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

Abstract

-Paratuberculosis, caused by Mycobacterium avium subsp. paratuberculosis (Map), is a chronic granulomatous enteric disease in cattle. Among molecular components of Map, protein p34 was identified as specific and immunodominant for bovine B cells. In order to determine if specific antibodies could influence the course of Map pathogenesis, the interaction between bacteria and bovine macrophages was studied. Bovine polyclonal antibodies from 3 calves vaccinated with protein p34-cx, 6 calves vaccinated with heatkilled Map, 8 naturally infected, and 3 healthy calves -as negative controls-were used. Specific anti-Map, -p34-cx and -PPA-3 antibodies were evaluated and isotype characterized. Infected and Map vaccinated animals showed similar IgG1 and IgG2 response against Map whole bacteria. When p34-cx was used as the antigen, mainly IgG1 and IgG3 were detected in infected and only IgG1 in p34-cx vaccinated animals. Bovine polyclonal antibodies from three animals of each category were isolated and affinity purified through Map and p34-cx columns. The effect of these antibodies in association with Map and a transformed bovine peritoneal macrophage's cell line (Bov-Mac) as well as activation of NF-κB transcription factor was studied. Our results show that association of Map significantly increased in vitro after pretreatment with bovine anti-Map or anti-p34-cx antibodies obtained from vaccinated or infected cattle when compared with those of controls. Improved activation of NF-κB was detected in macrophages that ingested Map opsonized with either anti-Map or anti-p34-cx specific antibodies of infected or vaccinated calves, suggesting that both anti-Map and IgG1 anti-p34-cx antibodies support Map-macrophage interactions.Mycobacterium avium subsp. paratuberculosis / p34 / IgG1 / macrophages / cattle

Antibodies Induced by Lipoarabinomannan in Bovines: Characterization and Effects on the Interaction betweenMycobacterium aviumSubsp.paratuberculosisand MacrophagesIn Vitro

“…MAP-phagocytosis enhancement by hyperimmune sera has been previously reported in Bomac cells [18] and bovine blood monocyte-derived macrophages (BMDMs) [17, 37]. The phagocytic levels detected herein were comparable to those published by Woo et al for Bomac cells [38], although our indexes were lower than those described for BMDM when ingesting MAP [17, 37].…”

“…The influence of opsonization with antibodies on MAP phagocytosis and intracellular viability has been previously evaluated [17, 18, 33, 37, 38]. However, published reports have been generally based on assays where total serum was examined as a source of antibodies.…”

“…An HRP-conjugated goat antibovine IgG (KPL) was added in a 1 : 1000 dilution. For specific isotype evaluation, HRP-conjugated sheep anti-bovine IgM, IgG1, and IgG2 antibodies (Behtyl Laboratories Inc., Montgomery, TX, USA) diluted 1 : 300 and rabbit antibovine IgG3 antibody [18] diluted 1 : 500 followed by HRP-conjugated goat antirabbit IgG (KPL) diluted 1 : 1000 were used. Plates were developed as described above.…”

“…A recent report has evaluated the effect of immune serum on the MAP macrophage interaction suggesting an active role of antibodies [17]. In addition, our group has previously reported that purified specific antibodies against MAP could enhance the MAP-macrophage interaction in vitro and improve the activation of the nuclear factor NF- κ B in infected cells [18]. …”

Effect of lipoarabinomannan from Mycobacterium avium subsp avium in Freund’s incomplete adjuvant on the immune response of cattle

“…Sample and control sera (1:500) were incubated for 1 h at 37°C. HRP-conjugated sheep anti-bovine IgM, IgG1 and IgG2 (1:300) (Bethyl Labs Inc., USA) and unlabeled rabbit anti-bovine IgG3 diluted 1:500 (23) were added and incubated for 1 h at 37°C. Then, HRP-conjugated goat anti-rabbit IgG (KPL) diluted 1:1000 was added to anti-IgG3 wells.…”