Cell lines matching the source epithelium are indispensable for investigating porcine intestinal transport and barrier properties on a subcellular or molecular level and furthermore help to reduce animal usage. The porcine jejunal cell line IPEC-J2 is established as an in vitro model for porcine infection studies but exhibits atypically high transepithelial resistances (TER) and only low active transport rates so that the effect of nutritional factors cannot be reliably investigated. This study aimed to properly remodel IPEC-J2 and then to re-characterize these cells regarding epithelial architecture, expression of barrier-relevant tight junction (TJ) proteins, adequate TER and transport function, and reaction to secretagogues. For this, IPEC-J2 monolayers were cultured on permeable supports, either under conventional (fetal bovine serum, FBS) or species-specific (porcine serum, PS) conditions. Porcine jejunal mucosa was analyzed for comparison. Main results were that under PS conditions (IPEC-J2/PS), compared to conventional FBS culture (IPEC-J2/FBS), the cell height increased 6-fold while the cell diameter was reduced by 50%. The apical cell membrane of IPEC-J2/PS exhibited typical microvilli. Most importantly, PS caused a one order of magnitude reduction of TER and of trans- and paracellular resistance, and a 2-fold increase in secretory response to forskolin when compared to FBS condition. TJ ultrastructure and appearance of TJ proteins changed dramatically in IPEC-J2/PS. Most parameters measured under PS conditions were much closer to those of typical pig jejunocytes than ever reported since the cell line’s initial establishment in 1989. In conclusion, IPEC-J2, if cultured under defined species-specific conditions, forms a suitable model for investigating porcine paracellular intestinal barrier function.

The trace element zinc is often used in the diet of weaned piglets, as high doses have resulted in positive effects on intestinal health. However, the majority of previous studies evaluated zinc supplementations for a short period only and focused on the small intestine. The hypothesis of the present study was that low, medium and high levels of dietary zinc (57, 164 and 2,425 mg Zn/kg from zinc oxide) would affect colonic morphology and innate host defense mechanisms across 4 weeks post-weaning. Histological examinations were conducted regarding the colonic morphology and neutral, acidic, sialylated and sulphated mucins. The mRNA expression levels of mucin (MUC) 1, 2, 13, 20, toll-like receptor (TLR) 2, 4, interleukin (IL)-1β, 8, 10, interferon-γ (IFN-γ) and transforming growth factor-β (TGF-β) were also measured. The colonic crypt area increased in an age-depending manner, and the greatest area was found with medium concentration of dietary zinc. With the high concentration of dietary zinc, the number of goblet cells containing mixed neutral-acidic mucins and total mucins increased. Sialomucin containing goblet cells increased age-dependently. The expression of MUC2 increased with age and reached the highest level at 47 days of age. The expression levels of TLR2 and 4 decreased with age. The mRNA expression of TLR4 and the pro-inflammatory cytokine IL-8 were down-regulated with high dietary zinc treatment, while piglets fed with medium dietary zinc had the highest expression. It is concluded that dietary zinc level had a clear impact on colonic morphology, mucin profiles and immunological traits in piglets after weaning. Those changes might support local defense mechanisms and affect colonic physiology and contribute to the reported reduction of post-weaning diarrhea.

The aim of this study was to evaluate age-related changes and the effect of dietary Zn concentration on morphological and immunological characteristics in the gastrointestinal tract of piglets. A total of 96 purebred Landrace piglets were weaned at the age of 26 ± 1 d, and randomly allocated into 3 treatment groups fed with low (57 mg Zn/kg), medium (164 mg Zn/kg), and high (2425 mg Zn/kg) dietary Zn (ZnO). Piglets (4 males and 4 females per treatment group) were killed at 33 ± 1, 40 ± 1, 47 ± 1, and 54 ± 1 d of age. In the jejunum, villus height, crypt depth, and the number of goblet cells producing neutral, acidic, sulfated, and sialylated mucins were measured. Intraepithelial lymphocytes were characterized by flow cytometry and the gene expression of mucin 2 (MUC2), mucin 20 (MUC20), β-defensin 3, and trefoil factor 3 (TFF3) was determined by reverse transcription quantitative PCR. Villus height and crypt depth in the jejunum showed age related differences (P < 0.01), whereas the dietary concentrations of Zn had no effect. The mucin types were modified mainly by age, and dietary Zn had no effect in the proximal jejunum. In the distal jejunum, age and Zn had effects on the mucin types. The abundance of sulfomucins decreased (P < 0.001) and sialomucins increased with age (P < 0.001), while high dietary ZnO reduced the sulfomucins (P < 0.001) and increased the sialomucins (P < 0.05) in the crypts. The phenotypes of lymphocytes in the epithelium of the proximal jejunum showed relatively constant percentages of T-cells, as well as natural killer cells. High dietary Zn treatment led to a reduced abundance of CD8⁺ γδ T-cells (P < 0.05). The apportionment of different cytotoxic T-cell was age dependent. Although the percentage of CD4^–CD8β⁺ increased (P < 0.01), the relative amount of CD4⁺CD8β⁺ decreased with age (P < 0.05). The expression of MUC2 and MUC20 was not influenced by age or dietary Zn concentration. High Zn intakes resulted in a reduced gene expression of β-defensin 3 (P < 0.05), but did not affect the expression of TFF3. It is concluded that Zn in the form of ZnO appears to have specific effects on the innate and adaptive gut associated immune system of piglets. These might contribute to the positive effects of Zn on the prevention of postweaning diarrhea.

Salmonellae are among the most widespread sources of foodborne infections and Salmonella Typhimurium, in particular, is correlated with human disease caused by the consumption of contaminated pork. Intraepithelial lymphocytes (IEL) have early contact with intestinal antigens and play an important role in the detection of pathogenic bacteria. The objective of this study was to determine whether a presumed probiotic Enterococcus faecium strain could improve histomorphological and immune system-related parameters of gut function after a Salmonella challenge in weaned pigs. In particular the morphological parameters villus length and width, crypt depth and width as well as the actual enlargement of the intestinal epithelial surface were calculated and the number of IEL was evaluated in sections of the porcine gut. Weaned piglets were challenged with Salmonella enterica serovar Typhimurium DT 104, and half of them also received Enterococcus faecium NCIMB 10415 in the diet. Animals were sacrificed at days post infection (DPI) 2 and 28. The effect of the factors "time post-infection/age" and "probiotic treatment" on jejunal morphology and IEL numbers and distribution was evaluated by light microscopy. The time post-infection had significant effects in both feeding groups. Animals sacrificed at DPI 28 had longer and wider villi, deeper and wider crypts, a higher villus enlargement factor, a higher ratio between villus and crypt enlargement factors as well as more IEL. Probiotic treatment resulted in longer villi, a higher ratio of villus surface/crypt circumference enlargement factors and significantly more IEL. The larger total number of IEL displayed by the probiotic group resulted from significantly higher numbers of IEL at the nuclear and apical levels of the intraepithelial compartment but not from the number of IEL situated at the basement membrane. The probiotic effects were only measurable 28 DPI. It is proposed that Enterococcus faecium NCIMB 10415 exerts an immune modulatory effect by increasing the numbers of intraepithelial lymphocytes.

Porcine intestinal mast cells. Evaluation of different fixatives for histochemical staining techniques considering tissue shrinkage