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Live-vaccine delivery systems expressing two model antigens from Mycoplasma hyopneumoniae, F2 P97 (Adh) and NrdF, were constructed using Salmonella enterica serovar Typhimurium aroA (STM-1), and immunogenicity in mice was evaluated. Recombinant plasmid-based expression (PBE) and chromosomally based expression (CBE) systems were constructed. The PBE system was formed by cloning both antigen genes into pJLA507 to create an operon downstream of temperature-inducible promoters. Constitutive CBE was achieved using a promoter-trapping technique whereby the promoterless operon was stably integrated into the chromosome of STM-1, and the expression of antigens was assessed. The chromosomal position of the operon was mapped in four clones. Inducible CBE was obtained by using the in vivo-induced sspA promoter and recombining the expression construct into aroD. Dual expression of the antigens was detected in all systems, with PBE producing much larger quantities of both antigens. The stability of antigen expression after in vivo passage was 100% for all CBE strains recovered. PBE and CBE strains were selected for comparison in a vaccination trial. The vaccine strains were delivered orally into mice, and significant systemic immunoglobulin M (IgM) and IgG responses against both antigens were detected among all CBE groups. No significant immune response was detected using PBE strains. Expression of recombinant antigens in S. enterica serovar Typhimurium aroA from chromosomally located strong promoters without the use of antibiotic resistance markers is a reliable and effective method of inducing a significant immune response.The use of live, attenuated bacteria as vaccine delivery systems for heterologous antigens has been extensively studied. In particular, attenuated Salmonella strains have been modified to express a wide range of antigens from bacterial, parasitic, and viral sources (reviewed in references 20, 29, and 41). After oral administration, Salmonella can penetrate the Peyer's patches via M cells and colonize the mesenteric lymph nodes, which contain various antigen-presenting cells (reviewed in reference 5). This can generate a range of immune responses, including systemic and mucosal responses at local and distal sites. Other advantages of using attenuated Salmonella include the ease of oral administration, which bypasses the need for needle administration; increased antigen presentation due to the use of a live-vector delivery system, the induction of both Th1 and Th2 immune responses; and the wide range of attenuated Salmonella and recombinant vectors available to researchers (19,20,51).However, there are a number of issues to overcome. Most methods for the expression of heterologous antigens in Salmonella use plasmids to express the antigenic proteins. This can have several drawbacks. The stable maintenance of the expression plasmid in vivo can be difficult to achieve. Tightly regulated promoters are often used to increase plasmid stability, and several in vivo-inducible promoters have delivered promising res...

The characterization of a polyelectrolyte complex material comprised of two biopolymers, a chitosan upper layer and a gellan gum under layer, is reported. It is shown that the upper layer of chitosan with incorporated levofloxacin displays an antibacterial activity, while the under layer of a gellan gum/TiO(2) composite supports the growth of fibroblastic cells.