Detection of porcine reproductive and respiratory syndrome virus and Mycoplasma hyorhinis antigens in pulmonary lesions of pigs suffering from respiratory distress.

Eleven field cases of a disease characterized by severe dyspnoea or abdominal breathing were examined post mortem. The affected pigs had antibody against porcine reproductive and respiratory syndrome virus (PRRSV). The predominant lung lesions were severe proliferative and interstitial pneumonia, and slight suppurative bronchopneumonia. The lesions were closely associated with the sites at which PRRSV and Mycoplasma hyorhinis antigens were detected. Four of five pigs inoculated with PRRSV developed slight pneumonitis. The fifth animal, which died of severe pneumonitis, yielded a heavy culture of M. hyorhinis. These findings demonstrate that dual infection with M. hyorhinis and PRRSV caused severe pulmonary lesions.

Isolation of porcine reproductive and respiratory syndrome (PRRS) virus from Heko-Heko disease of pigs.

Porcine reproductive and respiratory syndrome (PRRS) virus was isolated at high frequency from the sera and lungs of pigs affected with Heko-Heko disease. In addition, a considerable amount of Mycoplasma hyorhinis (Mhr) was also isolated from the lungs. Inoculation of gnotobiote pigs with the first isolate of PRRS virus resulted in the reproduction of proliferative and interstitial pneumonia. The virus was recovered from the inoculated pigs for long periods. Superinfection with PRRS virus and Mhr appeared to produce more serious pneumonia than inoculation with PRRS virus alone. In this study, the presence of PRRS virus was confirmed in Japan, and the PRRS virus was considered as being the most important pathogen for Heko-Heko disease.

Cytokine regulation of lactate dehydrogenase-elevating virus: inhibition of viral replication by interferon-gamma.

The mechanisms which regulate the replication of lactate dehydrogenase-elevating virus (LDV), a persistent murine model virus which infects macrophages, are unclear. For this study, the effects of murine recombinant interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) on LDV replication were examined. LDV permissiveness was reduced in macrophages obtained from uninfected mice treated with IFN-gamma prior to cell harvest and in vitro LDV infection. Virus inhibition by IFN-gamma was also observed when neonatal LDV-infected mice were injected with this cytokine prior to macrophage harvest and analysis of LDV replication-positive cells. Persistently LDV-infected mice demonstrated an increase in viremia levels following treatment with TNF-alpha. Neither IFN-gamma nor TNF-alpha had any direct in vitro effect on LDV replication in cultured macrophages, suggesting that the actions of these cytokines required secondary or accessory in vivo events. These results provide evidence for cytokine-mediated regulation of LDV infection and support a role for the immune system in the LDV-host relationship.

Porcine reproductive and respiratory syndrome virus: morphological, biochemical and serological characteristics of Quebec isolates associated with acute and chronic outbreaks of porcine reproductive and respiratory syndrome.

Cytolytic and noncytolytic strains of the porcine reproductive and respiratory syndrome virus (PRRSV) were isolated in primary cultures of porcine alveolar macrophages (PAM) from lung homogenates of stillborn fetuses or blood samples of dyspneic piglets collected from Quebec pig farms having experienced acute or chronic outbreaks of PRRS. Serological identification of the virus was confirmed by indirect immunofluorescence and indirect protein A-gold immunoelectron microscopy using reference antiserum prepared from experimentally-infected specific pathogen free (SPF) piglets and monoclonal antibodies (MoAbs) directed against the p15 nucleocapsid (N) protein of the reference ATCC-VR2332 isolate. Intracytoplasmic enveloped viral particles that tended to accumulate into cytoplasmic vesicles were observed in the infected PAM; no budding was demonstrated at the level of the cytoplasmic membrane. The extracellular virions appeared as pleomorphic but mostly spherical enveloped particles, 50-72 nm in diameter (averaged diameter of 50 particles was 58.3 nm), with an isometric core about 25-30 nm. Buoyant density of the virus in CsCL density gradients was estimated to 1.18-1.20 g/mL. No hemagglutinating activity was demonstrated. Analysis of semipurified virions of isolate IAF-exp91 by radioimmunoprecipitation (RIPA) and Western immunoblotting experiments, using reference rabbit and porcine hyperimmune sera, revealed four major viral proteins, a predominant 15 kD N protein and three other proteins with predicted M(r_ of 19, 26 and 42 kD. Progeny viral particles produced in PRRSV-infected PAM in the presence of tunicamycin lacked the 42 kD protein, thus confirming its N-glycosylated nature. Immunoprecipitation experiments using the anti-ATCC-VR2332 MoAbs confirmed the close antigenic relationships between Quebec and American reference isolates of PRRSV.

Interaction between Streptococcus suis serotype 2 and porcine reproductive and respiratory syndrome virus in specific pathogen-free piglets.

Secondary specific pathogen-free (sSPF) piglets were inoculated intranasally with Streptococcus suis serotype 2 alone, porcine reproductive and respiratory syndrome virus (PRRSV) alone, or with PRRSV followed by S suis. Uninfected piglets were used as controls. Pigs inoculated with PRRSV (ATCC VR-2332) followed by challenge with a virulent strain (87555) of S suis serotype 2 developed clinical signs, suppurative meningitis and large numbers of S suis in their tissues, including the brain and meninges. Pigs inoculated with PRRSV alone, S suis (87555) alone, or with PRRSV and the DH5 strain of S suis serotype 2 (lacking a protein associated with virulence) and the uninfected piglets did not develop clinical signs or lesions or have large numbers of bacteria in their tissues. The results suggest that PRRSV predisposes sSPF pigs to infection and disease caused by virulent S suis serotype 2. Co-infection of piglets with PRRSV and a virulent strain of S suis may provide a useful model for the study of S suis septicaemia and meningitis.

Immunohistochemical detection of porcine reproductive and respiratory syndrome virus using colloidal gold.

Two cytopathic agents were isolated on porcine alveolar macrophages following inoculation with homogenates of lung tissues from pigs showing respiratory problems. These isolates were identified as porcine reproductive and respiratory syndrome (PRRS) virus isolates by indirect immunofluorescence using a PRRS virus (PRRSV) specific monoclonal antibody (MAb) and were designated as LHVA-92-1 and LHVA-92-2. Immunogold electron microscopy using a porcine PRRS positive serum pool and protein A-gold resulted in an intense labelling of aggregates of viral particles. Dark specific cytoplasmic staining of porcine alveolar macrophages infected with both virus isolates could be observed by immunogold silver staining (IGSS) using the specific MAb. This method proved effective in detecting PRRSV antigens in several ethanol-fixed tissues of piglets intranasally inoculated with the supernatants of macrophages infected with each isolate. Immunogold silver staining was also successfully used for the detection of PRRSV antigens on sections of formalin-fixed paraffin-embedded lung tissues and on frozen sections of lungs. The present results indicate that colloidal gold may be useful for the identification and immunohistochemical detection of PRRSV in tissues.