Progression of bacterial growth to the bloodstream was monitored by blood samples obtained by cardiac puncture with a heparinized syringe. Samples were plated on blood agar and bacteraemia Bcl-2 inhibitor was reported as negative or positive haemocultures after incubation for 18 h at 37°C. Experiments were performed in triplicate and results were expressed as mean ± standard deviation (s.d.). Significant differences between means were determined by analysis of variance

(anova) with Fisher’s least significant difference (LSD) post hoc test using the StatGraphics software (Manugistics, Rockville, MD, USA). Differences were considered significant at P < 0·05. We evaluated administration of the probiotic strain L. casei by oral (O) and nasal (N) routes associated with nasal immunization with live (LL) and inactivated

(D-LL) recombinant strains. Results are shown in Fig. 1a and b and significant differences between groups on day 42 are shown in Table 1. The D-LL + Lc (N) (IgA: P < 0·001, IgG: P < 0·01), D-LL + Lc (O) (IgA: P < 0·01, IgG, P < 0·001) and LL + Lc (O) (IgA: P < 0·05, IgG: P < 0·001) groups showed the highest levels of IgA and IgG anti-PppA in bronchoalveolar lavages this website in comparison with the live vaccine. D-LL + Lc (N) induced the highest IgA levels in BAL, but without significant differences with the D-LL + Lc (O) and LL + Lc (O) groups. Although D-LL induced significantly high values of specific IgA (P < 0·05) and IgG (P < 0·05) antibodies compared to live vaccine (LL), IgA values

were lower than those obtained in the groups receiving the probiotic. The levels of specific anti-PppA IgM were increased slightly compared Niclosamide to those of LL in the groups that received Lc as an oral or nasal adjuvant associated with the inactivated vaccine, especially on day 28, although the differences were not significant (data not shown). Results showed that administration of the probiotic strain by both the oral and nasal routes exerted an important adjuvant effect on the humoral immune response in the lung compartment. This would provide an encouraging alternative for the use of vaccines involving the probiotic–inactivated recombinant bacterium association, with their associated advantages: adjuvant properties of the probiotic strain and safe application of an inactivated bacterium to human health. As expected, the groups that received only PBS, Lc (O) or Lc (N) showed no levels of specific anti-PppA antibodies. Nasal immunization with LL induced a good response of specific IgA, IgG and IgM antibodies in serum (Fig. 2a–c). The associated administration of the probiotic by the oral route did not induce a significant increase in the levels of these specific immunoglobulins in any of the assessed groups (Fig. 2).