Yanwei Li

Streptococcus agalactiae is a major contagious pathogen of bovine mastitis and causes substantial economic losses in the dairy industry. In this study, a dual-readout RPA-PfAgo detection platform targeting the conserved cfb gene of S. agalactiae was established and optimized. Seven pairs of RPA primers were designed and screened to construct the Basic-RPA assay, and six guide DNAs (gDNAs) together with a specific probe were evaluated for PfAgo-assisted detection. Field validation was performed using 153 bovine milk samples collected from five dairy-farming regions in China, and assay performance was compared with bacteriological culture and a standardized quantitative PCR (qPCR) assay. The Basic-RPA assay achieved optimal amplification at 37 °C for 30 min, with a detection limit of 1 × 10⁻³ ng/µL and no cross-reactivity with non-target bacteria. The optimized RPA-PfAgo-RTF assay detected as few as 10 copies/µL, whereas the RPA-PfAgo-LFD assay detected 100 copies/µL, and both formats showed high analytical specificity. In field milk samples, bacteriological culture detected 21 positive samples, whereas standardized real-time PCR (qPCR), RPA-PfAgo-RTF, and RPA-PfAgo-LFD each detected 33 positive samples. When compared with bacteriological culture as a conventional comparator, all three molecular assays showed 100.00% positive agreement, 90.91% negative agreement, and a Kappa value of 0.733. In addition, RPA-PfAgo-RTF and RPA-PfAgo-LFD were completely concordant with the standardized qPCR assay across all 153 samples. These results indicate that the dual-readout RPA-PfAgo platform is a rapid and reliable molecular tool for detection of S. agalactiae in bovine milk.

Background/Objectives: This study investigated the effects of chestnut tannic acid (TA) on the growth performance, the expression of tight junction proteins and the composition of the gut microbiota of weaned piglets, which could provide novel insights into the application of TA in swine production. Methods: In a 42-day trial, 180 healthy, 21-day-old Duroc × Landrace × Yorkshire piglets were randomly assigned to a Control group and four treatment groups (TA1–4), fed commercial diets supplemented with 0, 0.06%, 0.12%, 0.18% or 0.24% TA. Each group had six replicates of six pigs each. Results: The average daily gain in all TA groups, the jejunal and ileal villus height and the villus height-to-crypt depth ratio in the TA3 and TA4 groups were markedly increased (p < 0.05). The mRNA levels of MUC2 and ZO-1 were upregulated in the TA3 group, as were those of MUC4 in the jejunum and ileum and claudin in the duodenum and ileum; glutathione peroxidase and total antioxidant capacity were upregulated in the duodenum and jejunum in the TA3 group, and total superoxide dismutase was increased in all the TA2 groups (p < 0.05). Conversely, the malondialdehyde significantly decreased in all the TA groups (p < 0.05). TA supplementation improved the alpha diversity of the intestinal microflora and augmented probiotic abundance while reducing that of pathogenic bacteria. The contents of acetic, isobutyric, valeric, isovaleric, hexanoic and propionic acids, as well as total short-chain fatty acids (SCFA), were higher in the TA2 and TA3 groups (p < 0.05). Conclusions: TA inclusion in piglet diets improved the intestinal environment by upregulating the antioxidant enzymes, improving intestinal morphology and promoting probiotic growth and SCFA production while reducing pathogenic bacterial abundance, consequently enhancing the gut barrier and the growth of weaned piglets.

Deoxynivalenol (DON) is a secondary metabolite of fungi. Ingestion of feed containing DON causes severe intestinal damage in humans and animals, possibly due to cholesterol-enriched lipid raft abnormalities. Cholic acid (CA) and lithocholic acid (LCA) are metabolites of cholesterol transformation, which have been proven to benefit epithelial cell proliferation and reduce intestinal inflammation and lesions. Therefore, we aimed to study the protective roles of CA and LCA administration on the DON-exposed intestinal epithelial cells (IPI-2I) and the underlying mechanisms involved in cholesterol metabolism. We found that LCA pretreatment, but not CA, alleviated the reduction of cell numbers caused by DON exposure. Furthermore, we demonstrate that LCA restored the DON-induced cell apoptosis by reducing the cleaved caspase 3 and cleaved PARP-1 expression. DON-increased cellular cholesterol and bile acid contents were significantly reduced when LCA was co-treated. Further transcriptomic analysis revealed that the aberrant cholesterol homeostasis genes profile was observed in the cells exposed to DON or pretreated with LCA. We also validated that the key genes involved in cholesterol biosynthesis and transformation (cholesterol to bile acids) were strongly inhibited by the LCA treatment in the DON-exposed cells. Together, this study demonstrated that LCA ameliorated DON-caused toxic apoptosis in IPI-2I cells by maintaining cholesterol metabolism. We suggest that as an endogenous metabolite, LCA may be used as a therapeutic and/or integrated into a dietary intervention against mycotoxin toxicity.

Circadian rhythms exist in almost all types of cells in mammals. Thousands of genes exhibit approximately 24 h oscillations in their expression levels, making the circadian clock a crucial regulator of their normal functioning. In this regard, environmental factors to which internal physiological processes are synchronized (e.g., nutrition, feeding/eating patterns, timing and light exposure), become critical to optimize animal physiology, both by managing energy use and by realigning the incompatible processes. Once the circadian clock is disrupted, animals will face the increased risks of diseases, especially metabolic phenotypes. However, little is known about the molecular components of these clocks in domestic species and by which they respond to external stimuli. Here we review evidence for rhythmic control of livestock production and summarize the associated physiological functions, and the molecular mechanisms of the circadian regulation in pig, sheep and cattle. Identification of environmental and physiological inputs that affect circadian gene expressions will help development of novel targets and the corresponding approaches to optimize production efficiency in farm animals.

Nonalcoholic‐fatty‐liver‐disease (NAFLD) is the result of imbalances in hepatic lipid partitioning and is linked to dietary factors. We demonstrate that conjugated linoleic acid (CLA) when given to mice as a dietary supplement, induced an enlarged liver, hepatic steatosis, and increased plasma levels of fatty acid (FA), alanine transaminase, and triglycerides. The progression of NAFLD and insulin resistance was reversed by GW6471 a small‐molecule antagonist of peroxisome proliferator‐activated receptor α (PPARα). Transcriptional profiling of livers revealed that the genes involved in FA oxidation and lipogenesis as two core gene programs controlled by PPARα in response to CLA and GW6471 including Acaca and Acads. Bioinformatic analysis of PPARα ChIP‐seq data set and ChIP‐qPCR showed that GW6471 blocks PPARα binding to Acaca and Acads and abolishes the PPARα‐mediated local histone modifications of H3K27ac and H3K4me1 in CLA‐treated hepatocytes. Thus, our findings reveal a dual role of PPARα in the regulation of lipid homeostasis and highlight its druggable nature in NAFLD.

It has been reported that the proliferation and apoptosis of mammary epithelial cells affect milk production. Therefore, ensuring adequate mammary epithelial cells is expected to enhance milk production. This study is devoted to studying the effects of kisspeptin‐10 (Kp‐10), a peptide hormone composed of 10 amino acids, on bovine mammary epithelial cell (bMEC) proliferation and exploring the underlying mechanism of its action. bMECs were treated with various concentrations of Kp‐10 (1, 10, 100, and 1,000 nM), and 100 nM Kp‐10 promoted the proliferation of the bMECs. Kp‐10 promoted the cell cycle transition from G1 to the S and G2 phases, increased the protein levels of Cyclin D1 and Cyclin D3, and reduced the expression levels of the p21 gene. This study also showed that inhibition of G protein‐coupled receptor 54 (GPR54), AKT, mTOR, and ERK1/2 reduced the proliferation of the bMECs that had been induced by Kp‐10. In addition, Kp‐10 decreased the complexes formed by Rb and E2F1 and increased the expression levels of the E2F1 target genes. These results indicate that Kp‐10 promotes bMEC proliferation by activating GPR54 and its downstream signaling pathways.