In vivo-induced argininosuccinate lyase plays a role in the replication of Brucella abortus in RAW264.7 cells Brucellosis caused by Brucella species is a zoonotic disease with a serious impact on public health and the livestock industry. To better understand the pathogenesis of the disease, in vivo-induced antigen technology (IVIAT) was used to investigate the in vivo-induced antigens of Brucella abortus in this study. A genomic expression library of B. abortus was constructed and screened using pooled bovine B. abortus-positive sera by IVIAT. In total, 33 antigens were identified. Five antigens were further expressed and tested for their seroreactivity against 33 individual bovine B. abortus-positive sera by Western blot analysis. The results showed a highest positive rate of 32/33 for argininosuccinate lyase (ASL), indicating that ASL may be used as a candidate marker for serodiagnosis of brucellosis. Furthermore, an asl gene-deleted mutant strain S2308DASL was constructed, and the intracellular survival and replication of the mutant strain in RAW264.7 cells were investigated. Interestingly, the numbers of bacteria recovered from cells infected with mutant strain S2308DASL were similar at all time points observed from 0 h to 96 h post-infection, suggesting the asl gene plays an important role in the bacterial replication in RAW264.7 cells. Real-time quantitative PCR (qPCR) analysis showed that the mRNA levels in S2308DASL were decreased for BvrR, BvrS and virB5 when compared with those in S2308 (P,0.05). Our results not only expand the knowledge of Brucella intracellular replication but also expand the list of candidates for serodiagnostic markers of brucellosis.
INTRODUCTIONBrucella abortus is a Gram-negative, intracellular bacterium that can survive within a variety of cells and maintain a long-lasting interaction with the host cells (Han et al., 2012). Compared with other bacterial pathogens, B. abortus does not display classical virulence factors such as exotoxins, capsule, toxic LPS or plasmids. The virulence of B. abortus depends on its properties of survival and replication in host cells (Seleem et al., 2008). It is generally accepted that bacterial genes induced specifically during infection may be essential for bacterial survival and contribute to the pathogenesis of a disease. Therefore, an understanding of the genes involved in B. abortus-host interactions in vivo is crucial for understanding the pathogenesis of brucellosis. Recently, in vivo-induced antigen technology (IVIAT) has been developed to identify immunogenic bacterial genes expressed specifically during infection at the genomic level using sera from infected animals; this technique avoids the limitations of animal models (Gu et al., 2009;Hang et al., 2003;Harris et al., 2006;Kumar et al., 2011;Rollins et al., 2005Rollins et al., , 2008Zygmunt et al., 2006).In this study, IVIAT was used to identify B. abortus antigens induced in vivo, in which pooled bovine B. abortus-positive sera were used for the screening. In addition, selected antigen...