No abstract

The enteric pathogen Lawsonia intracellularis is one of the main causes of diarrhea and compromised weight gain in pigs worldwide. Traditional cell-line cultures have been used to study L. intracellularis pathogenesis. However, these systems fail to reproduce the epithelial changes observed in the intestines of L. intracellularis-infected pigs, specifically, the changes in intestinal cell constitution and gene expression. A more physiologically accurate and state-of-the-art model is provided by swine enteroids derived from stem cell-containing crypts from healthy pigs. The objective of this study was to verify the feasibility of two-dimensional swine enteroids as in vitro models for L. intracellularis infection. We established both three- and two-dimensional swine enteroid cultures derived from intestinal crypts. The two-dimensional swine enteroids were infected by L. intracellularis in four independent experiments. Enteroid-infected samples were collected 3 and 7 d postinfection for analysis using real-time quantitative PCR and L. intracellularis immunohistochemistry. In this study, we show that L. intracellularis is capable of infecting and replicating intracellularly in two-dimensional swine enteroids derived from ileum.

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.

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.

5-aza-2'-deoxycytidine (decitabine), is a chemotherapeutic DNA methyltransferase (DNMT) inhibitor widely used to treat myelodysplastic syndrome and acute myeloid leukemias. Decitabine’s anti-neoplastic activity is thought to result from inhibition of DNMTs leading to passive demethylation of 5’methylcytosines (5mC) in rapidly dividing tissues, resulting in cell death. However, we previously reported paradoxical effects on DNA methylation by decitabine in somatic tissues. Given the potential for lasting damage to DNA methylation in reproductive tissues from even short courses of decitabine in reproductive age humans, we chose to characterize its long-term effects here. Mice were treated with two clinically relevant doses of decitabine (0.15 mg/kg, 0.35 mg/kg) for 7 weeks and DNA methylation was assessed within female reproductive tract tissues. We found methylated cytosines within the ovary to be the least sensitive to decitabine exposure at both doses, whereas the uterus and the oviduct exhibited higher 5mC dysregulation, surprisingly biased towards hypermethylation at the 0.35 mg/kg dose. We identified the sites of differential methylation; revealing specific genes and pathways involved in cell differentiation, development, communication, and cell signaling that were universally altered in all tissues. In addition to our differential methylation data, we identified dysregulated transcription and pathways using RNAseq analyses. Overall, our findings show decitabine exposure causes an epigenetic insult to DNA methylation within female reproductive tissues. Our data provides evidence that further evaluation is needed to fully establish the long-term phenotypic effects post-decitabine exposure.