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Abstract. Porcine reproductive and respiratory syndrome virus (PRRSV) antigens were detected by a streptavidin-biotin complex technique in tissues of 3-week-old colostrum-deprived pigs that had been inoculated intranasally with PRRSV and had developed moderate respiratory disease. Moderate, multifocal, tan-colored consolidation of the lungs and severe enlargement of the lymph nodes were noted at necropsy. Severe interstitial pneumonia characterized by type 2 pneumocyte proliferation, septa1 infiltration with mononuclear cells, and accumulation of macrophages and necrotic cells in alveolar spaces was observed at 4 and 9 days postinoculation. Moderate multifocal perivascular lymphohistiocytic myocarditis was observed at 9 days postinoculation. Marked lymphoid follicular hyperplasia and follicular necrosis in the tonsil, spleen, and lymph nodes was observed. A monoclonal antibody that recognizes a conserved epitope of PRRSV nucleocapsid protein was used as primary antibody for immunohistochemistry. Antigen was readily detected in alveolar macrophages in the lung and in endothelial cells and macrophages in the heart. Macrophages and cells resembling dendritic cells in tonsil, lymph nodes, thymus, and spleen also stained intensely positive for viral antigen. PRRSV appears to replicate primarily within macrophages in the respiratory and lymphoid systems of the pig.

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Three groups of 5-week-old cesarian-derived, colostrum-deprived pigs were inoculated intranasally with either a high-virulence isolate (VR2385) or a low-virulence isolate (VR2431) of porcine reproductive and respiratory syndrome virus (PRRSV) or with uninfected cell culture and media. Formalin-fixed, paraffin-embedded tissues from pigs euthanatized at 10, 21, and 28 days post-inoculation were examined by in situ hybridization for PRRSV nucleic acid using a digoxigenin-labeled antisense RNA probe approximately 1,000 nucleotides in length. Alveolar macrophages were positive in the lungs of 9/9, 2/2, and 0/2 VR2385-inoculated pigs and 7/9, 1/2, and 2/3 VR2431-inoculated pigs at 10, 21, and 28 days post-inoculation, respectively. More positive cells were detected in lungs from VR2385-inoculated pigs compared to VR2431-inoculated pigs at 10 and 21 days post-inoculation. Positive cells within lymph nodes were tingible body macrophages in germinal centers and macrophages or interdigitating dendritic cells within the paracortical area. VR2385 was detected in the tracheobronchial lymph node (TBLN) and mediastinal lymph node (MLN) of 7/9 and 9/9 pigs at 10 days post-inoculation, but was only detected in the TBLN of 1/2 and 0/2 pigs and in the MLN of 0/2 and 1/2 pigs at 21 and 28 days post-inoculation, respectively. In contrast, VR2431 was detected in teh TBLN and MLN of 5/9 and 2/9 pigs at 10 days post-inoculation and in the TBLN of 0/2 and 1/3 pigs and in the MLN of 0/2 and 0/3 pigs at 21 and 28 days post-inoculation, respectively. There were more positive cells in TBLN and MLN in pigs inoculated with VR2385 at 10 days post-inoculation. Macrophages located at the epithelial-lymphoid interface of tonsilar crypts and within the paracortical areas were positive in tonsils of 9/9, 2/2, and 1/2 VR2385-inoculated pigs and 7/9, 1/2, and 1/3 VR2431-inoculated pigs at 10, 21, and 28 days post-inoculation, respectively. Positive cells in the thymic medulla were multinucleate and were only detected at 10 days post-inoculation in 2/9 VR2385-inoculated pigs and 4/9 VR2431-inoculated pigs. Positive cells within the spleen were few, spindle-shaped, located within smooth muscle trabecula, and only present at 10 days post-inoculation in 3/9 VR2385-inoculated pigs. We conclude that the tissue tropism and distribution of positive cells within tissues is similar for VR2385 and VR2431. However, tissues from more pigs and more cells within tissues were positive in pigs inoculated with VR2385 than VR2431 at 10 and 21 days post-inoculation. These findings indicate that the more virulent isolate VR2385 may replicate better in vivo than the less virulent isolate VR2431. This supports the hypothesis that an increased ability to replicate in vivo contributes to increased virulence of PRRSV.

Inducible nitric oxide synthase (iNOS) is important in the control of a number of intracellular pathogens, including mycobacteria, and is a marker of classic macrophage activation. In human granulomatous diseases such as leprosy, a spectrum of granulomatous lesions is described, ranging from the tuberculoid to lepromatous types. Tuberculoid granulomas are associated with enhanced iNOS production and improved clinical outcomes over the lepromatous types. The aim of this study is to determine whether an association exists between morphology of bovine Johne's disease granulomas and lesion macrophage effector functions. To accomplish this, we retrospectively evaluated 24 cases of bovine Johne's disease. In each case, we recorded the predominant granuloma morphology and evaluated iNOS immunoreactivity and bacterial burden by acid-fast stains and mycobacterial immunolabeling. The results of this study demonstrate that all cases had granulomas with features most similar to the lepromatous type. This morphology correlated with heavy bacterial burdens demonstrated by acid-fast staining and mycobacterial immunoreactivity. None of the cases had high expression of iNOS in mycobacterial-positive granulomas. When iNOS immunoreactivity was identified, it was usually located near the crypts and was distinct from the granulomatous foci. Inducible nitric oxide synthase (iNOS) plays a crucial role in host defense and is a major killing mechanism within macrophages. iNOS is one of three iso-forms of nitric oxide synthase that generates nitric oxide (NO) from L-arginine. iNOS expression is well described in macrophages and neutrophils; however, it has also been reported in several additional cell types including respiratory and digestive epithelia, cardiac myocytes, and endothelium. 10,16,34,41 Within seconds after formation, NO reacts with water and oxygen to form reactive intermediates including NO 2 , NO 3 , N 2 O 2 , and ON00. These reactive nitrogen intermedi-ates (RNI) are highly unstable and are directly toxic to many microorganisms. iNOS expression and RNI production are important for macrophage killing of a wide range of intracellular pathogens including His-toplasma capsulatum, Toxoplasma gondii, Listeria monocytogenes, Mycobacterium tuberculosis, and Ec-tromelia virus (reviewed in MacMicking et al. 30) iNOS expression is induced by several types of signals. Environmental factors such as hypoxia and bacterial components including lipopolysaccharide and mycobacterial lipoarabinomanins will lead to iNOS expression in macrophages. 23,37 In addition, iNOS is strongly induced by T-helper 1 (Th1)-type cytokines including interferon-(IFN), interleukin 2, and tumor necrosis factor-; therefore, iNOS expression is a marker of Th1-mediated macrophage activation. 15,20 Similar to other species of pathogenic mycobacteria, Mycobacterium avium subspecies paratuberculosis (M. a. ptb) is an intracellular pathogen of macrophages and monocytes. 46 The pathogenesis of M. a. ptb infection at the macrophage level is incompletely understood , wi...