At present， the remodeling of host metabolism by viral infection has been studied in a variety of viruses. As a porcine intestinal pathogenic coronavirus， the interaction between porcine epidemic diarrhea virus （PEDV） and host metabolic system has not been reported. Studies have shown that angiotensin-converting enzyme 2 （ACE2） is involved in the regulation of glucose metabolism in the pathophysiological state of the body and plays a role in anti-PEDV infection. In order to explore the regulatory effect of PEDV infection on intestinal glucose metabolism in piglets and the possible role of ACE2， the relationship between ACE2， PEDV infection and intestinal glucose metabolism in piglets was clarified. In this experiment， healthy and PED piglets were used as the research objects. By collecting blood， duodenum， jejunum and ileum tissue samples were isolated and intercepted for the following experiments： 1） The expression of PEDV M gene and N gene and protein in each segment of small intestine was detected. 2） Detection of glucose and lactic acid in blood； western blot was used to detect the expression of glucose transporter SGLT-1 and GLUT-2 protein. 3） RT-qPCR was used to detect the gene transcription of key enzymes in glycolysis and TCA cycle pathway in each intestinal segment； 4） ELISA and Western blot were used to analyze the content of Ang1-7， AngII and the protein expression of ACE2 and MasR in each intestinal segment； 5） Spearman method was used to analyze the correlation between ACE2 and PEDV infection and glucose metabolism-related enzyme expression changes.The results showed that ： 1） There was M gene expression in the jejunum tissue of diarrhea piglets， and a positive band appeared at about 552 bp. PEDV-N protein was expressed in duodenum， jejunum and ileum of piglets， with the highest expression in jejunum （P<0.01）. 2） Compared with the normal healthy group， the contents of glucose （P<0.05） and lactic acid （P<0.01） in the blood of PEDV-infected piglets were significantly or extremely significantly increased； the protein expressions of SGLT-1 and GLUT-2 in duodenum and jejunum were significantly or extremely significantly up-regulated （P<0.05 & P<0.01）. 3） The results demonstrated that the transcriptional levels of key glycolytic enzymes （HK2， PFK， PKM， and LDHA） were markedly elevated （P<0.01） in the small intestinal tissues of PEDV-infected piglets， whereas those of key TCA cycle enzymes （CS， OGDH， and IDH） were significantly suppressed （P<0.05 & P<0.01）.； 4） In the small intestinal tissues of PEDV-infected piglets， the protein expressions of ACE2 and MasR were significantly down-regulated （P<0.01）. In conjunction with these changes， the level of Ang1-7 was markedly decreased （P<0.01）， whereas that of AngII was considerably increased （P<0.01）； 5） PEDV infection was positively correlated with SGLT-1， GLUT-2， HK2， PKM and PFK in the small intestine of piglets， and negatively correlated with ACE2-Ang1-7-MasR axis. The results suggest that PEDV infection in intestinal tissues activates glycolysis and suppresses the TCA cycle， concomitant with a weakened ACE2-mediated ACE2-Ang1-7-MasR pathway. This metabolic rewiring manifests as a Warburg effect， characterized by increased glucose uptake and lactate production. Furthermore， this PEDV-induced remodeling of intestinal glucose metabolism is negatively correlated with ACE2 expression.

The study characterizes the pathogenicity and antimicrobial resistance profile of atypical enteropathogenic Escherichia coli （aEPEC） strain ZX185 isolated from sheep meat in Xinjiang， and evaluates its potential for foodborne transmission. Antimicrobial susceptibility was determined using the microbroth dilution method， and whole-genome sequencing was performed on the Illumina HiSeq platform. Bioinformatics analyses were conducted to investigate genomic features， including virulence and resistance determinants. A phylogenetic tree was constructed using core-genome single nucleotide polymorphisms （cgSNPs） to compare virulence gene content across closely related strains. The results indicated that strain ZX185 belongs to serotype O85：H4 and sequence type ST642. Although it was phenotypically susceptible to all 28 tested antibiotics， its genome harbored 43 antimicrobial resistance genes and an IncFIB plasmid replicon， suggesting potential for multidrug resistance. Additionally， this strain carries 91 virulence genes （including adhesion factors， type Ⅲ/Ⅵ secretion system effector proteins， etc.） and exhibits notable acid tolerance， with a survival rate exceeding 11% after 3 hours of treatment in simulated gastric fluid at pH≥3.5， indicating a strong potential for intestinal colonization and pathogenicity. Although cgSNP-based phylogenetic analysis indicated a close genetic relationship （26 SNPs apart） between strain ZX185 and the human-source U.S. strain MOD1-EC6173 （O85：H4， ST642）， ZX185 carries 35 additional virulence genes missing in MOD1-EC6173， predominantly associated with adhesion， T3SS/T6SS， and toxin synthesis. These findings indicate that although this sheep-derived aEPECstrain ZX185 shows phenotypic susceptibility， it possesses multidrug resistance genes and strong virulence traits， and exhibits high genomic similarity to a human-derived strain， indicating potential cross-host transmission risk. This study provides critical molecular insights into the pathogenesis of aEPEC， the evolution of antimicrobial resistance， and the foodborne transmission risk， supporting improved surveillance and control strategies.

This experiment aims to study the immune-enhancing effects of peptide gCK-16 combined with the attenuated vaccine of infectious bursal disease （IBD） and to evaluate the protective effects of the vaccination on SPF chickens（14-day-old）. To investigate the toxicity of the peptide gCK-16， the immune effect of vaccine and the levels of cytokines （IFN-γ， IL-4 and IL-6） in serum， the cells were stimulated by the different concentrations of peptide gCK-16 in vitro， and then the chickens were immunized with the different concentrations of peptide gCK-6 in combination with attenuated vaccine of IBD. Meanwhile， the challenge protection of infectious bursal disease virus （IBDV） was performed by detecting the survival rate， the bursa-weight/body-weight index （BBIX）， the splenic lymphocyte viability， the level of immunoglobulin （IgM and IgA）， the level of immunoglobulin （IgY） in the serum， the histopathological changes and the expression level of apoptosis-related proteins in the bursa. The results show that peptide gCK-16 has no obvious toxicity both in vivo and in vitro， and peptide gCK-16 combined with the attenuated vaccine of IBD can significantly increase the antibody titer of IBDV in the serum of chickens immunized with the attenuated vaccine of IBD and the level of cytokines （IFN-γ， IL-4 and IL-6） in the serum. Moreover， the peptide gCK-16 significantly promoted the stimulation index of LPS， ConA and IBDV to PBMCs. Immunization and challenge experiments also showed that the peptide gCK-16 significantly increased the survival rate and BBIX of chickens infected with IBDV， and reduced the pathological changes of bursa tissues， while the expression levels of pro-apoptotic proteins Bax， Caspase-3， Caspase-9 were significantly decreased， and the expression level of anti-apoptotic protein Bcl-2 was significantly increased. In conclusion， peptide gCK-16 can significantly enhance the humoral and cellular immune responses of the attenuated vaccine of IBD， and achieved an effective immune protection in chickens infected with IBDV， which provides theoretical reference and data support for the application of this peptide as an immunopotentiator of the attenuated vaccine of IBD.

The aim of this study was to investigate the effect of Bacillus subtilis （KC） in combination with different lactic acid bacterias on the quality and active effects of fermented Astragalus. In which the different lactic acid bacterias inludes Lactobacillus acidophilus （SS）， Lactobacillus rohitaensis （LY）， Lactobacillus plantarum （ZW）， and Streptococcus alactolyticus （FGM）. Four kinds of fermented Astragalus were prepared by the mixed probiotics （SS+KC， LY+KC， ZW+KC， and FGM+KC）， respectively. The negative control group （UNF） was the same preparation process with fermented Astragalus， but no probiotics. The quality of 4 fermented products was evaluated by measuring general physicochemical indicators （pH， ethanol content）， characteristic physicochemical indicators （total acid， organic acid， crude polysaccharide content）， total flavonoids， total saponins， total polyphenols， aflatoxin B₁ （AFB₁）， and deoxynivalenol （DON） content. HD11 cells were stimulated with different concentrations of 4 fermented products and UNF samples， respectively， and the cell proliferation activity was determined by the CCK8 method， and the phagocytic ability and cytokine expression were determined by immunofluorescence and ELISA methods. The results showed that the general physicochemical indicators and characteristic physicochemical indicators of LY+KC group， FGM+KC group， and ZW+KC group all met the requirements of plant enzyme （QB/T5323-2018）. Compared with UNF group， the total saponin content of LY+KC group was significantly reduced （P<0.05）， and the total flavonoid and total saponin contents of FGM+KC group and ZW+KC group were significantly increased （P<0.05）. Compared with UNF group， the AFB₁ and DON contents of FGM+KC group， ZW+KC group， and LY+KC group were significantly reduced （P<0.05）. At the concentration of 10 µg·mL^-1， four kinds of fermented Astragalus could promote the viability of HD11 cells， which was determined as the subsequent working concentration. After treated with ZW+KC and FGM+KC fermented Astragalus， the phagocytosis capacity and the expression of pro-inflammatory factors （IL-1β and IL-12） in HD11 cells were significantly higher than those in CON， UNF， SS+KC and LY+KC groups （P<0.05）. In conclusion， the selection of probitics is closely related to the quality and activity of fermented Astragalus. Among them， Astragalus membranaceus fermented by Bacillus subtilis combined with Lactobacillus plantarum or Streptococcus alactolyticus FGM has better quality， which can increase the content of total flavonoids and total saponins， degrade AFB₁ and DON， and improve the function of HD11 cells.

This study aimed to investigate the mechanism of Ziziphi Spinosae Semen in inhibiting LPS-induced bovine mastitis using network pharmacology， molecular docking and in vitro experiments. The core components and potential targets of Ziziphi Spinosae Semen were screened through TCMSP， Swiss Target Prediction and Pharm Mapper databases， while bovine mastitis-related targets were obtained from DisGeNET， OMIM and GeneCards databases. Venny， STRING database and Cytoscape 3.8.0 software were used for topological analysis to construct compound-target and disease-target interaction networks. Hub targets for treating bovine mastitis were screened and subjected to GO functional and KEGG pathway enrichment analyses. Molecular docking and visualization were performed between hub targets and active components using AutoDock. An LPS-induced inflammatory model was established in bMECs， with CCK-8 assay， qRT-PCR， Western blot and ELISA used to analyze the effects of Ziziphi Spinosae Semen on inflammatory factors （IL-1β， IL-6， TNF-α） and hub targets at mRNA and protein levels. Results showed that： 9 bioactive components of Ziziphi Spinosae Semen and 235 potential therapeutic targets for bovine mastitis were identified； PPI analysis revealed 4 hub targets： epidermal growth factor receptor （EGFR）， estrogen receptor 1 （ESR1）， peroxisome proliferator-activated receptor gamma （PPARG）， and phosphatidylinositol-4，5-bisphosphate 3-kinase catalytic subunit alpha （PIK3CA）； GO and KEGG enrichment analyses indicated predominant involvement in biological processes including positive regulation of cytosolic ion concentration， receptor complex formation， and steroid binding， with significant associations to pathways such as central carbon metabolism in cancer， endocrine resistance， insulin resistance， and EGFR tyrosine kinase inhibitor resistance； Molecular docking demonstrated high binding affinity between ESR1 and the key components of Ziziphi Spinosae Semen， betulinic acid and daucosterol. In vitro experiments confirmed that Ziziphi Spinosae Semen bioactive components significantly suppressed the LPS-induced upregulation of IL-1β， IL-6， TNF-α， and ESR1、PI3KCA、PPARG mRNA and ESR1 protein levels in bMECs. In conclusion， Ziziphi Spinosae Semen exerts therapeutic effects against bovine mastitis primarily through bioactive components like betulinic acid by modulating key targets including ESR1. This study provides a scientific foundation for developing Ziziphi Spinosae Semen based therapeutic strategies for bovine mastitis intervention.

This study aimed to investigate the mechanism of Banlian Zijin San （BLZJS） in preventing and treating heat stress in broilers using network pharmacology， molecular docking， and animal experiments. Active components and targets of BLZJS were screened using the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform （TCMSP）. Heat stress-related disease targets were identified via the GeneCards database. Venny and Cytoscape 3.7.2 software were used to map BLZJS-heat stress intersection targets and construct a "drug-component-disease-target" network. The STRING database established a protein-protein interaction （PPI） network. GO functional enrichment and KEGG pathway enrichment analyses of targets were performed using DAVID and Metascape databases. The results of network pharmacology showed that 122 targets were intersected between Banlian Zijinsan and heat stress， and the lipid and atherosclerosis and cancer pathways were the ones with higher number of enriched genes and significance， and the molecular docking results showed that the components of Banlian Zijinsan with higher number of potential targets bound better to the core targets of heat stress diseases；For the animal experiments， 300 1-day-old broiler chickens were randomly divided into 5 groups. Except for the control group， all other groups were subjected to heat stress treatment at an ambient temperature of 37±2 ℃ for 42 days. Three treatment groups were added with 0.5%， 1%， and 2% of traditional Chinese medicine compound to their basal diet， while the model group and control group were fed with basal diet. During the experiment， Banlian Zijin San could significantly reduce the levels of IL-6 and TNF-α in the serum of broiler chickens on days 14， 28， and 42 of heat stress （P<0.05）， significantly increased the level of IL-10 （P<0.05）， significantly reduced the levels of HSP70， HSP90， IL-6， and TNF-α in liver tissue （P<0.05）， and significantly increased the level of IL-10 （P<0.05）. Mild liver damage occurred in the model group on day 14， but not in BLZJS groups. Liver damage worsened in the model group on days 28 and 42， while BLZJS groups showed no significant damage.BLZJS alleviates heat stress-induced impairments in broiler growth performance and liver function. Its multiple active components likely regulate key targets （IL-6， IL-10， TNF-α， HSP70） and signaling pathways to mitigate heat stress.

The purpose of this study was to determine the circadian variation and differences of blood glucose and lipid metabolism， immunity， oxidation and antioxidation related indexes in postpartum healthy dairy cows and subclinical ketosis dairy cows， and to evaluate the effects of ketosis on the circadian physiological homeostasis of energy metabolism， immunity and oxidative stress in dairy cows from the perspective of the circadian clock. The blood samples of postpartum healthy group （postpartum 3-21 d， blood BHBA<1.2 mmol·L^-1， n=10） and subclinical ketosis group （postpartum 3-21 d， 1.2 mmol·L^-1<blood BHBA<3.0 mmol·L^-1， n=10） were collected at different time points （09：00， 17：00 and 01：00）. The blood ketone blood glucose meter and cholesterol determination kit were used to determine the diurnal changes of glucose and lipid metabolism indexes of dairy cows in each group. The white blood cells in the blood were isolated and total RNA was extracted. The circadian changes of inflammation-related genes and circadian clock genes in white blood cells were detected by real-time fluorescence quantitative PCR. The changes of blood oxidative stress indexes were detected by ELISA kit. The results showed that compared with the healthy group， the blood β-hydroxybutyrate （BHBA） content of dairy cows in the subclinical ketosis group was significantly increased （P<0.000 1）， the blood glucose concentration （P<0.01） and the plasma cholesterol content （P<0.000 1） were significantly decreased， and the blood glucose content （P<0.05） of dairy cows in the subclinical ketosis group exhibited significant difference between day and night. The changes of hydrogen peroxide （H₂O₂） （P<0.05）， catalase （CAT） （P<0.05）， superoxide dismutase （SOD） （P<0.05） and β-N-acetylglucosaminidase （NAG） （P<0.05） in plasma of healthy cows had significant diurnal differences. The changes of plasma H₂O₂ （P<0.05） and CAT （P<0.01） in subclinical ketosis group were significantly different between day and night. Compared with the healthy group， the contents of H₂O₂ （P<0.01） and malondialdehyde （MDA） （P<0.000 1）， and the activities of glutathione peroxidase （GSH-Px） （P<0.05） and NAG （P<0.000 1） in the plasma of dairy cows in the subclinical ketosis group were significantly increased. The qPCR results showed that the mRNA expression of circadian clock genes BMAL1 （P<0.01）， RORα （P<0.05） and PER2 （P<0.01） in white blood cells of healthy cows had significant day-night differences. The mRNA expression of circadian clock genes BMAL1 （P<0.05）， NR1D1 （P<0.05）， RORα （P<0.01） and PER2 （P<0.05） in white blood cells of subclinical ketosis cows had significant day-night differences. Compared with the healthy group， the circadian expression patterns of BMAL1 （P<0.05）， RORα （P<0.01） and PER2 （P<0.01） mRNA in blood leukocytes of subclinical ketosis cows were significantly changed. The mRNA expression of inflammatory cytokines interleukin-6 （IL-6） （P<0.000 1）， interleukin-8 （IL-8） （P<0.05）， interleukin-10 （IL-10） （P<0.05）， interleukin-1β （IL-1β） （P<0.01）， and chemokine ligand 5 （CCL5） （P<0.01） in the blood of healthy cows showed significant circadian differences. The mRNA expression of IL-6 （P<0.01）， IL-10 （P<0.01） and IL-1β （P<0.000 1） in blood leukocytes of dairy cows with subclinical ketosis showed significant difference between day and night. Compared with the healthy group， the circadian expression patterns of IL-6， IL-10 and IL-1β mRNA in the blood leukocytes of dairy cows in the subclinical ketosis group changed， and the circadian differences in the expression of IL-8 and CCL5 mRNA disappeared. In summary， there are circadian differences in some blood glucose and lipid metabolism indexes， oxidative stress indexes， blood leukocyte circadian clock genes and inflammatory factor gene expression between healthy cows and subclinical ketosis cows. However， the circadian changes of some blood glucose and lipid metabolism indexes， oxidative stress indexes， blood leukocyte circadian clock genes and inflammatory factor gene expression in subclinical ketosis cows are significantly changed. The steady-state of circadian changes of plasma oxidation and antioxidant system is abnormal， and the circadian changes of circadian clock genes and inflammatory related gene expression in blood leukocytes are abnormal. The decrease of body resistance associated with ketosis may be related to the abnormal circadian rhythm of inflammatory factor expression in peripheral blood leukocytes of dairy cows. The above research preliminarily analyzed the characteristic changes of blood indexes of ketosis cows from the perspective of circadian clock， which provided research basis and theoretical basis for the accurate prevention and control and accurate diagnosis of ketosis in dairy cows.

The aim of this study was to understand the genomic characteristics and pathogenicity of epidemic porcine reproductive and respiratory syndrome virus （PRRSV） strains in China， and to explore their recombination and related variations. Pathological samples were collected， processed， and subjected to three consecutive blind passages in Marc-145 and PAM cells. Plaque purification was performed post-indirect IFA. Complete genomic sequences of the isolated strains were sequenced and analyzed. Piglet infection experiment was conducted for systematic assessment of the strains’ pathogenicity. In this study， six PRRSV type 2 strains were isolated and named WH-2021A， GZ-2021W， SX-2022M， HB-2021A， H-1， and C-1. WH-2021A， GZ-2021W， and SX-2022M belong to NADC30-like strains of L1.8.GZ-2021W is a recombinant strain with NADC30 as the main parent and highly pathogenic strain HuN4 as the secondary parent. SX-2022M is a recombinant strain with NADC30 as the main parent and classical strain CH-1a as the secondary parent. HB-2021A belongs to the L3.5 QYYZ branch， while H-1 and C-1 belong to the L8.7 highly pathogenic strain and the L5.1 classical strain， respectively. Piglets infection experiments showed that WH-2021A had the strongest pathogenicity， and the mortality rate of piglets was 100%， followed by H-1， with a mortality rate of 80%. The two recombinant strains of GZ-2021W and SX-2022M could cause a series of clinical lesions， but no piglets died. Pathological examination showed that the lesions of lung， lymph nodes and tonsils in the infected group were obvious. In this study， 6 epidemic PRRSV strains were isolated， and their genomic characteristics and pathogenicity were explored， which provides early warning of PRRS immune prevention， as well as critical data support for the recombinantion and evolutionary analysis of RRRSV-2.

This study aimed to prepare a positive control for RT-qPCR detection of Porcine Reproductive and Respiratory Syndrome Virus genotype 2 （PRRSV-2） by utilizing the property of MS2 phage capsid protein to self-assemble and encapsulate exogenous nucleic acids into armored RNA， and to investigate its stability and RNase resistance. The specific target gene （conserved region of ORF7） of PRRSV-2 was inserted downstream of the PAC site in the pET28a（+）/CP-A recombinant plasmid to construct a recombinant vector. Through prokaryotic expression， ammonium sulfate precipitation， and gel filtration chromatography purification， uniform MS2 virus-like particles （VLPs） with a diameter of 20-28 nm were obtained. Benzonase nuclease digestion and RT-qPCR were used to verify its thermal stability and RNase resistance. The recombinant vector containing MS2 phage capsid protein， PAC site， and PRRSV-2 target gene was successfully constructed. After purification， armored RNA encapsulating the target gene was formed， which was resistant to RNase degradation and stable for 7 days at 37 ℃ under sterile conditions. This armored RNA can serve as a positive control for PRRSV-2 RT-qPCR detection. Compared with traditional synthetic nucleic acids， its advantages include： 1） simulating the nucleic acid state of real viruses and monitoring the entire detection process； 2） strong RNase resistance； 3） intra- and inter-batch coefficients of variation （CV） of <2% among 3 batches； 4） stable storage at 37 ℃ for 7 days. It provides a reliable quality control tool for PRRSV-2 detection.

This study aimed to establish a rapid and effective indirect ELISA method for detecting Listeria monocytogenes infection in samples. The InlB protein was expressed and purified in vitro using a prokaryotic expression system. The optimal parameters for the indirect ELISA method were determined through optimization， an indirect ELISA method was established. Soluble expression of InlB protein was achieved using the pCold II-10 × His vector. Optimal conditions determined by a grid-screen method were： InlB protein coating concentration of 4 μg·mL^-1， blocking with 10% skim milk powder at room temperature for 2 hours， optimal serum dilution of 1∶400， incubation at 37 ℃ for 1 hour， the optimal dilution for the secondary antibody was 1∶2 500， with incubation at 37 ℃ in the dark for 8 min. The positive/negative cutoff value was determined to be 0.37. The indirect ELISA method established in this study reacted only with L. monocytogenes-positive sera， maintaining positive detection at a serum dilution of 1∶800. The coefficient of variation （CV） for intra-batch replicates ranged from 2.79% to 6.64%， while the CV for inter-batch replicates ranged from 2.61% to 7.75%. Compared with a commercial kit， the overall agreement rate was 95.83%. The indirect ELISA method established in this study for detecting L. monocytogenes-infected samples exhibits high specificity， sensitivity， and reproducibility. It can be used for rapid detection of L.monocytogenes-infected samples， providing a reference for monitoring listeriosis.