Avian influenza viruses (AIVs) are recognized as persistent threats to poultry health and global food security. The H9N2 subtype is widely distributed in poultry. A nationwide epidemiological survey was conducted in South, North, Central, and Southwest China, including Muscovy duck farms and live poultry markets, from 2022 to 2024. A total of 1208 Muscovy duck serum samples, 1212 duck-derived oropharyngeal/cloacal swabs and lung/tracheal tissues, and 480 chicken-derived samples were collected. In addition, 31 H9N2 strains were isolated, 2 of which were duck derived and 29 of which were chicken derived. A novel coinfection challenge model combining ocularnasal inoculation of H9N2 with injection of Escherichia coli (E. coli) was established, which induced severe tracheal lesions that closely resembled naturally occurring field pathologies. The coinfection challenge model successfully replicated prevalent field scenarios of viral‒bacterial synergism, resulting in severe lung lesions and a significant increase in viral loads. The results demonstrated that inactivated and live-vector vaccines provided more than 80% protection, with significant reductions in viral shedding and clinical symptoms. This study underscores the urgent need for updated vaccines matching circulating strains and the establishment of a validated framework for evaluating poultry vaccines under real-world coinfection scenarios.

Streptococcus suis is a pathogenic bacterium primarily found in pigs, where it causes sudden death and meningitis, but it is also capable of causing septic shock and meningitis in humans, being the most important cause of adult bacterial meningitis in Southeast Asia. As the host's innate immune system serves as the first line of defense against microbial invaders, it is crucial to understand the intricate relationship between the bacterium and this system. Despite an increasing number of studies performed in the last 20 years, the pathogenesis of S. suis infection is not fully understood, and there is still an absence of effective vaccines on the market. Recent population-genomic studies have further highlighted that pathogenic S. suis is not a single uniform entity but comprises multiple lineages with distinct genome content and disease associations, raising the possibility that innate immune evasion mechanisms may not be equivalent across lineage. In the current article, we conducted a comprehensive review of the literature, focusing on the innate response developed mainly by phagocytes against this Gram-positive bacterium. Our aim was to summarize the last findings and to identify the main gaps in knowledge to guide future research efforts toward areas that require further investigation.

Porcine reproductive and respiratory syndrome (PRRS), caused by the PRRS virus (PRRSV), remains one of the most economically significant infectious diseases affecting the global swine industry, resulting in annual losses exceeding US $1.2 billion in the USA alone. PRRSV infects porcine alveolar macrophages (PAMs) and induces profound mitochondrial dysfunction, leading to the release of mitochondrial DNA (mtDNA). However, the mechanisms by which PRRSV-induced mitochondrial damage acts as a damage-associated molecular pattern (DAMP) to trigger inflammatory responses have not been fully elucidated. Here, we report that highly pathogenic PRRSV (HP-PRRSV) infection upregulates interleukin-1β (IL-1β) messenger RNA (mRNA) expression and protein secretion in a dose- and time-dependent manner. Transmission electron microscopy revealed progressive mitochondrial swelling, cristae disorganization, and matrix loss during HP-PRRSV infection. Using RNAscope combined with MitoGreen staining, we discovered that mitochondrial double-stranded RNA (mtdsRNA), generated from bidirectional transcription of mitochondrial DNA, is released into the cytosol during PRRSV infection. In vitro, transcription-driven mtdsRNA or mtdsRNA isolated from PAMs directly activated the NLRP3 inflammasome, inducing caspase-1 cleavage and IL-1β secretion. Mechanistically, the PRRSV-encoded nonstructural protein 11 (nsp11) inhibited NLRP3-dependent inflammatory responses by suppressing the oligomerization of the apoptosis-associated speck-like protein containing a caspase recruitment domain (CARD) (ASC), thereby constraining excessive inflammation and establishing a delicate balance between pro-inflammatory signaling and viral immune evasion. Collectively, our findings identify mtdsRNA as a previously unrecognized mitochondrial DAMP that links PRRSV-induced mitochondrial injury to NLRP3 inflammasome activation, while also revealing a counter-regulatory role for nsp11 in modulating inflammation.

Bovine coronavirus (BCoV), a primary etiological agent of calf diarrhea, exhibits dual tropism for the respiratory and enteric tracts. However, research on BCoV gene function and pathogenesis has been limited by the lack of convenient and flexible reverse genetics tools. Herein, we report a robust reverse genetics system for BCoV based on an optimized circular polymerase extension reaction (CPER) methodology. We successfully rescued recombinant BCoV (rBCoV) in HRT-18G cells and engineered recombinant BCoV expressing ZsGreen fluorescent protein (rBCoV/Zs-T2A-ns2). Phenotypic analysis demonstrated that both unmodified rBCoV and rBCoV/Zs-T2A-ns2 maintained growth kinetics comparable to the wild-type BCoV. Importantly, the reporter virus demonstrated remarkable genetic stability for over ten passages, identifying the ns2 locus as a suitable insertion site for stable foreign gene expression without compromising viral fitness or sequence integrity. As a proof-of-concept application, the rBCoV/Zs-T2A-ns2 was employed for antiviral compound evaluation, successfully identifying four compounds-camostat, nafamostat, niclosamide, and nelfinavir-capable of effectively inhibiting BCoV replication. Further time-of-addition assays revealed that nelfinavir and niclosamide targeted the BCoV replication stage, while camostat and nafamostat interfered with BCoV internalization. This versatile CPER-based reverse genetics system provides a critical platform for advanced BCoV pathogenesis research and accelerated development of novel vaccines and effective antiviral therapeutics, ultimately contributing to enhanced bovine health and sustainable livestock production.

Enterococcus spp. from food animals represent important reservoirs of antimicrobial resistance; however, the mechanisms underlying tigecycline resistance remain poorly understood. In this study, we investigated the genetic basis and evolutionary dynamics of tigecycline resistance in Enterococcus hirae. Among 118 Enterococcus isolates recovered from 150 swine-derived samples, E. hirae was the predominant species (68/118, 57.63%). Tigecycline resistance was identified in ten isolates (10/118, 8.47%), all belonging to a single E. hirae clone. Whole-genome sequencing of a representative isolate, EG2361, revealed three plasmids, among which pEG2361-2 harbored an IS1216E-associated multidrug resistance region capable of generating two circular translocatable units (TUs). TU1, carrying the promoter-variant tet(L) locus together with tet(M) and poxtA, underwent tandem amplification, leading to plasmid structural heterogeneity. Functional analyses demonstrated that tet(L) reduced tigecycline susceptibility, whereas enhanced resistance mainly associated with variation in the upstream promoter region rather than Tet(L) amino acid substitutions. Experimental evolution revealed a dynamic resistance trajectory. The initial increase in tigecycline resistance was associated with TU1 amplification and marked upregulation of tet(L) expression. Subsequently, tet(L) expression declined, whereas tigecycline minimum inhibitory concentrations (MICs) remained elevated and stable. Recurrent rpsJ mutations emerged independently across evolved lineages and were associated with high-level resistance. Together, these findings support a stepwise evolutionary model in which insertion sequence (IS)1216E-mediated amplification of the promoter-variant tet(L) locus drives rapid early adaptation, followed by the emergence of chromosomal rpsJ mutations associated with stable high-level resistance. This study expands current understanding of tigecycline resistance evolution in enterococci and highlights the interplay among mobile genetic elements, transcriptional regulation, and chromosomal adaptation in shaping antimicrobial resistance.

Bovine digital dermatitis (BDD) is a globally distributed infectious hoof disease that negatively impacts the wellbeing and productivity of the affected animal. Treponema species and in particular Treponema phagedenis are the microbes strongly associated with the disease. Recently, it has been reported that Bos indicus cattle are more resistant to BDD and to the colonization by Treponema phagedenis in contrast to many Bos taurus breeds. This study investigated whether Bos indicus mount a different immune response to Treponema phagedenis compared with Bos taurus cattle, to better understand the different disease outcome in the two subspecies. Therefore, we exposed freshly isolated primary bovine blood cells to Treponema phagedenis and dissected subsequent bovine immune responses. The comparison between the two subspecies revealed significant different immune responses to Treponema phagedenis. We observed a stronger Th1/proinflammatory cytokine induction in Bos taurus animals mainly driven by CD4[+] T and CD21[low] B cells in secreting IFN-γ. We assume that these distinct initial immune responses are linked to the control of Treponema phagedenis and the outcome of BDD. Additionally, we observed significant higher levels in the expression of the matrix metalloproteinase-9 by peripheral blood mononuclear cells in Bos indicus cattle, which likely benefits the host response to Treponema phagedenis.

Between 14 October 2024 and 24 March 2025, 43 cases of clade 2.3.4.4b highly pathogenic avian influenza (HPAI) type A (H5N1) infections in wild birds and 1 case in a leopard cat were detected through the national HPAI surveillance program. We performed whole-genome sequencing of 51 isolates and investigated their phylogenetic relationships. Phylogenetic analyses identified nine distinct genotypes, designated Kor24-25A to Kor24-25I. The most commonly detected genotype, Kor24-25A (n = 24), was frequently detected in Pelecaniformes and was most prevalent between December 2024 and January 2025. Bayesian continuous and discrete trait phylodynamic analyses using HA gene sequences identified the three most probable routes of viral introduction via Gyeonggi, Jeju, and the southern coastal areas of the Korean Peninsula and suggested that wild ducks served as the primary reservoirs facilitating viral transmission in the wild bird population. During the 2024-2025 season, great egrets (Ardea alba) appeared to be more frequently affected than other wild bird species, suggesting possible seasonal differences in species-specific disease outcomes among wild birds. This study provides valuable insights into the phylodynamics, host associations, and transmission patterns of HPAI H5N1 viruses. It emphasizes the importance of enhanced surveillance programs targeting key avian hosts and high-risk geographic areas.

Bovine viral diarrhea virus (BVDV) is an important infectious pathogen responsible for substantial economic losses in the global cattle industry. The structural protein E2 serves as a major protective antigen of BVDV and is the main target for host neutralizing immune responses. However, its antigenic structure and epitope distribution remain poorly understood. Here, five novel monoclonal antibodies (mAbs) against the E2 protein of BVDV-1 were generated using hybridoma technology, and their reactivity patterns, biological properties, and recognized epitopes were systematically characterized. As a result, one mAb, TBH033, specifically reacted with BVDV, while four mAbs, TBH002, TBH004, TBH008, and TBH009, exhibited broad-spectrum reactivity against different subgenotypes of classical swine fever virus (CSFV) and BVDV, as well as E2 proteins from eight pestivirus species (BVDV-1, BVDV-2, BVDV-3, CSFV, BDV, PPeV, GPeV, and ovIT PeV). All five mAbs exhibited high binding affinity to the E2 protein and potent cross-neutralizing activity against BVDV strains of different subgenotypes. Epitope mapping with chimeric recombinant and site-mutated E2 proteins revealed that TBH002, TBH004, TBH008, and TBH009 recognized a linear epitope in the DA domain, comprising [70]H, [72]R, [74]L and [76]T as critical residues; TBH033 targeted a BVDV-specific conformational epitope in the DB domain, defined by the conserved amino acid motifs [103]LCP[105], [123]GP[124]. These findings enrich our understanding of the antigenic structure of the E2 protein and may facilitate the design of E2-based diagnostic tools and vaccines.

Host defense peptides (HDPs) possess both direct bacteriocidal and immunomodulatory activities. In this study, chicken cathelicidin-1 (cCATH-1) and chicken cathelicidin-3 (cCATH-3) were selected based on their markedly different minimum inhibitory concentrations (MICs) against avian pathogenic Escherichia coli (APEC) strains, with MICs of cCATH-1 being 4- to 16-fold lower than those of cCATH-3. However, following intraperitoneal challenge with APEC strain E058 (4 × 10[6] CFU/mouse), 80% of cCATH-3 treated mice survived while 20% of cCATH-1 treated mice survived. Flow cytometry (FC) revealed significantly lower peripheral blood Ly6C[high] monocytes but higher peritoneal Ly6C[high] macrophages in cCATH-3-treated mice at 4 and 8 h post-treatment. Peritoneal CCL3 secretion was significantly upregulated in cCATH-3 treated mice at 8 h post-treatment. Upon knockout of macrophages, the protection of cCATH-3 in mice against APEC E058 challenge was markedly abolished. In vitro, peritoneal macrophages treated with cCATH-3 showed stronger bacteriocidal activity than those treated with cCATH-1. Mouse CCL-3 (mCCL3)-specific antibody neutralize the effect of mCCL3 on macrophages chemotaxis in vitro. mCCL3-specific antibody administration resulted in 0% survival in cCATH-3-treated mice versus 60% survival in cCATH-3-treated mice administered with mock antibody, while cCATH-1-treated mice showed 0% survival administered with either mCCL3-specific antibody or mock antibody after APEC E058 strain challenge. In summary, cCATH-3 provides robust protection against APEC infection compared with cCATH-1 in mice, partially through CCL3-mediated macrophage chemotaxis and enhanced bacteriocidal activity in activated macrophages, revealing a novel mechanism of avian cathelicidins and supporting HDPs as alternatives to antibiotics for controlling avian colibacillosis.

Actinobacillus pleuropneumoniae (A. pleuropneumoniae) is the etiologic agent of porcine pleuropneumonia, a severe respiratory disease characterized by hemorrhagic, necrotizing lung lesions that cause significant economic losses on the global swine industry. However, the molecular mechanisms underlying the lung pathogenicity of A. pleuropneumoniae remain unclear. Here we demonstrate that A. pleuropneumoniae infection induces lung injury through activating the NLRP3-mediated inflammatory response mediated by the YadA-like head region (Adh) of Apa1, a bacterial adhesin that mediates attachment to host cells. Importantly, we show that Adh triggers the NLRP3-mediated inflammatory response by engaging olfactory receptor family 5 subfamily M member 11 (OR5M11), a host receptor previously shown to interact with Adh. Furthermore, OR5M11 contributes to the Adh-activated inflammatory response by promoting mitochondrial DNA release. Taken together, our results provide new insights into the pathogenesis of A. pleuropneumoniae, particularly regarding NLRP3-mediated inflammatory pathology.

Mycoplasma hyopneumoniae (Mhp) is the primary etiological agent of porcine enzootic pneumonia and establishes infection through close association with the respiratory ciliated epithelium. Classical models of Mhp colonization have focused mainly on specific interactions between adhesins, particularly P97, and ciliary receptors. Although this framework has provided important insights into ciliary attachment, increasing evidence indicates that Mhp adhesion involves multiple surface proteins and host ligands, including sulfated glycosaminoglycans (GAGs), fibronectin, plasminogen (Plg) and extracellular actin. In this review, we synthesize current evidence and propose a glycocalyx-mediated infectious niche framework for early Mhp colonization. Within this framework, sulfated glycans within the respiratory glycocalyx may contribute to bacterial positioning at the airway interface, while multiligand adhesin interactions may contribute to attachment and Plg-binding surface proteins may facilitate host-factor recruitment. Among these interactions, plasminogen recruitment provides a potential link between adhesion and local interface modification, including possible extracellular matrix remodeling and epithelial injury. Other processes, including potential GAG-mediated local retention, host glycan-dependent variation in attachment and glycocalyx remodeling, remain hypotheses requiring further validation. By distinguishing experimentally supported interactions from mechanistic inferences, this review presents Mhp colonization as a dynamic host-pathogen interface process rather than a simple adhesin-receptor event. This framework provides a basis for future investigation of early colonization mechanisms and anti-adhesion strategies in porcine enzootic pneumonia.

Glässer's disease, caused by Glaesserella parasuis (GPS), causes substantial economic losses in the swine industry worldwide. This situation is largely attributable to the poor cross-protective efficacy of currently available vaccines. In this study, a multi-antigen cocktail subunit vaccine against GPS infection was developed. In silico antigenicity analysis, the immunoprotective efficacy, and antibody titers of 14 GPS antigens were assessed in a murine model. Finally, VacJ, PtsG, and GAPDH were identified as the component of the cocktail vaccine, with Gel-01 as the optimal adjuvant. Immunization of the cocktail vaccine induced robust humoral and cellular immune responses. The vaccine conferred effective cross-protection against lethal-dose GPS4 and GPS5 infections in mice. As supported by a pilot study, it also demonstrated favorable immunoprotective efficacy against GPS infection in piglets. Furthermore, immune sera from vaccinated mice significantly inhibited the growth of GPS4 and GPS5. Overall, these results suggested that the subunit cocktail vaccine established in this study exhibits great potential for preventing GPS infection under experimental conditions, and further large‑scale pig trials are still required to validate its practical applicability in field production.

Brucella is a successful intracellular bacteria, regulating various physiological activities within host cells by secreting multiple virulence factors. However, the specific molecular mechanisms underlying these processes remain unclear. Through interactions with host proteins, Brucella can influence cellular physiological processes to evade immune responses and enhance the bacteria survival and reproductive abilities. The type IV secretion system (T4SS) of Brucella plays a significant role in the process of Brucella infecting cells. This study revealed the interaction between BtpB and YWHAZ (14-3-3ζ) by analyzing the interaction network of 112 host proteins interacting with Brucella effector protein BtpB. Furthermore, we discovered that BtpB regulates the expression of YWHAZ protein and the deletion of its TIR domain abolishes this interaction as well as the effect on YWHAZ protein expression. Moreover, BtpB activates pro-apoptotic markers leading to apoptosis induction in cells. Our results provide mechanistic evidence that BtpB-dependent apoptosis regulation is contingent upon interaction with YWHAZ, and further characterize YWHAZ as a host factor governing Brucella's intracellular survival capacity. This study provides valuable understanding of the immune response mechanism against intracellular bacterial infections in hosts and offers a theoretical basis for future prevention and treatment strategies for Brucellosis.

Pathological changes and viral presence in the bursa of Fabricius (BF) have been reported, but the immunosuppression of infectious bronchitis virus (IBV) remains poorly characterized. Here, we systematically evaluated the pathogenicity of two GI-19 lineage IBV isolates, CK/CH/FJ-B3/2023 (bursal origin) and CK/CH/FJ-T5/2023 (tracheal origin), in 7-day-old specific pathogen-free (SPF) chicks, including further assessments of serum cytokines and the bursal apoptosis. We further assessed the impacts of these two IBV isolates on the Newcastle disease virus (NDV) inactivated vaccine-induced hemagglutination inhibition (HI) antibody response. This study reveals that both GI-19 lineage IBV isolates, which originated from the recombination of the 4/91 vaccine strain, exhibit bursa-tropism, inducing bursal pathological atrophy, prolonged viral detection [up to 35 days post-infection (dpi)] and delayed seroconversion until 14 dpi. Notably, IBV infection induces humoral immunosuppression in SPF chickens, evidenced by a ≥ 1log2 reduction in NDV-HI antibody titers at 14 and 28 dpi, with antibody production recovering to normal levels by 35 dpi. This is accompanied by a sustained systemic proinflammatory response characterized by persistent upregulation of tumor necrosis factor-α (TNF-α), interleukin 1β (IL-1β), IL-6, and interferon-gamma (IFN-γ) in the serum of infected chicks up to 28 dpi. Early-onset bursal apoptosis was detected as early as 1 dpi, with the bursal apoptosis index significantly higher than that of the negative control throughout the observation period. These findings highlight the humoral immunosuppression induced by IBV variants. Further characterization of IBV types is warranted to elucidate IBV immunosuppressive mechanisms.

Japanese encephalitis virus (JEV), a zoonotic mosquito-transmitted flavivirus, induces human encephalitis and swine reproductive diseases. No antiviral drugs or treatments exist for flavivirus infections, which cause severe symptoms. Even though some host proteins can suppress flavivirus proliferation, the virus can inhibit this antiviral effect. This study identified a host protein SAMSN1 (SAM domain, SH3 domain, and nuclear localization signal 1; also referred to as HACS1, SLY2, and NASH1) having antiviral activity during JEV replication. SAMSN1 was upregulated during JEV infection. The expression of SAMSN1 inhibits JEV replication by binding and degrading JEV-encoded nonstructural proteins (NS1, NS5) through the proteasome and the SAMSN1-TRIM21-p62 autophagy protein degradation pathways. SAMSN1 induced expression of type I interferon through the RIG-I/MyD88-TBK1-IRF3 axis in the JEV infection process. Current findings reveal a novel role of SAMSN1 in inhibiting JEV replication and proliferation, through viral protein degradation and interferon pathway upregulation.