BACKGROUND
if exposure to an antigen by the oral route fails to promote an oral immune response, any subsequent re-exposure  results in suppression of immunity; this process is known as oral tolerance.
oral tolerance is a major suppressive immunological process designed to prevent local and peripheral overreaction to innocuous antigens  <cit> .
commensal bacteria are critically required for proper gut-associated lymphoid tissue  development and induction of oral tolerance  <cit> .
mucosal dendritic cells  play an active role in inducing oral tolerance through mechanisms which require retinoic acid, vitamin d, interleukin - <dig>  transforming growth factor -β, and indoleamine- <dig> ,-dioxygenase  <cit> .
in the mesenteric lymph nodes , t regulatory  cells undergo differentiation and home back to the inductor site to induce and/or maintain antigen-specific oral tolerance  <cit> .
several physical barriers prevent antigen/pathogen contact with galt and subsequent penetration of the gut wall making targeted induction of oral immunity a significant challenge  <cit> .
the gut of the newborn piglet is uniquely designed to be semi-permeable or ‘leaky’ for a limited time to allow colostrum-derived cells, antibodies, and other macromolecules such as albumin, cytokines, antimicrobial peptides and many other bioactive products to be passively transferred to the piglets.
these maternally-derived cells and macromolecules traverse the gut wall then enter into the vasculature where they play a variety of roles including passive immunity against disease  <cit> .
‘gut closure’ occurs within a few days after birth in ruminants  <cit>  and pigs  <cit> , but it does not occur until after weaning  in rats and mice  <cit> .
in humans, a considerable amount of ‘gut-closure’ occurs both before birth and within a few days after birth but it may in fact take up to two years to reach the same level of impermeability that is observed in the adult gut  <cit> .
once across the gut wall, antigens may be able to interact with dcs within the sub-epithelial dome which can then present antigens to t cells within isolated lymphoid follicles and/or peyer’s patches to promote induction of oral immunity rather than being taken up by tolerogenic mucosal dcs which promote oral tolerance  <cit> .
despite the overwhelming propensity to respond to an oral antigen with tolerance, oral vaccines are highly sought because of their ease of administration.
they are needle-free and therefore present reduced risk of transmitting infections and less need for qualified personnel to administer the vaccine.
moreover, an estimated 90% of all pathogens invade through mucosal surfaces, therefore mucosal immunity  offer the potential to control pathogens at their point of entry.
previous work from our lab showed that rat pups and lambs orally vaccinated starting the day after birth with multiple doses of soluble ovalbumin  responded with immunity to subsequent intraperitoneal  immunization  <cit> .
in contrast, we showed that newborn piglets orally immunized within six hours after birth with single bolus of soluble ova then boosted through the i.p.
route one month later showed significantly lower anti-ova immunoglobulin  a titres and a strong trend towards lower anti-ova igm, igg <dig>  igg <dig> and igg titres relative to the i.p.
control group indicating induction of oral tolerance  <cit> .
these data showed agreement with haverson et al  <cit>  who demonstrated that newborn piglets orally vaccinated once with ova induced classical oral tolerance following a systemic challenge by showing reduced specific systemic igg responses.
when we included unmethylated oligonucleotides containing cg oligodeoxynucleotides  and soluble polyphosphazene in the oral vaccine administered at birth, the response to i.p.
immunization one month later was induction of immunity   <cit> .
clearly the components of the oral vaccine administered at birth impacted the response to the booster immunization one month later.
factors contributing to induction of oral tolerance include: the host’s immunological maturity at time of exposure, the timing and the frequency of exposure, and the nature of the antigen  <cit> .
we established that a single bolus of soluble ova with cpg odn and polyphosphazene adjuvants administered the day after birth induced oral immunity in piglets  <cit> .
our next step will be to establish whether inclusion of the antigen in a particulate form promotes oral immunity and whether the response could be observed at distal mucosal sites.
to test this hypothesis, conventionally reared neonatal piglets were gavaged within six hours after birth with  <dig>  mg or  <dig>  mg ova plus cpg odn in a soluble form as well as ova plus cpg odn encapsulated within a polyphosphazene microparticle   <cit> .
systemic and mucosal antibody titres and ex vivo cytokine production are assessed to determine antibody-mediated and cell-mediated immunity.
RESULTS
explanation of vaccination procedure
piglets were gavaged with a two-part vaccine consisting of soluble ova  with 50 μg soluble cpg  <dig>  as well as a mp encapsulating  <dig>  mg ova + 50 μg cpg  <dig> .
these vaccines will be referred to as  <dig>  mg ova/cpg + mp and  <dig>  mg ova/cpg + mp.
the experimental timeline is detailed in figure  <dig>  piglets were gavaged at less than six hours of age when their gut would be semi-permeable with the idea that both the soluble ova/cpg and the ova and cpg odn within the mp would cross the leaky gut wall.
polyphosphazene-based mps are water-soluble and would then dissolve over time to release the encapsulated ova and cpg odn thus acting like a prime-boost  <cit> .
piglets were bled three days after birth, day seven after birth, and weekly thereafter .
piglets were i.p.
immunized with 10 mg ova  plus incomplete freund’s adjuvant  at 28 days of age and all piglets were euthanized at 49 days of age.
the i.p.
control group received a saline gavage but piglets were boosted with the i.p.
vaccination  to act as our primary i.p.
vaccine control group.figure  <dig>
description and timeline of immunization protocol.
piglets  were gavaged with ovalbumin   + 50 μg unmethylated oligonucleotides containing cg oligodeoxynucleotides  as a soluble vaccine as well as with  <dig>  mg ova + 50 μg cpg  <dig> within a  <dig>  mg pcep polyphosphazene microparticles .
gavages took place within six hours of birth.
the negative control  group and the i.p.
control group were gavaged with saline.
with the exception of the saline control group, the remaining groups were i.p.
immunized at four weeks of age with 10 mg ova in incomplete freunds’ adjuvant .
at eight weeks of age, piglets were euthanized and lung lavages were harvested.
blood was obtained on day three, day seven and then weekly .
at time of death, blood was drawn for pbmc isolation.
newborn piglets vaccinated by oral gavage responded with significant serum anti-ova igm, iga, and igg <dig>  igg <dig> and igg production after re-exposure by the i.p.
route
the definition for oral tolerance is that oral exposure to antigen which is subsequently encountered via a systemic route triggers reduced immune responses  relative to animals exposed to antigen systemically without prior oral exposure  <cit> .
previous work in our laboratory showed that animals orally immunized six hours after birth with 5 mg or  <dig>  mg ova responded to i.p.
immunization at one month of age with oral tolerance, not oral immunity  <cit> .
therefore these groups  were not repeated here.
in the current trial, when we assessed the serum antibody titres in the piglets prior to weaning  for all groups, we observed negligible anti-ova antibodies of any isotype indicating that the sows did not pass any interfering ova-specific passive immunity to the piglets .
even one week after weaning , all isotypes of piglet serum anti-ova antibodies titres were negligible suggesting that oral gavage at birth with  <dig>  mg or  <dig>  mg ova/cpg + mp alone did not promote antibody-mediated immunity.
on day  <dig>  which was two weeks after the i.p.
booster immunization, we observed a significant increase in serum anti-ova igm , iga , igg <dig> , and igg <dig>  titres in the i.p.
control group relative to the saline control group.
the group gavaged with  <dig>  mg ova/cpg + mp showed significant induction of anti-ova igm , iga , igg , igg <dig> , and igg <dig>  relative to the saline control group suggesting that prior oral exposure to low dose ova circumvented induction of oral tolerance.
the animals gavaged with ten-fold higher dose of soluble ova  showed a strong trend towards increased anti-ova antibody production relative to the saline control group but the data were not statistically significant.figure  <dig>
ova-specific antibody-mediated immune responses in serum from newborn piglets gavaged with ova then i.p.
immunized with ova at four weeks of age.
piglets  were gavaged and i.p.
immunized as described in figure  <dig>  control newborn piglets were not gavaged or immunized with ova.
we measured serum anti-ova igm , iga , igg , igg <dig>  and igg <dig>  production on day  <dig>  day  <dig> and day  <dig> after birth.
each data point represents an individual animal and median values are indicated by horizontal lines.
*p <  <dig> ., **p <  <dig> .
on day  <dig>  serum titres from animals within the i.p.
control group showed a significant increase in anti-ova igg <dig> antibodies  relative to the saline control group.
in contrast, the group gavaged with  <dig>  mg ova/cpg + mp showed significantly more anti-ova igm , iga , igg <dig> , and igg <dig>  titres than what was observed in the saline control group.
the group gavaged at birth with  <dig>  mg ova/cpg + mp showed significantly more anti-ova igg <dig> , and igg <dig>  antibodies compared to the saline control group, but the other isotypes did not.
thus, unlike our previous data which showed that piglets orally vaccinated with soluble ova alone induced oral tolerance  <cit> , data from the current trial shows that piglets orally vaccinated with  <dig>  mg or  <dig>  mg ova/cpg + mp showed significant induction of anti-ova antibodies in serum indicating circumvention of oral tolerance.
newborn piglets vaccinated orally with low dose ova/cpg + mp responded with significant anti-ova igg <dig> and igg titres in lung lavage after re-exposure by the i.p.
route
according to the ‘common mucosal immune system’ theory, antigen-sensitized precursor b and t lymphocytes generated at one mucosal site  can be detected at anatomically remote and functionally distinct compartments   <cit> .
after 49 days, the pigs were euthanized and bronchoalveolar lavage was collected.
piglets gavaged with saline but injected with ova and ifa by the i.p.
route alone  failed to trigger significant anti-ova igm , iga , igg , igg <dig> , or igg  titres relative to the saline control group.
newborn piglets gavaged with  <dig>  mg ova/cpg + mp had very low titres of anti-ova igm , iga , and igg <dig>  but the titres for anti-ova igg  and igg <dig>  were statistically higher than the saline control group.
collectively, these results suggest that oral exposure to ova/cpg + mp triggered low level mucosal immunity at a distal site.figure  <dig>
ova-specific antibody-mediated immune responses in lung washes from newborn piglets gavaged with ova then i.p.
immunized with ova at four weeks of age.
piglets  were gavaged and i.p.
immunized as described in figure  <dig>  control newborn piglets were not gavaged or immunized with ova.
lung lavages were collected four weeks post i.p.
immunization and ova-specific serum igm , iga , igg , igg <dig> , and igg <dig>  titres were measured.
elisa titres are expressed as the reciprocal of the highest dilution resulting in a reading of two standard deviations above the negative control.
each data point represents an individual animal and median values are indicated by horizontal lines.
*p <  <dig> , **p <  <dig> .
neonatal piglets gavaged with ova/cpg + mp did not respond with induction of ova-specific cell-mediated immunity
finally, we sought to determine whether neonatal piglets orally gavaged with  <dig>  mg or  <dig>  mg ova/cpg + mp developed cell-mediated immunity as measured by ifn-γ ) cytokines and il- <dig> .
peripheral blood mononuclear cells  were collected at eight weeks of age and they were restimulated ex vivo with ova or media for 72 hours before the supernatants were collected and antibody titres were assessed.
pbmcs did not show ova-specific induction of ifn-γ for any group except in the presence of the mitogen concanavalin a .
interestingly, we observed a decrease in production of the anti-inflammatory cytokine il- <dig> relative to unstimulated  cells in all groups including the saline control groups.
these data indicate that oral gavage of newborn piglets did not promote cell-mediated immunity as measured at 8 weeks of age.
discussion
because the vast majority of infectious agents enter the body through mucosal routes, it is reasonable to assume that mucosal immunity which combats the infectious agent prior to colonization and penetration would be much more effective than systemic immunity.
but it has proven very challenging to design effective oral subunit vaccine without the use of very strong mucosal adjuvants such as cholera toxin  <cit> .
the majority of clinically approved oral vaccines for use in pigs are live attenuated viruses or bacteria .
one may speculate that in order to trigger an immune response instead of tolerance, the pathogen must traverse the gut wall and/or penetrate the epithelial cells lining the gut wall.
if this is the case, it is understandable that subunit vaccines that lack strong adjuvants such as cholera toxin should fail to promote immunity  <cit> .
however, such adjuvants cause significant side effects and are therefore not in clinical use.
due to the inherent risk of attenuated pathogen vaccines reverting to virulence, live attenuated vaccines against pathogens such as porcine reproductive and respiratory virus are not recommended for use in seronegative herds and are therefore a reactive vaccine instead of a proactive vaccine  <cit> .
previous work in our lab showed that oral administration of soluble ova in rat pups or lambs starting immediately after birth was sufficient to promote oral immunity or at minimum prevented induction of oral tolerance  <cit> .
in contrast, our research with piglets showed that soluble ova administered immediately after birth triggered induction of oral tolerance  <cit> .
however, if cpg odn and polyphosphazene adjuvants were included in the soluble oral vaccine, there was instead evidence of induction of serum antibody-mediated immunity in response to systemic re-exposure in later life  <cit> .
in the current study, we investigated whether oral immunization with a mixture of soluble and particulate ova plus cpg odn and polyphosphazene adjuvants could promote oral immunity.
we determined that systemic anti-ova antibody-mediated immune responses were induced in newborn piglets gavaged once on the day of birth with  <dig>  mg or  <dig>  mg ova/cpg + mp and subsequently boosted with ova and ifa by the i.p.
route one month later.
if oral tolerance to ova had been induced instead, re-exposure to the antigen by i.p.
injection should have resulted in a reduction of serum anti-ova antibody titres as observed in  <cit> .
anti-ova igg and igg <dig> titres in lung lavages were significantly induced in piglets gavaged at birth with  <dig>  mg ova/cpg + mp indicating induction of mucosal antibody-mediated immunity at a distal mucosal site.
igg has not traditionally been recognized as a major mucosal immunoglobulin, however there is growing evidence that oral vaccines can elevate local and systemic igg titres  <cit>  and that igg antibodies may play a role in passive transfer of luminal antigens across the gut wall using fcrn  <cit> .
therefore, our data shows that vaccination of newborn piglets with a joint soluble and particulate subunit vaccine triggered systemic antibody-mediated immunity which contrasts with what is reported in older piglets orally vaccinated with soluble antigens or newborn piglets gavaged with ova alone  <cit> .
results from our lab showed that oral vaccination of newborn lambs with ova produced minimal cell-mediated immunity as established by ifn-γ expression and lymphocyte proliferation in ex vivo stimulated splenocytes  <cit> .
similarly, ex vivo-stimulated mln cells from rat pups gavaged with ova after birth failed to produce significantly higher ifn-γ titres relative to cells from pups gavaged after birth with saline  <cit> .
in the current study, we gavaged piglets with soluble and particulate ova plus polyphosphazene and cpg odn, the latter of which is known to promote ifn-γ production  <cit> .
despite inclusion of cpg odn in the oral piglet vaccine, ifn-γ production was negligible suggesting that oral immunization of newborns may not induce significant cell-mediated immunity.
further studies must be undertaken to clarify the precise dose, vaccine formulation and timing of exposure required for induction of cellular immunity as well as including direct measurements of mucosal immunity.
experiments are underway to elucidate the kinetics of gut permeability and the impact this has on the mechanisms of antigen uptake and where antigen presentation to lymphocytes occurs .
should early life oral vaccination consistently circumvent induction of oral tolerance and/or promote oral immunity, it will have important implications for protecting against infectious diseases in the very young and it may reduce the number of carriers of disease-producing organisms within a herd.
CONCLUSIONS
in the present study, we determined that low dose ova/cpg + mp circumvented induction of oral tolerance with comparable serum anti-ova antibodies relative to the animals gavaged with  <dig>  mg ova/cpg + mp relative to the i.p.
control animals.
there was a trend towards induction of mucosal immunity but the results were not statistically significant.
these results are intriguing and should be studied further to establish whether it may be advisable to proactively orally vaccinate newborn piglets to prevent induction of oral tolerance to ensure that the they can respond appropriately to parenteral vaccines in later life.

 if exposure to an antigen by the oral route fails to promote an oral immune response, any subsequent re-exposure results in suppression of immunity; this process is known as oral tolerance. oral tolerance is a major suppressive immunological process designed to prevent local and peripheral overreaction to innocuous antigens. commensal bacteria are critically required for proper gut-associated lymphoid tissue development and induction of oral tolerance. mucosal dendritic cells play an active role in inducing oral tolerance through mechanisms which require retinoic acid, vitamin d, interleukin - transforming growth factor -β, and indoleamine-,-dioxygenase. in the mesenteric lymph nodes, t regulatory cells undergo differentiation and home back to the inductor site to induce and/or maintain antigen-specific oral tolerance. several physical barriers prevent antigen/pathogen contact with galt and subsequent penetration of the gut wall making targeted induction of oral immunity a significant challenge. the gut of the newborn piglet is uniquely designed to be semi-permeable or ‘leaky’ for a limited time to allow colostrum-derived cells, antibodies, and other macromolecules such as albumin, cytokines, antimicrobial peptides and many other bioactive products to be passively transferred to the piglets. these maternally-derived cells and macromolecules traverse the gut wall then enter into the vasculature where they play a variety of roles including passive immunity against disease. ‘gut closure’ occurs within a few days after birth in ruminants and pigs, but it does not occur until after weaning in rats and mice. in humans, a considerable amount of ‘gut-closure’ occurs both before birth and within a few days after birth but it may in fact take up to two years to reach the same level of impermeability that is observed in the adult gut. once across the gut wall, antigens may be able to interact with dcs within the sub-epithelial dome which can then present antigens to t cells within isolated lymphoid follicles and/or peyer’s patches to promote induction of oral immunity rather than being taken up by tolerogenic mucosal dcs which promote oral tolerance. despite the overwhelming propensity to respond to an oral antigen with tolerance, oral vaccines are highly sought because of their ease of administration. they are needle-free and therefore present reduced risk of transmitting infections and less need for qualified personnel to administer the vaccine. moreover, an estimated 90% of all pathogens invade through mucosal surfaces, therefore mucosal immunity offer the potential to control pathogens at their point of entry. previous work from our lab showed that rat pups and lambs orally vaccinated starting the day after birth with multiple doses of soluble ovalbumin responded with immunity to subsequent intraperitoneal immunization. in contrast, we showed that newborn piglets orally immunized within six hours after birth with single bolus of soluble ova then boosted through the i.p. route one month later showed significantly lower anti-ova immunoglobulin a titres and a strong trend towards lower anti-ova igm, igg igg and igg titres relative to the i.p. control group indicating induction of oral tolerance. these data showed agreement with haverson et al who demonstrated that newborn piglets orally vaccinated once with ova induced classical oral tolerance following a systemic challenge by showing reduced specific systemic igg responses. when we included unmethylated oligonucleotides containing cg oligodeoxynucleotides and soluble polyphosphazene in the oral vaccine administered at birth, the response to i.p. immunization one month later was induction of immunity. clearly the components of the oral vaccine administered at birth impacted the response to the booster immunization one month later. factors contributing to induction of oral tolerance include: the host’s immunological maturity at time of exposure, the timing and the frequency of exposure, and the nature of the antigen. we established that a single bolus of soluble ova with cpg odn and polyphosphazene adjuvants administered the day after birth induced oral immunity in piglets. our next step will be to establish whether inclusion of the antigen in a particulate form promotes oral immunity and whether the response could be observed at distal mucosal sites. to test this hypothesis, conventionally reared neonatal piglets were gavaged within six hours after birth with  mg or  mg ova plus cpg odn in a soluble form as well as ova plus cpg odn encapsulated within a polyphosphazene microparticle. systemic and mucosal antibody titres and ex vivo cytokine production are assessed to determine antibody-mediated and cell-mediated immunity.  explanation of vaccination procedure piglets were gavaged with a two-part vaccine consisting of soluble ova with 50 μg soluble cpg as well as a mp encapsulating  mg ova + 50 μg cpg. these vaccines will be referred to as  mg ova/cpg + mp and  mg ova/cpg + mp. the experimental timeline is detailed in figure  piglets were gavaged at less than six hours of age when their gut would be semi-permeable with the idea that both the soluble ova/cpg and the ova and cpg odn within the mp would cross the leaky gut wall. polyphosphazene-based mps are water-soluble and would then dissolve over time to release the encapsulated ova and cpg odn thus acting like a prime-boost. piglets were bled three days after birth, day seven after birth, and weekly thereafter. piglets were i.p. immunized with 10 mg ova plus incomplete freund’s adjuvant at 28 days of age and all piglets were euthanized at 49 days of age. the i.p. control group received a saline gavage but piglets were boosted with the i.p. vaccination to act as our primary i.p. vaccine control group.figure description and timeline of immunization protocol. piglets were gavaged with ovalbumin  + 50 μg unmethylated oligonucleotides containing cg oligodeoxynucleotides as a soluble vaccine as well as with  mg ova + 50 μg cpg within a  mg pcep polyphosphazene microparticles. gavages took place within six hours of birth. the negative control group and the i.p. control group were gavaged with saline. with the exception of the saline control group, the remaining groups were i.p. immunized at four weeks of age with 10 mg ova in incomplete freunds’ adjuvant. at eight weeks of age, piglets were euthanized and lung lavages were harvested. blood was obtained on day three, day seven and then weekly. at time of death, blood was drawn for pbmc isolation. newborn piglets vaccinated by oral gavage responded with significant serum anti-ova igm, iga, and igg igg and igg production after re-exposure by the i.p. route the definition for oral tolerance is that oral exposure to antigen which is subsequently encountered via a systemic route triggers reduced immune responses relative to animals exposed to antigen systemically without prior oral exposure. previous work in our laboratory showed that animals orally immunized six hours after birth with 5 mg or  mg ova responded to i.p. immunization at one month of age with oral tolerance, not oral immunity. therefore these groups were not repeated here. in the current trial, when we assessed the serum antibody titres in the piglets prior to weaning for all groups, we observed negligible anti-ova antibodies of any isotype indicating that the sows did not pass any interfering ova-specific passive immunity to the piglets. even one week after weaning, all isotypes of piglet serum anti-ova antibodies titres were negligible suggesting that oral gavage at birth with  mg or  mg ova/cpg + mp alone did not promote antibody-mediated immunity. on day which was two weeks after the i.p. booster immunization, we observed a significant increase in serum anti-ova igm, iga, igg, and igg titres in the i.p. control group relative to the saline control group. the group gavaged with  mg ova/cpg + mp showed significant induction of anti-ova igm, iga, igg, igg, and igg relative to the saline control group suggesting that prior oral exposure to low dose ova circumvented induction of oral tolerance. the animals gavaged with ten-fold higher dose of soluble ova showed a strong trend towards increased anti-ova antibody production relative to the saline control group but the data were not statistically significant.figure ova-specific antibody-mediated immune responses in serum from newborn piglets gavaged with ova then i.p. immunized with ova at four weeks of age. piglets were gavaged and i.p. immunized as described in figure  control newborn piglets were not gavaged or immunized with ova. we measured serum anti-ova igm, iga, igg, igg and igg production on day day and day after birth. each data point represents an individual animal and median values are indicated by horizontal lines. *p < ., **p < . on day serum titres from animals within the i.p. control group showed a significant increase in anti-ova igg antibodies relative to the saline control group. in contrast, the group gavaged with  mg ova/cpg + mp showed significantly more anti-ova igm, iga, igg, and igg titres than what was observed in the saline control group. the group gavaged at birth with  mg ova/cpg + mp showed significantly more anti-ova igg, and igg antibodies compared to the saline control group, but the other isotypes did not. thus, unlike our previous data which showed that piglets orally vaccinated with soluble ova alone induced oral tolerance, data from the current trial shows that piglets orally vaccinated with  mg or  mg ova/cpg + mp showed significant induction of anti-ova antibodies in serum indicating circumvention of oral tolerance. newborn piglets vaccinated orally with low dose ova/cpg + mp responded with significant anti-ova igg and igg titres in lung lavage after re-exposure by the i.p. route according to the ‘common mucosal immune system’ theory, antigen-sensitized precursor b and t lymphocytes generated at one mucosal site can be detected at anatomically remote and functionally distinct compartments. after 49 days, the pigs were euthanized and bronchoalveolar lavage was collected. piglets gavaged with saline but injected with ova and ifa by the i.p. route alone failed to trigger significant anti-ova igm, iga, igg, igg, or igg titres relative to the saline control group. newborn piglets gavaged with  mg ova/cpg + mp had very low titres of anti-ova igm, iga, and igg but the titres for anti-ova igg and igg were statistically higher than the saline control group. collectively, these results suggest that oral exposure to ova/cpg + mp triggered low level mucosal immunity at a distal site.figure ova-specific antibody-mediated immune responses in lung washes from newborn piglets gavaged with ova then i.p. immunized with ova at four weeks of age. piglets were gavaged and i.p. immunized as described in figure  control newborn piglets were not gavaged or immunized with ova. lung lavages were collected four weeks post i.p. immunization and ova-specific serum igm, iga, igg, igg, and igg titres were measured. elisa titres are expressed as the reciprocal of the highest dilution resulting in a reading of two standard deviations above the negative control. each data point represents an individual animal and median values are indicated by horizontal lines. *p < , **p < . neonatal piglets gavaged with ova/cpg + mp did not respond with induction of ova-specific cell-mediated immunity finally, we sought to determine whether neonatal piglets orally gavaged with  mg or  mg ova/cpg + mp developed cell-mediated immunity as measured by ifn-γ ) cytokines and il-. peripheral blood mononuclear cells were collected at eight weeks of age and they were restimulated ex vivo with ova or media for 72 hours before the supernatants were collected and antibody titres were assessed. pbmcs did not show ova-specific induction of ifn-γ for any group except in the presence of the mitogen concanavalin a. interestingly, we observed a decrease in production of the anti-inflammatory cytokine il- relative to unstimulated cells in all groups including the saline control groups. these data indicate that oral gavage of newborn piglets did not promote cell-mediated immunity as measured at 8 weeks of age. discussion because the vast majority of infectious agents enter the body through mucosal routes, it is reasonable to assume that mucosal immunity which combats the infectious agent prior to colonization and penetration would be much more effective than systemic immunity. but it has proven very challenging to design effective oral subunit vaccine without the use of very strong mucosal adjuvants such as cholera toxin. the majority of clinically approved oral vaccines for use in pigs are live attenuated viruses or bacteria. one may speculate that in order to trigger an immune response instead of tolerance, the pathogen must traverse the gut wall and/or penetrate the epithelial cells lining the gut wall. if this is the case, it is understandable that subunit vaccines that lack strong adjuvants such as cholera toxin should fail to promote immunity. however, such adjuvants cause significant side effects and are therefore not in clinical use. due to the inherent risk of attenuated pathogen vaccines reverting to virulence, live attenuated vaccines against pathogens such as porcine reproductive and respiratory virus are not recommended for use in seronegative herds and are therefore a reactive vaccine instead of a proactive vaccine. previous work in our lab showed that oral administration of soluble ova in rat pups or lambs starting immediately after birth was sufficient to promote oral immunity or at minimum prevented induction of oral tolerance. in contrast, our research with piglets showed that soluble ova administered immediately after birth triggered induction of oral tolerance. however, if cpg odn and polyphosphazene adjuvants were included in the soluble oral vaccine, there was instead evidence of induction of serum antibody-mediated immunity in response to systemic re-exposure in later life. in the current study, we investigated whether oral immunization with a mixture of soluble and particulate ova plus cpg odn and polyphosphazene adjuvants could promote oral immunity. we determined that systemic anti-ova antibody-mediated immune responses were induced in newborn piglets gavaged once on the day of birth with  mg or  mg ova/cpg + mp and subsequently boosted with ova and ifa by the i.p. route one month later. if oral tolerance to ova had been induced instead, re-exposure to the antigen by i.p. injection should have resulted in a reduction of serum anti-ova antibody titres as observed in. anti-ova igg and igg titres in lung lavages were significantly induced in piglets gavaged at birth with  mg ova/cpg + mp indicating induction of mucosal antibody-mediated immunity at a distal mucosal site. igg has not traditionally been recognized as a major mucosal immunoglobulin, however there is growing evidence that oral vaccines can elevate local and systemic igg titres and that igg antibodies may play a role in passive transfer of luminal antigens across the gut wall using fcrn. therefore, our data shows that vaccination of newborn piglets with a joint soluble and particulate subunit vaccine triggered systemic antibody-mediated immunity which contrasts with what is reported in older piglets orally vaccinated with soluble antigens or newborn piglets gavaged with ova alone. results from our lab showed that oral vaccination of newborn lambs with ova produced minimal cell-mediated immunity as established by ifn-γ expression and lymphocyte proliferation in ex vivo stimulated splenocytes. similarly, ex vivo-stimulated mln cells from rat pups gavaged with ova after birth failed to produce significantly higher ifn-γ titres relative to cells from pups gavaged after birth with saline. in the current study, we gavaged piglets with soluble and particulate ova plus polyphosphazene and cpg odn, the latter of which is known to promote ifn-γ production. despite inclusion of cpg odn in the oral piglet vaccine, ifn-γ production was negligible suggesting that oral immunization of newborns may not induce significant cell-mediated immunity. further studies must be undertaken to clarify the precise dose, vaccine formulation and timing of exposure required for induction of cellular immunity as well as including direct measurements of mucosal immunity. experiments are underway to elucidate the kinetics of gut permeability and the impact this has on the mechanisms of antigen uptake and where antigen presentation to lymphocytes occurs. should early life oral vaccination consistently circumvent induction of oral tolerance and/or promote oral immunity, it will have important implications for protecting against infectious diseases in the very young and it may reduce the number of carriers of disease-producing organisms within a herd.  in the present study, we determined that low dose ova/cpg + mp circumvented induction of oral tolerance with comparable serum anti-ova antibodies relative to the animals gavaged with  mg ova/cpg + mp relative to the i.p. control animals. there was a trend towards induction of mucosal immunity but the results were not statistically significant. these results are intriguing and should be studied further to establish whether it may be advisable to proactively orally vaccinate newborn piglets to prevent induction of oral tolerance to ensure that the they can respond appropriately to parenteral vaccines in later life. 
newborn piglets gavaged with low dose soluble and particulate ova plus cpg odn and polyphosphazene adjuvants produced antigen-specific antibodies in serum and lung after systemic re-exposure in later life. here, we will investigate whether newborn piglets gavaged with a vaccine comprised of ova plus unmethylated cpg oligodeoxynucleotides combined with ova plus cpg encapsulated within polyphosphazene microparticles responded with systemic and mucosal immunity. further, piglets gavaged with mg ova/cpg + mp generated significant anti-ova igg and igg titres in lung compared to saline control piglets but results were comparable to titres measured in parenteral control piglets. thus, a single oral exposure at birth to a combined soluble and particulate ova vaccine including adjuvants can circumvent induction of oral tolerance which impacts response to i.p. these data indicate circumvention of oral tolerance but not induction of oral immunity. newborn piglets were gavaged with a combined soluble and particulate vaccine consisting of ova plus μg cpg and mg ova plus μg cpg encapsulated within a polyphosphazene mp referred to as ova/cpg + mp. we observed significantly higher titres of serum anti-ova immunoglobulin igm, iga, igg, igg igg and igg in piglets immunized with mg ova/cpg + mp relative to saline control animals. we previously determined that newborn piglets orally gavaged with ovalbumin responded to systemic ova re-exposure with tolerance; if adjuvants were included in oral vaccine, piglets responded with antibody-mediated immunity:211– 2014).
 we previously determined that newborn piglets orally gavaged with ovalbumin responded to systemic ova re-exposure with tolerance; if adjuvants were included in oral vaccine, piglets responded with antibody-mediated immunity:211– 2014). here, we will investigate whether newborn piglets gavaged with a vaccine comprised of ova plus unmethylated cpg oligodeoxynucleotides combined with ova plus cpg encapsulated within polyphosphazene microparticles responded with systemic and mucosal immunity. to monitor the response to systemic antigen re-exposure, piglets were i.p.-immunized with ova plus incomplete freund’s adjuvant one month later.  newborn piglets were gavaged with a combined soluble and particulate vaccine consisting of ova plus μg cpg and mg ova plus μg cpg encapsulated within a polyphosphazene mp referred to as ova/cpg + mp. control piglets were gavaged with saline alone. piglets were i.p. immunized with mg ova in ifa at four weeks of age and then euthanized at eight weeks of age. we observed significantly higher titres of serum anti-ova immunoglobulin igm, iga, igg, igg igg and igg in piglets immunized with mg ova/cpg + mp relative to saline control animals. thus, a single oral exposure at birth to a combined soluble and particulate ova vaccine including adjuvants can circumvent induction of oral tolerance which impacts response to i.p. vaccination in later life. further, piglets gavaged with mg ova/cpg + mp generated significant anti-ova igg and igg titres in lung compared to saline control piglets but results were comparable to titres measured in parenteral control piglets. peripheral blood mononuclear cells ex vivo-stimulated with ova showed markedly decreased production of il- cytokine after hours relative to animal-matched cells incubated with media alone. no production of ifn-γ was observed from any groups.  newborn piglets gavaged with low dose soluble and particulate ova plus cpg odn and polyphosphazene adjuvants produced antigen-specific antibodies in serum and lung after systemic re-exposure in later life. these data indicate circumvention of oral tolerance but not induction of oral immunity. keywords pigletsneonateovalbuminoraltoleranceissue-copyright-statement© the author 2015 