Opsonization Effects onMycobacterium aviumsubsp.paratuberculosis-Macrophage Interactions

Abstract

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...

Bovine IgG1 antibodies againstMycobacterium aviumsubsp.paratuberculosisprotein p34-cx improve association of bacteria and macrophages

“…can be ingested in the presence or absence of serum and it has been suggested that different entry pathways could affect bacilli survival into the macrophages [13,39]. Besides, other authors described that complete immune sera and normal bovine sera were similar in the ability to promote macrophage uptake of Map in vitro [19].…”

“…Maps were stained with fluorescein isothiocyanate (FITC, Sigma Chemical Co.) as described previously [19]. Briefly, 4 × 10 7 broth-grown bacteria were suspended in carbonate buffer (0.001% FITC in a 0.2 M Na 2 CO 3 -NaHCO 3 , 150 mM NaCl buffer) pH 9.2 for 30 min.…”

Competition for Antigen between Th1 and Th2 Responses Determines the Timing of the Immune Response Switch during Mycobaterium avium Subspecies paratuberulosis Infection in Ruminants

“…At later stages of the infection animals with multi-bacillary lesions express predominantly a Th2-type immune response that is measured by the presence of MAP-specific IgG1 antibodies, production of which is driven by IL-4 or IL-10 producing CD4 T cells [18]–[20]. Although high levels of MAP-specific antibodies are detected in animals in late stages of the disease, these antibodies do not appear to be protective and may even be detrimental by increasing uptake of extracellular bacteria by macrophages [24]. Thus, experimental infections of sheep with MAP suggest a switch from dominance of the MAP-specific Th1 immune response in the early stages of the disease to a predominantly Th2 response at later stages of the disease (Figure 1A).…”

Mycobacterium avium subspecies paratuberculosis infection of cattle does not diminish peripheral blood-derived macrophage mycobactericidal activity

“…We next determined if these differences were attributable to variation in phagocytosis by measuring attachment/uptake of FITC labeled M. avium subsp. paratuberculosis by flow cytometry as previously described [5]. The level of attachment and uptake of M. avium subsp.…”