Racheal Aye was also supported by a grant from the German Academic Exchange Support (DAAD), in-region scholarship

Racheal Aye was also supported by a grant from the German Academic Exchange Support (DAAD), in-region scholarship. identify an antigen to any of the inhibitory antibodies. Thirteen anti-Mycoplasma mycoidessubsp.mycoides(AMMY) monoclonal antibodies (mAbs) inhibited adhesion by at least 30% and ten of the mAbs bound to multiple bands on Western blots suggesting that this antibodies bound to proteins of variable sizes. AMMY 10, a previously characterizedMmm- capsular polysaccharide (CPS) specific antibody, inhibited growth ofMmm in vitroand also caused agglutination ofMmmtotal cell lysate. AMMY 5, a 2-oxo acid dehydrogenase acyltransferase (Catalytic domain name) (MSC_0267) specific antibody, was identified and polyclonal rabbit serum against recombinant MSC_0267 blocked adhesion ofMmmto BoLEC by 41%. Antigens recognized by these antibodies could be vaccine candidate(s) and should be subsequently tested for their ability to induce a protective immune responsein vivo. == 1. Introduction == Many mycoplasmas that infect livestock adhere to and colonize epithelial surfaces of various tissues in their hosts. Adhesion is usually thought to be tissue and host specific, and a prerequisite for colonization by pathogenic mycoplasmas (Barile and Rottem, 1993). The loss of adhesion by mutation results in loss of infectivity, and reversion to the cytoadhering phenotype is usually accompanied by requisition of infectivity and virulence (Razin and Jacobs, 1992;Baseman and Tully, 1997). Cytoadherence of mycoplasmas to host cells may result in damage by interference with membrane receptors or alteration of transport mechanisms of the host cell (Debey and Ross, 1994), release of cytotoxic metabolites (Pilo et al., 2007) or hydrolysis of host cell phospholipids by potent Oridonin (Isodonol) membrane-bound phospholipases present in manyMycoplasmaspecies (Shibata et al., 1995). The cytoadherence mechanisms of manyMycoplasmaspecies, including those ofM.pneumoniae(Chaudhry et al., Oridonin (Isodonol) 2007),M.genitalium(Ueno et al., 2008),M.suis(Zhang et al., 2015),M.hominis(Brown et al., 2014),M.hyopneumonia(Burnett et al., 2006),M.agalactiae(Fleury et al., 2002) andM.bovis(Track et al., 2012) have been described. In these studies, adherence was shown to be a complex multifactorial process involving one or more adhesion molecules. In our previous study, we established a cytoadherence assay that measured the conversation ofMmmwith various host cells and showed thatMmmcytoadherence is usually tissue and host specific (Aye et al., 2015). In this study we further analyzed the Oridonin (Isodonol) adhesion ofMycoplasma mycoidessubspeciesmycoides(Mmm), the causative agent of contagious bovine pleuropneumonia (CBPP), an important disease of cattle in sub-Saharan Africa. The aim of this study was to identify potentialMmmvaccine targets by developing a panel Oridonin (Isodonol) of monoclonal antibodies againstMmm, and use them to investigate their inhibitory effect on the adherence ofMmmto primary bovine lung epithelial cells (BoLEC), their capacity to inhibitMmmgrowthin vitro, and to further identify an antigen to which any one of the inhibitory antibodies bound. Using an indirect flow cytometry assay, we identified 13 anti-Mycoplasma mycoidessubspeciesmycoidesantibodies (AMMY 113) that inhibited adhesion ofMmmto BoLEC. Subsequent to this initial work,Schieck et al (2016)characterized AMMY 10 as anMmm-specific capsular polysaccharide (CPS) antibody. We further identifiedpdhC, part of the pyruvate dehydrogenase (PDH) complex, as an antigen that bound to AMMY 5 and provided evidence of its possible involvement in the cytoadhesion.Mycoplasmamolecules involved in the adhesion process, as well as molecules essential for growth or important biological functions are candidate vaccine targets. == 2. Materials and methods == == 2.1.Mmm-specific monoclonal antibody production == Five 68 week old BALB/c mice were bHLHb24 immunized intraperitoneally with 100 gMmmstrain Afad whole cell lysate in 100 l PBS mixed with equal volumes of TitterMax adjuvant (Sigma) on days 0, 14 and 21. Seven days after the last immunization, antibody titers were measured by indirect enzyme-linked immunosorbent assay (ELISA-see below). Mice with highest antibody titers were given a booster dose of 100 gMmmlysate without adjuvant three days before aseptic spleen removal for fusion. Mice were used in accordance with the International Livestock Research Institute Institutional Care and Use Committee guidelines (IACUC Ref nr 2010.06) Fusion was carried out as previously described in detail (Naessens et al., 1985) using X63Ag8.653 murine myeloma cells. The hybridomas were cultured in 96-well Costar tissue culture plates Oridonin (Isodonol) (Sigma) in the presence of thymocytes, hypoxanthine, aminopterin, and thymidine (Sigma) at 37 C in 5% CO2for 1014 days. Hybridomas were screened for antibody secretion by standard indirect ELISA. Briefly, Immunlon 2 HB plates (Dynatech Laboratories) were coated with 5 g/mlMmmstrain Afad whole cell lysate (100.