Effect of bamboo vinegar powder on the expression of the immune-related genes MyD88 and CD14 in weaning piglets.

The aim was to explore the feasibility of using bamboo vinegar powder as an antibiotics substitute in weaning piglets. Forty-five healthy Duroc × Landrance × Yorshire piglets (weight 6.74 ± 0.17 kg; age 31 days) were randomly divided into the control group (basic diet), ANT group (basic diet + 0.12% compound antibiotics), BV1 group (basic diet + 0.1% bamboo vinegar powder), BV5 group (basic diet + 0.5% bamboo vinegar powder) and BV10 group (basic diet + 1% bamboo vinegar powder). MyD88 and CD14 expression in immune tissues was examined using real-time PCR. MyD88 expression in the control group were significantly lower than that in other groups in all tissues (p⟨0.05), while CD14 expression showed the opposite trend. MyD88 expression was significantly higher in the BV10 group than in other groups in lung tissue (P⟨0.05), significantly higher in the ANT group than in the BV1 group in the kidneys (P⟨0.05), significantly higher in the BV10 group than in the BV1 group in the thymus (P⟨0.05), and signifi- cantly higher in the BV1 group than in the BV10 group in the lymphatic tissue (P⟨0.05). These differences between experimental groups were not observed for the CD14 gene (P>0.05). Thus, adding bamboo vinegar powder to the basic diet of weaning piglets had immune effects similar to antibiotics and the effect was dose-dependent. Moreover, the MyD88 and CD14 genes appear to play a role in these immune effects.

Promoter identification and analysis of key glycosphingolipid biosynthesis-globo series pathway genes in piglets.

Glycosphingolipid biosynthesis-globo series pathway genes (FUT1, FUT2, ST3GAL1, HEXA, HEXB, B3GALNT1, and NAGA) play an important regulatory role in the defense against Escherichia coli F18 in piglets. In this study, we identified the transcription initiation site and promoter of this gene cluster by mined previous RNA-seq results using bioinformatics tools. The FUT1 transcription initiation region included five alternative splicing sites and two promoter regions, whereas each of the six other genes had one promoter. Dual luciferase reporter results revealed significantly higher transcriptional activity by FUT1 promoter 2, indicating that it played a more important role in transcription. The promoters of glycosphingolipid biosynthesis genes identified contained a CpG island within the first 500 bp, except for the B3GALNT1 promoter which included fewer CpG sites. These results provide a deeper insight into methylation and the regulatory mechanisms of glycosphingolipid biosynthesis-globo series pathway genes in piglets.

LBP gene methylation involved in mRNA expression and resistance to E. coli F18 in weaned piglets.

Lipopolysaccharide binding protein (LBP) plays an important role in recognizing and regulating endotoxin. In this study, we aimed at clarifying the relationship between the methylation of LBP gene and it's expression, to identify mechanisms involved in resistance to E. coli F18 in Sutai weaned piglets. LBP expression was detected by real-time PCR in duodenum and jejunum tissues from E. coli F18-sensitive or -resistant piglets. The LBP methylation status of the regions with many CG sites upstream of the transcription start site was analyzed by Bbisulfite Sequencing PCR (BSP) +Miseq in jejunum and duodenum tissue. The results showed that LBP expression was significantly higher in the sensitive group than the resistant group in duodenum tissue (p<0.05). There was a negative correlation between the methylation of CpG islands upstream of the LBP transcription start site and its expression; the methylation at two CpG sites in particular was significantly correlated with reduced LBP expression (CpG-1 and CpG-2; p<0.05 and p<0.01, respectively). These indicated that the methylation of CpG-1 and CpG-2 sites in the LBP region is involved in the regulation of LBP expression, and may provide key contributions to resisting E. coli F18 in Sutai weaned piglets.

Use of Fluorescence Quantitative Polymerase Chain Reaction (PCR) for the Detection of Escherichia coli Adhesion to Pig Intestinal Epithelial Cells.

An efficient and accurate method to test Escherichia coli (E. coli) adhesion to intestinal epithelial cells will contribute to the study of bacterial pathogenesis and the function of genes that encode receptors related to adhesion. This study used the quantitative real-time polymerase chain reaction (qPCR) method. qPCR primers were designed from the PILIN gene of E. coli F18ab, F18ac, and K88ac, and the pig ß-ACTIN gene. Total deoxyribonucleic acid (DNA) from E. coli and intestinal epithelial cells (IPEC-J2 cells) were used as templates for qPCR. The 2-ΔΔCt formula was used to calculate the relative number of bacteria in cultures of different areas. We found that the relative numbers of F18ab, F18ac, and K88ac that adhered to IPEC-J2 cells did not differ significantly in 6-, 12-, and 24-well culture plates. This finding indicated that there was no relationship between the relative adhesion number of E. coli and the area of cells, so the method of qPCR could accurately test the relative number of E. coli. This study provided a convenient and reliable testing method for experiments involving E. coli adhesion, and also provided innovative ideas for similar detection methods.

Developmental expression of LTßR and differential expression in Escherichia coli F18 resistant/sensitive piglets.

We analyzed LTßR mRNA expression in piglets from birth to weaning and compared the differential expression between Escherichia coli F18-resistant and sensitive populations to determine whether this gene could be used as a genetic marker for E. coli F18 resistance. Sutai piglets of different age groups (8, 18, 30, and 35 days; N = 4 each) and piglets demonstrating resistance/sensitivity to E. coli F18 were used. LTßR expression levels were determined by real-time PCR. The LTßR expression levels in the lymph node, duodenum, and jejunum were significantly higher in 8-day-old piglets than in the other age groups (P < 0.01), and the expression levels were significantly higher in the lungs of 8-day-old piglets than in 35-day-old piglets (P < 0.01) and 30 day-old piglets (P < 0.05). In liver tissue, the expression level was significantly higher in the 35-day-old piglets than in other age groups (P < 0.01). In the stomach tissue, the expression level was significantly higher in 35-day-old piglets than in 18-day-old piglets (P < 0.05). LTßR expression in the lymph nodes was significantly higher in the resistant group than in the sensitive group (P < 0.01), but there was no significant difference in the other tissues (P > 0.05). These results indicate that 8 days after birth is a crucial stage in the formation of mesentery lymph nodes and immune barriers in pigs, and increased expression of LTßR may be beneficial for developing resistance to E. coli F18.

Differential expression of Toll-like receptor 4 signaling pathway genes in Escherichia coli F18-resistant and - sensitive Meishan piglets.

The Toll-like receptor 4 (TLR4) signaling pathway is an important inflammatory pathways associated with the progression of numerous diseases. The aim of the present study was to investigate the relationship between TLR4 signaling and resistance to Escherichia coli F18 in locally weaned Meishan piglets. Using a real-time PCR approach, expression profiles were determined for key TLR4 signaling pathway genes TLR4, MyD88, CD14, IFN-α, IL-1ß and TNF-α in the spleen, thymus, lymph nodes, duodenum and jejunum of E. coli F18-resistant and -sensitive animals. TLR4 signaling pathway genes were expressed in all the immune organs and intestinal tissues, and the expression was generally higher in the spleen and lymph nodes. TLR4 transcription was higher in the spleen of sensitive piglets (p<0.05), but there was no significant difference in TLR4 mRNA levels in other tissues. Similarly, CD14 transcription was higher in lymph nodes of sensitive animals (p<0.05) but not in other tissues. IL-1ß expression was higher in the spleen and in the duodenum of resistant piglets (p<0.05, p<0.01, respectively), and there were no significant differences in other tissues. There were also no significant differences in the expression of MyD88, TNF-α and IFN-α between sensitive and resistant piglets (p>0.05). These results further confirm the involvement of the TLR4 signaling pathway in resistance to E. coli F18 in Meishan weaned piglets. The resistance appeared to be mediated via downregulation of TLR4 and CD14, and upregulation of MyD88 that may promote the release of cytokines TNF-α, IL-1ß, IFN-α and other inflammatory mediators which help to fight against E. coli F18 infection.

Differential expression of FUT1 and FUT2 in Large White, Meishan, and Sutai porcine breeds.

To assess the relationship between the expression of a(1,2)-fucosyltransferase (FUT1 and FUT2) genes and resistance to Escherichia coli F18 in weaned pigs, FUT1 and FUT2 expression levels in Large White, Meishan, and Sutai pigs (with resistance to E. coli F18) were determined using real-time PCR. The results revealed that FUT1 and FUT2 expression levels were higher in the liver, lungs, kidneys, stomach, duodenum, and jejunum than in the muscle and heart. Medium FUT2 expression levels were detected in the spleen, thymus, and lymph nodes. Intestinal FUT1 expression levels were higher in Sutai pigs than in Large White and Meishan pigs (P < 0.05). However, intestinal FUT2 expression levels were lower in Sutai pigs than in Large White and Meishan pigs (P < 0.05). FUT1 and FUT2 expression levels did not differ between Large White and Meishan pigs (P > 0.05). The results revealed that high FUT1 expression levels and low FUT2 expression levels in the intestines of Sutai pigs affected FUT1 and FUT2 enzymes, the synthesis of type 2 H and type 1 H antigens, and E. coli F18 adhesion. Moreover, low FUT2 expression levels conferred resistance to E. coli F18.