ABSTRACT
Background and purpose: The sequence of Omp25 is conserved in all Brucella species. The high antigenicity of the product of this gene stimulates the host's immune system. Using engineered probiotic bacteria is an appropriate method for vaccine transport. The aim of this study was to express the Omp25 of the Brucella abortus pathogenic bacterium in Lactococcus lactis probiotic bacterium. Materials and methods: In this experimental study, the required vector was designed and synthesized to include the gene of interest and a signal peptide (pNZ8148-Usp45-Omp25). E. coli strain TOP10F was transformed using the pNZ8148-Usp45-Omp25 expression vector based on induction by nisin. The recombinant plasmid was extracted from the transformed bacteria using a plasmid extraction kit. The L. lactis was transformed by pNZ8148-Usp45-Omp25 vector using electroporation. Evaluation of the expression of Omp25 gene at the RNA level was assessed by reverse transcription method and confirming the presence of recombinant Omp25 protein in the engineered bacteria using SDS-PAGE method. Results: Successful expression of B. abortus Omp25 in L. lactis was verified by RT-PCR. Subsequently, the proteins were separated based on molecular weight using sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE). The protein expression analysis showed the expression of Omp25 as a 25 kDa extra band in transformed L. lactis compared to the L. lactis receiving the vector lacking the target gene. Conclusion: This study shows that Omp25 is expressed in L. lactis transformed via pNZ8148-Usp45-Omp25 by electroporation. Transformed L. lactis can be successfully used as a subunit oral vaccine in prevention of Brucellosis.
ABSTRACT
Millions of Norway rats (Rattus norvegicus) inhabit New York City (NYC), presenting the potential for transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from humans to rats. We evaluated SARS-CoV-2 exposure among 79 rats captured from NYC during the fall of 2021. Our results showed that 13 of the 79 rats (16.5%) tested IgG- or IgM-positive, and partial SARS-CoV-2 genomes were recovered from all 4 rats that were qRT-PCR (reverse transcription-quantitative PCR)-positive. Genomic analyses suggest these viruses were associated with genetic lineage B, which was predominant in NYC in the spring of 2020 during the early pandemic period. To further investigate rat susceptibility to SARS-CoV-2 variants, we conducted a virus challenge study and showed that Alpha, Delta, and Omicron variants can cause infections in wild-type Sprague Dawley (SD) rats, including high replication levels in the upper and lower respiratory tracts and induction of both innate and adaptive immune responses. Additionally, the Delta variant resulted in the highest infectivity. In summary, our results indicate that rats are susceptible to infection with Alpha, Delta, and Omicron variants, and wild Norway rats in the NYC municipal sewer systems have been exposed to SARS-CoV-2. Our findings highlight the need for further monitoring of SARS-CoV-2 in urban rat populations and for evaluating the potential risk of secondary zoonotic transmission from these rat populations back to humans. IMPORTANCE The host tropism expansion of SARS-CoV-2 raises concern for the potential risk of reverse-zoonotic transmission of emerging variants into rodent species, including wild rat species. In this study, we present both genetic and serological evidence for SARS-CoV-2 exposure to the New York City wild rat population, and these viruses may be linked to the viruses that were circulating during the early stages of the pandemic. We also demonstrated that rats are susceptible to additional variants (i.e., Alpha, Delta, and Omicron) that have been predominant in humans and that susceptibility to infection varies by variant. Our findings highlight the reverse zoonosis of SARS-CoV-2 to urban rats and the need for further monitoring of SARS-CoV-2 in rat populations for potential secondary zoonotic transmission to humans.