Although breeding of F4 receptor − negative (F4R−) pigs may prevent post-weaning diarrhea, the underlying immunity is poorly understood. Here, various doses of a Bacillus licheniformis and Bacillus subtilis mixture (BLS-mix) were orally administered to F4ab/acR− pigs for 1 week before F4 (K88) − positive ETEC/VTEC/EPEC challenge. Administration of BLS-mix increased the percentage of Foxp3−IL-10+ T cells but not of Foxp3+IL-10+ regulatory T (Treg) cells among peripheral blood CD4+ T cells. A low dose of BLS-mix feeding resulted in increased the expression of IL-6, TNF-α, IL-10, and the transcription factors Foxp3 and T-bet mRNAs in the jejunum. Administration of either a low or high dose BLS-mix also led to an increase in the percentage of CD4+Foxp3+ Treg cells among intraepithelial lymphocytes and CD4+IL-10+ T cells in the small intestinal Peyer’s patches and the lamina propria of F4ab/acR− pigs following F4+ ETEC/VTEC/EPEC challenge. The increased number of IL-10–producing CD4+ T cells was attributed to an increase in the proportion of Foxp3−IL-10+ Treg cells rather than Foxp3+IL-10+ Treg cells. Our data indicate that oral administration of BLS-mix to newly weaned F4ab/acR− pigs ameliorates enteritis in an F4+ ETEC/VTEC/EPEC model; however, induction of IL-10–producing Foxp3− Treg cells by BLS-mix administration cannot account for the protection of newly weaned F4ab/acR− pigs from F4+ ETEC/VTEC/EPEC infection, and that excessive generation of CD4+IL-10+ T cells following consumption of BLS-mix during episodes of intestinal inflammation that is caused by enteric pathogens might prohibit clearance of the pathogen. Select probiotic mixtures may allow for tailoring strategies to prevent infectious diseases.

Extracellular vesicles (EVs) are membranous vesicles found in biological fluids with essential functions. However, milk-derived EV proteins from clinical mastitis (CM) and subclinical mastitis (SM) cows have yet to be studied in detail. In this study, milk-derived EVs of CM, SM, and Healthy cows were extracted using a combination of acetic acid/ultracentrifugation and density gradient ultracentrifugation and analyzed using a shotgun proteomic by data-independent acquisition mode. A total of 1253 milk exosome proteins were identified and quantified. Differently enriched (DE) proteins were identified as given a Benjamini–Hochberg adjusted p < 0.05 and a fold change of at least 2. There were 53 and 1 DE proteins in milk-derived EVs from CM and SM cows compared with healthy cows. Protein S100-A9, Protein S100-A8, Chitinase-3-like protein 1, Haptoglobin, Integrin beta-2, and Chloride intracellular channel protein 1 were more abundant in the CM group (adjusted p < 0.05). Still, their enrichment in the SM group was not significant as in the Healthy group. The enrichment of DE proteins between CM and Healthy group was consistent with elevated GO (Gene Ontology) processes—defense response, defense response to Gram-positive bacterium, granulocyte chemotaxis also contributed to Reactome pathways—neutrophil degranulation, innate immune system, and antimicrobial peptides in the CM group. These results provide essential information on mastitis-associated proteins in milk-derived EVs and indicate the biological functions of milk-derived EVs proteins require further elucidation.