Evaluation of the Immunity Responses in Mice to Recombinant Bacillus subtilis Displaying Newcastle Disease Virus HN Protein Truncations.

Bacillus subtilis, a probiotic bacterium with engineering potential, is widely used for the expression of exogenous proteins. In this study, we utilized the integrative plasmid pDG364 to integrate the hemagglutinin-neuraminidase (HN) gene from Newcastle disease virus (NDV) into the genome of the B. subtilis 168 model strain. We successfully constructed a recombinant B. subtilis strain (designated B. subtilis RH) that displays a truncated HN antigen fragment on the surface of its spores and further evaluated its immunogenic effects in mice. Using ELISA, we quantified the levels of IgG in serum and secretory IgA (sIgA) in intestinal contents. The results revealed that the recombinant B. subtilis RH elicited robust specific mucosal and humoral immune responses in mice. Furthermore, B. subtilis RH demonstrated potential mucosal immune adjuvant properties by fostering the development of immune organs and augmenting the number of lymphocytes in the small intestinal villi. Additionally, the strain significantly upregulated the relative expression of inflammatory cytokines such as IL-1ß, IL-6, IL-10, TNF-α, and IFN-γ in the small intestinal mucosa. In conclusion, the B. subtilis RH strain developed in this study exhibits promising mucosal immunogenic effects. It holds potential as a candidate for an anti-NDV mucosal subunit vaccine and offers a novel preventive strategy for the poultry industry against this disease.

Evaluation of probiotic properties of a Brevibacillus laterosporus strain.

Brevibacillus laterosporus is a strain of probiotic bacteria that has been widely used in pest control, cash crop, and other production areas. However, few studies have been conducted on its use as a feed additive in animals. Therefore, the probiotic potential of B. laterosporus PBC01 was evaluated by characterizing hydrophobicity, auto-aggregation activity, bile salt and simulated gastrointestinal fluid tolerance, bienzymatic, and antibacterial activity. Antibiotic susceptibility, hemolysis assays, and supplemental feeding of mice were also performed to evaluate safety features. Our results showed that B. laterosporus PBC01 had moderate hydrophobicity, high auto-agglutination ability. Meanwhile, B. laterosporus PBC01 had good tolerance to bile salt and simulated gastrointestinal fluid. It had the ability to secrete protease, cellulase, and to inhibit various pathogens. In addition, B. laterosporus PBC01 was sensitive to many antibiotics, and did not produce hemolysin. In the safety assessment of mice, it did not cause any deaths, nor did it affect the cell components of blood, antioxidant capacity, and reproductive health. The study indicated the great probiotic characteristics and safety of B. laterosporus PBC01. This may provide a theoretical basis for the clinical application and development of probiotic-based feed additives.

Targeted Screening of Fiber Degrading Bacteria with Probiotic Function in Herbivore Feces.

Cellulolytic bacteria with probiotic functions play a crucial role in promoting the intestinal health in herbivores. In this study, we aimed to correlate the 16S rRNA gene amplicon sequencing and fiber-degrading enzyme activity data from six different herbivore feces samples. By utilizing the separation and screening steps of probiotics, we targeted and screened high-efficiency fiber-degrading bacteria with probiotic functions. The animals included Maiwa Yak (MY), Holstein cow (CC), Tibetan sheep (TS), Southern Sichuan black goat (SG), Sichuan white rex rabbit (CR), and New Zealand white rabbit (ZR). The results showed that the enzymes associated with fiber degradation were higher in goat and sheep feces compared to cattle and rabbit's feces. Correlation analysis revealed that Bacillus and Fibrobacter were positively correlated with five types of fiber-degrading related enzymes. Notably, the relative abundance of Bacillus in the feces of Tibetan sheep was significantly higher than that of other five herbivores. A strain TS5 with good cellulose decomposition ability from the feces of Tibetan sheep by Congored staining, filter paper decomposition test, and enzyme activity determination was isolated. The strain was identified as Bacillus velezensis by biological characteristics, biochemical analysis, and 16S rRNA gene sequencing. To test the probiotic properties of Bacillus velezensis TS5, we evaluated its tolerance to acid and bile salt, production of digestive enzymes, antioxidants, antibacterial activity, and adhesion ability. The results showed that the strain had good tolerance to pH 2.0 and 0.3% bile salts, as well as good potential to produce cellulase, protease, amylase, and lipase. This strain also had good antioxidant capacity and the ability to antagonistic Staphylococcus aureus BJ216, Salmonella SC06, Enterotoxigenic Escherichia coli CVCC196, and Escherichia coli ATCC25922. More importantly, the strain had good self-aggregation and Caco-2 cell adhesion rate. In addition, we tested the safety of Bacillus velezensis TS5 by hemolysis test, antimicrobial susceptibility test, and acute toxicity test in mice. The results showed that the strain had no hemolytic phenotype, did not develop resistance to 19 commonly used antibiotics, had no cytotoxicity to Caco-2, and did not have acute toxic harm to mice. In summary, this study targeted isolated and screened a strain of Bacillus velezensis TS5 with high fiber-degrading ability and probiotic potency. This strain can be used as a potential probiotic for feeding microbial preparations for ruminants.

Complete genome analysis of Bacillus velezensis TS5 and its potential as a probiotic strain in mice.

Introduction: In recent years, a large number of studies have shown that Bacillus velezensis has the potential as an animal feed additive, and its potential probiotic properties have been gradually explored. Methods: In this study, Illumina NovaSeq PE150 and Oxford Nanopore ONT sequencing platforms were used to sequence the genome of Bacillus velezensis TS5, a fiber-degrading strain isolated from Tibetan sheep. To further investigate the potential of B. velezensis TS5 as a probiotic strain, in vivo experiments were conducted using 40 five-week-old male specific pathogen-free C57BL/6J mice. The mice were randomly divided into four groups: high fiber diet control group (H group), high fiber diet probiotics group (HT group), low fiber diet control group (L group), and low fiber diet probiotics group (LT group). The H and HT groups were fed high-fiber diet (30%), while the L and LT groups were fed low-fiber diet (5%). The total bacteria amount in the vegetative forms of B. velezensis TS5 per mouse in the HT and LT groups was 1 × 109 CFU per day, mice in the H and L groups were given the same volume of sterile physiological saline daily by gavage, and the experiment period lasted for 8 weeks. Results: The complete genome sequencing results of B. velezensis TS5 showed that it contained 3,929,788 nucleotides with a GC content of 46.50%. The strain encoded 3,873 genes that partially related to stress resistance, adhesion, and antioxidants, as well as the production of secondary metabolites, digestive enzymes, and other beneficial nutrients. The genes of this bacterium were mainly involved in carbohydrate metabolism, amino acid metabolism, vitamin and cofactor metabolism, biological process, and molecular function, as revealed by KEGG and GO databases. The results of mouse tests showed that B. velezensis TS5 could improve intestinal digestive enzyme activity, liver antioxidant capacity, small intestine morphology, and cecum microbiota structure in mice. Conclusion: These findings confirmed the probiotic effects of B. velezensis TS5 isolated from Tibetan sheep feces and provided the theoretical basis for the clinical application and development of new feed additives.

The panda-derived Lactiplantibacillus plantarum BSG201683 improves LPS-induced intestinal inflammation and epithelial barrier disruption in vitro.

Captive pandas are suffering from intestinal infection due to intestinal microbiota characterized by a high abundance of Enterobacteriaceae induced by long-term captivity. Probiotic supplements showed improvement in intestinal barrier function and inflammation. However, the effects of panda-derived probiotics on the intestinal epithelium and inflammation have not been elucidated. In the present study, lipopolysaccharide (LPS) impaired Caco-2 and RAW264.7 inflammatory models were applied to assess the protection of Lactiplantibacillus plantarum BSG201683 (L. plantarum G83) on barrier disruption and inflammation. The results showed that treatment with L. plantarum G83 significantly decreased the paracellular permeability to fluorescein isothiocyanate conjugated dextran (MW 4000, FITC-D4) after LPS induction. Meanwhile, L. plantarum G83 alleviated the reduction in tight junction (TJ) proteins and downregulated proinflammatory cytokines caused by LPS in Caco-2 cells. L. plantarum G83 also significantly decreased the expression and secretion of pro-inflammatory cytokines in LPS-induced RAW264.7 cells. In addition, the IL-10 increased in both Caco-2 and RAW264.7 cells after L. plantarum G83 treatment. The phagocytosis activity of RAW264.7 cells was significantly increased after L. plantarum G83 treatment. Toll-like receptor 4/ nuclear factor kappa-B (TLR4/NF-κB) signaling pathways were significantly down-regulated after L. plantarum G83 intervention, and the phosphorylation of NF-κB/p65 was consistent with this result. Our findings suggest that L. plantarum G83 improves intestinal inflammation and epithelial barrier disruption in vitro.

Prolonged fluoride exposure induces spatial-memory deficit and hippocampal dysfunction by inhibiting small heat shock protein 22 in mice.

Millions of residents in areas with high-fluoride drinking water supply ingest excessive levels of fluoride for long periods. This study investigated the mechanisms and impacts of lifelong exposure to naturally occurring moderate-high-fluoride drinking water on spatial-memory function by studying mice in controlled experiments. Spatial-memory deficits and disorders of hippocampal neuronal electrical activity were observed in mice exposed to 25-ppm or 50-ppm-fluoride drinking water for 56 weeks, but not in adult or old mice exposed to 50 ppm fluoride for 12 weeks. Ultrastructural analysis showed severely damaged hippocampal mitochondria, evidenced by reduced mitochondrial membrane potential and ATP content. Mitochondrial biogenesis was impaired in fluoride-exposed mice, manifesting as a significantly reduced mtDNA content, mtDNA-encoded subunits mtND6 and mtCO1, and respiratory complex activities. Fluoride reduced expression of Hsp22, a beneficial mediator of mitochondrial homeostasis, and decreased levels of signaling for the PGC-1α/TFAM pathway-which regulates mitochondrial biogenesis-and the NF-κß/STAT3 pathway-which regulates mitochondrial respiratory chain enzyme activity. Hippocampus-specific Hsp22-overexpression improved fluoride-induced spatial-memory deficits by activating the PGC-1α/TFAM and STAT3 signaling pathways, while Hsp22-silencing aggravated the deficits by inhibiting both pathways. Downregulation of Hsp22 plays a vital role in fluoride-induced spatial-memory deficits by impacting mtDNA-encoding subsets and mitochondrial respiratory chain enzyme activity.

Fluoride disrupts intestinal epithelial tight junction integrity through intracellular calcium-mediated RhoA/ROCK signaling and myosin light chain kinase.

Fluoride is a common contaminant of groundwater and agricultural commodity, which poses challenges to animal and human health. A wealth of research has demonstrated its detrimental effects on intestinal mucosal integrity; however, the underlying mechanisms remain obscure. This study aimed to investigate the role of the cytoskeleton in fluoride-induced barrier dysfunction. After sodium fluoride (NaF) treatment of the cultured Caco-2 cells, both cytotoxicity and cytomorphological changes (internal vacuoles or massive ablation) were observed. NaF lowered transepithelial electrical resistance (TEER) and enhanced paracellular permeation of fluorescein isothiocyanate dextran 4 (FD-4), indicating Caco-2 monolayers hyperpermeability. In the meantime, NaF treatment altered both the expression and distribution of the tight junction protein ZO-1. Fluoride exposure increased myosin light chain II (MLC2) phosphorylation and triggered actin filament (F-actin) remodeling. While inhibition of myosin II by Blebbistatin blocked NaF-induced barrier failure and ZO-1 discontinuity, the corresponding agonist Ionomycin had effects comparable to those of fluoride, suggesting that MLC2 serves as an effector. Given the mechanisms upstream of p-MLC2 regulation, further studies demonstrated that NaF activated RhoA/ROCK signaling pathway and myosin light chain kinase (MLCK), strikingly increasing the expression of both. Pharmacological inhibitors (Rhosin, Y-27632 and ML-7) reversed NaF-induced barrier breakdown and stress fiber formation. The role of intracellular calcium ions ([Ca2+]i) in NaF effects on Rho/ROCK pathway and MLCK was investigated. We found that NaF elevated [Ca2+]i, whereas chelator BAPTA-AM attenuated increased RhoA and MLCK expression as well as ZO-1 rupture, thus, restoring barrier function. Collectively, abovementioned results suggest that NaF induces barrier impairment via Ca2+-dependent RhoA/ROCK pathway and MLCK, which in turn triggers MLC2 phosphorylation and rearrangement of ZO-1 and F-actin. These results provide potential therapeutic targets for fluoride-induced intestinal injury.

Effects of Antimicrobial Peptides Gal-13 on the Growth Performance, Intestinal Microbiota, Digestive Enzyme Activities, Intestinal Morphology, Antioxidative Activities, and Immunity of Broilers.

To evaluate the application effect of antimicrobial peptides Gal-13 (AMP Gal-13) instead of antibiotic feed additives, 90 7-day-old Ross 308 broilers were randomly divided into 3 groups. Group A was fed a basic diet as the control, and Groups B and C were supplemented with AMP Gal-13 (100 mg/kg and 200 mg/kg, respectively). After a 35-day feeding experiment, the weight and average daily gain (ADG) of the broilers in Group B were significantly higher than those of the broilers in Group A. The Enterococcus sp. and Escherichia coli counts in the ileum and cecum in Group A were significantly higher than those in Groups B and C, while the Lactic acid bacteria (LAB) and Bifidobacterium sp. counts were significantly lower. The amylase activity of the jejunum in Group B was significantly higher than that in Group A. The villus length (VL): crypt depth (CD) ratios of the jejunum and ileum in Group B were significantly higher than those in Group A. The glutathione peroxidase (GSH-Px) activities in the liver and serum in Groups B and C were significantly higher than those in Group A, while the malondialdehyde (MDA) activity was significantly lower. The titers of Newcastle disease virus (NDV)-specific antibodies were elevated significantly in Group B at the age of 42 days. Additionally, the weights of the spleen and thymus were significantly increased. The expression levels of Il-2, Il-6, Tgf-ß4, Tnf-α, and Mif in the spleen in Groups B and C were significantly downregulated to different degrees; Il-4 expression in Group B was significantly upregulated, while Ifn-γ expression in Group C was significantly upregulated. The results suggested that adding AMP Gal-13 to the diet could improve intestinal digestion, the antioxidant capacity, and immune function, ultimately promoting the growth of broilers.

Promotion of Egg Production Rate and Quality Using Limosilactobacillus oris BSLO 1801, a Potential Probiotic Screened from Feces of Laying Hens with Higher Egg Productive Performance.

In this experiment, laying hens were divided into a high productive group (group H) and a low productive group (group L). The purpose of this experiment was to screen and isolate a potential probiotic associated with the laying rate from group H by comparing the results via 16S rDNA high-throughput sequencing. The high-throughput sequencing analysis results showed that there were some differences in the composition of the gut microbiome between groups H and L on the Phylum and Genus levels. Through isolation and identification, we screened 16 lactobacilli strains. Among the 16 strains, S5 showed good acid tolerance, bile salt tolerance, and cholesterol degradation. Therefore, we chose strain S5 (identified as Limosilactobacillus oris, named Limosilactobacillus oris BSLO 1801) as a potential probiotic to promote the productivity of ordinary laying hens. During the animal experiment, 288 Hy-line white hens (30 weeks old) were divided into four groups, with six replications (n = 12) per group. The control group received the basic diet, and the treatment groups received the same basic diet supplemented with 107 CFU/kg, 108 CFU/kg, and 109 CFU/kg of BSLO 1801. The laying hens were acclimated to the environment for 1 week before the initiation of the experiment. Dietary supplementation with 107 CFU/kg and 109 CFU/kg of BSLO 1801 increased the laying rate significantly, and the potential probiotic improved the egg weight in all treatment groups. Additionally, the cholesterol content of the yolk dropped significantly in the 109 CFU/kg group, and the weight of egg yolk was significantly increased in all treatment groups. However, no significant differences in eggshell strength, eggshell thickness, protein height, and Haugh unit were observed among the four groups. These results revealed that lactobacilli spp. are important bacteria of the intestinal microbiome in highly productive laying hens, and BSLO 1801 was isolated as a potential probiotic. Through these animal experiments, we also found that adding BSLO 1801 to the basic diet of laying hens could effectively improve the laying rate, average egg weight, and yolk weight and reduce the cholesterol content in egg yolk.

Compound Probiotics Improve Body Growth Performance by Enhancing Intestinal Development of Broilers with Subclinical Necrotic Enteritis.

The aim of this study is to explore whether or not the combined application of BS15 and H2 is capable to have a more effective control effect on SNE in broilers. A total of 240 1-day-old female chickens were randomly divided into 5 groups: (a) basal diet in negative control group (NC group); (b) basal diet + SNE infection (coccidiosis vaccine + CP) (PC group); (c) basal diet + SNE infection + H2 pre-treatment (BT group); (d) basal diet + SNE infection + BS15 pre-treatment (LT group); and (e) basal diet + SNE infection + H2 pre-treatment + BS15 pre-treatment (MT group). The results showed the MT group had the most positive effect on inhibiting the negative effect of growth performance at 42 days of age. In the detection of the NC, PC, and MT group indicators at 28 days of age, we found that MT group significantly promoted ileum tissue development of broilers, and the ileum of broilers in the MT group formed a flora structure different from NC and PC, although it was found that the MT group had no effect on the butyrate level in the cecum, but it could affect the serum immune level, such as significantly reducing the level of pro-inflammatory cytokine IL-8 and increasing the content of immunoglobulin IgM and IgG. In conclusion, the composite preparation of Lactobacillus johnsonii BS15 and Bacillus licheniformis H2 could effectively improve the growth performance against SNE broilers, which is possibly caused by the improvement of the immune levels, the reduction of inflammation levels, and the promotion of the intestinal development.

Effects of Bacillus methylotrophicus SY200 Supplementation on Growth Performance, Antioxidant Status, Intestinal Morphology, and Immune Function in Broiler Chickens.

The present study was focused on evaluating the effects of Bacillus methylotrophicus SY200 in broiler production. A total of 120 healthy 7-day-old broiler chicks were randomly assigned to four dietary treatments, which included basal diet supplemented with 0%, 0.10%, 0.25%, or 0.50% (w/w) B. methylotrophicus SY200 preparation (1.0 × 109 cfu/g), regarded as negative control group (NC), low-dose group (BML), medium-dose group (BMM), and high-dose group (BMH), respectively. Each treatment was fed the corresponding experimental diet for 35 days. Results showed that dietary supplementation of B. methylotrophicus SY200 could improve broiler weight gain, especially the finisher phase. Further studies suggested that a certain amount of B. methylotrophicus SY200 enhanced the broiler antioxidant status and improved the morphological development of jejunum. Besides, dietary supplementation of B. methylotrophicus SY200 especially in 0.50% levels significantly increased the relative weight of immune organs and Newcastle disease virus antibody titer, similarly, increased mRNA expression levels of claudin-1, claudin-3, zonula occluden-1, and zonula occluden-2 were observed in the jejunum of BMM group. Moreover, B. methylotrophicus SY200 also showed beneficial effects in improving broilers microbiota homeostasis by increasing the number of beneficial bacteria. Conclusively, B. methylotrophicus SY200 could effectively improve the antioxidant status, modulate the intestinal structure, enhance the intestinal mucosal barrier function, and regulate the immune function of broilers, which finally improves the performance of the chicken in the finisher period.

Multi-functional Potential of Five Lactic Acid Bacteria Strains Derived from Giant Panda (Ailuropoda melanoleuca).

The multi-functional properties of lactic acid bacteria (LAB) on host health have been a popular research topic. The aim of present study was to assess the multi-functional potential of five LAB strains isolated from giant panda. In this study, we analyzed five giant panda LAB strains (Weissella confuse WJ202003 (W3), WJ202009 (W9), WJ202021 (W21), BSP201703 (X3); Lactiplantibacillus plantarum BSGP201683 (G83)) and found that they exhibited rapid growth as well as strong acid production capacity. The five LAB strains possessed high cell surface hydrophobicity to the four tested solvents (xylene, hexadecane, chloroform, ethyl acetate; except strain W9), auto-aggregation ability, co-aggregation ability with three pathogens (Escherichia coli, Enterotoxigenic Escherichia coli, Salmonella), adhesion ability to Caco-2 cell line, and strongly biofilm formation ability, suggesting an adhesion property. As investigated for their antioxidative potential, all the strains showed good tolerance to H2O2, high scavenging ability against 1, 1-diphenyl-2-picrylhydrazyl (DPPH), and hydroxyl (OH-), and reduction ability. Furthermore, the five LAB strains could produce multiple probiotic substances, including exopolysaccharide (EPS), gamma-aminobutyric acid (GABA), bile salt hydrolase (BSH), cellulase (only strain G83), and protease (except strain X3), which was the first to report the production of EPS, GABA, BSH, cellulase, and protease in giant panda-derived LAB strain. These results demonstrated that strains W3, W9, W21, X3, and G83 had multi-functional potential and could be utilized as potential probiotics for giant panda.

Dynamic Monitoring of Changes in Fecal Flora of Giant Pandas in Mice: Co-Occurrence Network Reconstruction.

Giant pandas are uniquely vulnerable mammals in western China. It is important to develop an animal model to explore the intestinal flora of giant pandas to understand the relationship between digestive diseases and flora. Existing animal models of intestinal flora focus on human flora-associated animals, such as mice, and there is a very limited amount of knowledge regarding giant panda flora-associated animals. To fill this gap, fecal microorganisms from giant pandas were transplanted into pseudosterile and germfree mice using single and multiple gavages. Fecal samples were collected from mice at four time points after transplantation for microbial community analysis. We determined that compared to pseudosterile mice, the characteristics of intestinal flora in pandas were better reproduced in germfree mice. There was no significant difference in microbial diversity between germfree mice and giant panda gut microbes from day 3 to day 21. Germfree mice at the phylum level possessed large amounts of Firmicutes and Proteobacteria, and at the genus level, Escherichia-Shigella, Clostridium sensu stricto 1, and Streptococcus dominated the intestinal flora structure. The microbial community co-occurrence network based on indicator species indicated that germfree mice transplanted with fecal bacteria tended to form a microbial community co-occurrence network similar to that of giant pandas, while pseudosterile mice tended to restore the microbial community co-occurrence network originally present in these mice. Our data are helpful for the study of giant panda flora-associated animals and provide new insights for the in vitro study of giant panda intestinal flora. IMPORTANCE The giant panda is a unique vulnerable mammal in western China, and its main cause of death is digestive system diseases regardless of whether these animals are in the wild or in captivity. The relationship between the intestinal flora and the host exerts a significant impact on the nutrition and health of the giant pandas. However, the protected status of the giant panda has made in vivo, repeatable, and large-sample sampling studies of their intestinal flora difficult. This greatly hinders the research depth of the giant panda intestinal flora from the source. The development and utilization of specific animal models to simulate the structure and characteristics of the intestinal flora provide another means to deal with these research limitations. However, current research examining giant panda flora-associated animals is limited. This study is the first to reveal dynamic changes in the fecal flora of giant pandas in mice after transplantation.

Surface Display of porcine circovirus type 2 antigen protein cap on the spores of bacillus subtilis 168: An effective mucosal vaccine candidate.

The oral mucosal vaccine has great potential in preventing a series of diseases caused by porcine circovirus type 2 (PCV2) infection. This study constructed a recombinant Bacillus subtilis RB with PCV2 Capsid protein (Cap) on its spore surface and cotB as a fusion partner. The immune properties of the recombinant strain were evaluated in a mouse model. IgA in intestinal contents and IgG in serum were detected by enzyme-linked immunosorbent assay (ELISA). The results demonstrated that recombinant spores could activate strong specific mucosal and humoral immune responses. In addition, spores showed good mucosal immune adjuvant function, promoting the proliferation of CD3+, CD4+ and CD8+ T cells and other immune cells. We also found that the relative expression of inflammatory cytokines such as IL-1ß, IL-6, IL-10, TNF-α and IFN in the small intestinal mucosa was significantly up-regulated under the stimulation of recombinant bacteriophage. These effects are important for the balance of Th1/Th2-like responses. In summary, our results suggest that recombinant B. subtilis RB as a feed additive provides a new strategy for the development of novel and safe PCV2 mucosal subunit vaccines.

Lactobacillus johnsonii YH1136 plays a protective role against endogenous pathogenic bacteria induced intestinal dysfunction by reconstructing gut microbiota in mice exposed at high altitude.

Background: Intestinal microbiota plays an important role in maintaining the microecological balance of the gastrointestinal tract in various animals. Disturbances in the intestinal microbiota may lead to the proliferation of potentially pathogenic bacteria that become the dominant species, leading to intestinal immune disorders, intestinal inflammation, and other intestinal diseases. Numerous studies have been confirmed that high-altitude exposure affects the normal function of the intestine and the composition of the intestinal microbiota. However, it is still necessary to reveal the changes in intestinal microbiota in high-altitude exposure environments, and clarify the relationship between the proliferation of potentially pathogenic bacteria and intestinal injury in this environment. In addition, explored probiotics that may have preventive effects against intestinal diseases. Methods and results: C57BL/6 mice were randomly divided into three groups, a high-altitude group (HA), control group (C), and high-altitude probiotic group (HAP). The HA and HAP groups were subjected to hypoxia modeling for 14 days in a low-pressure oxygen chamber with daily gavage of 0.2 mL of normal saline (HA) and Lactobacillus johnsonii YH1136 bacterial fluid (HAP), while the control group was fed normally. L. johnsonii YH1136 was isolated from feces of a healthy Tibetan girl in Baingoin county, the Nagqu region of the Tibet Autonomous Region, at an altitude of 5000 meters. Our observations revealed that gavage of YH1136 was effective in improving the damage to the intestinal barrier caused by high-altitude exposure to hypoxic environments and helped to reduce the likelihood of pathogenic bacteria infection through the intestinal barrier. It also positively regulates the intestinal microbiota to the extent of Lactobacillus being the dominant microbiome and reducing the number of pathogenic bacteria. By analyzing the expression profile of ileal microRNAs and correlation analysis with intestinal microbiota, we found that Staphylococcus and Corynebacterium1 cooperated with miR-196a-1-3p and miR-3060-3p, respectively, to play a regulatory role in the process of high-altitude hypoxia-induced intestinal injury. Conclusion: These findings revealed the beneficial effect of L. johnsonii YH1136 in preventing potential endogenous pathogenic bacteria-induced intestinal dysfunction in high-altitude environments. The mechanism may be related to the regulation of intestinal injury from the perspective of the gut microbiota as well as miRNAs.

Ameliorating Effects of TRIM67 against Intestinal Inflammation and Barrier Dysfunction Induced by High Fat Diet in Obese Mice.

Tripartite Motif 67 (TRIM67) is an important member of TRIM family proteins, which participates in different cellular processes including immune response, proliferation, differentiation, carcinogenesis, and apoptosis. In recent years, a high fat diet (HFD) has remained one of the main causes of different metabolic diseases and increases in intestinal permeability as well as inducing intestinal inflammation. The current study investigated the protective effects of TRIM67 in the ileum and colon of obese mice. 4-week-old wild-type (WT) C57BL/6N mice and TRIM67 knockout (KO) C57BL/6N mice were selected and randomly divided into four sub-groups, which were fed with control diet (CTR) or HFD for 14 weeks. Samples were collected at the age of 18 weeks for analysis. To construct an in vitro obesity model, over-expressed IPEC-J2 cells (porcine intestinal cells) with Myc-TRIM67 were stimulated with palmitic acid (PA), and its effects on the expression level of TRM67, inflammatory cytokines, and barrier function were evaluated. The KO mice showed pathological lesions in the ileum and colon and this effect was more obvious in KO mice fed with HFD. In addition, KO mice fed with a HFD or CTR diet had increased intestinal inflammation, intestinal permeability, and oxidative stress compared to that WT mice fed with these diets, respectively. Moreover, IPEC-J2 cells were transfected with TRIM67 plasmid to perform the same experiments after stimulation with PA, and the results were found consistent with the in vivo evaluations. Taken together, our study proved for the first time that HFD and TRIM67 KO mice have synergistic damaging effects on the intestine, while TRIM67 plays an important protective role in HFD-induced intestinal damage.

Psychoactive Effects of Lactobacillus johnsonii BS15 on Preventing Memory Dysfunction Induced by Acute Ethanol Exposure Through Modulating Intestinal Microenvironment and Improving Alcohol Metabolic Level.

The negative effects of ethanol (EtOH) abuse on the body have been widely reported in recent years. Building on the microbiota-gut-brain axis hypothesis, our study aimed to demonstrate the potential psychobiotic role of Lactobacillus johnsonii BS15 in the preventive effects of acute EtOH intake on memory impairment. We also determined whether L. johnsonii BS15 intake could effectively improve resistance to acute drinking and alleviate the adverse effects of EtOH. Male mice were fed L. johnsonii BS15 orally with (Probiotic group) or without (Control and Alcohol groups) daily dose of 0.2 × 109 CFU/ml per mouse for 28 days. Gavage with L. johnsonii BS15 significantly modified the ileal microbial ecosystem (assessed by 16S rRNA gene sequencing) in favor of Firmicutes and Lactobacillus, indicating the ability of BS15 to restore the gut microbiota. The acute EtOH exposure model (7 g/kg EtOH per mice) was established by gavage, which was administered to the alcohol and probiotic groups on day 28 of the experiment. The L. johnsonii BS15 intake effectively reduced alcohol unconsciousness time, blood alcohol concentration, and serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels. Meanwhile, the improvement of ethanol resistance time and the activities of alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) in the liver were shown by BS15 in acute alcohol-induced mice. We found that acute EtOH exposure reduced the exploration ratio (assessed by the novel object recognition test), escape latency, number of errors (assessed by passive avoidance test), and spontaneous exploration (assessed by T-maze test) in mice, which were obviously improved by L. johnsonii BS15. In the hippocampus, L. johnsonii BS15 significantly reversed the decrease in antioxidant capacity of superoxide dismutase (SOD), malondialdehyde (MDA), and glutathione (GSH) and mRNA expression of memory-related functional proteins of brain-derived neurotrophic factor (BDNF) and cyclic ampresponse element binding protein (CREB) in the hippocampal tissue after acute EtOH exposure. In conclusion, L. johnsonii BS15 intake appears as a promising psychoactive therapy to ameliorate alcohol-mediated memory impairment by increasing EtOH metabolic levels.

Dietary Probiotic Supplementation Suppresses Subclinical Necrotic Enteritis in Broiler Chickens in a Microbiota-Dependent Manner.

Background: Chicken meat is one of the most consumed meats worldwide and poultry production is increasing at an exponential rate. Reducing antibiotic usage has resulted in the recurrence of subclinical necrotic enteritis again and influenced global poultry production. Probiotics are potential antibiotic substitutes that can be used to prevent subclinical necrotic enteriti. However, the precise mechanism of action of probiotics and information on which gut microbes confer this efficacy remain elusive. Methods and results: The subclinical necrotic enteritis animal model was used to reveal the mechanism underlying the effect of probiotics on intestinal health through RNA sequencing and 16S rDNA amplicon sequencing. Bacillus licheniformis H2 feeding significantly reduced the relative abundance of Clostridium perfringens in the ileum and markedly ameliorated the pathological damage in the ileum and liver. In addition, oral administration of B. licheniformis H2 contributed to the enhancement of the intestinal barrier function and epithelial renewal, reducing energy consumption, and improving enteral nutrition absorption. Probiotic B. licheniformis H2 also ameliorated the inflammatory response and increased the immunity of subclinical necrotic enteritis infected broilers. Finally, B. licheniformis H2 feeding regulated liver gene expression to suppress immune response and promoted growth and metabolism depending on the gut microbiota. Conclusions: These results indicated the mechanism of probiotic action of B. licheniformis H2 in maintaining intestinal health and thus promoting growth and B. licheniformis H2 may serve as an antibiotic substitute to prevent subclinical necrotic enteritis in poultry farming.

Assessment the role of some Bacillus strains in improvement rex rabbits resistance against ETEC challenge.

Increasing reports have indicated that specific strains of probiotic Bacillus have the potential to prevent diseases. The purpose of this study was to explore the effects of three Bacillus strains (Bacillus subtilis BSWJ2017001, Bacillus pumilus BSWJ2017002, and B. subtilis BSWJ2017003) mixture dietary supplementation on rex rabbits infected with enterotoxigenic Escherichia coli (ETEC). In this study, 60 35-day-old weaning rex rabbits were separated into two groups randomly: control group (fed basal diet with no antibiotics) and Bacillus strains group (fed basal diet containing 1.0 × 106 CFU/g Bacillus strains mixture). After 8 weeks of feeding, the rex rabbits were inoculated orally with 5.0 mL of ETEC (1.0 × 109 CFU/mL) and assessed at 0, 12, and 24 h. The Bacillus strains mixture attenuated the oxidative damage, diarrhea severity, and intestinal damage of ETEC infected rabbits. It also significantly increased the population of Lactobacillus spp., and Bifidobacterium spp., and decreased the population of Enterococcus spp.. Moreover, Bacillus strains group exhibited higher levels of toll-like receptor (TLR) 2, anti-inflammatory cytokines, secretory immunoglobulin A, and intestinal barrier-related genes than control group, as well as lower levels of TLR-4 and pro-inflammatory cytokines. These results demonstrated that Bacillus strains mixture could attenuate injury caused by ETEC and enhance disease resistance by improving specific intestinal microbiota members and immunity in weaning rex rabbits.

The Panda-Derived Lactobacillus plantarum G201683 Alleviates the Inflammatory Response in DSS-Induced Panda Microbiota-Associated Mice.

Intestinal diseases are one of the main causes of captive giant panda death. Their special dietary habits and gastrointestinal tract structure often lead to intestinal epithelium damage and secondary intestinal infection. The captive giant panda is predisposed to suffer from microbiota dysbiosis due to long-term artificial feeding and antibiotic misuse. However, there are few reported probiotics to treat giant panda enteritis and the associated dysbiosis. This study aims to elucidate the mechanism by which Lactobacillus plantarum G201683 (L. plantarum G83), a promising panda-derived probiotic, exerts a protective effect on intestinal inflammation in the dextran sulfate sodium- (DSS) induced panda microbiota-associated (DPMA) mouse model. The DPMA mouse was generated by antibiotic treatment and 5% DSS drinking water administration to assess the effect of L. plantarum G83 on intestinal inflammation and microbiota in vivo. Our results demonstrated the successful generation of a DPMA mouse model with Enterobacteriaceae enrichment, consistent with the giant panda intestinal microbiota. L. plantarum G83 decreased clinical and histological severity of intestinal inflammation, enhanced intestinal tight junction protein expression (ZO-1, Occludin) and alleviated inflammatory cytokine production (TNF-) in the colon of DPMA mice. The administration of L. plantarum G83 altered the microbiota composition by decreasing pathogen associated taxa such as E. coli and increasing abundance of beneficial bacteria including Bifidobacterium spp. These changes in microbiota composition were associated with an increased concentration of short chain fatty acids (SCFA), reduced NF-κB signaling, and an altered balance of T helper cell subsets. Our findings support L. plantarum G83 as a promising probiotic to treat intestinal inflammation in the giant panda.