E of 0.5? mm2 to better protect the antigen from degradation by minimising the time spent in the rumen. In sheep, particles with diameters larger than 1.18 mm transit through the rumen slower than smaller particles [32]; this has also been found in cattle with increased forage particle size improving fibre digestibility by increasing retention time in the rumen [33]. From the rumen, the vaccine transits through the reticulum and omasum before reaching the abomasum (true stomach) where enzymatic digestion of protein, carbohydrates and lipids is initiated. It is anticipated that breakdown of the plant cells encapsulating the rLTB antigen begins in the rumen and continues in the reticulum, the principal sites for cellulose digestion in ruminant species. It was in the abomasum mucus that antibody responses were first observed following administration of the LTBLeaf vaccine. This suggests that as the leaf material begins to degrade the antigen remains sufficiently protected during rumination, Pleuromutilin supplier presumably by the lipid coating provided by the oil formulation matrix. In contrast, the lack of antibody response in abomasum mucus from the LTB-HR vaccine suggests that root tissue may be comparatively more resistant to rumination and enzymatic digestion resulting in delayed antigen release. Although GALT is absent in the abomasum, immune responses can be induced when the mucosal epithelium is penetrated [2]. LTB is particularly efficient in crossing the epithelium from the lumen primarily via binding to ganglioside GM1 along with other mammalian galactoglycoprotein receptors [13,14]. Moreover, direct sampling of antigen from the mucosal lumen may also occur via intra- and sub-epithelial DCs [2,34]. Once the antigen has traversed the mucosal epithelium it is transported by DCs via the lymphatics to draining MLNs where antigen-specific B cells are generated and then returned to mucosal sites via the blood stream [2,35]. From the abomasum, the vaccine materials enter the small intestine. By this stage breakdown of the plant cells and formulation matrix should be completed, releasing the remainder of its antigenic cargo. It was in the small intestine that the most robust mucosal immune responses were detected from both the LTB-Leaf and LTB-HR vaccines, the leaf material producing elevated IgA titres compared to other treatments in all five sheep receiving this vaccine. It was of interest that section 4, the section further through the GIT, was the site where the most robust antigen-specific IgG responses were found while IgA responses expanded to earlier sites (sections 2 to 4). The consistency in theOral Immunogenicity of a Model PMV in Sheepimmune response observed at the small intestine, particularly for the LTB-Leaf group, is noteworthy given the potential for (-)-Calyculin A variable responses when using an outbred 
sample of sheep. LTB-specific IgA antibodies were absent in all sera, irrespective of vaccine treatment or number of doses administered. This is not unexpected as detection of antibody production in serum following mucosal immunisation can be typically difficult particularly when responses are low [24]. An alternative approach, previously validated in several studies, was utilised to detect antibodies secreted by MLNs using the ASC assay [23,36]. Elevated IgA titres were detected in the MLNs of two LTB-Leaf- and LTB-HRvaccinated sheep as compared to other treatments. In addition, MLN 2 was identified as the most active site for generating an IgG response.E of 0.5? mm2 to better protect the antigen from degradation by minimising the time spent in the rumen. In sheep, particles with diameters larger than 1.18 mm transit through the rumen slower than smaller particles [32]; this has also been found in cattle with increased forage particle size improving fibre digestibility by increasing retention time in the rumen [33]. From the rumen, the vaccine transits through the reticulum and omasum before reaching the abomasum (true stomach) where enzymatic digestion of protein, carbohydrates and lipids is initiated. It is anticipated that breakdown of the plant cells encapsulating the rLTB antigen begins in the rumen and continues in the reticulum, the principal sites for cellulose digestion in ruminant species. It was in the abomasum mucus that antibody responses were first observed following administration of the LTBLeaf vaccine. This suggests that as the leaf material begins to degrade the antigen remains sufficiently protected during rumination, presumably by the lipid coating provided by the oil formulation matrix. In contrast, the lack of antibody response in abomasum mucus from the LTB-HR vaccine suggests that root tissue may be comparatively more resistant to rumination and enzymatic digestion resulting in delayed antigen release. Although GALT is absent in the abomasum, immune responses can be induced when the mucosal epithelium is penetrated [2]. LTB is particularly efficient in crossing the epithelium from the lumen primarily via binding to ganglioside GM1 along with other mammalian galactoglycoprotein receptors [13,14]. Moreover, direct sampling of antigen from the mucosal lumen may also occur via intra- and sub-epithelial DCs [2,34]. Once the antigen has traversed the mucosal epithelium it is transported by DCs via the lymphatics to draining MLNs where antigen-specific B cells are generated and then returned to mucosal sites via the blood stream [2,35]. From the abomasum, the vaccine materials enter the small intestine. By this stage breakdown of the plant cells and formulation matrix should be completed, releasing the remainder of its antigenic cargo. It was in the small intestine that the most robust mucosal immune responses were detected from both the LTB-Leaf and LTB-HR vaccines, the leaf material producing elevated IgA titres compared to other treatments in all five sheep receiving this vaccine. It was of interest that section 4, the section 
further through the GIT, was the site where the most robust antigen-specific IgG responses were found while IgA responses expanded to earlier sites (sections 2 to 4). The consistency in theOral Immunogenicity of a Model PMV in Sheepimmune response observed at the small intestine, particularly for the LTB-Leaf group, is noteworthy given the potential for variable responses when using an outbred sample of sheep. LTB-specific IgA antibodies were absent in all sera, irrespective of vaccine treatment or number of doses administered. This is not unexpected as detection of antibody production in serum following mucosal immunisation can be typically difficult particularly when responses are low [24]. An alternative approach, previously validated in several studies, was utilised to detect antibodies secreted by MLNs using the ASC assay [23,36]. Elevated IgA titres were detected in the MLNs of two LTB-Leaf- and LTB-HRvaccinated sheep as compared to other treatments. In addition, MLN 2 was identified as the most active site for generating an IgG response.