HoBi-like pestivirus (HoBiPeV), as a newly discovered virus species in recent years, poses a serious threat to the global cattle industry due to its wide transmission and high pathogenicity. The genetic sequence of HoBiPeV exhibits significant differences when compared to the known strains of BVDV-1 (Pestivirus bovis) and BVDV-2 (Pestivirus tauri).Current detection methods are typically limited to identifying BVDV-1 and BVDV-2, and are unable to effectively differentiate HoBiPeV from the other two genotypes. To facilitate the rapid detection and differentiation of various genotypes, this study developed a pair of universal primers along with three TaqMan probes based on the 5’ UTR sequences of the BVDV-1, BVDV-2, and HoBiPeV available in GenBank. A one-step multiplex TaqMan RT-qPCR detection method was developed. The results showed that this method could specifically detect the three genotypes of BVDV without cross-reactivity. It exhibited high sensitivity, with a minimum detection limit of 1 × 10¹copies/µL. The amplification efficiencies were 104.6

Mycoplasma bovis is one of the primary pathogens associated with bovine respiratory disease. The clinical manifestations of this disease primarily include pneumonia, mastitis, and arthritis, which have resulted in significant economic losses worldwide. Currently, there is no effective vaccine available, and antibiotic treatment often yields unsatisfactory results. Therefore, the development of vaccines with high immunogenicity and effective protective capabilities is crucial for preventing and treating M. bovis outbreaks. This study aimed to develop a multiepitope vaccine for M. bovis and to evaluate its immunogenicity and protective efficacy in BALB/C mice and rabbits. On the basis of the three dominant antigens, MbovP274, MbovP570, and ENO1, the researchers designed the multiepitope tandem recombinant protein, MbovE3. The vaccine was prepared using prokaryotic expression vectors, baculovirus, and DNA vectors for expressing MbovE3, and BALB/C mice and rabbits were utilized as experimental models to verify vaccine effectiveness. The rMbovE3 vaccine, based on a prokaryotic expression system, induced high levels of specific IgG and cellular immune responses in both the BALB/C mice and rabbit models, significantly reducing M. bovis infection and tissue damage in rabbits. Additionally, MbovE3 expressed via baculovirus and the DNA vaccine DNAE3+GM-CSF, expressed by DNA vectors, both exhibited effective protective effects in rabbits. Overall, these findings suggest that rMbovE3, developed via a multiepitope approach, is a promising candidate vaccine against M. bovis infection. These findings provide a scientific foundation for the development of effective vaccines against M. bovis and will facilitate research on and the development of related vaccine products.

IL-10, an anti-inflammatory cytokine, plays a crucial role in limiting immune responses to pathogens, preventing host damage. However, the mechanisms underlying Brucella-mediated IL-10 production remain incompletely understood. In this study, we demonstrate that the proline racemase protein A (PrpA) of Brucella melitensis M5–90 induces macrophages to secrete IL-10 by activating the Tpl2-ERK signaling pathway, thereby promoting chronic infection. Moreover, Tpl2 deletion impairs macrophage bactericidal ability, accompanied by reduced TNF-α, IL-1β and IL-10 levels. Additionally, Trp309, Glu103, and Glu129 of PrpA participate in interaction with Tpl2, but these residues do not influence PrpA-mediated IL-10 production in macrophages. PrpA deletion enhances IFN-γ levels, specific anti-Brucella IgG, and CD4+ and CD8+ T cell numbers in mice. Furthermore, the Brucella melitensis M5–90 prpA mutant provides higher protection than the parental strain against virulent Brucella melitensis M28 infection in mice. Our findings suggest that Brucella PrpA promotes IL-10 secretion by macrophages through Tpl2 activation for bacterial survival and persistent infection, making the Brucella melitensis M5–90 prpA mutant a promising vaccine for enhanced protection.

Microparticles (MPs) serve as critical mediators of intercellular communication by shuttling bioactive molecules, holding significant potential for clinical diagnostics and next-generation vaccine development. Brucella, an intracellular pathogen responsible for severe zoonosis, evades host immunity through persistent infection; however, the role of Brucella-induced MPs in modulating adaptive immunity remains poorly defined. In this study, we demonstrate that the infection of RAW264.7 macrophages with the attenuated Brucella melitensis strain M5 triggers robust MP release. These MPs enhance host defense by promoting bacterial clearance and restricting intracellular survival. In murine models, MPs elicit potent Th1-polarized immunity, characterized by elevated IgG2a titers and increased IFN-γ production. Mechanistically, MPs derived from M5-infected macrophages activate the nuclear factor-κB (NF-κB) pathway in bone marrow-derived dendritic cells (BMDCs), augmenting the secretion of IL-12 and TNF-α. Critically, MPs prime cytotoxic T lymphocytes (CTLs) through both direct and indirect pathways, resulting in the specific lysis of Brucella-infected targets. Collectively, Brucella-induced MPs function as integrated immunogenic units that bridge innate recognition with adaptive effector responses, unveiling a novel host-pathogen interaction axis and advancing MPs-based strategies against brucellosis.

This study investigated DNA methylation-related changes in cows during early pregnancy and constructed gene pools associated with pregnancy. We used peripheral blood samples from six healthy, productive, and similar cows 30 days post-insemination (Pre 30) and six non-pregnant cows (Ctrl), and performed association analysis using Reduced Representation Bisulfite Sequencing (RRBS) and transcriptomic sequencing methods. Targeted bisulfite sequencing (TBS) and quantitative real-time PCR (qRT-PCR) methods were used to identify differentially methylated genes. An obvious trend of hypomethylation was observed within the whole genome of Pre 30 dairy cows. A total of 76,530 differentially methylated sites (DMCs) were identified, comprising 32,886 hypermethylated sites and 43,644 hypomethylated sites. Additionally, there were 16,411 differentially methylated regions (DMRs), including 8,455 hypomethylated regions and 7,956 hypermethylated regions. RNA-Seq identified 44 significantly differentially expressed genes (DEGs), including 25 up and 19 down-regulated DEGs. Gene ontology (GO) analysis revealed significant enrichment in the developmental process, cell differentiation, and nervous system development in biological processes; Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis demonstrated significant enrichment in cancer-related pathways, such as cancer overview and cellular and gastric cancer and immune disease signaling pathways. We validated the methylation patterns of 17 genes and showed that the overall trend was hypomethylation, consistent with sequencing results of RRBS. qRT-PCR showed that HBB, HBA1 and GPR4 were significantly increased in the Pre 30 group (P < 0.05), whereas DOK7 and ISG15 were significantly decreased (P < 0.05), consistent with transcriptome sequencing. Our data encompass a diverse range of transcriptional dynamics and DNA methylation alterations observed during the early stages of pregnancy in dairy cows, offering novel insights for subsequent investigations into epigenetic changes occurring during this critical period.

Bovine viral diarrhea virus (BVDV) continues to threaten the global cattle industry and can also infect other ruminants, including sheep. Currently available vaccines still have limitations in terms of safety, durability of immunity, and protective efficacy. To investigate a candidate multivalent BVDV subunit vaccine, four recombinant BVDV proteins (NS5A, E0, E2, and mE2T) were formulated as monovalent subunit vaccines, and a multivalent combined subunit vaccine (CSV) was further constructed by mixing the four antigens in equal proportions. Using a susceptible sheep model, we evaluated BVDV-specific antibody responses, lymphocyte proliferation, serum cytokine concentrations, neutralizing antibody activity, and whole-blood BVDV RNA loads following experimental challenge. The results showed that CSV significantly increased BVDV-specific IgG levels and the IgG2a/IgG1 ratio, enhanced peripheral blood lymphocyte proliferation, and increased serum concentrations of IFN-γ, TNF-α, and IL-4. Virus neutralization assays showed that sera from CSV-immunized sheep exhibited measurable neutralizing activity against the BVDV-1a NADL strain. Following intraperitoneal challenge with the cytopathic BVDV-1a NADL strain, the CSV group showed lower whole-blood BVDV RNA loads than the PBS group at the evaluated time points, while reduced RNA loads were also observed in the IB.V group. These findings show that CSV induced measurable immune responses and was associated with lower whole-blood BVDV RNA loads during the 2-week post-challenge observation period. This study supports the feasibility of a multivalent combination strategy for BVDV subunit vaccine development and provides an experimental basis for subsequent evaluation in cattle, including assessment of fetal protection and prevention of persistent infection.

Currently, many detection methods for porcine reproductive and respiratory syndrome virus have been developed, However, the optimal laboratory diagnostic method remains controversial. To evaluate the diagnostic accuracy of PRRSV detection methods based on systematic reviews and meta-analyses, and to determine the optimal strategy for laboratory detection of PRRSV. Articles published between 1 January 2015 and 1 January 2025 were retrieved from multiple databases. Based on different detection methods, the articles were divided into three categories: traditional immunological techniques, molecular amplification techniques, and convergent diagnostic technologies. The sensitivity and specificity of each study were calculated. Diagnostic accuracy was assessed using threshold value definitions, ROC curve analysis, and statistical methods. Meta-analysis was performed using a random-effects model and pooled SROC curves. Stratified analysis and meta-regression were used to address effect size variability caused by differences in detection targets, tissue samples tested, and control trial designs. A total of 55 articles on traditional immunological techniques (involving 17,359 samples), 90 articles on molecular amplification techniques (involving 21,362 samples), and 14 articles on convergent diagnostic technologies (involving 1,289 samples) were included in the meta-analysis. In the 55 studies on traditional immunological techniques, the overall sensitivity was 0.93–0.94 (95

Bovine viral diarrhea (BVD) poses a substantial economic threat to the cattle industry, necessitating vaccines with broad cross-protective immunity. Heterologous prime-boost immunization represents a promising strategy to broaden immune protection by engaging complementary immunological pathways across vaccine platforms. Here, we evaluated heterologous prime-boost regimens combining recombinant subunit, adenovirus-vectored, and DNA vaccines, all targeting the E2 glycoproteins of BVDV-1 (I E2) and BVDV-2 (II E2; I E2-II E2), in a BALB/c mouse model. The DNA/subunit and DNA/adenovirus regimens elicited potent cellular and humoral immunity. Crucially, DNA vaccine priming preferentially skewed the immune response toward a Th1-biased phenotype, optimizing the overall immune profile. Our findings confirm that heterologous prime-boost strategies can synergistically enhance the cross-protective efficacy of BVD vaccines, providing a solid foundation for future development, pending further validation in cattle.

Porcine reproductive and respiratory syndrome (PRRS) is one of the most economically devastating diseases affecting the global pig industry. Host microRNAs directly target viral gene regions to exert their disease-fighting effects. PRRS virus (PRRSV) infection upregulates miR-361-3p expression; however, it is unclear whether it can exert inhibitory effects by directly targeting viral genes. Bioinformatic and experimental findings revealed that miR-361-3p inhibited PRRSV replication by directly targeting the PRRSV ORF1b and ORF1a loci. Intramuscular injection of pcDNA3.1-pri-miR-361 verified the expression of miR-361-3p in mammals. In summary, miR-361-3p plays an important role in infection and may be a promising therapeutic target for PRRS, providing insights into possible drug therapies.

BACKGROUND:A secondary Pasteurella multocida (Pm) infection following Mycoplasma ovipneumoniae (Mo) challenge in sheep results in severe respiratory disease. Scavenger receptor A (SRA) is a key phagocytic receptor on macrophages, which facilitates microbial clearance. However, the role of sheep SRA in Mo-associated secondary Pm infection is less understood. METHODS AND RESULTS:The expression of SRA was found to be the highest in alveolar macrophages (AMs) compared with lung tissue and spleen using quantitative polymerase chain reaction. Also, SRA was expressed in various tissues of sheep. SRA overexpression significantly enhanced Pm uptake by CHO cells, whereas fucoidan (a type A scavenger receptor-specific blocker) markedly reduced bacterial phagocytosis by AMs. This indicated an essential role of sheep SRA in macrophage phagocytosis of Pm. However, Mo infection downregulated SRA expression and impaired AM-mediated phagocytosis of Pm; this effect was reversed by SRA overexpression. Furthermore, we presumed that this might be due to the inhibition of SRA expression by tumor necrosis factor-alpha and interleukin 6, thereby suppressing SRA-dependent phagocytosis. CONCLUSIONS:The findings of this study elucidate the role of SRA in Mo-induced secondary Pm infection and provide some support for in-depth exploration in subsequent studies.

Porcine reproductive and respiratory syndrome virus (PRRSV) infection inflicts enormous economic losses on the global swine industry and imposes significant pressure on agricultural production. However, there are currently no clinically approved effective therapeutics specifically targeting PRRSV. Accordingly, the development of novel antiviral agents against PRRSV is urgently needed. Notably, the structural glycoprotein 4 (GP4) of PRRSV—which plays a crucial role in viral entry into host cells—represents a promising target for antiviral development. Nanobodies, characterized by their small size, structural stability, high affinity, and excellent solubility, have emerged as attractive candidates for next-generation therapeutic development. Yet, to date, no specific nanobodies targeting PRRSV GP4 have been reported. In this study, we isolated GP4-specific nanobodies using phage display technology and investigated their mechanisms underlying viral suppression through a series of in vitro functional assays. Our results demonstrate that Nb6, Nb31, and Nb85 significantly inhibit PRRSV infection by disrupting both viral attachment to host cells and subsequent internalization processes. Collectively, these findings indicate that Nb6, Nb31, and Nb85 hold substantial potential for development as antiviral agents against PRRSV infection.

Bovine viral diarrhea virus (BVDV) is a major pathogen responsible for significant economic losses in the global cattle industry. The diverse transmission routes and the characteristics of asymptomatic infections make it difficult to contain the spread; there is an urgent need to develop new effective antiviral strategies. Nanobodies (Nbs) have become a promising new type of antiviral agent due to their advantages, including small molecular size, stable structure, high specificity, and ease of production. This study successfully screened a specific nanobody, Nb7, targeting the key functional protein NS5A of BVDV using phage display technology. Furthermore, the nanobody was effectively delivered into Madin–Darby bovine kidney (MDBK) cells by fusing it with the cell-penetrating peptide TAT. The results demonstrate that TAT-Nb7, specifically targeting the non-structural protein NS5A of BVDV, significantly inhibits viral replication in MDBK cells. In conclusion, this study indicates that TAT-Nb7 holds promise as a therapeutic candidate for the prevention and control of BVDV infection.

Infection with Mycoplasma hyopneumoniae (M. hyopneumoniae) leads to chronic infectious pneumonia in pigs, resulting in significant distress and economic losses in the global pig industry. The pathogen secretes various proteins, including toxins, adhesins, and virulence-related enzymes, which facilitate adhesion, invasion, and immune evasion processes between bacteria and the host. However, the effector proteins of M. hyopneumoniae are predominantly uncharacterized. In this study, we demonstrate that the nuclease Mhp597 functions as a potential effector protein of M. hyopneumoniae, and we elucidate its mechanism of action in facilitating immune evasion. Our findings indicate that Mhp597 exhibits high expression efficiency in host cells and significantly inhibits IFN-alpha and IFN-beta protein expression. Using yeast two-hybrid and co-immunoprecipitation experiments, we established that Mhp597 interacts with porcine alveolar macrophage vimentin (Vim) via specific amino acid residues (Arg 232, Lys 256, Phe 263, and Lys 317). Further analysis revealed that Mhp597 inhibited the phosphorylation of TBK1 and IRF3 via Vim, thereby suppressing type I interferon (IFN-I) production and promoting the proliferation of M. hyopneumoniae within host cells. In conclusion, this study provides the first detailed account of the molecular mechanism by which Mhp597 negatively regulates the TBK1-IRF3-IFN-I signaling pathway through Vim, thus facilitating immune evasion and proliferation of M. hyopneumoniae within host cells. These findings enhance our understanding of the pathogenic mechanisms of M. hyopneumoniae and suggest potential molecular targets for the development of novel therapeutic strategies.

In recent years, the roles of Exosomes in tumors and infectious diseases have been found to have potential application value in disease diagnosis and treatment. However, whether Exosomes are involved in host-acquired immune responses against Brucella has not been reported. This study explored the significance of Exosomes in the context of Brucella infection and their influence on host immune responses. Additionally, we assessed the immunoprotective efficacy of these Exosomes in a murine model. The findings demonstrate that Exosomes played an important role in immune regulation during Brucella infection, enhancing the host's anti-Brucella immune response and inhibiting intracellular bacterial survival. A novel mechanism by which antigens are transmitted between immune cells through Exosomes to initiate an adaptive immune response against Brucella was also identified. These insights contribute to a deeper understanding of Brucella pathogenesis and host immune regulation, offering a promising avenue for the development of innovative Brucella vaccines or immune adjuvants for brucellosis.

Mixed infections of Mannheimia haemolytica and Mycoplasma bovis are relatively common in bovine respiratory diseases, presenting severe respiratory symptoms and high mortality that severely endanger the cattle industry. In this study, a serotype A1 strain of Mannheimia haemolytica, designated as XJCJMh1, was isolated and identified from the lung tissue of a hybrid Simmental calf infected with Mycoplasma bovis. The pathogenicity of this strain was evaluated using Kunming mice as a model. The results indicated that infection with XJCJMh1 caused pathological manifestations such as pulmonary hemorrhage and edema in mice. Subsequently, the genome of this strain was sequenced and assembled using Illumina sequencing to obtain general genomic features. The genome was annotated and analyzed for gene functions using the Swiss-Prot, NR, GO, COG, KEGG, CAZy, TCDB, and Pfam databases. Additionally, the virulence factors and resistance genes of this strain were annotated using the PHI, VFDB, and CARD databases. The genome of Mannheimia haemolytica XJCJMh1 is 2,595,489 base pairs (bp) in length, with a GC content of 40.93%. Notably, this strain exhibits three distinct genomic islands and contains 98 effectors associated with the type III secretion system (T3SS). The XJCJMh1 strain harbors 74 virulence genes and 45 resistance genes. We annotated the proteins, genes, and associated GO and KEGG pathways of the XJCJMh1 strain; exploring the relationship between these annotations and the strain’s pathogenicity is of considerable value. This study is of great significance for clarifying the pathogenic mechanism and genetic characteristics of the Mannheimia haemolytica strain XJCJMh1 in cattle, and its results provide a scientific reference for analyzing the genomic basis of pathogenicity and drug resistance of Mannheimia haemolytica under co-infection conditions.

Mycoplasma bovis is an important pathogen that is associated with respiratory diseases, mastitis, and arthritis in cattle, leading to significant economic losses in the global cattle industry. Most notably in this study, we pioneer the discovery that its secreted effector ENO1 (α-enolase) directly targets host cytoskeletal proteins for metabolic–immune regulation. Using an innovative GST pull-down/mass spectrometry approach, we made the seminal discovery of β-actin (ACTB) as the primary host target of ENO1—the first reported bacterial effector–cytoskeleton interaction mediating metabolic reprogramming. ENO1–ACTB binding depends on a hydrogen bond network involving ACTB’s 117Glu and 372Arg residues. This interaction triggers (1) glycolytic activation via Glut1 upregulation, establishing Warburg effect characteristics (lactic acid accumulation/ATP inhibition), and (2) ROS-mediated activation of dual inflammatory axes (HIF-1α/IL-1β and IL-6/TNF-α). This work establishes three groundbreaking concepts: (1) the first evidence of a pathogen effector hijacking host ACTB for metabolic manipulation, (2) a novel ‘glycolysis–ACTB–ROS-inflammation’ axis, and (3) the first demonstration of bacterial proteins coordinating a Warburg effect with cytokine storms. These findings provide new targets for anti-infection therapies against Mycoplasma bovis.

Interleukin-6 (IL-6) is a crucial cytokine involved in inflammation and immune regulation. However, the detection of IL-6 with ultrasensitivity and high specificity remains a significant challenge due to the inherent complexity of biofluids. Herein, we present a digital surface enhanced Raman scattering (SERS) immunoassay using core-shell Au@Ag-Au nanotags for IL-6 detection with ultrasensitivity and high reliability. A low-cost silicon chip was functionalized as capture substrates, employing novel SERS nanotags that exhibit strong, robust and reproducible signals at single-nanoparticle resolution as the amplification element. We proposed two analytical methods to validate single-molecule events follow a Poisson distribution and to quantify protein biomarkers over a broad linear dynamic range, respectively. The strong alignment between theoretical and experimental results enhances the method's reliability. Our assay provides two readouts: colorimetric analysis by naked eyes for high concentrations (>1 ng/mL) and digital SERS analysis for low concentrations. Following method optimization, we obtained a linear range from 100 fg/mL to 1 ng/mL (R2 = 0.994) with a limit of detection (LOD) of 12.4 fg/mL, suitable for clinical applications. The method was tested for IL-6 quantification in healthy human serum and saliva, with recoveries from 92.4% to 105.3%. Finally, the immunoassay demonstrated strong consistency with the standard clinical laboratory method when tested with clinical serum samples. Thus, our proposed the digital SERS immunoassay is a promising tool for the precision clinical diagnosis of IL-6-related diseases or other conditions.

BACKGROUND:Brucellosis is a zoonotic disease that poses a significant threat to both animal husbandry and public health. Currently available vaccines for brucellosis are all live attenuated forms, which carry the risk of potential infectivity and provide a relatively limited range of protection. In contrast, inactivated vaccines are perceived to exhibit poor protective efficacy and fail to elicit effective cellular immunity. This study aimed to comprehensively evaluate the efficacy of the Brucella inactivated vaccine (CF) with the objective of developing a safer and more effective candidate for brucellosis vaccination. METHODS:Firstly, we evaluated the safety of CF in mice. Subsequently, we immunized mice with CF using various doses and methods, determining the optimal immunization dose and method through challenge testing. We evaluated the vaccine's immunogenicity by detecting the cellular and humoral immune levels induced by CF in mice, and assessed the vaccine's protective effect based on the post challenge organ bacterial load. Additionally, we evaluated the protective effects of dual doses and secondary immunization in guinea pigs. RESULTS:The results indicate that CF is safe and non-toxic; it induced significant increases in specific IgG antibody levels against Brucella during the early stages and markedly enhanced the T cell immune response, thereby promoting a Th1-biased immune response in mice. Following the challenge, CF demonstrated protective efficacy comparable to that of the S2 vaccine against B. melitensis biovar 3 infection in mice. CF-immunized guinea pigs were able to resist infection by B. melitensis M28 and B. melitensis biovar 3. CONCLUSIONS:In summary, CF significantly induces both humoral and cellular immunity in mice. This study reports for the first time that a safe and effective inactivated Brucella vaccine (CF) can induce cellular immune responses and effectively prevent animal brucellosis.

Brucellosis is one of the most common zoonotic diseases caused by Brucella spp. However, there is currently no Brucella vaccine available for humans. Although some attenuated live vaccines have been approved for animals, their protective efficacy is suboptimal. In previous studies, we utilized an epitope- and structure-based vaccinology platform to identify the immunodominant epitopes of Brucella antigens OMP19, OMP16, OMP25, and L7/L12, and constructed the multi-epitope vaccine MEV-Fc against Brucella. In this study, OMP19, OMP16, OMP25, and L7/L12, and MEV-Fc was expressed and purified via an Escherichia coli expression system, which validated that MEV-Fc possesses high immunological efficacy and exerts a significant protective effect in BALB/c mice within the Brucella infection model. MEV-Fc enhanced Th1 and Th2 immune responses and strongly induced the production of the pro-inflammatory cytokine IFN-γ. Furthermore, MEV-Fc protected mice against Brucella infection compared to control group (PBS). In conclusion, our results provide new insights and data support for the development of human Brucella vaccines.

Bovine viral diarrhea virus (BVDV) is one of the major viral pathogens responsible for respiratory disease complexes in cattle and other ruminants; it has spread worldwide and poses a significant threat to the cattle industry. To understand the prevalence and genetic diversity of BVDV in northern China, this study conducted an epidemiological survey of BVDV in dairy cows across 13 provinces in northern China from June 2022 to June 2024. A total of 2,199 nasal swab samples were analyzed by RT-PCR. The results revealed an overall positive rate of 6.05