Shijun Bao

Mycoplasmopsis synoviae is an important pathogen in poultry that primarily causes synovitis, tenosynovitis, eggshell abnormalities, and respiratory diseases, resulting in significant economic losses to the poultry industry worldwide. The aim of this study was to systematically screen and characterize the proteins secreted by M. synoviae and to investigate the role of SSB in M. synoviae infection. We first identified 13 proteins secreted by M. synoviae using liquid chromatography‒tandem mass spectrometry analysis. Bioinformatic analysis of SSBs revealed that they lack a transmembrane domain and the characteristic features of a cleavable signal peptide and are potential virulence factors. After induced expression and purification of SSB from Escherichia coli , rabbit anti-SSB serum was prepared. Subcellular localization analysis by immunogold labeling and Western blotting further confirmed that SSB is present mainly in the cytoplasm and culture supernatant of M. synoviae . ELISA and indirect immunofluorescence analyses revealed that SSB binds to membrane proteins and adheres to the surface of chicken DF-1 cells and that rabbit anti-SSB serum significantly inhibits M. synoviae adhesion to DF-1 cells. In vitro experiments confirmed that SSB could interact directly with plasminogen and fibronectin in a dose-dependent manner. In addition, our study revealed that SSB significantly promoted Annexin A2 (AnxA2) protein expression in DF-1 cells during adhesion, significantly colocalized with AnxA2, and promoted its enrichment at the membrane. These findings offer a novel experimental foundation for understanding the molecular mechanisms underlying the role of the secreted protein SSB in M. synoviae infection. IMPORTANCE: Pathogenic bacteria transport proteins to the extracellular compartment through a variety of secretion systems, and most of these secreted proteins act as adhesins, toxins, or immunomodulatory molecules in host cells, playing an important role in bacterial pathogenesis. However, studies on secreted proteins of Mycoplasmopsis synoviae are still incomplete. In our study, we systematically screened and identified the secreted proteins of M. synoviae strain WVU1853 for the first time and conducted an in-depth investigation of the role of SSB, one of the secreted proteins, in the infection process, in vitro . Our results not only reveal the key role of SSB in the infection process of M. synoviae but also provide a new theoretical basis for studying the invasion mechanisms of Mycoplasma -secreted proteins and related processes.

Mycoplasma synoviae (M. synoviae) is an avian pathogen responsible for respiratory disease and synovitis. The VlhA (variable lipoprotein hemagglutinin) family of surface adhesins plays a critical role in host cell attachment, yet the specific residues and structural determinants governing this interaction remain incompletely understood. In this study, we selected the C-terminal lipid-associated membrane hemagglutinin (LAM HA) domain within the VlhA family as the bait protein, based on its conserved C-terminal region. Yeast two-hybrid screening identified 18 LAM HA-interacting host proteins, and molecular docking pinpointed five residues (S83, R85, Y88, N124, K192) as the most frequent interaction hotspots. To assess their collective functional relevance, we constructed a combined deletion mutant lacking these five candidate residues. At the optimal pH of 6.0–6.5, wild-type LAM HA showed the highest titer (1:2), significantly exceeding that at pH 7.0–7.5 (no activity) and pH 5.0–5.5 (1:1) (p < 0.05), whereas the deletion mutant displayed a reduced titer (from 2 to 1; p = 0.05). Secondary structure analysis at the same pH revealed decreased α-helix content (from 7.90% to 7.57%), along with increased β-sheet (from 38.92% to 38.83%) and random coil (from 10.83% to 10.44%). Molecular dynamics simulations revealed that the deletion mutant exhibited elevated RMSF (from 2.83 ± 1.88 Å to 4.18 ± 2.65 Å) and Rg (from 40.48 ± 1.31 Å to 54.16 ± 4.47 Å) at pH 6.0–6.5, while RMSD showed a slight decrease (11.17 ± 1.33 Å vs. 12.14 ± 0.21 Å), collectively indicating compromised structural stability of the mutant. Collectively, these findings suggest that the LAM HA domain contributes to hemagglutination activity through pH-sensitive conformational stability and specific residue clusters—a property that may be relevant to M. synoviae colonization in the acidic microenvironments of the respiratory tract and synovial fluid during infection.

ISG15 is an interferon-induced ubiquitin-like protein that exerts diverse functions during viral infection. We previously reported that ovine ISG15 (oISG15) promoted bluetongue virus (BTV) replication by stabilizing viral NS1 and VP4 in an ISGylation-independent manner, although the mechanism remained unknown. Here, using the generated anti-oISG15 antibody, we performed immunoprecipitation (IP) coupled with label-free Liquid Chromatography-Tandem Mass Spectrometry (LC–MS/MS) to characterize the oISG15-associated proteome in BTV-infected and uninfected cells. The results showed that BTV infection markedly remodeled the oISG15 association landscape, with decreased enrichment of several autophagy- and trafficking-related proteins and increased enrichment of mitochondrial metabolic proteins in oISG15 immunoprecipitates. Functionally, oISG15 overexpression induced modest alterations in autophagy-related markers in uninfected cells yet attenuated these markers’ abundance during BTV infection. Together, this study revealed BTV-induced changes in the oISG15-associated proteome that coincided with alterations in autophagy-related markers.

Introduction: This study aimed to evaluate the effects of biochar as a feed supplement for growing pigs. Biochar, a carbon-rich material produced by pyrolysis of biomass under low- or no-oxygen conditions, is widely used in agriculture due to its porous structure and high adsorption capacity. Recently, it has gained attention as a potential feed additive for livestock because of its ability to bind toxins and gut microbiota modulation. Although several farm trials have reported improved animal productivity with biochar supplementation, the underlying biological function remains largely unclear. Methods: To identify candidate pathways associated with biochar supplementation, we performed transcriptomic (RNA-Seq) and proteomic (LC-MS) analyses on blood samples collected from pigs in 3% biochar-supplemented group and control group. Results: In this study, supplementing the basal diet with 3% biochar significantly reduced mortality ( P < 0. 01) and improved the average daily gain (ADG) ( P < 0. 01), leading to better overall growth performance in growing pigs. The results of transcriptomic (RNA-Seq) and proteomic (LC-MS) analyses demonstrated that biochar supplementation enhanced immune function. RNA-Seq analysis identified 524 differentially expressed genes (413 down-regulated and 111 up-regulated). Up-regulated genes were significantly enriched in the IL-17 signaling pathway and the complement and coagulation cascades, whereas the most prominently down-regulated pathway was related to the cytoskeleton in muscle cells. Proteomic analysis identified a total of 892 proteins, of which 192 were up-regulated and 186 were down-regulated in the biochar group. Pathways associated with cytokine-cytokine receptor interaction and complement and coagulation cascades were up-regulated in proteomic analysis. In contrast, down-regulated proteins were primarily enriched in cytoskeleton-related processes in muscle cells, Salmonella infection, and IgSF CAM signaling pathways. Discussion: These findings indicate that dietary biochar supplementation improves the health and performance of growing pigs, at least in part, by modulating immune-related pathways. The combined transcriptomic and proteomic results provide new molecular insights into how biochar exerts its beneficial effects as a feed additive.

African swine fever virus (ASFV) is the causative agent of African swine fever (ASF), a fatal and highly contagious disease, resulting in enormous losses to the global swine industry. No licensed vaccines or effective therapeutics are currently available to control ASFV infection. Interferons (IFNs) serve as key mediators of host antiviral immunity by inducing interferon-stimulated genes (ISGs), but the specific mechanisms by which individual ISGs restrict ASFV replication remain unclear. Interferon-induced protein with tetratricopeptide repeats 3 (IFIT3, also called ISG60) has been shown to exhibit antiviral activity against various viruses, but its role in ASFV infection has not been previously studied. Here, we used porcine alveolar macrophages (PAMs), the primary target cells of ASFV, to investigate IFIT3’s function in ASFV replication. We found that overexpression of IFIT3 inhibited ASFV replication, while its knockdown enhanced viral propagation. Mechanistically, IFIT3 directly blocked ASFV adsorption to host cells, thereby suppressing all subsequent stages of the viral cycle. IFIT3 also specifically interacted with ASFV F334L, an early viral gene product that encodes the small subunit of ribonucleotide reductase, a key enzyme for viral DNA synthesis. Additionally, IFIT3 positively regulated the STAT1/TBK1/IRF3 signaling axis: its overexpression increased phosphorylation of TBK1 and IRF3, as well as the protein level of STAT1, while IFIT3 knockdown attenuated activation of these molecules. Transcriptomic analysis of IFIT3-knockout PAMs revealed significant suppression of innate immune pathways, including type I interferon, JAK-STAT, and RIG-I-like receptor pathways, along with downregulated expression of core antiviral molecules such as ISG15, MX1, and STAT1. Conversely, pathways related to viral adsorption, endocytosis, and cytoskeleton were activated, and pathways involved in protein translation initiation, endoplasmic reticulum stress, and autophagy were dysregulated, creating a favorable intracellular environment for ASFV replication. In conclusion, IFIT3 restricts ASFV replication possibly by inhibiting viral adsorption and promoting innate immune signaling, identifying it as a potential therapeutic target against ASFV. This study’s limitation is its in vitro PAM model; future work will validate IFIT3’s role in vivo and develop targeted inhibitors.

Purpose: Bovine parainfluenza virus type 3 (BPIV3) is an important pathogen in the bovine respiratory disease complex. The purpose of this study was to develop an efficient and rapid serological detection method, an indirect enzyme-linked immunosorbent assay (iELISA), for BPIV3. Methods: The full-length N gene of BPIV3 was amplified from reverse-transcribed BPIV3 cDNA and ligated into the pET-28a prokaryotic expression vector. The recombinant target N protein was correctly expressed. After purification, the target N protein was used to immunize BALB/c mice to prepare polyclonal antibodies. After experimental optimization, an iELISA for detecting antibodies against the BPIV3 N protein was established using purified protein and prepared polyclonal antibodies. Results: The recombinant N protein was efficiently expressed after induction with isopropyl β-D-1-thiogalactopyranoside at 32°C for 16 h. Immunization of BALB/c mice with the purified recombinant N protein elicited polyclonal antibodies that specifically reacted with BPIV3, with an antibody titer of 1:256000. The optimized iELISA conditions were as follows. Microtiter plates were coated with antigen at 5× 10 ⁻² μg/mL, incubated overnight at 4 °C, and blocked with 1% bovine serum albumin for 2 h at 37 °C. Subsequently, plates were incubated with primary polyclonal antibodies at a working dilution of 1:8000 and horseradish peroxidase-conjugated secondary antibodies at a dilution of 1:8000 for 1 h at 37 °C. Finally, the colorimetric reaction was allowed to proceed for 10 min. Specificity tests showed that this method had no serological cross-reaction with other pathogens. Both intra-assay and inter-assay coefficients of variation were lower than 10%. Positive signals were still detected when the clinical-positive BPIV3 serum was diluted 1:6400. The concordance rate between this method and the virus neutralization test for clinical serum samples was 95.8%. A total of 176 cattle serum samples were tested using this iELISA method, yielding a serum antibody positivity rate of 94.9%. Conclusion: The N protein polyclonal antibody prepared in this study provides an important biological basis for research on the mechanism of BPIV3 infection. The establishment of an iELISA can enable the rapid screening of clinical samples, thereby providing reliable technical support for the epidemiological investigation of BPIV3.

The “Short Beak and Dwarfism Syndrome” (SBDS), also termed “duck macroglossia,” is a contagious disease caused by novel goose parvovirus (NGPV)

Bovine coronavirus(BCoV) is a significant pathogen causing substantial economic losses in the cattle industry through increased calf mortality, reduced growth performance, and decreased milk yield. Rapid and accurate diagnostic methods are therefore essential for controlling BCoV transmission. Current diagnostic methods comprise two primary categories: conventional techniques and cutting-edge innovations. Conventional approaches, including molecular methods like RT-PCR/qRT-PCR and immunological assays such as ELISA and neutralization tests, remain the main diagnostic methods. However, they are limited by laboratory dependency as well as the necessary balance between speed and sensitivity. These limitations have promoted the development of innovative methods, including isothermal amplification, CRISPR/Cas systems, droplet digital PCR, and integrated platforms. This review comprehensively analyzes the advantages, limitations, and applications of current diagnostic methods, highlighting integrated platforms such as RPA-CRISPR-LFA and microfluidics-based LFA. These innovations bridge critical performance gaps by enhancing sensitivity and specificity while enabling field application, demonstrating significant potential as next-generation point-of-care diagnostics for managing this economically critical pathogen.

Mycoplasma bovis (M. bovis) has caused huge economic losses to the cattle industry. The interaction between M. bovis and host cells is elucidated by screening and identifying the target protein of M. bovis adhesin on the surface of the host cell membrane. However, the response mechanism of embryonic bovine lung (EBL) cells to M. bovis infection is not yet fully understood. Additionally, it is necessary to further explore whether infection with M. bovis induces oxidative stress and mitochondrial damage in EBL cells. In this study, oxidation reaction, mitochondrial membrane potential, mitochondrial structure, and apoptosis ability of EBL cells infected with M. bovis were assessed at different times (12, 24, 48 h post-infection; hpi). Then, the differential proteomic analysis of M. bovis-infected EBL cells at 12 h and 24 h was performed with uninfected cells as the control. The results showed that M. bovis infection reduced the antioxidant capacity of EBL cells, increased ROS levels, and decreased mitochondrial membrane potential. The mitochondrial membrane of EBL cells was damaged, and the ridge arrangement was disordered after infection by transmission electron microscopy. With the increase in infection time, the mitochondrial matrix partially dissolved and spilled. The apoptosis rate of EBL cells increased with the increase in infection time of M. bovis. Furthermore, proteomic analysis identified 268 and 2061 differentially expressed proteins (DEPs) at 12 hpi and 24 hpi, respectively, compared with the uninfected cells. According to GO analysis, these DEPs were involved in the mitosis and negative regulation of cell growth. Additionally, the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated the following pathways were linked to mitochondrial damage or cell growth regulation, including glycolysis/gluconeogenesis, pentose phosphate pathway, oxidative phosphorylation, AMPK, cGMP-PKG, cAMP, calcium, Wnt, Phospholipase D, apoptosis, MAPK, cell cycle, Ras, PI3K-Akt, mTOR, HIF-1. PPI results indicated that YWHAZ, PIK3CA, HSP90AB1, RAP1A, TXN, RAF1, MAPK1, PKM, PGK1, and GAPDH might be involved in mitochondrial pathway apoptosis induced by M. bovis infection. This study offers helpful data toward understanding the response of mitochondria of EBL cells to M. bovis infection.