Antibodies Induced by Lipoarabinomannan in Bovines: Characterization and Effects on the Interaction between Mycobacterium Avium Subsp. Paratuberculosis and Macrophages In Vitro

Abstract

Lipoarabinomannan (LAM) is a major glycolipidic antigen on the mycobacterial envelope. The aim of this study was to characterize the humoral immune response induced by immunization with a LAM extract in bovines and to evaluate the role of the generated antibodies in the in vitro infection of macrophages with Mycobacterium avium subsp. paratuberculosis (MAP). Sera from fourteen calves immunized with LAM extract or PBS emulsified in Freund's Incomplete Adjuvant and from five paratuberculosis-infected bovines were studied. LAM-immunized calves developed specific antibodies with IgG1 as the predominant isotype. Serum immunoglobulins were isolated and their effect was examined in MAP ingestion and viability assays using a bovine macrophage cell line. Our results show that the antibodies generated by LAM immunization significantly increase MAP ingestion and reduce its intracellular viability, suggesting an active role in this model.

Figure 1

Relative levels of LAM extract-specific IgG isotypes expressed as ELISA Units (EUs) ± standard deviation of two independent measurements for each animal at 1 : 100 dilution of sera.

Figure 2

(a) Characterization of LAM extract by SDS-PAGE (lane 1: purified LAM of M. tuberculosis, lane 2: LAM extract, both silver-stained for carbohydrate detection) and immunoblot. (lane 3: LAM extract reactivity assessed against one representative serum from the infected control group). (b) Immunoblot of bovine sera reactivity against LAM extract. Left side of the dotted line: LAM extract was run as antigen. D and UD indicate protein-digested or undigested antigen. 1, 2 and 3 show results of three representative sera from LAM-immunized group. Normal control shows results representative for sera in that group. Right side of the dotted line: ovalbumin (OVA) was run as antigen and reactivity assessed against serum of an OVA-immunized bovine, as a positive control of protein digestion. MWM: molecular weight markers.

Figure 3

Functional effects of antibodies: (a) on MAP ingestion. Results are expressed as mean phagocytic index for each treatment ± standard deviation; (b) on MAP intracellular viability. Results are expressed as % change in MAP viability 72 h after the ingestion by Bomac cells. Boxes are representative of electrophoretic (lane 1) and SDS-PAGE (lane 2) pattern of precipitated and purified Igs. α and γ indicate alpha- and gamma- globulins bands. MWM: molecular weight markers. Error bars indicate standard deviation of three independent assays and different letters or numbers over bars represent significant differences between groups.