Background: pUL21 is a conserved protein of Alphaherpesvirinae that performs multiple important functions. The C-terminus of pUL21 in other members of this subfamily has RNA-binding ability; this domain contributes to pseudorabies virus (PRV) retrograde axonal transport in vitro and in vivo and participates in newly replicated viral DNA packaging and intracellular virus transport. However, knowledge regarding duck enteritis virus (DEV) pUL21 is limited. Results: We verified that DEV UL21 is a γ2 gene that encodes a structural protein. Moreover, we observed that pUL21 localized to the nucleus and cytoplasm. DEV pUL21 interacted with pUL16 and formed a complex in transfected human embryonic kidney (HEK) 293 T cells and DEV-infected duck embryo fibroblasts (DEFs). These results were further confirmed by CO-IP assays. Conclusions: The DEV UL21 gene is a late gene, and pUL21 localizes to the nucleus and cytoplasm. DEV UL21 is a virion component. In addition, pUL21 can interact with pUL16. These findings provide insight into the characteristics of UL21 and the interaction between pUL21 and its binding partner pUL16. Our study enhances the understanding of DEV pUL21.

Brucella, a zoonotic facultative intracellular pathogenic bacterium, poses a significant threat both to human health and to the development of the livestock industry. Alanine racemase (Alr), the enzyme responsible for alanine racemization, plays a pivotal role in regulating virulence in this bacterium. Moreover, Brucella mutants with alr gene deletions (Δalr) exhibit potential as vaccine candidates. However, the mechanisms that underlie the detrimental effects of alr knockouts on Brucella pathogenicity remain elusive. Here, initially, we conducted a bioinformatics analysis of Alr, which demonstrated a high degree of conservation of the protein within Brucella spp. Subsequent metabolomics studies unveiled alterations in amino acid pathways following deletion of the alr gene. Furthermore, alr deletion in Brucella suis S2 induced decreased resistance to stress, antibiotics, and other factors. Transmission electron microscopy of simulated macrophage intracellular infection revealed damage to the cell wall in the Δalr strain, whereas propidium iodide staining and alkaline phosphatase and lactate dehydrogenase assays demonstrated alterations in cell membrane permeability. Changes in cell wall properties were revealed by measurements of cell surface hydrophobicity and zeta potential. Finally, the diminished adhesion capacity of the Δalr strain was shown by immunofluorescence and bacterial enumeration assays. In summary, our findings indicate that the alr gene that regulates amino acid metabolism in Brucella influences the properties of the cell wall, which modulates bacterial adherence capability. This study is the first demonstration that Alr impacts virulence by modulating bacterial metabolism, thereby providing novel insights into the pathogenic mechanisms of Brucella spp.

Brucella suis, the causative agent of brucellosis, poses a significant public health and animal husbandry threat. However, the role of the alanine racemase (alr) gene, which encodes alanine racemase in Brucella, remains unclear. Here, we analyzed an alr deletion mutant and a complemented strain of Brucella suis S2. The knockout strain displayed an unaltered, smooth phenotype in acriflavine agglutination tests but lacked the core polysaccharide portion of lipopolysaccharide (LPS). Genes involved in the LPS synthesis were significantly upregulated in the deletion mutant. The alr deletion strain exhibited reduced intracellular viability in the macrophages, increased macrophage-mediated killing, and upregulation of the apoptosis markers. Bcl2, an anti-apoptotic protein, was downregulated, while the pro-apoptotic proteins, Bax, Caspase-9, and Caspase-3, were upregulated in the macrophages infected with the deletion strain. The infected macrophages showed increased mitochondrial membrane permeability, Cytochrome C release, and reactive oxygen species, activating the mitochondrial apoptosis pathway. These findings revealed that alanine racemase was dispensable in B. suis S2 but influenced the strain’s rough features and triggered the mitochondrial apoptosis pathway during macrophage invasion. The deletion of the alr gene reduced the intracellular survival and virulence. This study enhances our understanding of the molecular mechanism underlying Brucella’s survival and virulence and, specifically, how alr gene affects host immune evasion by regulating bacterial LPS biosynthesis.

To establish a rapid nucleic acid detection method for field screening of Brucella spp., a visual assay based on recombinase polymerase amplification (RPA) combined with CRISPR/Cas12a was developed. Four pairs of RPA primers and two crRNAs were designed according to the conserved Brucella bcsp31 sequence. The optimal RPA primer–crRNA combination was screened using agarose gel electrophoresis and fluorescence readout, and the concentrations of Cas12a protein, crRNA, and ssDNA fluorescent reporter were optimized. RPA primer pair 2 showed the best amplification performance, and RPA2-crRNA1 was selected as the optimal combination for the assay. The final reaction conditions were 200 nM Cas12a protein, 200 nM crRNA, and 250 nM ssDNA fluorescent reporter. The limit of detection of the established method was 2.47 × 10⁰ copies/μL. No cross-reactivity was observed with Mycoplasma capricolum subsp. capripneumoniae, Chlamydia abortus, Chlamydia psittaci, or Salmonella enterica serovar Typhimurium. Among 56 clinical sheep nasal swab samples collected during routine surveillance, qPCR identified 22 positive and 34 negative samples, while the RPA-CRISPR/Cas12a assay identified 20 positive and 36 negative samples. The positive agreement, negative agreement, overall agreement, positive predictive value, negative predictive value, and Cohen’s kappa value were 90.91%, 100.00%, 96.43%, 100.00%, 94.44%, and 0.924, respectively. These findings provide preliminary evidence that the developed bcsp31-targeted RPA-CRISPR/Cas12a assay is rapid and visually interpretable and may be useful for preliminary Brucella spp. screening in routine veterinary surveillance settings. Larger-scale validation using different sample types and samples from different regions is still required.

Brucellosis is an important zoonotic disease caused by Brucella spp., posing significant impacts on animal health, livestock production, and public health. Infected dairy animals can shed the pathogen in milk, making milk a noninvasive and readily accessible sample for disease surveillance. Therefore, detection of Brucella in milk can be used for monitoring brucellosis in dairy herds. In this study, polyclonal antibodies were produced by immunizing camels with lipopolysaccharide (LPS) derived from Brucella abortus vaccine strain 19 (A19). These antibodies were subsequently conjugated to magnetic beads to develop immunomagnetic beads (IMBs). The IMBs were combined with a sandwich ELISA for the enrichment and detection of Brucella in milk samples. The IMB-sELISA showed a limit of detection (LOD) of 1 × 10⁴ CFU/mL. The method reduced matrix interference and showed satisfactory analytical performance. This approach can be applied for on-farm sample screening and herd-level surveillance to support the diagnosis and control of brucellosis in dairy animals.

Brucellosis remains an important zoonotic disease for livestock production and public health, creating a need for rapid molecular screening methods with limited equipment requirements. We developed a visual recombinase polymerase amplification (RPA)-CRISPR/Cas12a assay targeting a 642-bp region of the Brucella omp25 gene. Comparative sequence analysis was used to assess target conservation, after which four RPA primer pairs and two CRISPR RNAs (crRNAs) were experimentally screened. RPA3-crRNA1 produced the strongest fluorescence response and was used for assay optimization. The final Cas12a reaction contained 250 nM Cas12a, 200 nM crRNA, and 200 nM single-stranded DNA fluorescent reporter. RPA amplification and Cas12a detection required 20 min each, and positive reactions produced visible yellow-green fluorescence under 470-nm blue light. No cross-reactivity was observed with the non-target pathogens included in the experimental panel. In silico comparisons with selected phylogenetically related taxa identified sequence differences within the final RPA3 primer-binding sites and crRNA1 recognition region, supporting a low predicted risk of sequence-based cross-reactivity for the taxa examined. Using purified plasmid DNA, the lowest DNA concentration detected under the experimental conditions tested was 2.72 copies/μL of input template, corresponding to an estimated 13.6 copies per RPA reaction. Among 96 bovine nasal swab samples collected during routine surveillance, agreement analysis against the qPCR comparator showed concordant results for 93 of 96 samples, with positive percent agreement (PPA), negative percent agreement (NPA), and overall percent agreement (OPA) of 100.00, 93.75, and 96.88%, respectively; three qPCR-negative samples showed weak RPA-CRISPR/Cas12a fluorescence and could not be independently resolved. Because the target region is conserved in the vaccine strains A19, S19, and RB51, the assay is intended for preliminary screening of brucellosis-associated Brucella DNA rather than species identification or differentiation of vaccine-derived and wild-type DNA. Broader validation with additional specimen types and specificity panels is required before routine diagnostic application.