, 2000; Cheung & Bolin, 2002). PCV2 vaccines under development include inactivated vaccine (Opriessnig et al., 2009), DNA vaccines (Kamstrup et al., 2004; An et al., 2008; Fan et al., 2008b), recombinant virus-vectored

vaccines (Ju et al., 2005; Wang et al., 2007; Fan et al., 2008a) and bacterial-vectored vaccines (Wang et al., 2008). SEZ is a widespread pathogen associated with swine streptococcal diseases. The M-like protein (SzP) is a cell surface-anchored protein of SEZ that conveys phagocytosis resistance (Hong-Jie et al., 2009) and is an excellent vaccine target. Meehan et al. (1998) immunized mice with the recombinant SzP derived from SEZ strain W60, which protected them from intraperitoneal challenge with the homologous strain. An SzP-deletion SEZ strain was attenuated and was able to elicit Selleckchem Panobinostat good protective immunity against challenge with the wild-type strain (Hong-Jie et al., 2009). An acapsular attenuated vaccine strain C55138 ΔhasB was constructed in our laboratory and showed good protection this website efficiency against SEZ challenge (data not shown). It is thus hypothesized that incorporation of PCV2 capsid protein into SzP of SEZ strain C55138 ΔhasB (SEZ-Cap) could further attenuate the virulence of this stain and also elicit an immune response against PCV2 capsid

protein at the same time. In this study, we used SEZ as a bacterial vector for expression of PCV2 capsid protein and verified this hypothesis. SEZ strain C55138 (China Institute of Veterinary Drug Control, Beijing, China) was originally recovered from a diseased pig Wilson disease protein with septicemia in Sichuan, China. It was grown on tryptone soya broth (TSB) (Oxoid, Wesel, Germany) or tryptone soya agar (TSA) (Difco Laboratories, Detroit, MI) plus 5% newborn calf serum at 37 °C under aerobic conditions. The capsule-deficient

mutant ΔhasB was constructed in our laboratory by disruption of the capsule synthesis hasB gene (data not shown). A thermosensitive broad-host-range vector pG+host5 (Appligene, Illkirch, France) was used to construct the SEZ-Cap recombinant strain (Biswas et al., 1993). The primers used in this study are detailed in Table 1. The corresponding position of the primers on the genome of SEZ is illustrated in Fig. 1a. When necessary, erythromycin was added to the culture media at the following concentrations: 150 μg mL−1 for Eschrichia coli and 5 μg mL−1 for SEZ. Five 4- to 6-week-old BALB/c mice were immunized twice at 2-week intervals by intraperitoneal injection with commercially available PCV2-inactive vaccine (Nannong Hi-tech Co. Ltd, Nanjing, China). Serum samples were tested using a commercial PCV2 ELISA IgG kit (Ingezim Circovirus IgG, Ingenasa, Madrid, Spain). When the serum sample with a sample/positive (S/P) ratio reached 1.