[Background]Mycoplasma contamination poses a persistent challenge in biological research and biopharmaceutical production.Conventional detection methods,such as classical culture assays,are time-consuming and lack sensitivity,while the standard PCR is prone to inhibitor interference and incapable of quantification,failing to meet the demand for rapid and precise quality control in vaccine production or laboratory settings.[Objective]This study aimed to establish a universal,highly specific,and sensitive quantitative real-time PCR(qPCR)method for efficient screening of mycoplasma contamination in cell cultures,live viral vaccines,and biological raw materials.[Method]The SILVA_138.1_SSURef database,encompassing 16S/18S rRNA sequences of bacteria,archaea,and fungi,was utilized to extract 181 non-redundant 16S rRNA sequences from classified mycoplasma species.The hypervariable V6-V8 region was identified as the optimal target via multiple sequence alignment(MEGA 11.0).Three primers(forward primers MF1:5'-GCAAARCTATRGARAYATAGYVGAG-3';MF2:5'-GCAAAGGCT TAGAAATAAGTTCGGAG-3;reverse primer MR:5'-CCARCTCYCATRGTKTGACGG-3')and a dual-quenched TaqMan probe(5'-FAM-ACAGRTGGTGCATGGYTGTCGTCAGCTC-BHQ1-3')were designed using Primer Premier 5.0,with primer-probe ratios and annealing temperatures optimized to establish the qPCR assay.Validation included:(1)primer-probe specificity testing against 11 Mycoplasma/Acholeplasma species(e.g.,Mycoplasma anatis,Mycoplasma bovis,Ureaplasma urealyticum);(2)sensitivity assessment via 10-fold serial dilutions(1.0×108-1.0×101 copies/μL)of Mycoplasma synoviae BHQ03,Mycoplasma gallisepticum,and Mycoplasma hyopneumoniae,with standard curves generated;(3)specificity evaluation against four common bacteria(Salmonella,Clostridium perfringens,Escherichia coli,Brucella)and eight animal cell lines(Marc145,Vero,CEF,etc.);(4)repeatability analysis(intra-and inter-assay variability)using M.synoviae BHQ03 dilutions(1.0×108-1.0×101 copies/μL);(5)parallel testing of 24 live viral vaccine batches(poultry,swine,and canine),20 cell culture samples(8 types),and 8 viral seed stocks via qPCR,conventional PCR,and classical culture methods.[Result]The optimized qPCR protocol employed a two-step amplification program(56℃annealing temperature).Specificity testing confirmed positive detection of all 11 Mycoplasma/Acholeplasma strains and no cross-reactivity with non-target bacteria or cell lines.Repeatability tests showed coefficient of variation(CV)values<2%for Ct values across replicates.Sensitivity assays demonstrated limits of detection(LOD)of 1-2 copies/μL for M.synoviae,M.gallisepticum,and M.hyopneumoniae.Comparative analysis of clinical samples revealed high concordance between qPCR,conventional PCR,and culture methods,with qPCR exhibiting superior sensitivity.[Conclusion]The universal qPCR method developed in this study provided an accurate,reliable,and rapid detection tool for potential mycoplasma contamination in cell cultures and live viral vaccines.

Brucellosis remains an important zoonosis, but evidence on the diagnostic accuracy of serological assays in Bactrian camels is limited. We conducted a cross-sectional study involving 1091 Bactrian camels from 24 herds in Alxa Right Banner, Inner Mongolia, China. Serum samples were tested using the Rose Bengal test (RBT), indirect ELISA (i-ELISA), fluorescence polarization assay (FPA), and complement fixation test (CFT). A Bayesian latent class model accounting for conditional dependence among the antibody-based assays was used to estimate individual-animal sensitivity (Se) and specificity (Sp), the true prevalence of antibodies to Brucella spp. within each herd, and individual-animal predictive performance under serial and parallel interpretation rules. FPA showed the highest sensitivity (median 0.946; 95% posterior credible interval [PCI]: 0.919-0.966), followed by CFT (0.857; 0.818-0.891), RBT (0.843; 0.803-0.878), and i-ELISA (0.781; 0.737-0.820). Specificity was highest for CFT (0.995; 0.987-0.999), followed by RBT (0.987; 0.976-0.995), i-ELISA (0.959; 0.942-0.973), and FPA (0.927; 0.903-0.949). The sample-size-weighted true prevalence of antibodies to Brucella spp. among the sampled camels was 36.4% (95% PCI: 33.9-38.8%). Parallel interpretation of RBT and FPA achieved a sensitivity of 0.989 (95% PCI: 0.981-0.994). The principal assay rankings were stable under alternative prior assumptions, single-herd omissions, and alternative conditional-dependence specifications. No single assay achieved both high sensitivity and high specificity. At the individual-animal level, parallel interpretation of RBT and FPA may be considered when minimizing false-negative classifications is the priority, whereas CFT may be preferred when higher specificity is required.

The existing risk stratification for acute myeloid leukemia (AML) reveals considerable heterogeneity in patient prognosis, underscoring the necessity for innovative risk stratification methodologies to optimize treatment responses. In this multicohort study, we explored the potential of carbohydrate metabolism and autophagy-related genes (CARGs) to enhance prognostic classification in AML patients. Employing univariate regression and least absolute shrinkage and selection operator (LASSO)-Cox stepwise regression analysis, we constructed a prognostic signature involving four genes related to CARGs in AML patients. By leveraging data from the TCGA cohort with 117 patients, the Gene Expression Omnibus (GEO) public data cohort with 1,431 patients, and our internal cohort of 117 patients, we showcased the robustness and accuracy of the CARG signature in forecasting survival outcomes among a collective sample of 1,665 non-Acute Promyelocytic Leukemia (APL) patients. Patients were categorized into high-risk and low-risk groups based on median risk score. The overall survival (OS) was significantly shorter in the high-risk group compared to the low-risk group. Differentially expressed genes (DEGs) were identified. Gene Ontology (GO) and Gene Set Enrichment Analysis (GSEA) analysis revealed that the DEGs were primarily associated with immune response signaling pathways. Immune-related analysis indicated that patients classified in the high-risk group exhibited a suppressive immune microenvironment. The results of the potential drugs for the risk groups demonstrated that inhibitors of PI3K/AKT/mTOR signaling pathway were effective. The novel risk model based on CARGs proposed in our study shows promise in prognostic classifications in AML, potentially providing new insights for the development of precise targeted cancer therapies.

Feline calicivirus (FCV) is a highly variable RNA virus that infects domestic cats and circulates endemically within feline populations, causing a wide spectrum of clinical manifestations, from asymptomatic infections to severe disease. Genomic analysis of 69 FCV strains revealed a high prevalence of the virus across multiple provinces in China. In vitro infection of CRFK cells with laboratory isolates FCV-BJ616 and FCV-BJDX40 resulted in significant cytotoxic effects. Serum proteomic analysis identified 221 upregulated and 123 downregulated proteins following infection with FCV-BJ616, and 233 upregulated and 165 downregulated proteins following infection with FCV-BJDX40. Among these, 215 proteins exhibited shared differential expression. Functional analyses revealed enriched pathways, including TNF signaling and ferroptosis. Notably, upregulation of Acyl-CoA Synthetase Long-Chain Family Member 4 (ACSL4) was correlated with lung injury, while downregulation of S100 Calcium Binding Protein A2 (S100A2) was associated with poor prognosis in FCV-associated oral disease. The differential expression of ACSL4 and S100A2 was further validated through Western blot analysis. These results suggest that ACSL4 and S100A2 are promising candidate biomarkers for monitoring FCV infection and disease progression, laying a foundation for future diagnostic and prognostic applications.

The unfolded protein response (UPR) is a cellular stress response mechanism that maintains endoplasmic reticulum (ER) homeostasis through three signaling pathways mediated by IRE1α, PERK, and ATF6 sensors. While UPR's role in viral infections has been well documented, recent studies indicate that intracellular bacterial pathogens have evolved specific mechanisms to hijack UPR signaling for survival and replication. This review examines UPR manipulation strategies employed by major bacterial pathogens, including Brucella, Mycobacterium tuberculosis, Legionella, and Salmonella. These pathogens utilize effector proteins that target specific UPR components: Brucella effectors VceC, BspB, TcpB, and BspL interact with ER chaperones and ERAD machinery; M. tuberculosis proteins Rv0297, ESAT-6, HBHA, and CdhM disrupt calcium homeostasis and alter ER morphology; Legionella Lpg0519 activates atypical ATF6 signaling; and bacterial toxins including cholera toxin bind IRE1α structural motifs for pathway activation. The molecular basis of UPR manipulation includes direct protein-protein interactions, calcium signaling interference, ER morphological disruption, and transcriptional program modulation. Bacterial hijacking of UPR pathways affects ER-phagy processes and host immune responses, facilitating intracellular survival. UPR pathway components serve as potential targets for host-directed therapy against persistent and drug-resistant infections. Small molecule modulators targeting IRE1α kinase activity, PERK inhibitors, and ATF6 pathway regulators may complement conventional antimicrobial approaches. Characterization of these host-pathogen interactions provides insights for developing therapeutic strategies that target bacterial dependencies on cellular stress responses.

Feline calicivirus (FCV) is a primary cause of upper respiratory tract infections and oral ulcerative disease in cats and exhibits substantial genetic diversity that complicates prevention and control. In this study, we isolated the FCV-BJ616 strain, established a reverse-genetics system, and investigated its pathogenic mechanisms, thereby providing a foundation for antibody-based therapies and broad-spectrum vaccine development. The virus was purified by three rounds of plaque cloning, and its morphology was examined by electron microscopy. VP1 expression was confirmed by immunofluorescence and Western blotting. Using integrated systems-biology and reverse-genetics approaches, an infectious clone of rFCV-BJ616 was successfully assembled and rescued, exhibiting genetic stability comparable to that of the parental strain. In vivo infection experiments showed that rFCV-BJ616 retained wild-type virulence, causing persistent high fever, weight loss, and multiorgan pathology in infected cats. Proteomic analysis indicated that infection with FCV-BJ616 or rFCV-BJ616 markedly activated cytokine-mediated inflammatory signaling pathways. Both FCV-BJ616 and rFCV-BJ616 significantly upregulated the expression of IL-8, S100A8/A9, and TLR3, which are associated with acute inflammation and tissue damage. Furthermore, elevated IFN-β levels concomitant with STAT1 downregulation suggested a transient attenuation of antiviral signaling during early immune activation. These findings were corroborated by ELISA-based validation of serum cytokine profiles. Collectively, this study provides new insights into the molecular pathogenesis and evolution of FCV-BJ616 and establishes a robust reverse-genetics platform for precise genome manipulation and future vaccine development.

Effective mucosal vaccines are critical for controlling infectious diseases in poultry, yet limitations in antigen delivery systems hinder their development. Here, we present a transgenic Eimeria tenella platform engineered to express multicopy viral capsid protein 1 (VP1) and viral capsid protein 2 (VP2) antigens of chicken infectious anemia virus (CIAV), a major immunosuppressive pathogen in poultry. Using a quadripartite co-transfection system driven by high-activity promoters, we achieved stable integration and expression of heterologous antigens, confirmed via PCR and Western blotting. Fluorescence-assisted cell sorting increased the proportion of recombinant parasites to >90%. Despite modestly reduced fecundity, the engineered strain (Et-TetVP1VP2) retained immunogenicity and induced robust humoral and cellular immune responses in vivo. Oral immunization of chickens conferred protection, reducing viral load and pathological lesions upon CIAV challenge. This work establishes E. tenella as a promising oral vaccine vector, offering a scalable and cost-effective platform for antigen delivery against avian pathogens, with broader implications for mucosal vaccine design.

Brucellosis, a significant zoonotic disease caused by Brucella spp., remains a major global challenge to both animal production and public health. Current live attenuated vaccines, such as Brucella abortus A19, are limited in their application due to residual virulence and interference with serodiagnosis. The intracellular survival and immune evasion of Brucella critically depend on effector proteins delivered by the Type IV Secretion System (T4SS), yet the functions of many of these effectors remain poorly defined. In this study, we constructed a markerless deletion mutant, A19Δ bpe275 , in the B. abortus A19 background and comprehensively evaluated its phenotype, virulence, and vaccine potential. The A19Δ bpe275 mutant retained wild-type morphology, smooth lipopolysaccharide (LPS) structure, in vitro growth kinetics, and genetic stability, but exhibited significantly impaired long-term intracellular survival in macrophages. In murine infection models, A19Δ bpe275 displayed markedly attenuated virulence, characterized by consistently lower splenic bacterial loads, milder histopathological lesions, and accelerated clearance compared to the parental A19 strain. Immunologically, infection with A19Δ bpe275 elicited a robust and sustained Th1-biased immune response. Notably, A19Δ bpe275 conferred comparable protective efficacy with improved safety against challenge with the virulent B. abortus 2308 and demonstrated cross-protection against B. melitensis 16M . Uniquely, the bpe275 deletion provides a molecular signature for a DIVA-compatible real-time PCR assay, enabling differentiation between vaccinated and infected animals. Collectively, by achieving an optimal balance between enhanced safety and preserved immunogenicity, A19Δ bpe275 emerges as a promising candidate for next-generation live attenuated brucellosis vaccines.

Abortion in cattle entails substantial economic loss, and rapid identification of abortigenic pathogens is critical for timely on-farm response and reduction in human exposure risk. In 2024, two Holstein cows from a small farm in Inner Mongolia aborted in close succession without an obvious cause. Vulvar swabs from both cows, one afterbirth sample, and whole blood from one aborted fetus were collected. Shotgun metagenomic sequencing was performed, followed by host-read removal, taxonomic profiling with Kraken2, de novo assembly of Brucella-aligned reads, and whole-genome comparison. Serological tests, Gram-stained smears, and Brucella genus- and species-specific qPCR assays were used as orthogonal verification. Putative resistance and virulence determinants were screened against CARD and VFDB. Brucella reads were detected in all samples, with the highest relative abundance in the 138-afterbirth (96%). qPCR assays detected Brucella DNA and B. abortus-specific signals in all four samples. A draft Brucella genome was assembled from the 138-afterbirth sample and was phylogenetically placed within B. abortus, showing relatedness to previously circulating Chinese lineages. Cows 138 and 198 were RBT-positive with SAT titres of 1:100 (++). No acquired Brucella resistance genes were identified in CARD. Within 72 h of sample receipt, B. abortus was reported to the farm and local authorities and emergency biosecurity measures were implemented. This field investigation shows that metagenomic sequencing, when combined with conventional serology, microscopy, and targeted qPCR, can support rapid etiological investigation when culture is delayed, hazardous, or biosafety level 3 facilities are unavailable.

HLA class I presentation of pathogen-derived peptides is essential for CD8+ T-cell recognition of Toxoplasma gondii. While in vitro MHC ligands have been documented, the in vivo ligandome during infection progression remains poorly characterized. Here, we employed an MS-based immunopeptidomics approach to directly profile the T. gondii immunopeptidome presented by HLA-A *02:01 in transgenic mice. By employing a hierarchical discovery funnel, our analysis identified a comprehensive repertoire of 3,744 unique T. gondii-derived peptides. Subsequent filtering for canonical 8-12mers, matching the typical binding length for HLA-A *02:01 ligands, established a high-confidence foundational ligandome of 3,433 peptides. Source protein analysis revealed that these peptides originate from diverse parasite proteins, including a substantial proportion of previously uncharacterized hypothetical proteins. Notably, specific ligands were consistently detected across both acute and chronic stages, suggesting stable MHC-I presentation throughout the infection cycle. By integrating in silico predictions with experimental validation, we prioritized 73 high-affinity candidates, five of which exhibited robust HLA-A *02:01 binding capacity in vitro and in vivo. Specifically, we identified a novel ligand derived from glycogen synthase (PGS) and determined its co-crystal structure with HLA-A *02:01, revealing favorable binding architecture. Overall, these findings expand the known HLA-A *02:01-restricted ligand landscape of T. gondii and provide a high-priority list of candidates for future functional validation of CD8+ T-cell immunogenicity.

Brucellosis remains a major global challenge to both animal production and public health. Brucella abortus A19, are limited in their application due to residual virulence and interference with serodiagnosis. The intracellular survival and immune evasion of Brucella critically depend on effector proteins delivered by the Type IV Secretion System (T4SS), yet the functions of many of these effectors remain poorly defined. In this study, we constructed a markerless deletion mutant A19Δbpe275, and comprehensively evaluated its phenotype, virulence, and vaccine potential. The A19Δbpe275 mutant retained smooth lipopolysaccharide (LPS) structure, in vitro growth kinetics, and genetic stability, but exhibited significantly impaired long-term intracellular survival in macrophages. In murine infection models, A19Δbpe275 displayed markedly attenuated virulence, characterized by consistently lower splenic bacterial loads, milder histopathological lesions, and accelerated clearance. Immunologically, infection with A19Δbpe275 was associated with a robust and sustained immune profile characterized by elevated Th1-associated cytokines. A19Δbpe275 conferred comparable protective efficacy an improved safety profile in the non-pregnant murine model against challenge with the virulent B. abortus 2308 and demonstrated cross-protection against B. melitensis 16 M. Collectively, by achieving an optimal balance between attenuated virulence and preserved immunogenicity, A19Δbpe275 emerges as a promising candidate for next-generation live attenuated brucellosis vaccines.

BACKGROUND:Brucellosis poses a significant threat to animal and human health globally. However, how Brucella subverts the immune response to establish persistent infections remains unclear. METHODS:We utilized single-cell RNA sequencing (scRNA-seq) to decipher the immune landscape of mice infected with Brucella abortus. Flow cytometry, a transgenic cell line and mouse, and antibody blockage were utilized to explore the relevant mechanisms. RESULTS:Brucella infection induced significant changes in the composition and signaling pathways of immune cells, and flow cytometry analysis further confirmed the scRNA-seq data. An in-depth analysis of macrophages, the main target cell for Brucella, demonstrated activation of type I interferon (IFN) and type II IFN signaling, tumor necrosis factor production, diverse cell deaths, etc. Specifically, Vir-2308 Brucella infection induced IFN-β expression, primarily originating from macrophages. In vitro, a significantly lower level of intracellular Brucella survival was observed in ifnar1-/- macrophages. In vivo, ifnar1 genetic deficiency rendered the mice less susceptible to Brucella challenge resulting in a lower bacterial load and higher levels of macrophages and neutrophils. Interestingly, Brucella infection induced a dramatic reduction of NK cells along with the upregulation of CD94:NKG2A, one typical immune checkpoint module of NK cells. Further blockage of the NKG2A receptor in mice significantly reduced the bacterial load in the tissues, concurrent with a higher ratio of mature dendritic cells and a lower proportion of B cells. CONCLUSIONS:scRNA-seq revealed that Brucella infection significantly alters the immune microenvironment in mice, providing insight into a better understanding of brucellosis pathogenesis and the immune evasion strategies of this sophisticated pathogen.

For Brucella spp., the ability to invade and survive within host macrophages is essential for causing chronic infections in their mammalian hosts. In this study, a genome-wide CRISPR knockout screen was performed for the first time in human THP-1 macrophages to identify host genes mediating resistance to Brucella invasion and intracellular survival. Results showed that the screening identified 35 candidate genes, 11 of which were selected to generate monoclonal knockout cell lines for functional validation. This study demonstrated that knockout of WDR4, ZNF532, or MTHFD1 significantly restricted Brucella invasion and early intracellular survival. In addition, TRAPPC2 knockout restricted Brucella invasion and, crucially, its intracellular survival throughout infection, exerting the most potent antibacterial effect. Mechanistically, TRAPPC2 deficiency suppresses Brucella infection by inhibiting autophagosome formation in macrophages. Furthermore, TRAPPC2 knockout decreases macrophage apoptosis and improves host cell viability following Brucella infection. These results provide therapeutic targets for combating Brucella infection and offer novel insights into the molecular mechanisms associated with Brucella-induced chronic infections.

Clostridium perfringens ( C. perfringens ) is an opportunistic pathogen, widely distributed in the environment. Epsilon toxin secreted by B and D type C. perfringens can cause enterotoxemia or necrotic enteritis in goats, sheep and cattle. Vaccination is one of the most effective measures to prevent such diseases. However, current toxoid vaccines still exhibit some problems, thus this study aims to engineer C. perfringens epsilon-ferritin nanoparticles antigen and evaluate its immunogenicity in mice and sheep. The r-epsilon successfully assembled with r-ferritin to form epsilon-ferritin nanoparticles via SDS-PAGE, transmission election microscopy and dynamic light scattering test. In the mouse experiment, epsilon-ferritin nanoparticles could significantly improve specific antibody levels including serum IgG, IgG subtypes antibodies, the ratio of IgG1/IgG2a and neutralizing antibodies. Moreover, epsilon-ferritin nanoparticles elicited more production of IFN-γ cytokines and more activation of memory CD8 + T cells in mice. In the sheep experiment, epsilon-ferritin nanoparticles could enhance the level of neutralizing antibodies and more production of IFN-γ cytokines. The present study revealed that epsilon-ferritin nanoparticles remarkably enhanced the immunogenicity of r-epsilon in mice and sheep, providing a new strategy for the development of C. perfringens vaccines.

Schmallenberg virus (SBV) is a transboundary animal pathogen that causes reproductive disorders in ruminants, necessitating standardized molecular surveillance. Adhering to the Chinese national standard GB/T 43159—2023, we developed armored RNA quality control materials using MS2 bacteriophage technology targeting the conserved SBV S segment and comprehensively characterized their physicochemical properties. Transmission electron microscopy (TEM) showed icosahedral symmetric virus-like particles (VLPs, approximately 25 nm), with the stock concentration determined to be 3.48 × 1010 copies/mL via digital PCR (dPCR). The armored RNA control effectively withstood RNase A degradation and remained stable at 37 °C for over 30 days. In bovine serum matrix simulation assays at identical medium concentrations, the serum armored RNA group (Ct 26.57) was detected 4.89 cycles earlier than the degraded serum naked RNA group (Ct 31.46). This confirms the physical protective efficacy of the MS2 capsid. Concurrently, the serum armored RNA group showed a delay of only 2.17 cycles compared to the aqueous armored RNA group (Ct 24.40), exhibiting a typical matrix effect. Overall, this armored RNA enables full-process quality control encompassing extraction, reverse transcription, and amplification, providing a safe and stable technical reference to support molecular surveillance and diagnostic preparedness for cross-border SBV.

Brucellosis, caused by Brucella spp., is a globally zoonotic disease that results in substantial economic losses and public health concerns. Although antibiotic-resistant Brucella strains have been reported worldwide, the current status and underlying mechanisms of resistance among Chinese isolates remain poorly characterized. In this study, we analyzed 636 clinical human isolates of B. melitensis from China using genomic sequencing, transcriptomic sequencing, and neural network prediction to identify key determinants and mechanisms of antibiotic resistance. Functional validations were performed using gene editing and protein-protein interaction assays. We found a gradual increase in resistance to trimethoprim-sulfamethoxazole (SXT) among Chinese isolates in recent years, despite the absence of known antibiotic resistance genes. Comparative genomic analyses between high- and low-minimum inhibitory concentration (MIC) isolates revealed specific single nucleotide polymorphisms (SNPs) that were present only in high-MIC isolates. Transcriptomic analysis demonstrated that high-MIC and low-MIC isolates activated distinct metabolic pathways in response to SXT exposure. Notably, genes influenced by specific SNPs exhibited opposing expression patterns after SXT treatment. Gene-editing experiments revealed that deletion of the glycoside hydrolase family 25 (GH25) gene, which was identified through SNP analysis, was associated with SXT resistance and notably altered Brucella energy metabolism, although it did not impact virulence in host cells. Further, we identified a direct interaction between GH25 and XylF. Collectively, our study reveals a novel genetic mechanism driving SXT resistance in B. melitensis. These findings highlight the critical need for vigilant surveillance of antibiotic resistance to mitigate public health risks associated with the potential widespread emergence of antibiotic resistance.

African swine fever (ASF), caused by the African swine fever virus (ASFV), is a highly contagious and fatal disease. Accurate detection in the early stages of an outbreak relies on molecular methods, but serological monitoring at the population level is also crucial for assessing the extent of exposure and past infections. This experiment developed an indirect enzyme-linked immunosorbent assay (ELISA) to detect antibodies against ASFV, using three ASFV RNA polymerase subunits (H359L, C147L, and D339L) as coating antigens. The recombinant proteins were successfully expressed in Escherichia coli and purified. Using a checkerboard titration method, we systematically optimized key assay parameters, determining the optimal coating conditions to be a mixture of H359L, C147L, and D339L at a volume ratio of 1:2:2, with individual concentrations of 1 μg/mL, 0.4 μg/mL, and 0.5 μg/mL, respectively. Other optimized parameters included a serum dilution of 1:200, a blocking buffer containing 5% skim milk, and specific incubation conditions for the secondary antibody and substrate. The cut-off value was established at 0.430 (x¯ + 4SD) based on 30 negative sera. The established triple-antigen indirect ELISA exhibited high sensitivity (detecting positives at dilutions up to 1:3200) and excellent specificity (no cross-reactivity with antisera against CSFV, PRRSV, PRV, PCV2, and PEDV. Both intra and inter assay repeatability were confirmed, with coefficients of variation ranging from 1.020% to 7.600%. Validation with 123 clinical serum samples demonstrated a 96.75% concordance rate with a commercial kit. In conclusion, the three-antigen indirect ELISA established in this study exhibits high specificity and sensitivity, making it suitable for serological surveillance and exposure assessment of ASFV antibodies. It can be combined with molecular detection for epidemiological investigations and integrated prevention and control measures.

We report a rare occurrence of genetic material from both Brucella canis and B. melitensis detected in a domesticated dog from Jiangsu Province, China. The detection was confirmed through serological assays, quantitative PCR (qPCR), metagenomic and genomic sequencing. Epidemiological evidence and whole genome sequencing suggested zoonotic transmission from the dog to its owner, who was diagnosed with brucellosis. Although isolation of both Brucella species was unsuccessful, the presence of dual genetic material highlights diagnostic complexities and underscores the zoonotic risks posed by companion animals, necessitating enhanced diagnostic awareness and preventive measures.

Brucella suis is a zoonotic intracellular pathogen responsible for brucellosis, mainly in swine and humans. Although numerous genome sequences are publicly available, an integrative genomic analysis combining pan-genome architecture, structural organization, evolutionary relationships, and vaccine-associated targets remains limited. In this study, we analyzed 91 publicly available B.suis genomes to characterize their pan-genome composition and genomic structure. The pan-genome exhibited an open configuration, indicating continued genomic diversification. A total of 2,146 core genes were identified, representing conserved functions essential for species maintenance, while the accessory genome reflected strain-level variability. Phylogenetic reconstruction based on single-copy orthologs revealed distinct evolutionary clades among the strains. A complementary phylogenetic analysis of pan-genome gene presence–absence patterns further supported clade differentiation and highlighted variation in accessory gene repertoires. Comparative synteny and genome structural analyses demonstrated largely conserved chromosomal organization with localized rearrangements across strains. Screening of the core proteome identified 64 putative antigenic proteins with predicted surface localization and immunogenic properties. Additionally, resistance-associated determinants related to tetracycline and doxycycline were detected in one genome within the dataset. This comprehensive genomic analysis defines the pan-genome structure, evolutionary relationships, and genome organization of B.suis. The integration of core and pan-genome-based phylogenies provides complementary insights into strain diversification, while the identified conserved antigenic candidates offer a foundation for future experimental validation and rational vaccine development strategies.