an increasing number of OsHV-1-positive cells in spat prior to a marked increase in mortality indicated the strong likelihood of an on-going and active viral infection in some oysters. Further studies are recommended to elucidate OsHV-1 pathogenesis in Pacific oysters in association with other potentially causal variables, such as elevated temperature and interaction with Vibrio spp. bacteria.

Keywords: Crassostrea gigas; In situ hybridization; Microvariant; Ostreid herpesvirus-1; Pacific oysters; μVar.