This experiment evaluated dry matter intake (DMI), ruminal in situ nutrient disappearance, total-tract apparent nutrient digestibility, microbiome, and ruminal fermentation parameters of crossbred beef cows offered forage-based diets and a Bacillus-based direct-fed microbial (DFM). Fifteen rumen-cannulated Angus × Hereford cows were used in a crossover design. Treatments consisted of forage-based diets plus 500 g of protein-mineral supplement mixed with 1) 3 g of a Bacillus-based DFM containing Bacillus licheniformis and B. subtilis (BAC; n = 15; 2.2 × 109 CFU of the mixture/g; Bovacillus, Novonesis, Lyngby, Denmark), or 2) without BAC (CON; n = 15). Each experimental period lasted 33 d with 30 d of wash-out between periods. Cows were fed daily with chopped grass-mixed hay [Idaho fescue (Festuca idahoensis) and Bluebunch wheatgrass (Pseudoroegneria spicata)]. From days 17 to 26, 5 g of titanium dioxide (TiO2) was dosed twice daily, at 12-h intervals, as an external marker of fecal output. From days 22 to 26, fecal spot samples were collected at 12-h intervals. On day 27, ruminal fluid was collected prior to feeding (0 h) and at 4, 8, 12, 16, and 20 h post-feeding to evaluate ruminal pH, volatile fatty acids (VFA), and NH3-N. From days 28 to 32, 4 g of grass-mixed hay was placed in Dacron bags and introduced through the cannulas for 0, 12, 24, 48, 72, and 96 h. A treatment effect was observed (P = 0.01) for DMI, which was greater for cows supplemented with BAC vs. CON. Cows supplemented with BAC tended (P > 0.06) to have greater dry matter (DM) and neutral detergent fiber (NDF) disappearance vs. CON. A treatment effect was observed (P ≤ 0.02) for total-tract apparent DM and CP digestibility, which were greater for BAC vs. CON cows. Also, cows supplemented with BAC tended (P ≥ 0.06) to have a greater total-tract NDF and acid detergent fiber (ADF) digestibility vs. CON cohorts. Therefore, cows supplemented with BAC had greater (P < 0.01) amounts of DM, CP, NDF, and ADF digested vs. CON cows. A treatment × time effect was observed (P = 0.02) for ruminal pH, which increased at 16 and 20 h of collection for BAC vs. CON cows. Treatment effects were not (P ≥ 0.18) observed for VFA, N-NH3, and plasma concentrations of glucose, whereas plasma concentration of urea tended (P = 0.06) to be reduced for cows supplemented with BAC vs. CON. Hence, supplementation with a Bacillus-based DFM increased forage intake, the digestibility of DM and CP, and the amount of nutrients digested by rumen-cannulated cows receiving a forage-based diet.

Direct-fed microbials (DFMs) have shown the potential to improve livestock performance and overall health. Extensive research has been conducted to identify new DFMs and understand their mechanisms of action in the gut. Bacillus species are multifunctional spore-forming bacteria that exhibit resilience to harsh conditions, making them ideal candidates for applications in the feed industry and livestock production. This study investigates the mode of action of B. licheniformis and B. subtilis in the rumen using diverse in vitro techniques. Our results revealed that both strains germinated and grew in sterile rumen and intestinal contents from dairy cows and bulls. Gas composition analysis of in vitro cultures in a medium containing 40% rumen fluid demonstrated that germination of B. licheniformis and B. subtilis strains reduced oxygen levels, promoting an anaerobic environment favorable to rumen microbes. Enzymatic activity assays showed that B. licheniformis released sugars from complex substrates and purified polysaccharides in filtered rumen content. Additionally, the combination of B. licheniformis and B. subtilis survived and grew in the presence of a commercial monensin dose in rumen fluid media. The effects of B. licheniformis and B. subtilis on rumen fermentation activity and microbiota were studied using an in vitro batch fermentation assay. In fermenters that received a combination of B. licheniformis and B. subtilis, less CO2 was produced while dry matter degradation and CH4 production was comparable to the control condition, indicating better efficiency of dry matter utilization by the microbiota. The investigation of microbiota composition between supplemented and control fermenters showed no significant effect on alpha and beta diversity. However, the differential analysis highlighted changes in several taxa between the two conditions. Altogether, our data suggests that the administration of these strains of Bacillus could have a beneficial impact on rumen function, and consequently, on health and performance of ruminants.

Abstract Managing bacterial infections is of great importance in cattle production, particularly those caused by Salmonella enterica serovars Typhimurium or Dublin, which can impact both animal health and human food safety. Direct-fed microbial (DFM) can support gastrointestinal health and alleviate potential bacterial infections. In the present study, the capacity of a bacteria-based DFM product [BOVAMINE DEFEND Plus; BDP; Novonesis, Denmark (BDP)] containing Lactobacillus animalis, Propionibacterium freudenreichii, Bacillus licheniformis, and B. subtilis to reduce Salmonella Typhimurium ATCC14028 invasion was investigated using the HT29-MTX cell line (Exp. 1) and also to evaluate the possible antagonistic effect against S. Dublin using an in vitro agar well diffusion method (Exp. 2). Briefly, in Exp. 1, after a 30 min pre-incubation with 1.0 ×108 CFU of BDP and/or 2.5 × 106 CFU of S. Typhimurium were added to each transwell. The quantity of S. Typhimurium invading the cells was measured after 90 min of co-incubation using CFU counts. As a control, the viability of the total S. Typhimurium was assessed to confirm the mode of action of BDP targets the pathogen invasion properties. In Exp. 2, the antagonistic efficacy of BDP against S. Dublin CHCC41286, isolated from cattle, was tested by adding BDP to wells in CLED agar plates casted with the pathogen above, zones of inhibition measured after 48 h incubation. In Exp. 1, BDP significantly (P < 0.05) reduced by 80.6 % the invasion of S. typhimurium into HT-29-MTX cells as well as from 16 % the viability of the pathogen. The antagonistic properties of BDP towards S. Dublin were confirmed by clear inhibition zones (8.6 mm). These two in vitro findings set the stage for exploring the potential benefits of using a novel DFM as a promising tool and strategy to mitigate Salmonella infections in ruminants and improve animal health, food safety, and public health. Further in vivo confirmation needs to be developed to validate these preliminary in vitro results.

Three experiments were designed to evaluate the in vitro effects of a multispecies bacterial-based direct-fed microbials (DFM) containing Lactobacillus animalis 506, Propionibacterium freudenreichii 507, Bacillus licheniformis 809, and Bacillus subtilis 597 against the pathogens Salmonella enterica serovar Typhimurium and Dublin. The DFM reduced the invasion of Salmonella into intestinal epithelial cells, while also supporting the integrity of the same cells under laboratorial settings. These results indicate that the bacterial-based DFM containing Lactobacillus animalis 506, P. freudenreichii 507, B. licheniformis 809, and B. subtilis 597 could be a good candidate to alleviate potential adverse effects of S. enterica in ruminants.

Enterotoxigenic Escherichia coli (ETEC) is the main infectious agent responsible for piglet post-weaning diarrhea with high mortality rates. Antimicrobials represent the current principal strategy for treating ETEC infections in pig farms, but the occurrence of multi-resistant bacterial strains has considerably increased in the last decades. Thus, finding non-antibiotic alternatives becomes a real emergency. In this context, we investigated the effect of a live yeast strain, Saccharomyces cerevisiae var boulardii CNCM I-1079 (SB) in an in vitro model of the weaning piglet colon implemented with a mucus phase (MPigut-IVM) inoculated with ETEC and coupled with an intestinal porcine cell line IPI-2I. We showed that SB was able to modulate the in vitro microbiota through an increase in Bacteroidiaceae and a decrease in Prevotellaceae families. Effluents collected from the SB treated bioreactors were able to mitigate the expression level of genes encoding non-gel forming mucins, tight junction proteins, innate immune pathway, and pro-inflammatory response in IPI-2I cells. Furthermore, SB exerted a significant protective effect against ETEC adhesion on porcine IPEC-J2 intestinal cells in a dose-dependent manner and showed a positive effect on ETEC-challenged IPEC-J2 by lowering expression of genes involved in pro-inflammatory immune responses. Our results showed that the strain SB CNCM I-1079 could prevent microbiota dysbiosis associated with weaning and protect porcine enterocytes from ETEC infections by reducing bacterial adhesion and modulating the inflammatory response.

Enterotoxigenic Escherichia coli (ETEC) is the principal pathogen responsible for post-weaning diarrhea in newly weaned piglets. Expansion of ETEC at weaning is thought to be the consequence of various stress factors such as transient anorexia, dietary change or increase in intestinal inflammation and permeability, but the exact mechanisms remain to be elucidated. As the use of animal experiments raise more and more ethical concerns, we used a recently developed in vitro model of piglet colonic microbiome and mucobiome, the MPigut-IVM, to evaluate the effects of a simulated weaning transition and pathogen challenge at weaning. Our data suggested that the tested factors impacted the composition and functionality of the MPigut-IVM microbiota. The simulation of weaning transition led to an increase in relative abundance of the Prevotellaceae family which was further promoted by the presence of the ETEC strain. In contrast, several beneficial families such as Bacteroidiaceae or Ruminococcaceae and gut health related short chain fatty acids like butyrate or acetate were reduced upon simulated weaning. Moreover, the incubation of MPigut-IVM filtrated effluents with porcine intestinal cell cultures showed that ETEC challenge in the in vitro model led to an increased expression of pro-inflammatory genes by the porcine cells. This study provides insights about the etiology of a dysbiotic microbiota in post-weaning piglets.

Background Risk factors for the etiology of post-weaning diarrhea, a major problem in swine industry associated with enormous economic losses, remain to be fully elucidated. In concordance with the ethical concerns raised by animal experiments, we developed a new in vitro model of the weaning piglet colon (MPigut-IVM) including a mucin bead compartment to reproduce the mucus surface from the gut to which gut microbes can adhere. Results Our results indicated that the MPigut-IVM is able to establish a representative piglet archaeal and bacterial colon microbiota in terms of taxonomic composition and function. The MPigut-IVM was consequently used to investigate the potential effects of feed deprivation, a common consequence of weaning in piglets, on the microbiota. The lack of nutrients in the MPigut-IVM led to an increased abundance of Prevotellaceae and Escherichia-Shigella and a decrease in Bacteroidiaceae and confirms previous in vivo findings . On top of a strong increase in redox potential, the feed deprivation stress induced modifications of microbial metabolite production such as a decrease in acetate and an increase in proportional valerate, isovalerate and isobutyrate production. Conclusions The MPigut-IVM is able to simulate luminal and mucosal piglet microbiota and represent an innovative tool for comparative studies to investigate the impact of weaning stressors on piglet microbiota. Besides, weaning-associated feed deprivation in piglets provokes disruptions of MPigut-IVM microbiota composition and functionality and could be implicated in the onset of post-weaning dysbiosis in piglets.

Dietary, environmental, and social stresses induced by weaning transition in pig production are associated with alterations of gut microbiota, diarrhea, and enteric infections. With the boom of -omic technologies, numerous studies have investigated the dynamics of fecal bacterial communities of piglets throughout weaning but much less research has been focused on the composition and functional properties of microbial communities inhabiting other gastrointestinal segments. The objective of the present study was to bring additional information about the piglet bacterial and archaeal microbiota throughout the entire digestive tract, both at the structural level by using quantitative PCR and high-throughput sequencing, and on functionality by measurement of short-chain fatty acids and predictions using Tax4Fun tool. Our results highlighted strong structural and functional differences between microbial communities inhabiting the fore and the lower gut as well as a quantitatively important archaeal community in the hindgut. The presence of opportunistic pathogens was also noticed throughout the entire digestive tract and could trigger infection emergence. Understanding the role of the intestinal piglet microbiota at weaning could provide further information about the etiology of post-weaning infections and lead to the development of effective preventive solutions.

A close symbiotic relationship exists between the intestinal microbiota and its host. A critical component of gut homeostasis is the presence of a mucus layer covering the gastrointestinal tract. Mucus is a viscoelastic gel at the interface between the luminal content and the host tissue that provides a habitat to the gut microbiota and protects the intestinal epithelium. The review starts by setting up the biological context underpinning the need for experimental models to study gut bacteria-mucus interactions in the digestive environment. We provide an overview of the structure and function of intestinal mucus and mucins, their interactions with intestinal bacteria (including commensal, probiotics and pathogenic microorganisms) and their role in modulating health and disease states. We then describe the characteristics and potentials of experimental models currently available to study the mechanisms underpinning the interaction of mucus with gut microbes, including in vitro, ex vivo and in vivo models. We then discuss the limitations and challenges facing this field of research.

The effects of gut microbiota on human traits are expected to be small to moderate and adding the complexity of the human diseases, microbiome research demands big sample sizes. Fecal samples for such studies are mostly self-collected by participants at home. This imposes an extra level of complexity as sample collection and storage can be challenging. Effective, low-burden collection and storage methods allowing fecal samples to be transported properly and ensuring optimal quality and quantity of bacterial DNA for upstream analyses are necessary. Moreover, accurate assessment of the microbiome composition also depends on bacterial DNA extraction method. The aim of this study was to evaluate the reliability and efficiency of the OMNIgene•GUT kit as a participant-fecal friendly collection method (storage at room temperature for 24 h (O24h) or 7 days (O7d)) in comparison to the standard collection method (Fresh, storage at 4 °C for less than 24 h) in terms of amount of variability and information content accounting for two common DNA extraction methods.

Weaning is a critical event in the pig's life cycle, frequently associated with severe enteric infections and overuse of antibiotics; this raises serious economic and public health concerns. In this review, we explain why gut microbiota dysbiosis, induced by abrupt changes in the diet and environment of piglets, emerges as a leading cause of post-weaning diarrhea, even if the exact underlying mechanisms remain unclear. Then, we focus on nonantimicrobial alternatives, such as zinc oxide, essential oils, and prebiotics or probiotics, which are currently evaluated to restore intestinal balance and allow a better management of the crucial weaning transition. Finally, we discuss how in vitro models of the piglet gut could be advantageously used as a complement to ex vivo and in vivo studies for the development and testing of new feed additives.