Bacillus subtilis Expressing Chicken NK-2 Peptide Enhances the Efficacy of EF-1α Vaccination in Eimeria maxima-Challenged Broiler Chickens

This study was conducted to investigate the synergistic effects of orally delivered B. subtilis-cNK-2 on vaccination with rEF-1α against E. maxima infection in broiler chickens. Chickens were assigned into the following five groups: control (CON, no Eimeria infection), non-immunized control (NC, PBS), component 1 (COM1, rEF-1α only), component 2 (COM2, rEF-1α plus B. subtilis empty vector), and component 3 (COM3, rEF-1α plus B. subtilis-NK-2). The first immunization was administered intramuscularly on day 4, and the second immunization was given one week later with the same concentration of components as the primary immunization. The immunization of B. subtilis spores (COM2 and COM3) was performed by oral administration given for 5 consecutive days a week later than the second immunization. On day 19, all the chickens except the CON group were orally challenged with E. maxima oocysts (1.0 × 104/chicken). The results of the in vivo vaccination showed that all the chickens immunized with rEF-1α (COM1, COM2, and COM3) produced higher (p < 0.05) serum antibodies against EF-1α on 12 days post-E. maxima infection (dpi). The COM3 group showed a significantly (p < 0.05) higher average body weight gain (BWG) on 0-6, 6-9, and 0-12 dpi compared to those of the non-immunized chickens (NC). Immunization with rEF-1α alone (COM1) reduced the gut lesion score on 6 dpi and the fecal oocyst shedding on 9 dpi, whereas co-administration with B. subtilis spores (COM2 or COM3) led to further reduction in the lesion score. E. maxima infection increased the expression levels of IFN-γ and IL-17β in the jejunum, but these expressions were downregulated in the rEF-1α immunized (COM1) group and in the groups immunized with rEF-1α and orally treated with B. subtilis spores (COM2 or COM3) at 4 dpi. A reduced gene expression of occludin in the jejunum of the E. maxima-infected chickens on 4 dpi was upregulated following the immunization with COM2. Collectively, rEF-1α vaccination induced significant protection against E. maxima infection in the broiler chickens, and the efficacy of rEF-1α vaccination was further enhanced by co-administration with orally delivered B. subtilis spores expressing cNK-2.