Objective The outbreaks of infectious bovine rhinotracheitis (IBR) have been reported in multiple regions across China. To systematically characterize the molecular features and biological properties of the predominant strains of infectious bovine rhinotracheitis virus (IBRV) and provide etiological insights for evidence-based prevention and control against IBR. Methods We collected the bovine lung tissue for detection of common bovine respiratory viruses by PCR. Viral isolation was performed with MDBK cells, and then metagenomic sequencing was conducted to determine and analyze complete genome sequences of the viruses. Viruses were purified via the plaque assay and subcultured to the 9th generation (F9) for determination of the 50% tissue culture infectious dose (TCID₅₀), monitoring of one-step growth kinetics, and observation of viral particle morphology via electron microscopy. Two IBRV-seronegative healthy adults of cattle were intranasally inoculated with the F9 suspension (2.5 mL/nostril), while one additional head of cattle was housed in close contact. The clinical manifestations were monitored, including body temperature fluctuations and viral shedding dynamics. Results PCR detection revealed the presence of both IBRV and bovine coronavirus (BCoV) in the bovine lung tissue. After inoculation into MDBK cells, obvious cytopathic effects were observed at the third passage. Metagenomic sequencing confirmed the virus as IBRV, with a whole genome length of 134 678 bp. This isolate was designated as IBRV GSLT/04/2024. The TCID₅₀ of F9 was 10^5.5 TCID₅₀/mL, and no mutation was detected in the gB, gC, gD, or gE gene. Based on the gC gene and whole genome sequences, this strain was classified into the IBRV 1.2b subtype lineage. Viral shedding began on day 5 post-inoculation in the inoculated cattle and on day 10 in the contact cattle, lasting for approximately 10 days. The amount of viral shedding followed the order of nasal swabs>oral swabs>rectal swabs>ocular swabs. On day 30 post-inoculation, IBRV genes were only detected in the colon tissue. IBR-specific antibodies were detected on approximately day 7 in the inoculated cattle and on day 10 in the contact cattle. Conclusion We successfully isolated a novel IBRV subtype 1.2b strain with high infectivity in adult cattle. The findings provide epidemiological and etiological evidence to trace the recent surge in IBRV prevalence across China.

Objective To analyze the activity of ribose-5-phosphate isomerase B (RpiB) encoded by lmo0736 and explore its effect on the infection of Listeria monocytogenes (LM). Methods The recombinant protein Lmo0736 was obtained by prokaryotic expression and purification, and its catalytic activity for substrates was verified by the enzyme activity assay. The LM strain with lmo0736 knockout (LM Δlmo0736) and the complementary strain (LM CΔlmo0736) were constructed by bacterial homologous recombination. The growth curves of bacteria in vitro were plotted. The adhesion, invasion, and intercellular migration of bacteria were evaluated by in vitro cell infection models (Caco-2 intestinal epithelial cells and L929 fibroblasts). The ICR mouse infection model was used to measure the 7 d survival rate and 48 h organ load of each strain, and thus the pathogenicity of strains in mice was evaluated. Results Lmo0736 had typical RpiB activity and catalyzed the conversion of d-ribose-5-phosphate to d-ribulose-5-phosphate, with V_max=0.366 mmol/(L·min), K_m=4.489 mmol/L, k_cat=12.300 s^-1, and k_cat/K_m=2.740 L/(mmol·s). The growth rate of LM Δlmo0736 was not significantly different from that of the wild type EGD-e and LM CΔlmo0736in vitro, indicating that the deletion of lmo0736 did not affect the basic growth of bacteria. LM Δlmo0736 demonstrated significantly decreased adhesion and invasion in Caco-2 cells and intercellular migration in L929 cells and weakened colonization in mice, which indicated that lmo0736 regulated the pathogenicity of LM through a RpiB-dependent metabolic pathway. Conclusion This study reveals for the first time that the Lmo0736 of LM has typical RpiB activity. Although the functional loss of Lmo0736 does not directly affect the basic growth of the bacteria, it significantly attenuates the pathogenicity by weakening the adhesion, invasion, and intracellular migration in host cells and the colonization in vivo. The results accumulate experimental data for in-depth exploration of the biological functions of RpiB in LM. From the perspective of the association between metabolism and virulence, this study provides an experimental basis for delving into the infection mechanism of foodborne pathogens.

Objective To study the endophytic microbiota in different ecological niches of pepper (Capsicum annuum L.) seedlings at various stages following Ralstonia solanacearum infection, aiming to explore the endophyte migration dynamics triggered by R. solanacearum infection. Methods The copy number of R. solanacearum was quantified by quantitative real-time PCR (qPCR). Samples from roots, stems, and leaves of both infected and healthy pepper plants were collected at 1, 4, and 7 days post-inoculation (dpi). High-throughput amplicon sequencing was employed to analyze the endophytic bacterial and fungal communities. Dual-dimensional analyses integrating microbial copy number and community structures were conducted to elucidate the spatiotemporal dynamics of endophytic microbiota and pathogen proliferation characteristics under R. solanacearum invasion. Results The copy number of R. solanacearum continuously increased in roots and stems post-inoculation, peaking in leaves at 4 dpi and then significantly declining. The infection induced pronounced alterations in endophytic bacterial communities across all tissues, with temporal effects outweighing tissue-specific variations. Roots exhibited heightened sensitivity to pathogen invasion. At 7 dpi, the relative abundance of Bacillota in endophytic bacteria in the plants increased significantly. At the genus level, the relative abundance of Rhizobium, Pseudomonas, and endogenous fungi Fusarium and Aspergillus increased significantly compared with that in the control group. The beta diversity indices and structures of endophytic microbiota in all tissues underwent marked changes during infection. Pseudomonas emerged as a signature bacterial genus in roots, while Aspergillus dominated stems and leaves as a fungal indicator. The co-occurrence network analysis revealed greater complexity of the endophytic microbiota in infected plants, with an elevated proportion of negative correlation edges. Ascomycota served as pivotal network hubs, reflecting enhanced antagonistic interactions and inter-microbial associations under pathogen stress. Ten potential antagonistic microbial taxa were identified, including six bacterial taxa of Clostridia (Bacillota). Conclusion This study delineates the proliferation pattern of R. solanacearum in pepper seedlings and characterizes the structural and migration dynamics of endophytic microbiota following pathogen invasion.

Objective To breed Bifidobacterium adolescentis strains that can adapt to the gastrointestinal environment of felines and have strong intestinal colonization capabilities. Methods B. adolescentis was isolated from the feces of felines with long and regular life spans. The original strain QC-Y (with the life span extension rate reaching 33.85%) was selected through biomass assessments and mouse life span experiments and it was identified as B. adolescentis. After radiation-induced mutation, QC-Y-09 was screened out by the biomass assessment, gastrointestinal tolerance domestication, and evaluation. Results QC-Y-09 showed the biomass 55.667 times and the tolerance score 5.66 times that of QC-Y. Moreover, the tolerance of the strain to the feline gastrointestinal environment showed good genetic stability. The survival rates of the 10th generation of QC-Y-09 in the artificial gastric juice, intestinal juice, anaerobic, and micro-aerobic environments were 18.80%, 41.60%, 93.26%, and 48.39%, respectively, which were 62.67%, 108.53%, 97.92%, and 94.40% of those of the original generation. The intestinal colonization test showed that the colonization ability of QC-Y-09 in felines was significantly stronger than that of QC-Y and human-derived B. adolescentis. Seven days after the feeding of the microbial inoculum was stopped, the viable count of B. adolescentis in the feline feces of the QC-Y-09 group still reached 4.37 lg CFU/g, which was 1.74 lg CFU/g and 3.02 lg CFU/g, respectively, higher than those of the QC-Y group and the human-derived B. adolescentis group. In addition, QC-Y-09 had a good relieving effect on the feline food change stress, reducing the feline food change stress rate by 85.71%. The results of SNP analysis showed that QC-Y-09 was significantly different from QC-Y at the gene level, and the differentially expressed genes were mainly enriched in the ribosome structure and aminoacyl-tRNA biosynthesis pathway. Conclusion Feline-derived B. adolescentis QC-Y-09 bred in this study can effectively adapt to the gastrointestinal environment and colonize the intestines of felines. This study provides both theoretical and practical bases for the application of B. adolescentis in functional food for felines.

Objective To study the protective effect of baicalin on mice infected by porcine extraintestinal pathogenic Escherichia coli (ExPEC) PCN033 strain and explore the underlying mechanism. Methods The mouse infection model and Western blotting were employed to determine the clinical features, survival rate, bacterial loads in different tissue samples, pathological changes, and expression levels of P65, IκBα, NLRP3, ASC, and Caspase-1 of mice in the infection group and the baicalin treatment group. Results After baicalin treatment, the mental state of mice in the baicalin treatment group was obviously better than that in the infection group. The survival rate of mice in the baicalin treatment group was higher than that in the infection group, and the colonization ability of PCN033 in the blood, brain, and lung of mice in the baicalin treatment group was lower than that in the infection group. Further studies showed that baicalin inhibited the PCN033 infection-induced activation of phosphorylation of P65 and IκBα and down-regulated the expression levels of NLRP3, ASC, and Caspase-1 in mice. Conclusion Baicalin alleviated the inflammatory response caused by porcine ExPEC infection by inhibiting NF-κB signaling pathway and blocking the activation of NLRP3 inflammasome, thereby reducing the clinical symptoms and tissue damage in mice. It is suggested that baicalin may be a potential drug to prevent and treat porcine ExPEC infection by regulating the inflammatory response.

Objective As a zoonotic pathogen, Proteus mirabilis poses a serious challenge to public health due to its multi-antibiotic resistance and the synergistic effect of virulence genes. To characterize the antibiotic resistance transmission of bacteria in the food chain in Zhejiang Province, this study systematically monitored the antibiotic resistance phenotypes and genes of isolates from cattle slaughterhouses and farmers’ markets, and analyzed the distribution differences of antibiotic resistance genes (ARGs), virulence genes (VGs), and mobile genetic elements (MGEs). Methods A total of 384 samples (feces, carcasses, environment, etc.) were collected from cattle slaughterhouses and farmers’ markets, and the strains were identified by 16S rRNA gene sequencing. Twenty ARGs and 10 VGs were detected by the K-B disc diffusion method and PCR, and the ARGs and VGs carried by P. mirabilis were analyzed. The ARG clusters were analyzed by sequencing of integron gene cassettes, and the co-occurrence network of ARGs, VGs, and MGEs was constructed. Subsequently, conjugative transfer experiments were carried out to explore the horizontal transmission potential of ARGs. Results A total of 101 strains of P. mirabilis were isolated, with the total isolation rate of 26.30%. The isolation rate of strains from slaughterhouses (33.85%) was significantly higher than that from farmers’ markets (18.75%). The resistance rates to ceftriaxone sodium, amoxicillin, and erythromycin were all over 90.00%. Among the ARGs, bla_TEM (89.09%), sul1 (77.71%), and tetA (63.29%) had the highest detection rates, and the distribution of ARGs in slaughterhouses was more complex. The VGs fliL (92.08%) and zapA (80.20%) were highly expressed in the isolates, which suggested potential pathogenicity. The detection rate of integrons in slaughterhouses was significantly higher than that in farmers’ markets, and PCR amplification results showed that there were a variety of ARGs, including aminoglycoside and trimethoprim resistance genes. Co-occurrence network analysis showed that ARGs, VGs, and MGEs had significantly positive correlations, and type I integron (intI1) was the hub gene. Conjugative transfer experiments confirmed that bla_TEM could be transmitted across species via horizontal transmission. Conclusion Compared with farmers’ markets, slaughterhouses are key nodes in the spread of antibiotic resistance due to the antibiotic exposure pressure, high organism density, and rich mobile components. The findings emphasize the importance of strengthening antibiotic management and monitoring the transmission chain of ARGs, providing a scientific basis for the prevention and control of antibiotic resistance under the framework of “One Health”.

Objective To elucidate and compare the diversity, structure, and functional characteristics of gut microbiota in sympatric fish species from a perspective of microbial ecology and explore the role of gut microbiota in feeding habit and ecological niche differentiation. Methods Foregut, midgut, and hindgut samples from Gymnocypris eckloni and Schizopygopsis pylzovi, along with their aquatic environmental samples, were collected from the upper Yellow River. The gut microbiota and potential functions were compared by 16S rRNA gene high-throughput sequencing and multiple bioinformatics approaches. Results Microbial alpha diversity followed the trend of aquatic environment>S. pylzovi>G. eckloni (P<0.05). In G. eckloni, alpha diversity was highest in the foregut, whereas in S. pylzovi, it decreased progressively from the foregut to the midgut and then to the hindgut. Beta diversity analysis based on clustering and CPCoA demonstrated that microbial communities derived from different gut segments of the same species were more similar to each other than those derived from the same gut segment of different species, indicating that the differences attributable to species were greater than those attributable to gut segments (P<0.001). Pseudomonadota accounted for over 50.00% of the gut microbiota in all sample types, while Bacillota comprised more than 25.00% in the guts of both fish species, which was significantly higher than that (3.80%) observed in the aquatic environment. Fusobacteriota and Cetobacterium were nearly absent in water and S. pylzovi but showed high abundance in the midgut and hindgut of G. eckloni. Enterococcus was specifically enriched in the hindgut of G. eckloni, while Lactococcus were predominantly found in the hindgut of S. pylzovi. PICRUS2 functional prediction revealed that gut microbiota of both species primarily enriched amino acid and carbohydrate metabolism pathways. The enriched metabolic pathways varied significantly across different gut segments of S. pylzovi, while significant differences in signaling molecules and interaction, cardiovascular diseases, and metabolism of terpenoids and polyketides were noted for the same gut segments between the two species. Conclusion There were significant differences in the microbiota composition and diversity in the gut between the two fish species and their aquatic environments, with distinct gut microbiota functions for each species. This study establishes a micrological foundation for research on the feeding habit and ecological niche differentiation of fish and provides theoretical support for exploitation of gut microbial resources and conservation and resource management of plateau fish species.

Objective The mesophilic salt-tolerant xylanase XynRBM26, a member of the GH10 family, holds significant application value in industrial fields such as animal feed. This study improved the thermostability of this enzyme by protein modification via rational design, aiming to lay a foundation for the industrial application of XynRBM26 preparations. Methods The bioinformatics software FoldX was used to conduct positionscan of the 3D structure predicted by AlphaFold 2.0 for XynRBM26. The mutants with free energy changes less than -0.5 kcal/mol were selected to construct an initial electronic library. According to the Pro effect and screening principles for potential mutants, an electronic library composed of Pro mutations was subsequently established. Finally, site-directed mutagenesis was employed to construct mutant genes, and positive mutants were screened after heterologous expression, purification, and experimental verification. Results After screening of the initial potential mutants, a small and precise mutant library consisting of 21 Pro effect mutants was constructed. All the mutants were experimentally validated, and positive single-point mutants D222P, V182P, D344P, and A352P with significantly increased T_m values were screened out. Through subsequent stacking screening, a three-point stacked Pro effect mutant with superior properties was obtained. The combination of this mutant with the experimentally screened positive mutant G115D produced the most stable mutant M4 (G115D-D222P-D344P-A352P). Compared with wild-type XynRBM26, M4 showed increases of 6.5 °C and 5 °C in T_m and optimal temperature, respectively. Moreover, M4 presented the half-life (t_1/2) at 55 °C 7.5-fold longer than the wild type, and its relative activity at the optimal temperature was 3.44 folds that of the wild type. Conclusion Screening thermostable mutants of the salt-tolerant xylanase XynRBM26 of the GH10 family based on the Pro effect and two different effect superimposing strategies is an effective approach.

Objective To investigate the effect of Lactobacillus reuteri CCTCC M 2016546 on chronic unpredictable mild stress (CUMS)-induced depression in rats and explore its potential mechanism. Methods Sixty male Sprague-Dawley (SD) rats were initially randomized into three groups: blank control (n=8), CUMS model (n=43), and prevention (n=9). The other groups except the blank control group received three CUMS stimuli daily. The prevention group was administrated with the L. reuteri CCTCC M 2016546 suspension (1×10⁹ CFU/d) via oral gavage prior to daily stress induction. After 4 weeks, depressive behaviors were assessed by sucrose preference, forced swimming, and open field tests. Successfully modeled rats (n=43) were re-randomized into five groups: model control (n=8), fluoxetine (2.1 mg/kg, n=8), combined therapy (2.1 mg/kg fluoxetine+1×10⁹ CFU/d CCTCC M 2016546, n=9), high-dose CCTCC M 2016546 (5×10⁹ CFU/d L. reuteri CCTCC M 2016546, n=9), and low-dose CCTCC M 2016546 (1×10⁹ CFU/d CCTCC M 2016546, n=9). Rats were administrated with corresponding agents daily for 4 weeks and then subjected to behavioral tests again. The levels of 5-hydroxytryptamine (5-HT) in the hippocampus and adrenocorticotropic hormone (ACTH) and cortisol (CORT) in the serum were quantified via ELISA. Western blotting was performed to determine the protein levels of brain-derived neurotrophic factor (BDNF), tyrosine kinase receptor B (TrkB), protein kinase B (Akt), phosphatidylinisitol 3-kinase (PI3K), extracellular signal-regulated kinase (ERK), and cAMP-response element binding protein (CREB) in the brain tissue. Results Compared with the model control group, all treatment modalities (fluoxetine, combined therapy, and CCTCC M 2016546) alleviated depressive behaviors: increasing sucrose preference (P<0.01, P<0.001), reducing immobility time in suspension (P<0.01, P<0.05), and enhancing horizontal locomotion distance/central zone exploration (P<0.01, P<0.05). Biochemical analyses revealed that treatments reversed the CUMS-induced alterations in 5-HT, CORT, and ACTH levels (P<0.01, P<0.05). Western blotting demonstrated upregulated protein levels of BDNF, PI3K, CREB, TrkB, Akt, and ERK in the fluoxetine and combined therapy groups (P<0.01, P<0.05). High-dose CCTCC M 2016546 elevated Akt, BDNF, CREB, and PI3K levels (P<0.01, P<0.05), while low-dose CCTCC M 2016546 raised Akt and BDNF levels (P<0.01). Prophylactic CCTCC M 2016546 administration primarily enhanced TrkB expression (P<0.05). Conclusion L. reuteri CCTCC M 2016546 ameliorates CUMS-induced depression in rats, potentially by modulating the hypothalamic-pituitary-adrenal axis hyperactivity and activating the PI3K/Akt/CREB/BDNF signaling pathway.

Sulfate-reducing bacteria (SRB) with unique reductive capabilities enable simultaneous sulfate reduction and heavy metal removal, demonstrating potential in heavy metal pollution remediation. Objective To isolate efficient SRB strains from marine sediments and investigate their reductive characteristics and application prospects in Cr(VI) contamination remediation. Methods We enriched and screened out an efficient SRB strain and systematically analyzed its sulfate reduction efficiency, Cr(VI) removal efficiency, and metabolic responses under environmental stressors (such as pH, sulfate concentration, and heavy metal concentration). This strain was then used to synthesize biological iron sulfide composite, the physicochemical characteristics of which were then investigated by scanning electron microscopy (SEM), X-ray diffraction (XRD), and Fourier-transform infrared spectroscopy (FTIR). Furthermore, the feasibility of applying the biological iron sulfide composite in the remediation of Cr(VI)-contaminated environments was explored. Results Strain S5 was identified as Desulfovibrio sp. with GenBank accession number OR140726. Its protein concentration and sulfate reduction followed an S-shaped curve. This strain exhibited a certain degree of acid tolerance, with the OD₆₀₀ and sulfate reduction rate reaching 0.16±0.01 and (83.71±1.49)% at pH 5.0, respectively. The strain exhibited sulfate reduction capability in the presence of 0.5-1.3 g/L sulfate, achieving a maximum reduction rate of (92.27±1.20)%. In the presence of 10-30 mg/L Cr(VI), strain S5 demonstrated efficient Cr(VI) removal. However, when the Cr(VI) concentration was higher than 30 mg/L, the Cr(VI) removal rate of this strain decreased significantly. The biological iron sulfide composite prepared based on strain S5 was porous, amorphous, and rich in functional groups such as C=O, N-H, and Fe-S, and its Cr(VI) removal rates were above 85% and did not differ significantly when exposed to Cr(VI) at high concentrations. Conclusion Desulfovibrio sp. S5 is a strain with high efficiency of sulfate reduction and Cr(VI) removal, and the biological iron sulfide composite prepared with it can overcome the limitation of higher Cr(VI) concentration and maintain high Cr(VI) removal rate, which has obvious advantages in the remediation of Cr(VI) pollution. The results of this study can provide a scientific basis for the application of SRB in the bioremediation of Cr(VI)-polluted environments.