23 g OVA for 3 days (Group B), 0.023 OVA for 9 days (Group C) and the parenteral control group generated a trend towards increased free overnight delivery anti-OVA IgG titres in lung washes compared to control lambs although the data did not meet the criteria set for statistical significance (Figure 2B, p<0.065). Lambs gavaged with a single bolus of 2.27 g OVA prior to i.p. immunization showed negligible titres of anti-OVA IgG in the lung lavage which may indicate induction of oral tolerance. Collectively, these results suggest that prior oral antigen exposure in newborn lambs may impact the magnitude of response to i.p. immunization. Figure 2 OVA-specific humoral immune responses in lung washes from newborn lambs gavaged with OVA then i.p. immunized with OVA at 4 weeks of age. Lambs (n=4/group) were gavaged and i.

p. immunized as described in Figure 1A. Control … Gavage of neonatal lambs with OVA did not further promote induction of OVA-specific cell-mediated immunity Finally, we sought to determine whether lambs orally gavaged with OVA starting the day after birth developed cell-mediated immunity. Spleens were excised from each lamb at 7 weeks of age and splenocytes were restimulated with OVA or media. Splenocytes did not show OVA-specific induction of lymphocyte proliferation or interleukin (IL)-4 cytokine production in any groups (Figure 3A and data not shown). However, splenocytes from lambs gavaged with 0.23 g OVA over 3 days (Group B) generated significant antigen-specific IFN-�� production compared to unstimulated cells (Figure 3B, p<0.05). Splenocytes from lambs in Group C (p<0.

11) also showed a trend towards OVA-specific IFN-�� production but this data was not statistically significant. From the lambs in Group A and the parenteral control lambs, two had splenocytes which produced IFN-�� in response to ex vivo antigen-exposure. It is possible that lambs exposed for the shorter period with the higher dose (Group A) experienced limited induction of cellular immunity but not a humoral response. More animals will be needed to establish whether this is indeed the case. These data indicate that oral gavage of newborn lambs did not have any additive effect over what was observed in the group immunized via the i.p. route alone. Figure 3 OVA-specific cytokine production by splenocytes from lambs gavaged with OVA then i.p. immunized with OVA at 4 weeks of age.

Lambs AV-951 (n=4/group) were gavaged and i.p. immunized as described in Figure 1A. Lymphocyte proliferation … Discussion The present investigation showed that both systemic and mucosal humoral immune responses were induced following oral immunization of conventionally reared newborn lambs repeatedly exposed to 0.023 g OVA and primed with OVA in IFA by the i.p. route. Traditionally, immunization of the very young has been avoided because it was presumed that the neonatal immune system was too immature to respond.