Immunohistologic diagnosis of pseudorabies (Aujeszky's disease) using monoclonal antibodies.

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Pseudorabies (PR) (Aujesky’s disease) has been seen in several domestic species, such as pigs, ruminants, dogs, and horses. The laboratory diagnosis of PR has been investigated using various approaches. The most frequent have been immunofluorescence (IF) and immunoperoxidase methods, histopathologic investigation, tissue culture inoculation, and in situ hybridization. Immunoperoxidase staining can also be used to diagnose virus-induced nonsuppurative encephalitis, which is hard to differentiate histopathologically. Eighty monoclonal antibodies (MAbs) against PR virus (PRV) were produced at the Istituto Zooprofilattico to develop a competitive enzyme-linked immunosorbent assay to differentiate antibodies elicited by glycoprotein (G)F-negative strain vaccines from antibodies resulting from infection with wild type viruses and to develop a suitable diagnostic tool for the identification of PRV either directly from infected tissues or from inoculated cell cultures. Eight of the 80 MAbs reacted strongly in the IF test performed on frozen tissue sections (E. Brocchi, personal communication). The reactivity of these MAbs with paraffin-embedded sections was examined to evaluate immunoperoxidase staining for PRV diagnosis in routinely samples. Moreover, a retrospective study was conducted on animals submitted for necropsy because of clinical neurologic signs. The 8 MAbs specific for PRV (Table 1) were tested immunohistochemically with formalin-fixed, paraffin-embedded pig tonsils (for which PRV had been previously detected by IF on cryostatic sections). The following procedures were used for immunohistochemical staining. Five-micrometerthick paraffin-embedded sections were dewaxed in 2 changes of xilene for 15 minute each and hydrated through graded alcohols. All sections were treated with 3% hydrogen peroxide in methanol for 30-minutes at room temperature followed by several washes in Tris-buffered saline (TBS). Nonspecific antibody binding was blocked by 30 minutes of incubation with normal horse serum 1:75 diluted in TBS. As primary antibodies, ascitic fluid TBS dilutions (1:200, 1:400, 1:800, 1:1,000) of each MAb were incubated in 2 different ways, overnight at 4 C and 1 hour at 37 C. After MAb incubation, the sections were washed with TBS and sequentially incubated with biotinylated horse anti-mouse IgG 1: 200 diluted and streptoavidin peroxidase 1500 diluted for 30 minutes each, with rinses in TBS between the incubations. Diaminobenzidine was used as substrate of the peroxidase