Zinc (Zn) is an essential mineral and its deficiency manifests in non-specific clinical signs that require long time to develop. The response of swine intestine to Zn restriction was evaluated to identify early changes that can be indicative of Zn deficiency. Twenty-seven pigs (body weight = 77⋅5 ± 2⋅5 kg) were assigned to one of three diets: diet without added Zn (Zn-restricted diet, ZnR), and ZnR-supplemented with either 50 (Zn50) or 100 mg of Zn/kg of diet (Zn100) of Zn supplied by ZnCl₂. After 32 d consuming the diets, serum Zn concentration in ZnR pigs was below the range of 0⋅59–1⋅37 μg/ml considered sufficient, thereby confirming subclinical Zn deficiency. Pigs showed no obvious health or growth changes. RNA-seq analysis followed by qPCR showed decreased expression of metallothionein-1 (MT1) (P < 0⋅05) and increased expression of Zn transporter ZIP4 (P < 0⋅05) in jejunum and ileum of ZnR pigs compared with Zn-supplemented pigs. Ingenuity pathway analysis revealed that Zn50 and Zn100 induced changes in genes related to nucleotide excision repair and integrin signalling pathways. The top gene network in the ZnR group compared with Zn100 was related to lipid and drug metabolism; and compared with Zn50, was related to cellular proliferation, assembly and organisation. Dietary Zn concentrations resulted in differences in genes related to immune pathways. Our analysis showed that small intestine presents changes associated with Zn deficiency after 32 d of Zn restriction, suggesting that the intestine could be a sentinel organ for Zn deficiency.

Lawsonia intracellularis, an obligate intracellular bacterium, is an important enteric pathogen in pig herds and horse farms worldwide. The hallmark feature of L. intracellularis infection is the proliferation of epithelial cells in intestinal crypts. A major limitation to the study of L. intracellularis infection is the lack of an in vitro model that reproduces the changes observed in proliferative enteropathy. Here we investigated the suitability of mouse enteroids as a model to study L. intracellularis infection. Mouse enteroids were microinjected with L. intracellularis, filter-sterilized L. intracellularis culture supernatant, or sterile cell culture media (DMEM). L. intracellularis antigen was detected in mouse enteroids by immunohistochemistry and was located mostly in the basal region of the epithelium. There was no differential growth of enteroids among treatment groups, and cellular proliferation was not increased in L. intracellularis-infected enteroids in relation to non-infected enteroids based on immunofluorescence staining. L. intracellularis infection did not induce changes in gene expression of Ki-67 (proliferation marker), Sox9 (marker for transit amplifying cells) and Muc2 (marker for goblet cells). These results indicate that although L. intracellularis antigen is detectable in mouse enteroids, indicating susceptibility to infection, mouse enteroids fail to replicate the cellular proliferation and gene expression changes observed in proliferative enteropathy. Nevertheless, we have successfully demonstrated that mouse enteroids can be used to model days-long intracellular pathogen infection, serving as potential models for the study of other pathogens of interest in veterinary medicine.

Animals receiving Zinc (Zn) dietary supplementation with organic sources respond better to stress than inorganic Zn sources supplementation. The study aimed to identify the effect of different Zn sources on intestinal epithelial gene expression. In total, 45 pigs (9 per treatment) (77.5 ± 2.5 kg weight) were fed for 32 days, a corn-soybean meal diet without supplemented Zn (ZnR) or supplemented with 50 and 100 ppm of inorganic ZnCl₂ (Zn50 and Zn100), and amino acid-bound organic Zn sources (LQ50 and LQ100). Gene expression changes form RNA-seq in ileum tissues of ZnR revealed changes associated with Zn insufficiency. Comparing organic with inorganic Zn sources by one-way ANOVA, pro-inflammatory cytokine interleukin 18 (IL18) was downregulated (p = 0.03) and Toll-like receptor 2 (TLR2) upregulated (p = 0.02). To determine the role of epithelial cells in response to dietary Zn, swine intestinal organoids (enteroids) were exposed to Zn restriction, ZnCl₂ or LQ-Zn. In enteroids, ZIP4 expression decreased with added Zn compared with Zn-restriction (p = 0.006) but Zn sources did not affect (p > 0.05) IL18 or TLR2 expression. These results suggest that organic Zn may stimulate TLR2 signaling possibly affecting immune response, while decreasing the proinflammatory cytokine IL18 expression in non-epithelial cells of intestinal mucosa.

Background: The human genetic disorder Microvillus Inclusion Disease (MVID) results from inactivating mutations in Myosin Vb (MYO5B). MVID is characterized by severe, unremitting diarrhea that typically presents shortly after birth. The necessity of implementing total parenteral nutrition (TPN) and the long‐term administration of TPN in neonates with MVID presents challenges to elucidating the complex pathogenesis of MVID in humans. Thus, a large animal model of MVID that closely recapitulates human physiology is needed. To better understand the pathophysiology of MVID we created a large animal model that closely mimics human MVID. Methods: Pigs were cloned using somatic cell nuclear transfer of primary pig fibroblasts edited with TALEN to express a P663L mutation of the MYO5B gene. This swine P663L mutation, referred to as MYO5B(P663L), corresponds to the Navajo P660L mutation seen in MVID patients. Neonatal pigs were euthanized within 1 day of birth and tissue was collected for analysis and generation of intestinal enteroids. Results: The MYO5B(P663L) pigs developed diarrhea shortly after birth, similar to patients with MVID. Immunostaining for brush border components F‐actin, ezrin, CD10 and DPPIV showed the presence of subapical intracellular inclusions in enterocytes of MYO5B P663L pigs. Transmission electron microscopy confirmed the presence of inclusions lined with microvilli in the MYO5B(P663L) pigs. Immunostaining for NHE3 and SGLT1, drivers of intestinal sodium and water absorption, showed decreased apical expression and diffuse sub‐apical expression in the MYO5B(P663L) pigs compared to control pigs. In contrast to decreased expression of sodium transporters, CFTR was maintained on the apical membrane of MYO5B(P663L) pigs. Enteroids generated from the proximal small intestine of neonatal control and MYO5B(P663L) pigs grew at similar rates, but showed differences in the brush border. Enteroids derived from MYO5B(P663L) pigs had sub‐apical expression of intestinal alkaline phosphatase and DPPIV and decreased apical expression of NHE3 compared to control enteroids. Administration of forskolin resulted in increased swelling in MYO5B(P663L) enteroids (170.8% ± 15.9) compared to control enteroids (90.4% ± 4.6), indicative of functional Cl ⁻ secretion. Conclusions: Collectively, these data suggest that decreased apical expression of NHE3 and SGLT1, which are required for water absorption, are likely responsible for the MVID diarrhea disorder. Moreover, maintenance of functional CFTR on the apical membrane of enterocytes in the MYO5B(P663L) pig may further exacerbate the development of diarrhea by actively secreting Cl ⁻ , thus driving further water loss by facilitating fluid secretion. This novel large animal model of MVID could provide an important vehicle for testing novel therapeutic approaches for patients with MVID. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .