At 37uC for 1 h and antibodies diluted in 1 dry skim milk powder (DM) in PBST. Following all incubations, plates were washed three times with PBST. Plates were blocked with 5 DM in PBST before a 2 h room temperature (22?5uC) incubation with serially diluted crude plant extract starting with 1:100 in PBS. Plates were then incubated with 1:2,000 rabbit 
anti-LTB (Benchmark Biolabs), then 1:15,000 goat anti-rabbit IgG HRP conjugate (Sigma-Aldrich). Bound LTBFigure 1. LTB-specific IgG antibody titres in serum 1676428 collected from sheep before immunisation with LTB-Leaf (A), LTB-HR (B) or control vaccines (C). Black symbols denote positive (-)-Indolactam V responders defined as sheep with antibody titres at least three standard deviations above the control mean, non-responders are indicated by grey symbols. The horizontal lines represent geometric means, statistical analysis (Student’s t-test determined a significant difference between the means of the control and LTB-Leaf groups after four doses, p,0.05). doi:10.1371/journal.pone.0052907.gspecific antibodies were visualised using TMB-peroxidase substrate (Bio-Rad Laboratories) according to manufacturer’s instructions. The amount of rLTB in the freeze-dried plant BTZ-043 materials was calculated against a Pichia pastoris-made rLTB (Sigma-Aldrich)Oral Immunogenicity of a Model 
PMV in SheepFigure 2. LTB-specific antibody titres in MLNs collected 25837696 from successive sites along the small intestine of the sheep GIT following oral immunisation with four doses of LTB-Leaf (A and D, IgG and IgA respectively), LTB-HR (B and E, IgG and IgA respectively) or control (C and F, IgG and IgA respectively) plant materials. MLN 1 was sampled from the abomasum/duodenum junction, MLN 2? were the next three lymph nodes sampled from the first 0.5 m of the small intestine. 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.gstandard. Accumulation of the functional pentameric form of rLTB was confirmed by western blot [3].Mucosal vaccination of sheepOutbred, male sheep (Ovis aries, Merion/Merino) aged between 4.5 to 12 months were obtained from the Commercial Registered Pfizer Animal Health Woodend Farm and housed at the MonashOral Immunogenicity of a Model PMV in Sheepworm domestic sheep flocks. At trial termination (day 42), sheep were humanely killed by intravenous injection with a lethal dose of lethobabarb (100 mg/kg bodyweight).Collection and processing of biological specimensSerum collection. Blood samples were taken from the jugular vein using an 18 G needle immediately before the first immunisation (pre-immune), 14 days after each of the first three doses and four days (at trial termination) after the boost. The blood was clotted at room temperature (20?2uC) overnight and serum separated by centrifugation at 400 g for 10 min and stored at 220uC until required for LTB-specific antibody detection by ELISA. Sampling and in vitro culture of mesenteric lymph nodes. At post-mortem, four lymph nodes were taken fromthe mesentery, the first at the abomasum/duodenum junction (MLN 1) and the next three along the first 0.5 m of the small intestine (MLN 2?). MLNs were subjected to an antigen-specific antibody secreting cell (ASC) assay for detection of LTB-specific antibody responses using a protocol modified from those previously described [23,24]. MLNs were dissected into small pieces.At 37uC for 1 h and antibodies diluted in 1 dry skim milk powder (DM) in PBST. Following all incubations, plates were washed three times with PBST. Plates were blocked with 5 DM in PBST before a 2 h room temperature (22?5uC) incubation with serially diluted crude plant extract starting with 1:100 in PBS. Plates were then incubated with 1:2,000 rabbit anti-LTB (Benchmark Biolabs), then 1:15,000 goat anti-rabbit IgG HRP conjugate (Sigma-Aldrich). Bound LTBFigure 1. LTB-specific IgG antibody titres in serum 1676428 collected from sheep before immunisation with LTB-Leaf (A), LTB-HR (B) or control vaccines (C). 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. The horizontal lines represent geometric means, statistical analysis (Student’s t-test determined a significant difference between the means of the control and LTB-Leaf groups after four doses, p,0.05). doi:10.1371/journal.pone.0052907.gspecific antibodies were visualised using TMB-peroxidase substrate (Bio-Rad Laboratories) according to manufacturer’s instructions. The amount of rLTB in the freeze-dried plant materials was calculated against a Pichia pastoris-made rLTB (Sigma-Aldrich)Oral Immunogenicity of a Model PMV in SheepFigure 2. LTB-specific antibody titres in MLNs collected 25837696 from successive sites along the small intestine of the sheep GIT following oral immunisation with four doses of LTB-Leaf (A and D, IgG and IgA respectively), LTB-HR (B and E, IgG and IgA respectively) or control (C and F, IgG and IgA respectively) plant materials. MLN 1 was sampled from the abomasum/duodenum junction, MLN 2? were the next three lymph nodes sampled from the first 0.5 m of the small intestine. 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.gstandard. Accumulation of the functional pentameric form of rLTB was confirmed by western blot [3].Mucosal vaccination of sheepOutbred, male sheep (Ovis aries, Merion/Merino) aged between 4.5 to 12 months were obtained from the Commercial Registered Pfizer Animal Health Woodend Farm and housed at the MonashOral Immunogenicity of a Model PMV in Sheepworm domestic sheep flocks. At trial termination (day 42), sheep were humanely killed by intravenous injection with a lethal dose of lethobabarb (100 mg/kg bodyweight).Collection and processing of biological specimensSerum collection. Blood samples were taken from the jugular vein using an 18 G needle immediately before the first immunisation (pre-immune), 14 days after each of the first three doses and four days (at trial termination) after the boost. The blood was clotted at room temperature (20?2uC) overnight and serum separated by centrifugation at 400 g for 10 min and stored at 220uC until required for LTB-specific antibody detection by ELISA. Sampling and in vitro culture of mesenteric lymph nodes. At post-mortem, four lymph nodes were taken fromthe mesentery, the first at the abomasum/duodenum junction (MLN 1) and the next three along the first 0.5 m of the small intestine (MLN 2?). MLNs were subjected to an antigen-specific antibody secreting cell (ASC) assay for detection of LTB-specific antibody responses using a protocol modified from those previously described [23,24]. MLNs were dissected into small pieces.
