Alfalfa is conserved in silo-type bunkers in the cold and humid regions of Inner Mongolia, China. Its quality is essential to ensure a healthy and sustainable dairy production. However, the impact of environmental factors on the microbiota and fermentation products of alfalfa silage remains unclear. The present research examined changes in the microbiota and fermentation products and their association with environmental parameters in 72 samples collected from 12 farms located at 4 different latitudes and longitudes across four regions. The samples were labeled with distinct codes, A, B, and C, from the cold–rainy region, D, E, and F, from the warm–rainy region, G, H, and I from the cold–dry region, and J, K, and L from the warm–dry region. The lactic acid levels ranged from 14.25 to 24.27 g/kg of DM across all samples. The pH and concentrations of NH3-N and butyric acid in samples A, B, and H were higher (p < 0.01) than in the other samples. Samples D and E had higher acetic acid concentrations and 1, 2-propanediol content (p < 0.01). The fresh material was dominated by Pantoea and Pseudomonas, whereas Lactobacillus was the most dominant genus in all silages, except for the B silage. The A, B, and H silages contained more Clostridium but less Lactobacillus than the other silages. The lactic acid levels were strongly associated with Lactobacillus plantarum, Weissella paramesenteroides, Lactobacillus acetotolerans, Pedobacter borvungensis, and Lactobacillus brevis (p < 0.01). In contrast, the pH and the NH3-N and butyric acid concentrations were strongly associated (p < 0.01) with the presence of Clostridium estertheticum. A correlation analysis revealed that precipitation, temperature, longitude, and latitude were the most critical factors influencing epiphytic microbes in the fresh material. After silage fermentation, low-temperature conditions significantly affected the fermentation products and microbial community composition. In conclusion, the microbial community of silages is distinctive in cold and humid regions, and climatic parameters ultimately affect the microbiota and fermentation products. Furthermore, the findings of this study demonstrate that Illumina MiSeq sequencing combined with environmental factor assessment might provide new information about the microbiota composition and fermentation quality of silages, facilitating the achievement of high-quality silage.

Brucella is a globally distributed zoonotic disease that can cause abortion and changes in immune function in humans and animals. At present, there is no good treatment plan for Brucella, and animals can only be treated harmlessly once they become ill, resulting in huge economic losses. Therefore, the prevention of Brucella infection is a very crucial step. Although a variety of Brucella vaccines have been widely used, they have varying degrees of shortcomings. For example, some Brucella vaccines have residual virulence, which leads to the emergence of Brucella in animals during the immunization process. Bacillus infection and other conditions occur. To further reduce the toxicity of the Brucella vaccine and enhance its protective effect on animals, this study used Antigen 85A (Ag85A) as a carrier of the Brucella vaccine to fuse with the Brucella S2 vaccine. The results of the study found that the S2-Ag85A oral Brucella vaccine could effectively reduce the toxicity residue of the S2 vaccine, stimulate the mice to produce a better immunogenic response, and effectively activate the expression levels of Brucella heterozygous IgG1 and IgG2a. Experiments have shown that the expression of IFN-γ in the peripheral blood serum and spleen of mice is significantly increased, and the expression levels of IL-1β, TNF-α, and IL-6 are significantly reduced, which may indicate that S2-Ag85A oral Brucella vaccine could induce the expression of IFN-γ, thus downregulating the expression levels of IL-6 and TNF-α in the spleen tissue. The above results indicate that the S2-Ag85A oral vaccine is an effective attenuated vaccine for preventing Brucella infection.

Brucellosis is a common zoonotic disease caused by Brucella, which causes enormous economic loss and public burden to the epidemic areas. Earlier and precise diagnosis and timely culling of infected animals are crucial to prevent the infection of Brucella and the spread of the disease. In recent years, RNA-guided CRISPR/Cas12a nucleases have shown great promise in nucleic acid detection. This research aims to develop a CRISPR/CAST (CRISPR/Cas12a Test strip) package that can rapidly detect Brucella nucleic acid on-site screening, especially on the remote family pasture. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and its associated protein 12a (Cas12a), the CRISPR/Cas12a system combined with recombinase polymerase amplification(RPA), and lateral flow read-out. The CRISPR/CAST package can complete the assay of Brucella nucleic acid within 30 min under isothermal temperature conditions, with a sensitivity of 10 copies/μl, and no antigen cross-reacting against Yersinia enterocolitica O:9 , Escherichia coli O157 , Salmonella enterica serovar Urbana O:30 , and Francisella tularensis. The serum samples of 398 sheep and 100 cattle were tested by CRISPR/CAST package, of which 31 sheep and 8 cattle were Brucella DNA positive. The detection rate was consistent with the qPCR and higher than the Rose Bengal Test (RBT, 19 sheep, and 5 cattle were serum positive). CRISPR/CAST package can accurately detect the infected livestock’s Brucella DNA and accomplish within 30 min, which has the advantages of simple, fast, high sensitivity, and strong specificity, with no window period. Besides, the package needs no expensive equipment, standard laboratory, or professional operators. It is an effective tool for field screening and earlier, rapid diagnosis of Brucella infection. A package is an efficient tool for epidemic prevention and control.