Bevis Miller

Specific Immunological Unresponsiveness following Active Primary Responses to Proteins in the Weaning Diet of Piglets

Young piglets weaned onto soya diets frequently develop diarrhoea which may have a dietary and/or immunological component. Piglets abruptly weaned onto soya at 3 weeks of age developed levels of serum IgG anti-soya antibodies almost comparable to those induced by injection with soya protein in adjuvant at 7 weeks. In the piglets primed by feeding, no significant further increase in antibody occurred after subsequent systemic injection. In contrast, secondary responses were observed in age-matched animals, previously primed by injection, and primary responses were obtained in previously naive piglets. The results demonstrate the development of specific unresponsiveness to soya proteins in neonates fed soya, despite the occurrence of an initial vigorous immune response to the fed protein.

Direct experimental evidence that early-life farm environment influences regulation of immune responses

To cite this article: Lewis MC, Inman CF, Patel D, Schmidt B, Mulder I, Miller B, Gill BP, Pluske J, Kelly D, Stokes CR, Bailey M. Direct experimental evidence that early‐life farm environment influences regulation of immune responses. Pediatr Allergy Immunol 2012: 23: 265–269.

Rearing environment affects development of the immune system in neonates

Early‐life exposure to appropriate microbial flora drives expansion and development of an efficient immune system. Aberrant development results in increased likelihood of allergic disease or increased susceptibility to infection. Thus, factors affecting microbial colonization may also affect the direction of immune responses in later life. There is a need for a manipulable animal model of environmental influences on the development of microbiota and the immune system during early life. We assessed the effects of rearing under low‐ (farm, sow) and high‐hygiene (isolator, milk formula) conditions on intestinal microbiota and immune development in neonatal piglets, because they can be removed from the mother in the first 24 h for rearing under controlled conditions and, due to placental structure, neither antibody nor antigen is transferred in utero. Microbiota in both groups was similar between 2 and 5 days. However, by 12–28 days, piglets reared on the mother had more diverse flora than siblings reared in isolators. Dendritic cells accumulated in the intestinal mucosa in both groups, but more rapidly in isolator piglets. Importantly, the minority of 2–5‐day‐old farm piglets whose microbiota resembled that of an older (12–28‐day‐old) pig also accumulated dendritic cells earlier than the other farm‐reared piglets. Consistent with dendritic cell control of T cell function, the effects on T cells occurred at later time‐points, and mucosal T cells from high‐hygiene, isolator pigs made less interleukin (IL)‐4 while systemic T cells made more IL‐2. Neonatal piglets may be a valuable model for studies of the effects of interaction between microbiota and immune development on allergy.

Altered immune response to proteins fed after neonatal exposure of piglets to the antigen.

The weaning of piglets onto soya proteins at 3 weeks old normally results in an active response to the fed protein, as determined by the appearance of serum IgG antisoya antibody. This system thus allows the effects of manipulation on the response to a fed protein to be studied. In animals previously given 1 g of soya protein at birth, the magnitude of the antibody response to soya fed at 3 weeks was decreased, although similar amounts of the fed protein could be detected in serum. In addition, the relative affinity of the dominant interaction between antigen and antibody was reduced in these piglets by almost an order of magnitude. By comparison, the ability of piglets given soya at birth to respond to injected soya was not significantly reduced. These results indicate that the regulation of responses to fed and systemic antigens is largely separate. Very early oral exposure to antigen may affect the ability of neonatal animals to mount immune responses to, specifically, fed proteins while leaving the response to systemic antigen largely intact.

The immune response to dietary antigens and its influence on disease susceptibility in farm animals

Transient hypersensitivity reactions of the intestinal immune system to dietary antigens result in increases in enterocyte turnover and villous atrophy. These changes occur in the intestine of the post weaned piglet and precede the proliferation of E. coli and the development of post-weaning diarrhea. We therefore postulated that a transient cell mediated immune response to dietary antigens may increase susceptibility to disease. The interaction of dietary and microbial antigens upon the gut immune system has been investigated in mice and pigs and it has been shown that both exert powerful regulatory effects upon each other.

Dietary Antigen Handling by Mother and Offspring

Sows were Ted ovalbumin (OvA) as a novel protein antigen either throughout gestation and lactation (G + L) or during tactation only (L). This resulted in a significant uptake of OvA into blood, colostrum and milk along with a specific IgG response. In piglets from the G + L group. OvA and antibodies to OvA were detected in serum after ingestion of colostrum. In a large proportion of these piglets OvA was still detected at 3 weeks of age. In she L group a significant proportion of the piglets responded to OvA whilst still suckling their mother.

Effects of infection with transmissible gastroenteritis virus on concomitant immune responses to dietary and injected antigens.

ABSTRACT Normal piglets weaned onto soy- or egg-based diets generated antibody responses to fed protein. Concurrent infection with transmissible gastroenteritis virus (TGEV) did not affect the responses to dietary antigens at weaning, nor did it affect the subsequent development of tolerance. However, TGEV infection did enhance the primary immunoglobulin M (IgM) and IgG1, but not IgG2, antibody responses to injected soy in comparison to those of uninfected animals. Paradoxically, TGEV-infected animals showed an enhanced primary IgG1 antibody response to injected soy at 4 weeks of age, but they subsequently showed a reduced secondary response after an intraperitoneal challenge at 9 weeks of age in comparison to uninfected animals. The results suggest that an enteric virus, either used as a vaccine vector or present as a subclinical infection, may not have significant effects on the development of dietary allergies but may have effects both on the primary response and on the subsequent recall response to systemic antigens to which the animal is exposed concurrently with virus antigens.

Histochemistry of mucins of pig intestinal secretory epithelial cells before and after weaning

Two histochemical staining techniques, Alcian blue/PAS and high-iron-diamine/Alcian blue, which differentiated neutral from sulphated and sialylated acid mucins were applied to sections of duodenum, ileum, caecum, colon and rectum from pigs aged one day, 3 weeks and 10 weeks. In each age group, sulphated-acid mucins predominated at all sites, particularly in the large intestine. In both the small and the large intestine, non-sulphated mucins occurred mainly at the bases of the crypts. Neutral mucins occurred in a few goblet cells in crypts and villi, either alone or together with acid mucins; neutral mucins increased away from the bases of the crypts. No changes were noted in the caecum, colon or rectum of pigs one, two, 5 or 13 days after weaning onto a diet containing soya protein. In the small intestine, there was a transient increase in the numbers of goblet cells in the crypts and villi, a relative increase in sulphated and a decrease in non-sulphated acid mucins and a change in the distribution of sulphated mucins in the crypts. No change in proportions of neutral and acid mucins was detected.

Effect of Oral Antigen and Antibody Exposure at Birth on Subsequent Immune Status: A Study in Neonatal Pigs

Background: The purpose of this work was to investigate the effects of early low-level exposure to either antigen or antibody alone on subsequent immune responses in entirely immunologically naïve animals. This is impossible in species with a permeable placenta such as rodents or humans, where both antigen and antibody can be transferred in utero. It is, however, possible in pigs, due to the impermeable placenta of the sow. Thus, neonatal piglets were used for this study. Methods: Newborn piglets were exposed to ovalbumin (OVA) at dosages similar to those used in rodents to sensitise, as well as to serum containing anti-OVA antibodies. Results: Both single low doses of OVA (10 and 1,000 mg per animal) induced classical oral tolerance following a systemic challenge: both doses reduced specific systemic IgG responses and tertiary in vitro recall proliferative responses by splenocytes and especially by mesenteric lymph node (MLN) cells. Additionally, dietary challenge had phenotypic effects on helper T cells in MLN, which could be reversed by OVA at birth. In contrast, giving antibody as serum collected from hyperimmune or orally tolerant pigs had no functional effects. Conclusions: Overall, our data support the hypothesis that contrary to previous work in rodents, very early exposure of neonatal pigs to a single small dose of antigen can reduce subsequent immune responses. This may have implications for human health. However, although these data point to a reducing/regulatory effect of low doses of antigen in very young animals, they cannot be extrapolated directly to allergy.

Impact of diet composition on ileal digestibility and small intestinal morphology in early-weaned pigs fitted with a T-cannula

Piglets, separated from their dam at 12 days of age and fed a milk substitute hourly, were used as a model for suckling. Animals were fitted with a terminal ileal T-cannula and a jugular vein catheter. At 28 days of age, half of the pigs had a dietary change to a cereal-based weaner diet fed as slurry, and the others remained on milk substitute. Animals were labelled by oral administration of 15N-labelled yeast for 10 days (days 15 to 25). Blood samples were taken twice a day to monitor 15N enrichment of the blood plasma. Diets included polyethylenglycol (PEG 4000) to allow calculation of apparent ileal digestibility of nitrogen and individual amino acids. Ileal bacterial nitrogen was calculated from D-alanine content of the digesta. Furthermore, small intestinal (SI) villus height and crypt depth were measured. Feed intake was increased by the dietary change. The total nitrogen flow was 3.2 ± 0.4 g/day and 5.9 ± 0.4 for the milk and weaner diet, respectively. Endogenous nitrogen flow at the terminal ileum was similar for both groups (milk diet 2.4 ± 0.4 v. weaner diet 2.2 ± 0.3 g/day), whereas the bacterial nitrogen content (0.08 ± 0.01 g/day milk diet v. 0.15 ± 0.01 g/day weaner diet, P < 0.01) and exogenous nitrogen flow (0.94 ± 0.16 g/day milk diet v. 3.29 ± 0.12 g/day weaner diet, P < 0.001) increased significantly in the weaner-diet group. The ileal apparent digestibility coefficient of protein was 0.81 ± 0.06 and 0.68 ± 0.01 for the milk replacer and the weaner diet, respectively. Morphology measurements made along the SI at 25%, 50% and 75% were similar between piglets fed milk replacer and those fed a cereal-based weaner diet. The only statistical effect (P < 0.01) of dietary change was an increase in crypt depth in the weaner-diet group. In conclusion, pigs, following a dietary change analogous to weaning, lack the capacity to fully digest a standard weaner diet. This may result in an increased nutrient content entering the large intestine and an altered microbiota. In the absence of a period of anorexia, often associated with traditional weaning, we saw no evidence of villous atrophy, but report here a significant crypt hyperplasia, especially at the 75% level, as a result of dietary change.