Brucellosis is caused by Brucella spp.; it can result in fetal loss and abortion, resulting in economic losses and negative effects on human health. Herein, a cross-sectional study on the epidemiology of Brucella spp. in aborted livestock in Ningxia from 2022 to 2023 was conducted. A total of 749 aborted tissue samples from 215 cattle and 534 sheep were collected from farmers who reported abortions that were supported by veterinarians trained in biosecurity. The samples were analyzed using qPCR and were cultured for Brucella spp. when a positive result was obtained; the samples were speciated using AMOS-PCR. MLST and MLVA were employed for genotype identification. The results demonstrated that 8.68% of the samples were identified as being positive for Brucella spp. based on qPCR results. In total, 14 field strains of Brucella spp. were subsequently isolated, resulting in 11 B. melitensis, 2 B. abortus, and 1 B. suis. being identified via AMOS-PCR. Four sequence types were identified via MLST—ST7 and ST8 (B. melitensis), ST2 (B. abortus), and ST14 (B. suis)—with ST8 predominating. Five MLVA-8 genotypes and seven MLVA-11 genotypes were identified, with MLVA-11 GT116 predominating in livestock. Thus, at least three Brucella species are circulating in aborted livestock in Ningxia. This suggests a significant risk of transmission to other animals and humans. Therefore, disinfection and safe treatment procedures for aborted livestock and their products should be carried out to interrupt the transmission pathway; aborted livestock should be examined to determine zoonotic causes and targeted surveillance should be strengthened to improve the early detection of infectious causes, which will be of benefit to the breeding industry and public health security.

Mycobacterium tuberculosis (Mtb) infection can trigger immune responses in macrophages during its autophagy and apoptosis, as two crucial cellular processes which are essential for the host to clear the pathogen. Resveratrol (RES) is a natural polyphenolic compound with various biological functions such as anti-inflammatory, antioxidant, and immunomodulatory activities. However, the specific mechanism by which it regulates autophagy and apoptosis in a Bacillus Calmette-Guérin (BCG)-infected macrophage model remains to be elucidated. This study aimed to analyze the effects of RSE on BCG-induced autophagy and apoptosis in macrophages and explore the underlying molecular mechanisms. The mouse macrophage cell line RAW264.7 was used to establish a BCG infection model and evaluate the interventional effects of RES. Cell viability was detected by CCK-8 assay. The expression levels of autophagy- and apoptosis-related proteins were analyzed by Western blotting. LC3 puncta formation was observed by immunofluorescence. Autophagy rate and apoptosis rate were determined by flow cytometry. The ultrastructure of autophagosomes and autolysosomes was observed by transmission electron microscopy. The results showed that BCG infection significantly increased the expression of LC3-II, Beclin1, Atg5, and Atg7 in macrophages; decreased p62 levels; and increased the number of intracellular autophagosomes and autolysosomes. Concurrently, BCG infection also induced apoptosis, including upregulation of Bax, Caspase 3, Caspase 9, and PARP1 expression; downregulation of Bcl-2 expression; and an increased apoptosis rate. RES promoted autophagosome accumulation and inhibited apoptosis in BCG-infected macrophages in a concentration-dependent manner; however, these effects were significantly attenuated after SIRT1 gene silencing by siRNA and inhibition by EX527. Therefore, it is speculated that RES may regulate autophagy and apoptosis through the SIRT1/FOXO1 pathway. These findings provide a potential experimental basis for developing RES into a host-directed therapeutic (HDT) agent, and offer new insights into the mechanisms by which natural products participate in host defense.

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.