[Objective] To investigate the effects of Weizmannia coagulans BC-G44 on the intestinal microbiota structure, barrier function, and inflammatory response in the colon of the rat model of antibiotic-associated diarrhea (AAD). [Methods] A total of 30 five-week-old Sprague-Dawley (SD) rats with similar body weights were randomized into five groups (n=6): control (Con), model (Mod), low-dose W. coagulans BC-G44 (LBC-G44), medium-dose W. coagulans BC-G44 (MBC-G44), and high-dose W. coagulans BC-G44 (HBC-G44). The experiment encompassed a modeling period (7 days) and a recovery period (12 days). During the modeling period, rats in the Con group were administrated with normal saline at 2 mL/d by gavage, while those in the Mod, LBC-G44, MBC-G44, and HBC-G44 groups were administrated with a mixture containing clindamycin, ampicillin, and streptomycin (2 mL/d) by gavage to induce AAD. During the recovery period, the Con and Mod groups continued to receive normal saline, while the LBC-G44, MBC-G44, and HBC-G44 groups received W. coagulans BC-G44 suspensions at 10⁷, 10⁸, and 10⁹ CFU/d, respectively. On day 19, colonic tissue samples were collected for histological examination, and the concentrations of cytokines, and the expression levels of barrier proteins and inflammation-related genes in the colonic mucosa were measured. Furthermore, the microbiota composition and metabolites in the colonic chyme were analyzed. [Results] On day 7 of modeling, rats in the Mod, LBC-G44, MBC-G44, and HBC-G44 groups exhibited diarrhea, weight losses, and reduced food intake compared with those in the Con group (P<0.05). On day 19, compared with the Mod group, the MBC-G44 and HBC-G44 groups showed increases in the colonic mucosa thickness and goblet cell number (P<0.05). The HBC-G44 group showed increased relative abundance of Bacteroides and concentrations of lactate in the colonic chyme (P<0.05), elevated levels of d-lactic acid (d-LA) and diamine oxidase (DAO) in the colonic mucosa, and up-regulated relative mRNA levels of Claudin-1, Occludin, and MUC2 in the colonic mucosa (P<0.05). Meanwhile, compared with the Mod group, the MBC-G44 and HBC-G44 groups showed down-regulated relative mRNA levels of Toll-like receptor 4 gene (TLR4) and nuclear factor-kappa B gene (NF-κB) (P<0.05), and reduced concentrations of tumor necrosis factor (TNF)-α and interleukin (IL)-1β (P<0.05) in the colonic mucosa. [Conclusion] W. coagulans BC-G44 can ameliorate antibiotic-induced colonic injury, regulate the colonic microbiota composition and metabolites, enhance intestinal barrier function, and reduce intestinal inflammatory responses in rats, thus alleviating the symptoms of AAD.

[Objective] To develop a DNA vaccine by loading avian pathogenic Escherichia coli ghosts with the fimbrium gene flfA of Gallibacterium anatis and evaluate the immune effect of this vaccine in chickens. [Methods] Taking flfA of G. anatis as the target gene, we constructed the eukaryotic expression plasmid pCAGGS-flfA from the plasmid pCAGGS-HA carrying the chicken β-actin promoter, which enhanced the efficacy of vaccine. The temperature-sensitive plasmid PBV-E-SN was then constructed and transformed into the ampicillin-sensitive avian pathogenic E. coli isolate to prepare a bacterial ghost. Finally, pCAGGS-flfA was loaded into the bacterial ghost to prepare a bacterial ghost vaccine. Seven-day-old chickens were assigned into five groups: bacterial ghost loading pCAGGS-flfA, pCAGGS-flfA, empty bacterial ghost, PBS, and normal control. The primary vaccination was carried out for 7-day-old chickens, and the booster immunization was performed two weeks after the primary immunization. The chickens were challenged with G. anatis four weeks after the primary immunization, and the immune effect of the vaccine was evaluated. [Results] The eukaryotic expression plasmid pCAGGS-flfA was successfully expressed in cells in vitro. The lysis rate of the constructed avian pathogenic E. coli ghosts heated at 42 °C for 210 min reached 99.94%. The specific IgG antibody titer measured by ELISA, the number of shedding bacteria in chicken cloacal swabs and throat swabs, and the bacterial loads in tissue and organs of challenged chickens showed that the immune effects in the bacterial ghost loading pCAGGS-flfA group and the pCAGGS-flfA group were much higher than those in other groups. Moreover, the bacterial ghost loading pCAGGS-flfA showed stronger immune effect than pCAGGS-flfA. [Conclusion] Bacterial ghosts as the carriers of pCAGGS-flfA significantly enhanced the immune effect of the DNA vaccine.

[Objective] To study the probiotic properties and safety of Limosilactobacillus reuteri CHF7-2 isolated from Chahua chicken, providing a theoretical basis for the development of this strain as a microecological feed additive. [Methods] In vitro assays were conducted to evaluate the adhension, enzyme production, antibacterial activity, and antioxidant activity of strain CHF7-2. PacBio Sequel II and Illumina NovaSeq 6000 were used for whole genome sequencing of strain CHF7-2. Bioinformatics tools and databases were then used for genome annotation to explore the probiotic mechanisms and safety of this strain at the molecular level. [Results] Strain CHF7-2 possessed significant probiotic properties and safety. It exhibited high surface hydrophobicity, self-coagulation, and antioxidant activity. The strain inhibited the growth of Escherichia coli K88, Staphylococcus aureus ATCC 49521, Salmonella gallinarum CICC 21510, and Salmonella choleraesuis CVCC 3383. Strain CHF7-2 produced protease and lipase, and it was safe for use in feed since it did not exhibit hemolytic activity. Whole genome sequencing revealed that the genome size of CHF7-2 was 2 116 761 bp, with the G+C content of 38.8%, encoding 2 067 genes. Additionally, the genome carried the biosynthetic gene cluster of the class III bacteriocin EnlA and multiple genes involved in the acid tolerance, bile salt tolerance, heat stress tolerance, cold stress tolerance, adhesion, antioxidation, and organic acid synthesis, with no virulence or antibiotic resistance genes detected. [Conclusion] L. reuteri CHF7-2 is a potential probiotic strain and a promising candidate for use as a microecological feed additive.

[Objective] To evaluate the colonization ability and biocontrol effects of Streptomyces sp. ZH-356 with antagonistic effects on plant pathogenic fungi and reveal the biocontrol mechanism of Streptomyces sp. ZH-356 by omics analysis. [Methods] The colonization of Streptomyces sp. ZH-356 in plants was detected by the GFP fluorescent labeling method. The biocontrol effects and potential of Streptomyces sp. ZH-356 on plant fungal diseases were evaluated based on the biocontrol effects of the inoculant in different dosage forms (seed coating agent, wettable powder, gum inoculant, and bone glue inoculant). The whole genome information of Streptomyces sp. ZH-356 was analyzed by third-generation sequencing, and its gene functions were annotated. The comparative transcriptome analysis was performed to screen the differentially expressed genes during the antagonizing process of Streptomyces sp. ZH-356 against plant pathogenic fungi, and thus the genes involved in the synthesis of antagonistic substances were predicted. [Results] Streptomyces sp. ZH-356 stably colonized the roots and stems of tomato and wheat plants. Different dosage forms of inoculants prepared based on Streptomyces sp. ZH-356 demonstrated strong control effects on tomato early blight and apple valsa canker. Among them, the seed coating agent prepared with Streptomyces sp. ZH-356 did not affect the germination rate of tomato seeds after treatment while protecting tomato seedlings from the infection of Alternaria solani. The wettable powder prepared with Streptomyces sp. ZH-356 showed both prevention and treatment effects on tomato early blight, with the prevention effect stronger than the treatment effect. The liquid inoculants prepared from Streptomyces sp. ZH-356 had control effects on apple Valsa canker, regardless of whether the diseased bark was scraped or not, while the control effect was better when the diseased bark was scraped and better than that of thiophanate-methyl. The whole genome sequencing results showed that Streptomyces sp. ZH-356 contained only one linear chromosome with a size of 9 435 898 bp and the average G+C content of 70.82%. A total of 8 432 coding genes, 69 tRNA genes, and 18 rRNA genes were predicted. Species annotation results showed that Streptomyces sp. ZH-356 did not belong to any Streptomyces species whose genome has been sequenced. Genome-wide analysis showed that there were 32 biosynthetic gene clusters (BGCs) for secondary metabolites in ZH-356. Transcriptomic analysis showed that the expression of the NRPS/T1PKS gene cluster ZH_356_GM000343-ZH_356_GM000422 was significantly up-regulated in the process of antagonizing plant pathogenic fungi, suggesting that it may be a BGC mediating the biosynthesis of active substances against plant pathogenic fungi in Streptomyces sp. ZH-356. Moreover, ZH_356_GM000409 may be the core biosynthetic gene of the active substances. [Conclusion] Streptomyces sp. ZH-356 can colonize plants, and the biocontrol agents prepared based on this strain demonstrate good control effects on plant fungal diseases. The active substances for the antagonistic effects may be synthesized by the gene cluster ZH_356_GM000343-ZH_356_GM000422. The above work lays a foundation for the industrial application of strain ZH-356 and the research on the mechanism of antagonizing plant pathogenic fungi.

[Objective] Biofilm formation and adhesion are important indicators for evaluating the beneficial effects of probiotics. However, the relationship of specific genes with the biofilm formation and adhesion of Lactiplantibacillus remains unclear. The rbk gene encodes ribokinase, which is involved in ribose metabolism and may be related to biofilm formation and adhesion. This study aims to analyze the effects of rbk overexpression on the biofilm formation and adhesion of Lactiplantibacillus paraplantrum LR-1, explore the role of this gene in the regulation of quorum sensing (QS) and expression of related genes, and reveal the influencing mechanism of rbk overexpression in bacteria from a metabolic profile perspective. [Methods] L. paraplantarum LR-1 was selected as the target strain, and the shuttle vector pMG76e was used to construct the recombinant strain rbk-pMG76e-LR-1. The overexpression of rbk was confirmed by qRT-PCR and the enzyme activity assay. Crystal violet staining, cell adhesion assay, and qRT-PCR were employed to evaluate the effects of rbk overexpression on biofilm formation, adhesion, and expression of tuf, luxS, and rpoN. Furthermore, untargeted metabolomics analysis was conducted to assess the effect of rbk overexpression on the metabolic profile. Finally, the effect on the biofilm formation and adhesion of LR-1 was verified by exogenous addition of metabolites. [Results] The overexpression of rbk increased the biofilm formation of LR-1 and the adhesion to HT-29 cells by 1.55-2.34 folds and 3.58 folds, respectively. Moreover, the overexpression of rbk up-regulated the expression levels of tuf, luxS, and rpoN by 70.30, 96.94, and 45.61 folds, respectively. The untargeted metabolomics analysis revealed that rbk overexpression led to changes in the abundance of 145 metabolites. Finally, the exogenous addition of l-proline, rhamnose, and nicotinamide adenine dinucleotide (NADH) increased the biofilm formation of LR-1 by 1.27, 1.39, and 1.25 folds and the adhesion by 1.40, 1.41, and 1.52 folds, respectively. [Conclusion] This study demonstrates that rbk can serve as a key target for enhancing the biofilm formation and adhesion of Lactiplantibacillus.

Zearalenone (ZEN) is a secondary metabolite produced by the filamentous fungi of Fusarium, causing serious harm to human and animal health. [Objective] To identify a ZEN-degrading strain and investigate its growth and degradation characteristics under different conditions. [Methods] A bacterial strain that can efficiently degrade ZEN was screened out with ZEN as the only carbon source from soil samples of a field with continuous maize cropping in Anhui Province, China. The strain was identified by morphological observation, biochemical tests, and phylogenetic analysis based on 16S rRNA gene sequences. The effects of temperature, pH, and incubation time on the growth rate and ZEN degradation efficiency of the strain were studied. Furthermore, the efficiency of different active components of the strain on ZEN degradation was measured, and the locations of the active components were determined. [Results] A total of 21 ZEN-degrading strains were isolated from the soil samples, among which strain DC-R2 showed the strongest degradation effect and it was identified as Gordonia sp. BHI medium was the optimal medium for the growth of this strain. The optimal culture conditions of the strain for ZEN degradation were 37 ℃ and pH 8.0, under which 100% ZEN (5 μg/mL) was degraded within 6 h. Intracellular enzymes were the main contributors in DC-R2 to ZEN degradation. [Conclusion] We isolated a strain Gordonia sp. DC-R2 capable of efficiently degrading ZEN. The intracellular enzyme present in this strain is the key to the degradation of ZEN. This provides a foundation for the purification of the key degrading enzyme in subsequent work and the potential application.

[Objective] Mumps virus (MuV) is the causative agent of mumps. Nowadays, genotype F is widely prevalent in China, while genotype G appears in localized areas and is exhibiting a trend of gradual expansion. To understand the genetic characteristics of genotype G strains in China, we selected two genotype G MuV strains that were isolated from Dalian, Liaoning for analysis. [Methods] The whole genomes of the two strains were sequenced, and the genotypes were determined according to the WHO reference strains. Furthermore, we compared the molecular characteristics among different genotypes and within genotype G. By comparison with the sequences of genotype G strains in other areas of China, we analyzed the features of genotype G MuVs in China, as well as the genetic distance and variations of key antigenic sites between the wild-type genotype G strains and the existing vaccine strains. [Results] The strains isolated in this study both belonged to genotype G, and they showed the nucleotide differences ranging from 4.2% to 6.9% from other 12 genotypes, with the greatest divergence from genotype A. Among the protein coding genes, the SH coding gene exhibited the largest variation, while the NP, M, and L coding genes were conserved. The P, F and HN coding genes demonstrated significant differences among different genotypes. The genotype G strains isolated in this study were closely related to the strain isolated in Jiangsu Province (Jiangsu.CHN/22.13/2), while they were distinct from the strains previously isolated in Liaoning Province. The genotype G strains isolated in this study lacked a N-glycosylation site (aa 12-14) but gained a N-glycosylation site (aa 464-466) in the key epitope of HN protein. In addition, the genotype G strains showed considerable differences in terms of neutralizing epitopes from the genotype A vaccine strain. These differences suggested that the mutations of these sites may potentially reduce the cross-protection effects of vaccine strains against wild-type MuV strains. Although there were some mutations in F protein, the functional region was conserved. [Conclusion] This study details the genotypic characteristics of genotype G MuVs in Dalian, highlighting their high similarities to the genotype G strains in China and the WHO reference strains, while underscoring significant differences from the genotype A vaccine strain used worldwide. These findings suggest the necessity of continuous surveillance of MuV strains in China and further studies of their epidemiology and virology, which could provide references for tracing MuVs, cutting the transmission route, and developing immunization strategies in China.

[Objective] To explore the effects of freeze-thaw on oat silage during the aerobic exposure stage. [Methods] Oat silage was stored at a constant temperature (20 ℃, group 20, control) and freeze-thaw conditions (20 ℃ and -5 ℃ alternating every 12 h, group S) for 60 days and then subjected to aerobic exposure. Samples were collected on day 60 of ensiling and after aerobic exposure for 1, 3, and 5 days, respectively, for the determination of fermentation quality and nutritional quality as well as for 16S rRNA gene and ITS sequencing. [Results] The pH rose as the aerobic exposure was prolonged. The samples subjected to aerobic exposure at room temperature showed more rapid increases in ammonia nitrogen content and pH and more rapid decreases in lactic acid and acetic acid content than those subjected to aerobic exposure at freeze-thaw temperatures. Propionic acid and butyric acid were produced more rapidly in the S group, especially in the case of aerobic exposure at room temperature. Enterobacteria and yeast increased while lactic acid bacteria decreased as the aerobic exposure was prolonged (P<0.05). Shannon and Simpson indices of bacteria increased during aerobic exposure, and the relative abundance of lactic acid bacteria kept decreasing. The bacterial community structure presented a significant difference on days 3 of aerobic exposure, and microbial succession was accelerated by aerobic exposure at room temperature compared with that at the freeze-thaw temperature, with more species and higher counts of yeast and molds indicative of spoilage in the S group. [Conclusion] Freeze-thaw accelerated aerobic deterioration, especially aerobic exposure at room temperature. This study provides theoretical guidance for high-quality modulation and storage of oat silage in alpine areas.

[Objective] To establish a dual detection method for contaminations by six foodborne pathogens (Cronobacter, Escherichia coli O157:H7, Bacillus cereus, Staphylococcus aureus, Salmonella, and Listeria monocytogenes) in formula milk powder in a rapid manner. [Methods] Enzymatic recombinase amplification (ERA) is a novel isothermal amplification technology that exponentially amplifies trace amounts of DNA or RNA in 10-30 min at 25-42 ℃. The primers and probe of ERA for the detection of Cronobacter were designed. Meanwhile, the ERA primers and probes suitable for the detection of E. coli O157:H7, B. cereus, S. aureus, Salmonella, and L. monocytogenes were screened. Further, through pairwise combination and cross-reactivity analysis, as well as method optimization, the dual ERA detection system was established. The limit of detection and accuracy of the method were determined by application of this method in the detection of simulated contaminations and actual samples. [Results] Three groups of dual ERA systems were established, achieving the detection of six pathogens in 16 min 10 s. The established method showed the sensitivity of 1 ng/μL in the DNA detection of the combinations of Cronobacter with E. coli O157:H7, B. cereus, and S. aureus, while it showed the sensitivity of 10^-1 ng/μL in the DNA detection of Salmonella and L. monocytogenes. The results of the simulation contaminations showed that the limit of detection of the method was 1 CFU/mL. The dual ERA method established in this study was then adopted to detect 37 commercially available formula milk powder samples near the expiration date. The detection rates of B. cereus and L. monocytogenes were 37.84% and 21.62%, respectively. The results were consistent with those of the real-time PCR (industry standard method), confirming the accuracy of the dual ERA method established in this study. [Conclusion] The dual ERA method established in this study exhibits high specificity and high sensitivity. Moreover, it takes merely approximately 25 min from DNA extraction to obtaining the detection results, and it is capable of simultaneously detecting six pathogens, demonstrating high efficiency. This method is of importance for the rapid screening of foodborne pathogens.

Lactic acid bacteria (LAB) have extensive applications in food and medicine fields. They are used as starters and functional probiotics in food fermentation. The activity and performance of LAB are influenced by various environmental stresses involving osmotic pressure, temperature, oxygen, acidity, and bile salts. Edible LAB can provide numerous health benefits. However, their viable counts decrease during production, storage, and digestion. This paper systematically discusses the different stressful environments faced by LAB during production, storage, and digestion, as well as their stress responses. Furthermore, this paper summarizes existing high activity protection strategies and mechanisms from two aspects: isolating from stressful environments and enhancing strain resistance. This review aims to provide theoretical support for LAB strain engineering and product development.