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Carboxylesterases, historically referred as non-specific esterases, are ubiquitous hydrolases with high catalytic efficiency. Without exceptions, all mammalian species studied contain multiple forms of carboxylesterases. While having been widely studied in humans and experimental animals, these enzymes remain to be characterized in farm animals. In this study, we showed that pig liver esterase 1 (PLE1) and pig liver esterase 6 (PLE6) were highly active toward amoxicillin (AMO) and ampicillin (AMP), two major antibiotics that are widely used in food-supplements. Mass-spectrometric analysis established that the hydrolysis occurred at the β-lactam amide bond and the hydrolysis drastically decreased or completely eliminated the antibacterial activity. Furthermore, hydrolytic activity and proteomic analysis suggested that trace PLEs existed in pig plasma and contributed little to the hydrolysis of AMO and AMP. These results suggested that carboxylesterases-based hydrolysis determines the therapeutic intensity of these and related antibiotics and the magnitude of the determination occurs in a species-dependent manner.

This study aimed to identify the heat-resistant bioactive components of Enterococcus faecium HDRsEf1 (HDRsEf1) and investigate their beneficial mechanism. Heat-treated culture supernatants of HDRsEf1 significantly suppressed CXCL-1 expression in LPS-stimulated MODE-K cells (p < 0.001), indicating the presence of heat-resistant anti-inflammatory components. Crude protein (P-Ef1) and crude expolysaccharide (EPS-Ef1) were isolated from an HDRsEf1 culture supernatant using ammonium sulfate and ethanal precipitation. Critically, only crude EPS-Ef1 retained an anti-inflammatory effect after heat treatment, while crude P-Ef1 lost this activity. Further investigation revealed that crude EPS-Ef1 (25 μg/mL) promoted MODE-K cell proliferation via EdU assays (p < 0.001), potentially through an upregulation of PCNA mRNA expression (p < 0.001). Animal studies demonstrated that an oral administration of crude EPS-Ef1 (4 mg/kg bw, 14 days) significantly increased body weight gain and jejunal crypt depth (p < 0.05) while reducing intestinal CXCL-1 mRNA levels (p < 0.001). These in vivo findings are consistent with in vitro observations. A structural analysis using HPAEC and SEC-MALLS-RI characterized crude EPS-Ef1 as a heteropolysaccharide (Mw 80.3 kDa) with a near-spherical conformation (slope 0.13) composed of mannose, glucose, glucuronic acid, and galactose (5.4:4.4:1.2:1). In summary, this study identifies crude EPS-Ef1 as the heat-resistant postbiotic component. Crude EPS-Ef1 possesses the dual effects of suppressing intestinal inflammation and promoting intestinal epithelial cell proliferation, which provides a theoretical foundation for a crude EPS-Ef1-based postbiotic.

The focal point of probiotic efficacy and a crucial factor influencing poultry cultivation lies in the level of intestinal inflammation. In conventional farming processes, the reduction of intestinal inflammation generally proves advantageous for poultry growth. This study investigated the impact of Bacillus amyloliquefaciens TL (B.A.-TL) on inflammatory factor expression at both tissue and cellular levels, alongside an exploration of main active secondary metabolites. The results demonstrated that broiler feeding with a basal diet containing 4 × 109 CFU/kg B.A.-TL markedly enhanced chicken growth performance, concomitant with a significant decrease in the expression of genes encoding inflammatory cytokines (e.g., CCL4 , CCR5 , XCL1 , IL-1β , IL-6 , IL-8 , LITAF , and LYZ ) in jejunum and ileum tissues. The extracellular polysaccharides of B.A.-TL (EPS-TL) exhibited notable suppression of elevated inflammatory cytokine expression induced by Escherichia coli O55 lipopolysaccharides (LPS) in chicken macrophage-like cells (HD11) and primary chicken embryonic small intestinal epithelial cells (PCIECs). Moreover, EPS-TL demonstrated inhibitory effect on NF-κB signaling pathway activation. These findings suggested that the metabolic product of B.A.-TL (i.e., EPS-TL) could partly mitigate the enhanced expression of inflammatory factors induced by LPS stimulation, indicating its potential as a key component contributing to the anti-inflammatory effects of B.A.-TL.

The Riemerella anatipestifer bacterium is known to cause infectious serositis in ducklings. Moreover, its adherence to the host’s respiratory mucosa is a critical step in pathogenesis. Membrane cofactor protein (MCP; CD46) is a complement regulatory factor on the surface of eukaryotic cell membranes. Bacteria have been found to bind to this protein on host cells. Outer membrane proteins (OMPs) are necessary for adhesion, colonisation, and pathogenicity of Gram-negative bacteria; however, the mechanism by which R. anatipestifer adheres to duck cells remains unclear. In this study, pull-down assays and LC–MS/MS identified eleven OMPs interacting with duck CD46 (dCD46), with OMP71 exhibiting the strongest binding. The ability of an omp71 gene deletion strain to bind dCD46 is weaker than that of the wild-type strain, suggesting that this interaction is important. Further evidence of this interaction was obtained by synthesising OMP71 using an Escherichia coli recombinant protein expression system. Adhesion and invasion assays and protein and antibody blocking assays confirmed that OMP71 promoted the R. anatipestifer YM strain (RA-YM) adhesion to duck embryo fibroblasts (DEFs) by binding to CD46. Tests of the pathogenicity of a Δ omp71 mutant strain of RA-YM on ducks compared to the wild-type parent supported the hypothesis that OMP71 was a key virulence factor of RA-YM. In summary, the finding that R. anatipestifer exploits CD46 to bind to host cells via OMP71 increases our understanding of the molecular mechanism of R. anatipestifer invasion. The finding suggests potential targets for preventing and treating diseases related to R. anatipestifer infection.

Mycoplasma synoviae ( M. synoviae ) is a major bacterial pathogen that causes serious economic losses in the global poultry industry. Systemic changes in specific pathogen free White Leghorn egg-laying hens after M. synoviae infection were investigated using intra-tracheally inoculated animals. Samples were collected 10 days post-infection (dpi) (204-day-old) and 52 dpi (246-day-old). Infection caused air sac lesion, footpad swelling and oviduct atrophy. The qPCR and in situ hybridization showed that bacteria colonized the trachea and oviduct, and that bacterial loads in the magnum and uterus were significantly higher than in the infundibulum and isthmus. Histopathological examination revealed increased tracheal mucosal thickening accompanied by inflammatory cell infiltration, and that tubular glands of the uterus were edematous or dissolved. Infection also induced decreased egg production and eggshell strength, and eggshell apex abnormalities appeared at 14 dpi. Plasma metabolomics of hens analyzed by liquid chromatography-tandem mass spectrometry showed 168 and 128 differentially-expressed metabolites (DEM) at 10 and 52 dpi, respectively. Pathway analysis revealed that DEM at 10 dpi were enriched in five distinctive pathways: regulation of the actin cytoskeleton, neuroactive ligand-receptor interaction, sphingolipid metabolism, gap junctions, and necroptosis. In contrast, DEM at 52 dpi were enriched in fifteen pathways involved in steroid hormone biosynthesis, ferroptosis, the calcium signaling pathway, apelin signaling pathway, progesterone-mediated oocyte maturation, and oocyte meiosis. Combined metabolic analysis demonstrated that changes in ethylsalicylate, nicotinamide, (3-Methoxy-4-hydroxyphenyl) ethylene glycol sulfate, sphingosine-1-phosphate (d18:1), carnitine C24:6, and 15(R)-prostaglandin E1 correlated the best with M. synoviae infection. This study provides new insights into understanding pathogen mechanisms and signposts novel treatments for M. synoviae infection in poultry.

Riemerella anatipestifer infection is a critical disease that is a major threat to the poultry industry worldwide. The adhesion and invasion of host cells are key steps in the primary stages of bacterial infection. However, the outer membrane proteins that mediate these events in R. anatipestifer are poorly characterized. In this study, the PorV and PosF proteins, as well as the previously described OMP71 protein, were identified as important mediators of the adhesion and invasion of duck embryo fibroblast (DEF) cells by R. anatipestifer . Affinity chromatography-based surface proteomics was used to screen for adhesion proteins. The surface proteins on DEF cells were labelled with biotin-avidin to enrich for outer membrane proteins of R. anatipestifer, which generated 11 candidate proteins that were tested further. Protein adhesion and blocking assays and polyclonal antiserum inhibition analysis revealed that the PorV, PosF, and OMP71 proteins are adhesion factors. Knockout of porV or posF reduced the adhesion and invasion of R. anatipestifer in DEF cells. Moreover, the pathogenicity of the mutant strains was significantly attenuated, which supports the hypothesis that PorV and PosF are important virulence factors required for the pathogenicity of R. anatipestifer . The PorV protein is a key component of the type IX secretory system and is responsible for transporting effector substrates to the extracellular environment, whereas PosF belongs to the porin superfamily of barrel-shaped transmembrane proteins. This is the first description that PorV is an adhesin involved in host‒microbial interactions, which represents a breakthrough in pathogenicity studies of R. anatipestifer and other members of Flavobacteriaceae .

Background Campylobacter jejuni ( C. jejuni ), a major global cause of foodborne bacterial diseases, accounts for more than 90% of all reported cases. Poultry is considered a major reservoir for the transmission of Campylobacter to humans. While extensive research has been conducted abroad on the occurrence and epidemiology of C. jejuni in laying hens, there are scant reports on its prevalence in layer chickens in China. The present study was designed to isolate C. jejuni from 482 cloacal swabs collected from seven laying hen farms located in Hubei Province between January and March 2024. Furthermore, the study aimed to explore the genetic diversity, antibiotic resistance, and virulence gene profiles of the isolated strains. Results The overall prevalence of C. jejuni amounted to 4.36% (21/482). Whole-genome sequencing of these 21 isolates revealed 11 distinct sequence types (STs) and eight clonal complexes (CCs), with ST-6522 and CC-443 emerging as the predominant genotypes. Antimicrobial susceptibility testing against 11 antibiotics revealed high resistance rates among C. jejuni isolates, particularly towards ceftriaxone and enrofloxacin, where resistance was universal (100%). Similarly, high resistance levels were also observed for doxycycline (95.24%), ceftiofur (80.95%), tilmicosin (76.19%), and amoxicillin-clavulanic acid (57.14%). Through genomic resistance gene prediction, a total of eighteen resistance genes were identified within the 21 C. jejuni isolates. The most frequently occurring resistance genes were the gyrA (T86I) point mutation (95.14%), cmeR (95.14%), and tet(O) (95.14%). Notably, a robust correlation was discernibled between enrofloxacin resistance and the gyrA (T86I) point mutation, as well as between resistance to ceftriaxone and tilmicosin and the presence of the cmeR gene. Conversely, the correlations between other antibiotic resistance phenotypes and their corresponding resistance genes were less robust. A comprehensive analysis of virulence genes isolated from C. jejuni strains revealed a total of 117 virulence genes, categorized according to their functional roles. These categories encompass adhesion ( cadF , jlpA , porA , pebA ), invasion ( ciaB ), motility ( flaA , flgB , flhB ), toxin production ( cdtA , cdtB , cdtC ), and immune modulation ( htrB , wlaN ). Conclusions Our results revealed the high resistance rate of C. jejuni from laying-hens in hubei Province, China, which will help the farms take the necessary action to develop effective mitigation strategies for reducing Campylobacter infection in poultry.

Heat stress (HS) is a major stressor for laying hens. It causes enormous financial losses worldwide annually. The purpose of this study was to investigate the effect and mechanism of a probiotic mixture of Bacillus subtilis and Enterococcus faecium on the performance of laying hens under HS. Eight hundred and fifty-six commercial laying hens (Hy-Line Brown, aged 40 weeks) were randomly allocated to three groups, Group C (26 °C), Group H (33 °C) and Group H + P M (33 °C + probiotic mixture), respectively. The trial lasted for 20 days. Compared with Group H, we demonstrated that treatment with the probiotic mixture significantly increased the egg production rate, average daily feed intake (ADFI), and egg weight of laying hens under HS (p < .05). In addition, the eggshell thickness, eggshell strength and albumen height also improved. We further showed that these probiotics improved gut microbiota as well as enhanced intestinal integrity, which may be responsible for inhibiting the invasion of bacteria and improving the performance indices. The study suggests that this probiotic mixture may be an effective strategy for treating laying hens under HS.

The probiotic Enterococcus faecium HDRsEf1 (Ef1) has been shown to have positive effects on piglet diarrhoea, but the mechanism has not yet been elucidated. In this study, using the IPEC-J2 cell line to mimic intestinal epithelial cells and enterotoxigenic Escherichia coli (ETEC) K88ac as a representative intestinal pathogen, the mechanism underlying Ef1 protection against an enteropathogen was investigated. The results demonstrated that Ef1 was effective in displacing K88ac from the IPEC-J2 cell layer. Moreover, Ef1 and its cell-free supernatant (S-Ef1) modulate IL-8 released by IPEC-J2 cells. Ef1 and its cell-free supernatant showed the potential to protect enterocytes from an acute inflammatory response. In addition, Ef1 and its cell-free supernatant increased the transepithelial electrical resistance (TEER) of the enterocyte monolayer, thus strengthening the intestinal barrier against ETEC. These results may contribute to the development of therapeutic interventions using Ef1 in intestinal disorders of piglets.