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Sheep colibacillosis is one of the most common bacterial diseases found at large-scale sheep farms. The aim of this study was to employ RNA-seq to screen differentially expressed (DE) long non-coding RNAs (lncRNAs) that impart antagonistic or sensitive effects on Escherichia coli F17. In this study, individuals who had antagonistic or sensitive responses to E. coli F17 were identified by feeding E. coli F17 strains to Hu lambs. The sensitive group had higher levels of intestinal bacteria than that in the antagonistic group (P < 0.05), the jejunum showed various levels of mucosal tissue damage and had a dark colour, and disintegration of part of the small intestinal villi was observed. Totals of 34 DE lncRNAs and 703 DE mRNAs in two groups were identified. qRT-PCR results for 12 randomly selected DE lncRNAs and DE mRNAs were consistent with the RNA-seq data. Gene Ontology (GO), KEGG Pathway enrichment and lncRNA-mRNA interaction analyses identified 6 co-expressed genes, namely, MYO1G, TIMM29, CARM1, ADGRB1, SEPT4, and DESI2. This is the first study that has performed expression profiling of lncRNAs in the spleen of antagonistic and sensitive lambs. The identification of DE lncRNAs can facilitate investigations into the molecular mechanism underlying resistance to diarrhoea in sheep.

Sheep colibacillosis is one of the most common bacterial diseases in large-scale sheep farms. In this study, we orally administered Escherichia coli F17 (E. coli F17) to lambs to obtain antagonistic and sensitive individuals. We used RNA-seq to screen for differential circRNAs in the spleens of both antagonist and sensitive individuals to explore the effect of circRNA on anti-diarrhoea in sheep. The results showed that 60 differentially expressed (DE) circRNAs were screened by RNA-seq in the spleen of antagonistic and sensitive lambs, among which 31 were up-regulated and 29 were down-regulated; q-PCR was used to validate the relative expression levels of six randomly selected circRNAs in antagonist and susceptible lambs and found to be consistent with the results of RNA-seq. Using Miranda analysis of circRNA-miRNA-mRNA interactions, we found a certain target relationship between 6 circRNAs, 5 miRNAs and 9 mRNAs. The relative expression levels of mRNA in antagonistic and sensitive lambs were verified by q-PCR and were consistent with the results of RNA-seq. This study explored the expression profile of circRNA in the spleen of an antagonistic and susceptible lamb with diarrhoea and found that differentially expressed circRNAs were helpful for determining how the lambs resist the pathogenesis of diarrhoea and provided a scientific basis for lambs to resist diarrhoea.

Simple Summary: Sheep diarrhea is one of the most common illnesses in sheep farming and diarrhea in sheep caused by E. coli occupies a significant proportion of them. E. coli F17 is one of the main members of E. coli causing diarrhea in sheep. This study is devoted to improving the resistance of sheep itself to E. coli F17. This study provides theoretical support for the solution of E. coli F17-caused diarrhea in sheep. Diarrhea is the most common issue in sheep farms, typically due to pathogenic Escherichia coli (E. coli) infections, such as E. coli F17. microRNA, a primary type of non-coding RNA, has been shown to be involved in diarrhea caused by pathogenic E. coli. To elucidate the profound mechanisms of miRNA in E. coli F17 infections, methods such as E. coli F17 adhesion assay, colony counting assay, relative quantification of bacterial E. coli fimbriae gene expression, indirect immune fluorescence (IF), Cell Counting Kit-8 (CCK-8), 5-ethynyl-2′-deoxyuridine (EdU), Western blotting (WB), and scratch assay were conducted to investigate the effect of miR-329b-5p overexpression/knock-down on E. coli F17 susceptibility of sheep intestinal epithelial cells (IECs). The findings indicated that miR-329b-5p enhances the E. coli F17 resistance of sheep IECs to E.coli F17 by promoting adhesion between E. coli F17 and IEC, as well as IEC proliferation and migration. In summary, miR-329b-5p plays a crucial role in the defense of sheep IECs against E. coli F17 infection, providing valuable insights into its mechanism of action. [ABSTRACT FROM AUTHOR]

Escherichia coli (E. coli) F17 is one of the main pathogens causing diarrhea in young livestock. The specific F17 fimbriae and lipopolysaccharide (LPS) in the surface components of E. coli F17 induces immune activation via interacting with the intestinal epithelial cells (IECs)-expressed innate immune toll-like receptors (TLRs) signaling pathway. In this study, the expression patterns of eight canonical genes from the TLR signaling pathway (IL-6, IL-8, IL-1β, TLR4, MyD88, CD14, TNF-α and TRAF6) were analyzed in LPS-induced IECs, E. coli F17-infected IECs and ileum tissue of E. coli F17-infected lambs. The results showed that increased expression levels of all the studied genes were observed following post-LPS-induced and E. coli F17-infected treatment, with TLR4 having the highest up-regulated expression multiple (compared to NC, fold change = 17.94 and 20.11, respectively), and CD14 having the lowest up-regulated expression multiple (fold change = 2.68 and 1.59, respectively), and higher expression levels of all the studied TLR signaling pathway genes were observed in ileum tissue of E. coli F17 antagonistic (AN) lambs than in E. coli F17 sensitive (SE) lambs. Furthermore, when compared to LPS-induced IECs, E. coli F17-infected IECs showed a more pronounced increase in the expression of IL6, TLR4 and TNF-α, indicating the different roles of these genes in the IECs resistance to E. coli F17 infection. Our results demonstrate that the TLR signaling pathway likely promotes immune activation and provide the first evidence that TLRs have a significant potential to protect against E. coli F17 infections. [ABSTRACT FROM AUTHOR]

Simple Summary: Escherichia coli (E. coli) F17 is one of the major pathogenic bacteria responsible for diarrhea in farm animals; however, little is known about the effect of E. coli F17 infection on the intestinal microbiota. The aim of this study was to investigate the intestinal microbiota in lambs with different susceptibilities to E. coli F17. By conducting an E. coli F17 challenge experiment, lambs sensitive/resistant (SE/AN) to E. coli F17 were identified, and 16S rRNA gene sequencing was performed to evaluate the intestinal microbiota in SE/AN lambs. The results showed that a relatively higher level of richness and diversity were characterized in the bacterial communities in the AN lambs than in the SE lambs, while the abundance of Lactococcus and Megasphaera elsdenii was found to be significantly different between the AN and SE lambs. Furthermore, our results indicated that the Bacteroidetes:Firmicutes ratio can serve as a promising bacterial biomarker for E. coli F17 infection. Our results can help in the development of new insights for the treatment of farm animals infected by E. coli F17. Diarrhea is one of the most commonly reported diseases in young farm animals. Escherichia coli (E. coli) F17 is one of the major pathogenic bacteria responsible for diarrhea. However, the pathogenicity of diarrhea in lambs involving E. coli F17 strains and how E. coli F17 infection modifies lambs' intestinal microbiota are largely unknown. To evaluate diarrhea in newborn lambs with an infection of E. coli F17, 50 lambs were selected for challenge experiments and divided into four groups, namely, a high-dose challenge group, low-dose challenge group, positive control group, and negative control group. The E. coli F17 challenge experiments caused diarrhea and increased mortality in the experimental lamb population, with a higher prevalence (90%), mortality (35%), and rapid onset time (4–12 h) being observed in the high-dose challenge group than the results observed in the low-dose challenge group (75%, 10%, 6–24 h, respectively). After the challenge experiment, healthy lambs in the high-dose challenge group and severely diarrheic lamb in the low-dose challenge group were identified as lambs sensitive/resistant to E. coli F17 (E. coli F17 -resistant/-sensitive candidate, AN/SE) according to the histopathological detection. Results of intestinal contents bacteria plate counting revealed that the number of bacteria in the intestinal contents of SE lambs was 102~3-fold greater than that of the AN lambs, especially in the jejunum. Then, 16S rRNA sequencing was conducted to profile the intestinal microbiota using the jejunal contents, and the results showed that SE lambs had higher Lactococcus and a lower Bacteroidetes:Firmicutes ratio and intestinal microbiota diversity in the jejunum than AN lambs. Notably, high abundance of Megasphaera elsdenii was revealed in AN lambs, which indicated that Megasphaera elsdenii may serve as a potential probiotic for E. coli F17 infection. Our study provides an alternative challenge model for the identification of E. coli F17-sensitive/-resistant lambs and contributes to the basic understandings of intestinal microbiota in lambs with different susceptibilities to E. coli F17. [ABSTRACT FROM AUTHOR]

Simple Summary: Diarrhea and vomiting caused by Escherichia coli (E. coli) F17 are considered significant threats to animal farming. In the present study, RNA-Seq was performed to investigate the potential circRNA and miRNA biomarkers for E. coli F17-antagonism (AN) and -sensitive (SE) lambs. The results indicated that circRNA and miRNA expression is closely associated with the susceptibility of E. coli F17 in lambs. Numbers of circRNAs and miRNAs may serve as potential biomarkers for intestinal inflammatory response against E. coli F17 infection. Our study can provide a preliminary understanding of the underlying mechanisms of intestinal immunity. It has long been recognized that enterotoxigenic Escherichia coli (ETEC) is the major pathogen responsible for vomiting and diarrhea. E. coli F17, a main subtype of ETEC, is characterized by high morbidity and mortality in young livestock. However, the transcriptomic basis underlying E. coli F17 infection has not been fully understood. In this study, RNA sequencing was performed to explore the expression profiles of circRNAs and miRNAs in the jejunum of E. coli F17-antagonism (AN) and -sensitive (SE) lambs. A total of 16,534 circRNAs and 271 miRNAs (125 novel miRNAs and 146 annotated miRNAs) were screened, and 214 differentially expressed (DE) circRNAs and 53 DE miRNAs were detected between the AN and SE lambs (i.e., novel_circ_0025840, novel_circ_0022779, novel_miR_107, miR-10b). Functional enrichment analyses showed that source genes of DE circRNAs were mainly involved in metabolic-related pathways, while target genes of DE miRNAs were mainly enriched in the immune response pathways. Then, a two-step machine learning approach combining Random Forest (RF) and XGBoost (candidates were first selected by RF and further assessed by XGBoost) was performed, which identified 44 circRNAs and 39 miRNAs as potential biomarkers (i.e., novel_circ_0000180, novel_circ_0000365, novel_miR_192, oar-miR-496-3p) for E. coli infection. Furthermore, circRNA-related and lncRNA-related ceRNA networks were constructed, containing 46 circRNA-miRNA-mRNA competing triplets and 630 lncRNA-miRNA-mRNA competing triplets, respectively. By conducting a serious of bioinformatic analyses, our results revealed important circRNAs and miRNAs that could be potentially developed as candidate biomarkers for intestinal inflammatory response against E. coli F17 infection; our study can provide novel insights into the underlying mechanisms of intestinal immunity. [ABSTRACT FROM AUTHOR]

Simple Summary: This study was conducted to explore the antibacterial ability of sheep β-defensin 2 (SBD-2) against E. coli F17 infection in ovine intestinal epithelial cells (OIECs). Our data revealed that E. coli F17 induces SBD-2 expression in OIECs in vitro, which appears to be mediated through the activation of the signaling pathways NF-κB and MAPK. Our results provide a novel insight for the functionality of SBD-2, which could be useful for developing anti-infective drugs and/or breeding for E. coli diarrhea disease-resistant sheep. Escherichia coli (E. coli) F17 is a member of enterotoxigenic Escherichia coli, which can cause massive diarrhea and high mortality in newborn lambs. β-defensin is mainly produced by the epithelial tissue of the gastrointestinal tract in response to microbial infection. However, the molecular mechanism of sheep β-defensin 2 (SBD-2) against E. coli F17 remains unclear. This study aims to reveal the antibacterial ability of SBD-2 against E. coli F17 infection in sheep. Firstly, we established the culture system of ovine intestinal epithelial cells (OIECs) in vitro, treated with different concentrations of E. coli F17 for an indicated time. Secondly, we performed RNA interference and overexpression to investigate the effect of SBD-2 expression on E. coli F17 adhesion to OIECs. Finally, inhibitors of NF-κB and MAPK pathways were pre-treated to explore the possible relationship involving in E. coli F17 infection regulating SBD-2 expression. The results showed that E. coli F17 markedly (p < 0.01) upregulated the expression levels of SBD-2 mRNA and protein in a concentration- and time-dependent manner. Overexpression of SBD-2 contributed to enhancing E. coli F17 resistance in OIECs, while silencing SBD-2 dramatically improved the adhesion of E. coli F17 to OIECs (p < 0.05 or p < 0.01). Furthermore, E. coli F17 stimulated SBD-2 expression was obviously decreased by pre-treatment with NF-κB inhibitor PDTC, p38 MAPK inhibitor SB202190 and ERK1/2 MAPK inhibitor PD98095 (p < 0.05 or p < 0.01). Interestingly, adhesion of E. coli F17 to OIECs were highly enhanced by pre-treated with PDTC, SB202190 and PD98095. Our data suggested that SBD-2 could inhibit E. coli F17 infection in OIECs, possibly through NF-κB and MAPK signaling pathways. Our results provide useful theoretical basis on developing anti-infective drug and breeding for E. coli diarrhea disease-resistant sheep. [ABSTRACT FROM AUTHOR]