Escherichia coli (E. coli), a main mastitis-causing pathogen in sows, leads to mammary tissue damage. Here, we explored the effects of Lactobacillus johnsonii L531 on attenuating E. coli-induced inflammatory damage in porcine mammary epithelial cells (PMECs). L. johnsonii L531 pretreatment reduced E. coli adhesion to PMECs by competitive exclusion and the production of inhibitory factors and decreased E. coli-induced destruction of cellular morphology and ultrastructure. E. coli induced activation of NLRP3 inflammasome associated with increased expression of NLRP3, ASC, and cleaved caspase-1, however, L. johnsonii L531 inhibited E. coli-induced activation of NLRP3 inflammasome. Up-regulation of interleukin (Il)-1β, Il-6, Il-8, Il-18, tumor necrosis factor alpha, and chemokine Cxcl2 expression after E. coli infection was attenuated by L. johnsonii L531. E. coli infection inhibited autophagy, whereas L. johnsonii L531 reversed the inhibitory effect of E. coli on autophagy by decreasing the expression of autophagic receptor SQSTM1/p62 and increasing the expression of autophagy-related proteins ATG5, ATG16L1, and light chain 3 protein by Western blotting analysis. Our findings suggest that L. johnsonii L531 pretreatment restricts NLRP3 inflammasome activity and induces autophagy through promoting ATG5/ATG16L1-mediated autophagy, thereby protecting against E. coli-induced inflammation and cell damage in PMECs.

Coliforms and Staphylococcus spp. infections are the leading causes of bovine mastitis. Despite extensive research and development in antibiotics, they have remained inadequately effective in treating bovine mastitis induced by multiple pathogen infection. In the present study, we showed the protective effect of Zophobas morio (Z. morio) hemolymph on bovine mammary epithelial cells against bacterial infection. Z. morio hemolymph directly kills both Gram-positive and Gram-negative bacteria through membrane permeation and prevents the adhesion of E. coli or the clinically isolated S. simulans strain to bovine mammary epithelial (MAC-T) cells. In addition, Z. morio hemolymph downregulates the expression of nucleotide-binding oligomerization domain (NOD)-like receptor family member pyrin domain-containing protein 3 (NLRP3), caspase-1, and NLRP6, as well as inhibits the secretion of interleukin-1β (IL-1β) and IL-18, which attenuates E. coli or S. simulans-induced pyroptosis. Overall, our results suggest the potential role of Z. morio hemolymph as a novel therapeutic candidate for bovine mastitis.

Escherichia coli (E. coli) is a major environmental pathogen causing coliform mastitis, characterized by cell death and mammary tissue damage. Our previous study has shown the antimicrobial effect of Zophobas morio (Z. morio) hemolymph against mastitis pathogens. In this study, we established E. coli-induced cellular and animal models for mastitis, aiming to evaluate the protective effect of Z. morio hemolymph against E. coli-induced mastitis in vivo and in vitro. In mice with E. coli, Z. morio hemolymph attenuated bacterial burden and histopathological impairment, reduced the production of interleukin (IL)-1β, IL-18, tumor necrosis factor-α (TNF-α) and the ratio of CD4⁺ T/CD8⁺ T, and increased the production of IL-2 triggered by E. coli. Z. morio hemolymph also enhanced the integrity of the blood-milk barrier in E. coli-induced mastitis. In E. coli-stimulated porcine mammary epithelial cells, Z. morio hemolymph inhibited E. coli-induced inflammatory responses and upregulated tight junction proteins (ZO-1, Claudin-3 and Occludin). Moreover, we found that the anti-inflammatory effect of Z. morio hemolymph was mediated by inhibiting E. coli-induced NLRP3 inflammasome assembly, Caspase-1 activation, and reversing the inhibitory effect of E. coli on autophagy. Besides, Z. morio hemolymph augmented ATG5/ATG16L1-mediated autophagy activation, negatively regulated NLRP3 inflammasome activation. Our results reveal that Z. morio hemolymph alleviates E. coli-induced mastitis via lessening the inflammatory response by regulating the NLRP3 and ATG5/ATG16L1 signaling pathway, as well as repairing the blood-milk barrier.

Background: Post-weaning diarrhea (PWD) in piglets, often caused by F4⁺ enterotoxigenic Escherichia coli (ETEC), poses significant challenges in pig production. Traditional solutions like antibiotics and zinc oxide face increasing restrictions due to growing concerns over antibiotic resistance and environmental sustainability. This study investigates the application of bivalent heavy chain variable domain (V_HH) constructs (BL1.2 and BL2.2) targeting ETEC virulence factors, administered in feed to mitigate ETEC-induced PWD in weaned piglets. Results: The supplementation of BL1.2 and BL2.2 in both mash and pelleted feed significantly reduced the diarrhea incidence and fecal shedding of F4⁺ ETEC in challenged piglets. Pelleted feed containing V_HH constructs helped to preserve gut barrier integrity by maintaining levels of the tight junction protein occludin in the small intestine. Additionally, the constructs maintained blood granulocyte counts at a similar level to the non-challenged control group, including neutrophils, and ameliorated the acute phase protein response after challenge. Notably, even at low feed intake immediately after weaning, V_HH constructs helped maintain piglet health by mitigating ETEC-induced inflammation and the resulting diarrhea. Conclusions: Our findings demonstrated that using V_HH constructs as feed additives could serve as an effective strategy to help manage ETEC-associated PWD, by reducing F4⁺ ETEC gut colonization and supporting gut barrier function of weaned piglets. The high stability of these V_HH constructs supports their incorporation into industrial feed manufacturing processes, offering a more sustainable preventive strategy compared to traditional antimicrobial interventions, which could contribute to sustainable farming practices.

The effect of feeding fermented liquid feed (FLF) with added Pediococcus acidilactici to weaning piglets challenged with enterotoxigenic Escherichia coli (ETEC) F4 on aspects of diarrhea, performance, immune responses, and intestinal epithelial barrier function was investigated. A total of 46 weaners (weaning at 27–30 days of age) were assigned to four treatments: (1) Non-challenged and dry feed (Non-Dry); (2) Challenged and dry feed (Ch-Dry); (3) Non-challenged and FLF (Non-Ferm); (4) Challenged and FLF (Ch-Ferm). All groups received the same feed, either dry (Non-Dry and Ch-Dry), or in liquid form (Non-Ferm and Ch-Ferm) in which the cereals with added P. acidilactici (10⁶ CFU/g cereals) had been fermented for 24 h at 30°C. On day 1 and 2 post weaning, Ch-Dry and Ch-Ferm were orally inoculated with 5 mL × 10⁹ CFU ETEC F4/mL, whereas the Non-Dry and Non-Ferm received the same amount of saline. Fecal samples and blood samples were collected through the study period. The microbial composition, concentration of microbial metabolites and nutrient composition indicated that the quality of the FLF was high. In the first week, ADFI of both non-challenged groups was significantly higher (p < 0.05) than that of the Ch-Ferm group. The two challenged groups had higher fecal levels of FaeG gene (ETEC F4 fimbriae) from day 2 to 6 post weaning (p < 0.01), and higher risk of having ETEC F4 present in feces from day 3 to 5 post weaning (p < 0.05) compared to non-challenged groups, indicating the validity of the ETEC challenge model. Generally, ADG of the two groups fed FLF were numerically higher than those fed dry feed. Neither challenge nor FLF affected diarrhea. No significant differences were measured between Ch-Ferm and Ch-Dry regarding the level of plasma haptoglobin and C-reactive protein, hematological parameters or parameters related to epithelial barrier. The data indicated a low level of infection caused by the ETEC challenge, while recovery from weaning stress could be observed. The study showed that a strategy like this can be a way of providing a high level of probiotics to pigs by allowing their proliferation during fermentation.

For the long-standing riddle of metallic passivation, the second law of thermodynamics helps little. But this study shows that Ti passivation layer coming from room-temperature spontaneous oxidation possesses a dissipative structure which naturally extends from the lattice structure of Ti matrix. Macroscopically, its oxidation degree, oxygen content and amorphous structures exhibit a gradient decline from its surface to its bottom. Beneath a stable chemisorbed oxygen-metal atom monolayer, residue Ti lattice structures resemble stones and amorphous structures composed of TiO2, Ti2O3 and TiO resemble mortar, together they forming a heterogeneous stone-wall like structure. The rapid formation of the stable chemisorbed monolayer passivates the outmost Ti atoms to start Ti passivation, and subsequently a dissipative mechanism of oxidation cracks the Ti lattice structures and thus the oxide layer is formed and effect passivation. The solid phase formed by the open, stable, macroscopically-ordered, non-equilibrium oxide film, which can be aptly called "the dissipative oxidation phase", theoretically does not end in complete oxidation of thermodynamic stability. This study derails both the adsorption theory of passivation and the theory of passivity film, and thus sheds new light on the metallic passivation.