Secondary metabolites of Bacillus subtilis L2 show antiviral activity against pseudorabies virus.

Bacillus subtilis (B. subtilis) is a commercially important probiotic known to produce secondary metabolites with antibacterial, antifungal and anti-inflammatory activities. However, the potential ability of B. subtilis to combat viruses, especially DNA viruses, has not been extensively investigated. In this study, we identified two distinct B. subtilis strains and examined the efficiency of their secondary metabolites against pseudorabies virus (PRV), a swine herpesvirus resulting in economic losses worldwide. We found that treatment with the secondary metabolites of B. subtilis L2, but not the metabolites of B. subtilis V11, significantly inhibited PRV replication in multiple cells. Notably, the antiviral activity of the metabolites of B. subtilis L2 was thermal stable, resistant to protease digestion. Moreover, these metabolites effectively impeded PRV binding, entry and replication. Importantly, oral administration of the metabolites of B. subtilis L2 protected mice from lethal PRV infection, rescuing weight loss and reducing the viral load in vivo. In summary, our results reveal that the metabolites of B. subtilis L2 exhibit anti-PRV activity both in vitro and in vivo, providing a potential candidate for novel antiviral drugs.

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.

Nematicidal Volatiles from Bacillus atrophaeus GBSC56 Promote Growth and Stimulate Induced Systemic Resistance in Tomato against Meloidogyne incognita.

Bacillus volatiles to control plant nematodes is a topic of great interest among researchers due to its safe and environmentally friendly nature. Bacillus strain GBSC56 isolated from the Tibet region of China showed high nematicidal activity against M. incognita, with 90% mortality as compared with control in a partition plate experiment. Pure volatiles produced by GBSC56 were identified through gas chromatography and mass spectrometry (GC-MS). Among 10 volatile organic compounds (VOCs), 3 volatiles, i.e., dimethyl disulfide (DMDS), methyl isovalerate (MIV), and 2-undecanone (2-UD) showed strong nematicidal activity with a mortality rate of 87%, 83%, and 80%, respectively, against M. incognita. The VOCs induced severe oxidative stress in nematodes, which caused rapid death. Moreover, in the presence of volatiles, the activity of antioxidant enzymes, i.e., SOD, CAT, POD, and APX, was observed to be enhanced in M. incognita-infested roots, which might reduce the adverse effect of oxidative stress-induced after infection. Moreover, genes responsible for plant growth promotion SlCKX1, SlIAA1, and Exp18 showed an upsurge in expression, while AC01 was downregulated in infested plants. Furthermore, the defense-related genes (PR1, PR5, and SlLOX1) in infested tomato plants were upregulated after treatment with MIV and 2-UD. These findings suggest that GBSC56 possesses excellent biocontrol potential against M. incognita. Furthermore, the study provides new insight into the mechanism by which GBSC56 nematicidal volatiles regulate antioxidant enzymes, the key genes involved in plant growth promotion, and the defense mechanism M. incognita-infested tomato plants use to efficiently manage root-knot disease.

[High expression and immunogenicity analysis of HA globular head domain of H5 subtype avian influenza virus produced in Pichia pastoris].

To evaluate the immunogenicity of HA globular head domain of H5 subtype influenza virus (H5HA), the gene of H5HA was optimized and the recombinant pPICZaA-H5HA expressing vector was constructed and transfected into Pichia pastoris. The expression of the recombinant H5HA was confirmed by SDS-PAGE and Western blotting and the results demonstrated that the recombinant H5HA (37 kDa) was highly expressed in Pichia pastoris with concentration of 0.2 mg/mL in medium. The recombinant H5HA was concentrated and purified using Ni-NTA affinity chromatography. The immunogenicity of H5HA was evaluated by immunizing eight groups of chicken through intranasal or intramuscular injection with different doses of purified H5HA combined with different adjuvants, respectively. The results showed that the recombinant H5HA could induce high level IgG (HI titer was 1:64 and neutralizing antibody titer was 1:218) and the optimal dosage of the recombinant H5HA was 50 µg combined with oil. In addition, intramuscular injection was better than nasal immunization. This study provided a theoretical support for subunit vaccine development.

Preparation of anti-gatifloxacin antibody and development of an indirect competitive enzyme-linked immunosorbent assay for the detection of gatifloxacin residue in milk.

A polyclonal anti-gatifloxacin antibody has been prepared, and an indirect competitive enzyme-linked immunosorbent assay (cELISA) was developed on the basis of the antibody prepared for the first time. The antibody shows high sensitivity with an IC50 value of 2.6 ppb and excellent specificity with only a minor cross-reaction with lomefloxacin (3.0%) among common (fluoro)quinolones evaluated in this study. The high specificity of the antibody was explained by the molecular structures of related drugs by comparison with published research. The cELISA test kit developed has a detection limit of 0.05 ppb and could be used as a screening method to detect and regulate illegal use of gatifloxacin in food and food products. The test kit was applied to the detection of milk samples spiked by gatifloxacin. The recovery rates were in the range of 86-106%, whereas the intra- and interassay coefficients of variation were <14.3 and <19.6%, respectively.