Face with a glass slide. Mucus scrapings were prepared for ELISA as described by [25]. Abomasal scrapings were washed off the slide into a 50 ml tube with 3 ml PBST supplemented with 2x Roche Complete Protease Inhibitor Cocktail Fasudil (Hydrochloride) site tablets (PBST2I). The supernatant was collected following centrifugation at 9000 g for 15 min at 4uC and stored at 220uC until required.Figure 3. LTB-specific IgG (A) and IgA (B) antibody titres in abomasum mucus following oral immunisation with four doses of control or LTB-transgenic plant materials. The horizontal lines represent geometric means. Black symbols denote positive responders defined as sheep with antibody titres at least three standard deviations above the control mean, non-responders are indicated by grey symbols. doi:10.1371/journal.pone.0052907.gSmall intestine washes to sample intestinal secretions. Four sections of the small intestine were excised,University Werribee Animal Facility under conditions approved by the Monash University Animal Ethics Committee (AEC SOBSA/P/2009/98). Sheep were provided with water and standard feed ad lib and fasted 16 h before oral immunisation. Sheep were randomly assigned into four groups of 2? animals each (Table 1). A single sheep from the transgenic rLTB expressing leaf vaccine group (LTB-Leaf) developed balanopsthitis (pizzle rot) 14 days after beginning the trial and was treated with a testosterone implant. This sheep was not excluded from analyses. Sheep were immunised on days 0, 14 and 28 followed by a boost dose on day 38, four days before sacrifice. Vaccine materials were formulated immediately before delivery by mixing 19 g freezedried plant material with 200 ml of an oil based emulsion (125 ml peanut oil:75 ml dH2O). When receiving the transgenic rLTB plant-based vaccines (LTB-HR or LTB-Leaf), each dose was sufficient to deliver 5 mg rLTB. Sheep receiving the CtHR or CtLeaf vaccines were immunised with the equivalent volume of formulated control plant materials. The formulated vaccines were administered orally to sheep by gavage directly into the rumen to simulate drenching, a common delivery system used routinely toeach section measured 0.5 m in length and was taken 3 m apart, beginning at the abomasum/duodenum junction (section 1, 0?0.5 m). Sections 24195657 2? were sampled at 3.5? m, 7?.5 m and 10.5?11 m respectively. Each segment was flushed with 20 ml saline then incubated for 30 min with 10 ml saline and gentle rocking. Each end of the intestinal segments was clamped during washes to prevent leakage. Washes containing intestinal secretions were collected and stored at 220uC until required. Faecal sampling. Faecal samples were collected before vaccination on day 0 and again at day 16 and 36 h after immunisation with the second oral dose allowing administered vaccine material to complete transit through the sheep GIT [26]. Faecal matter was homogenised in 1 ml/g PBST2I with two 3 mm tungsten carbide beads for 1 min at a frequency of 28/s in a Qiagen Mixer Mill. The homogenate was cleared by centrifugation at 13,000 rpm at 4uC for 10 min and capture ELISA performed (as described above) to detect and quantify LTB in the supernatant.ELISA to determine LTB-specific IgG and IgA antibody titreLTB-specific ELISA was used to assess IgG and IgA antibody responses in immunised sheep. Costar 9018 96-well microtitre plates (Corning Life Sciences) were coated with 50 ml/well FGF-401 web chicken CTB antibody (Sigma-Aldrich) diluted 1:5,000 in PBS. Plates wereOral Immunogenicity of.Face with a glass slide. Mucus scrapings were prepared for ELISA as described by [25]. Abomasal scrapings were washed off the slide into a 50 ml tube with 3 ml PBST supplemented with 2x Roche Complete Protease Inhibitor Cocktail tablets (PBST2I). The supernatant was collected following centrifugation at 9000 g for 15 min at 4uC and stored at 220uC until required.Figure 3. LTB-specific IgG (A) and IgA (B) antibody titres in abomasum mucus following oral immunisation with four doses of control or LTB-transgenic plant materials. The horizontal lines represent geometric means. Black symbols denote positive responders defined as sheep with antibody titres at least three standard deviations above the control mean, non-responders are indicated by grey symbols. doi:10.1371/journal.pone.0052907.gSmall intestine washes to sample intestinal secretions. Four sections of the small intestine were excised,University Werribee Animal Facility under conditions approved by the Monash University Animal Ethics Committee (AEC SOBSA/P/2009/98). Sheep were provided with water and standard feed ad lib and fasted 16 h before oral immunisation. Sheep were randomly assigned into four groups of 2? animals each (Table 1). A single sheep from the transgenic rLTB expressing leaf vaccine group (LTB-Leaf) developed balanopsthitis (pizzle rot) 14 days after beginning the trial and was treated with a testosterone implant. This sheep was not excluded from analyses. Sheep were immunised on days 0, 14 and 28 followed by a boost dose on day 38, four days before sacrifice. Vaccine materials were formulated immediately before delivery by mixing 19 g freezedried plant material with 200 ml of an oil based emulsion (125 ml peanut oil:75 ml dH2O). When receiving the transgenic rLTB plant-based vaccines (LTB-HR or LTB-Leaf), each dose was sufficient to deliver 5 mg rLTB. Sheep receiving the CtHR or CtLeaf vaccines were immunised with the equivalent volume of formulated control plant materials. The formulated vaccines were administered orally to sheep by gavage directly into the rumen to simulate drenching, a common delivery system used routinely toeach section measured 0.5 m in length and was taken 3 m apart, beginning at the abomasum/duodenum junction (section 1, 0?0.5 m). Sections 24195657 2? were sampled at 3.5? m, 7?.5 m and 10.5?11 m respectively. Each segment was flushed with 20 ml saline then incubated for 30 min with 10 ml saline and gentle rocking. Each end of the intestinal segments was clamped during washes to prevent leakage. Washes containing intestinal secretions were collected and stored at 220uC until required. Faecal sampling. Faecal samples were collected before vaccination on day 0 and again at day 16 and 36 h after immunisation with the second oral dose allowing administered vaccine material to complete transit through the sheep GIT [26]. Faecal matter was homogenised in 1 ml/g PBST2I with two 3 mm tungsten carbide beads for 1 min at a frequency of 28/s in a Qiagen Mixer Mill. The homogenate was cleared by centrifugation at 13,000 rpm at 4uC for 10 min and capture ELISA performed (as described above) to detect and quantify LTB in the supernatant.ELISA to determine LTB-specific IgG and IgA antibody titreLTB-specific ELISA was used to assess IgG and IgA antibody responses in immunised sheep. Costar 9018 96-well microtitre plates (Corning Life Sciences) were coated with 50 ml/well chicken CTB antibody (Sigma-Aldrich) diluted 1:5,000 in PBS. Plates wereOral Immunogenicity of.
