Gang Zhang

Dairy cows are economically significant ruminants in China, and the dairy industry is closely linked to food safety and the agricultural economy. However, various factors such as pathogenic microorganisms often lead to frequent diseases in dairy cows. Furthermore, as potential hosts for diverse viruses, dairy cows can harbor zoonotic pathogens, which pose a threat to public health. The Shaanxi–Gansu–Ningxia region boasts abundant natural resources and extensive pastures. It is a major animal husbandry base in Northwest China, and dairy farming plays a significant role in the local economy. However, research on dairy cow virus diversity in this region remains limited; epidemic prevention and control capabilities are constrained, and the risk of disease outbreaks is elevated. In this study, 790 dairy cow samples were collected from 13 large-scale farms and free-range households in the Shaanxi–Gansu–Ningxia region from 2021 to 2023. Sample types consisted of nasal and anal swabs. Six viral metagenomic libraries were constructed and analyzed using high-throughput sequencing and bioinformatics methods, leading to the identification of 51 viral families. These comprised 16 positive-sense single-stranded RNA virus families, one Retroviridae family, four double-stranded RNA virus families, 21 double-stranded DNA virus families, and nine single-stranded DNA virus families. Among these, RNA viruses were represented by families such as Astroviridae, Coronaviridae, Caliciviridae, Picornaviridae, and Picobirnaviridae; DNA viruses were primarily detected in Circoviridae, Papillomaviridae, Genomoviridae, and Smacoviridae. Alpha diversity analysis revealed no significant differences in viral diversity and abundance among the three regions (p > 0.05); however, significant differences were observed in the read counts and proportions of RNA and DNA viruses across the provinces. Phylogenetic analysis further indicated that viruses carried by dairy cows exhibit considerable genetic diversity and pose potential cross-species transmission risks. This study established a reference database for the dairy cow virome in the Shaanxi–Gansu–Ningxia region, elucidated the phylogenetic relationships of key viruses, and provided a scientific basis for future monitoring and prevention of dairy cow viruses.

Background/Objectives: Mycobacterium tuberculosis is the causative agent of tuberculosis and the leading cause of death from a single infection with the microorganism. Tuberculosis remains globally one of the major diseases leading to high mortality rates, with serious implications for public health and economic development. Therefore, tuberculosis prevention and control is crucial for global health and socio-economic stability. The development of effective preventive vaccines remains an urgent task in the fight against tuberculosis. Methods: The Mycobacterium tuberculosis antigen PPE18 was fused to Zera, and Bacmid was extracted and transfected into Sf9, which was purified and characterized for the formation of nanoparticle protein bodies. BALB/c mice and calves were immunized, and the immunogenicity of the nanoparticle vaccine was assessed by serum antibodies and splenic lymphocytes. Results: Zera-71CA-mCherry can be expressed in Sf9 cells, forming 0.5–1.2 μm protein bodies. Excising the mCherry sequence, Zera-71CA/Zera-PPE18 candidate nanoparticle-immunized mice were able to elicit serum antibody levels and the proliferation of splenic lymphocytes, and immunized calves were determined to have high levels of serum antibody levels, and IFN-γ and TNF-α levels. Conclusions: The results indicated that Zera-71CA/Zera-PPE18 recombinant nanoparticles had good immunogenicity as a subunit vaccine in both BALB/c mice and calves and are potential candidates for further development as effective subunit vaccines.

Tuberculosis (TB), a human and animal disease caused by Mycobacterium tuberculosis (M.tb), has the highest global mortality rate after coronavirus disease 2019 (COVID-19) and poses a major public health threat globally. Since 1890, vaccine candidates for various forms of TB have been developed for different age groups, but these vaccine candidates have not provided intended protection in adolescents and adults in clinical trials. To help prevent and control the spread of TB, the development of a safe and effective TB vaccine is imperative. The MTB39A protein and the molecular adjuvant MTB32C protein were expressed by an insect-baculovirus expression system, and the recombinant baculovirus surface-displayed particles were evaluated for their immunogenicity in BALB/c mice and calves. The results showed that the rvAc-71CA/rvAc-MTB39A recombinant baculovirus surface-displayed particles exhibited good immunogenicity in mice and calves and could be further developed as potential candidates.

The objective of this study was to develop an indirect ELISA utilizing a polyclonal antibody against bovine rotavirus (BRV) VP6 protein. To achieve this, pcDNA3.1-VP6, a recombinant eukaryotic expression plasmid, was constructed based on the sequence of the conserved BRV gene VP6 and was transfected into CHO-K1 cells using the transient transfection method. The VP6 protein was purified as the coating antigen using nickel ion affinity chromatography, and an indirect ELISA was subsequently established. The study found that the optimal concentration of coating for the VP6 protein was 1 μg/mL. The optimal blocking solution was 3% skim milk, and the blocking time was 120 min. The secondary antibody was diluted to 1:4000, and the incubation time for the secondary antibody was 30 min. A positive result was indicated when the serum OD450 was greater than or equal to 0.357. The coefficients of variation were less than 10% both within and between batches, indicating the good reproducibility of the method. The study found that the test result was positive when the serum dilution was 2¹⁷, indicating the high sensitivity of the method. A total of 24 positive sera and 40 negative sera were tested using the well-established ELISA. The study also established an indirect ELISA assay with good specificity and sensitivity for the detection of antibodies to bovine rotavirus. Overall, the results suggest that the indirect ELISA method developed in this study is an effective test for detecting such antibodies.

Bovine diarrhea virus (BVDV) is considered to be the most common pathogen of severe diarrhea in cattle worldwide, with clinical manifestations of fever, diarrhea, ulcers, and abortions, which cause significant economic losses to the cattle industry. The establishment of an efficient, rapid and sensitive assay suitable for field conditions is conducive to the early detection of pathogens and the implementation of relevant treatments. Recombinase polymerase amplification (RPA) is a novel nucleic acid amplification method that has been widely used in the diagnosis of infectious diseases. In this paper, an RPAS assay for recombinase polymerase amplification combined with SYBR Green I was developed for the rapid detection of BVDV. The assay was completed at a constant temperature of 37℃ for 25 min, and the minimum detection limit of RPA was 1×10¹ copies/µL for gel electrophoresis. Under sunlight, the minimum detection limit of BVDV RPAS visualization was 1×10⁹ copies/µL; Under UV, the minimum detection limit of BVDV RPAS was 1×10⁵ copies/µL. The assay has no cross-reactivity with other viruses causing bovine gastrointestinal and respiratory tract viruses. Clinical samples had equal BVDV RPA, RPAS, and PCR detection thresholds, and SYBR Green I visualization was evident. In conclusion, the BVDV-RPAS established in this study, with high sensitivity and specificity, has the potential to be used as a powerful tool for BVD prevention and control.