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Currently, it is not known whether commercial pasteurization effectively kills Mycobacterium paratuberculosis in contaminated raw milk. Results from holder test tube experiments indicated that a residual population of viable bacteria remained after treatment at 65, 72, 74, or 76°C for 0 to 30 min. Use of a laboratory-scale pasteurizer unit demonstrated that treatment of raw milk at 72°C for 15 s effectively killed all M. paratuberculosis.

High antibody titers in ruminants infected with Mycobacterium avium subsp. paratuberculosis correlates with disease progression. Effects of humoral responses during mycobacterial infection are not completely understood. This study suggests that activation status may be an important factor in determining macrophage ability to limit proliferation of opsonized M. avium subsp. paratuberculosis.Generally, the humoral response present during mycobacterial infections has been considered nonprotective; however, the function and influence of the generated antibodies on the course of infection are incompletely understood. Development of high antibody levels in ruminants infected with Mycobacterium avium subsp. paratuberculosis correlates with deterioration of cell-mediated immunity and development of extensive multibacillary granulomatous lesions (9). The goal of the present study was to test the hypothesis that antibody opsonization of M. avium subsp. paratuberculosis would not enhance mycobactericidal responses in macrophages. We compared the interaction of cultured naive bovine macrophages with M. avium subsp. paratuberculosis opsonized with serum from naive or infected animals with high anti-M. avium subsp. paratuberculosis titers.Immune serum was pooled from five adult bovine cases of M. avium subsp. paratuberculosis infection that were confirmed by serum enzyme-linked immunosorbent assay for anti-M. avium subsp. paratuberculosis antibodies and gross and microscopic pathology findings. Anti-M. avium subsp. paratuberculosis serum levels were determined for each animal by the Iowa State University Veterinary Diagnostic Laboratory using an IDDEX system according to the manufacturer's instructions. We further titrated the positive serum samples to an endpoint opsonizing activity (indirect immunofluorescence) at Ͼ1:500 dilutions for each. Naive serum was pooled from two adult bovines maintained free of infection. Monocytes were generated from peripheral blood mononuclear cells and differentiated into macrophages by 7 days of culture. The 19698 strain of M. avium subsp. paratuberculosis was obtained from the American Type Culture Collection (Manassas, Va.) and conjugated with fluorescein isothiocyanate (FITC) by a previously described method (1). Bacteria were opsonized for 1 h at 37°C with complete or heat-inactivated serum.We used fluorescence microscopy to determine bacterial uptake/adherence by scoring 100 macrophages cultured on chambered slides (Nalge Nunc International, Naperville, IL) as either containing or lacking FITC-tagged M. avium subsp.paratuberculosis at 1 h postinfection. As shown in Fig. 1, opsonization with naive serum (NS), immune serum (IS), and heat-inactivated immune serum (HIS) led to similar uptake, which was increased over nonopsonized bacteria. Opsonization with heat-inactivated NS (HNS) led to similar uptake as nonopsonized bacteria (P Ͻ 0.05). Based on these results, complete IS and NS were similar in the ability to promote macrophage uptake of M. avium subsp. paratuberculosis, as were compleme...

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...