Detection of European porcine reproductive and respiratory syndrome virus in porcine alveolar macrophages by two-colour immunofluorescence and in-situ hybridization-immunohistochemistry double labelling

Two groups of five pigs aged 6 weeks were each infected oronasally with one of two different European isolates of porcine reproductive and respiratory syndrome virus (PRRSV). The animals were killed sequentially at 4, 7, 14 o r 21 days post-inoculation for examination. The methods used consisted of h istopathology, and mono- and double-labelling techniques based on in-situ h ybridization, immunofluorescence and immunohistochemistry. Porcine alveolar macrophages (PAMs) contained large amounts of PRRSV anti-en and PRRSV RNA, as shown by double labelling with (1) either PRRSV immunofluorescence or P RRSV-specific in-situ hybridization with digoxigenin-labeled riboprobes. an d (2) immunolabelling with Mac 387 antibody for calprotectin. Expression of PRRSV-RNA was not detectable in cytokeratin-positive hypertrophic and prol iferating pneumocytes or in cells of alveolar ducts or bronchiolar epitheli um. The use of two-colour immunofluorescence with confocal laser scanning m icroscopy and double labelling with in-situ hybridization-immunohistochemis try showed that PAMs,here the only pulmonary target cells. This contradicts earlier reports that epithelial pulmonary cells may also be infected by PR RSV. (C) 2001 Harcourt Publishers Ltd.