Fig 1.
Experimental design.
Fig 2.
Geometric mean titers (GMT) of hemagglutination inhibition (HAI) antibodies against influenza virus subtypes H5 or H1 in serum samples of vaccinated pregnant sheep (A) and goats (B) at 0, 21, 42, 63 days post-vaccination (DPV). Pregnant sheep and goats in the group I (Flu-BA_Omp19-SOD) were immunized twice concurrently via the subcutaneous and conjunctival routes of administration at an interval of 21 days with vaccines generated from the influenza viral vectors (IVV) subtypes H5N1 (prime vaccination) and H1N1 (booster vaccination). The vaccination of animals of group II (Flu-BA_Omp19-SOD_TV) was carried out in the same way as in group I, but only the vaccine generated from IVV subtypes H5N1 was used, which was administered three times at 21 days intervals. Sheep and goats in the negative control group (IV) were vaccinated with 20% Montanide Gel01 adjuvant in PBS three times at 21 days intervals. Data are presented as GMT with 95% confidence interval; ND–not detected; * P = 0.0002 to P<0.0001 vs. appropriate control group; † P = 0.04 vs. Day 63 DPV of the Flu-BA_Omp19-SOD_TV sheep group. Statistical analysis was performed using two-way ANOVA followed by Sidak's multiple comparisons test. P values < 0.05 were considered significant.
Fig 3.
IgG, IgG1 and IgG2a antibody responses against total mixed and individual Brucella L7/L12, Omp16, Omp19 and SOD proteins in pregnant sheep (A) and goats (B) at 0, 21, 42, 63 days post-vaccination (DPV) by ELISA. Pregnant sheep and goats in the group I (Flu-BA_Omp19-SOD) were immunized twice concurrently via the subcutaneous and conjunctival routes at an interval of 21 days with vaccines generated from the influenza viral vectors (IVV) subtypes H5N1 (prime vaccination) and H1N1 (booster vaccination). The vaccination of animals of group II (Flu-BA_Omp19-SOD_TV) was carried out in the same way as in group I, but only the vaccine generated from IVV subtypes H5N1 was used, which was administered three times at 21 days intervals. Sheep and goats in the negative control group (IV) were vaccinated with 20% Montanide Gel01 adjuvant in PBS three times at 21 days intervals. Data are presented as optical density (OD) ± standard deviations; * P = 0.04 to P<0.0001 vs. appropriate controls; ** P = 0.04 vs. IgG1. Statistical analysis was performed using two-way ANOVA followed by Sidak's multiple comparisons test. P values < 0.05 were considered significant. NI—not investigated.
Fig 4.
Lymphocyte stimulation index (A, C) and levels of IFN-γ (B, D) in the supernatants of PBMCs of pregnant sheep and goats at 42 and 63 days post-vaccination (DPV). Pregnant sheep and goats in the group I (Flu-BA_Omp19-SOD) were immunized twice concurrently via the subcutaneous and conjunctival routes at an interval of 21 days with vaccines generated from the influenza viral vectors (IVV) subtypes H5N1 (prime vaccination) and H1N1 (booster vaccination). The vaccination of animals of group II (Flu-BA_Omp19-SOD_TV) was carried out in the same way as in group I, but only the vaccine generated from IVV subtypes H5N1 was used, which was administered three times at 21 days intervals. Sheep and goats in the negative control group (IV) were vaccinated with 20% Montanide Gel01 adjuvant in PBS three times at 21 days intervals. All data are presented as mean ± standard error; * P = 0.02 to P<0.0001 vs. appropriate control group; † P = 0.02–0.01 vs. Day 42 DPV. Statistical analysis was performed using two-way ANOVA followed by Tukey's multiple comparisons test. P values < 0.05 were considered significant. NI—not investigated.
Table 1.
Rates of abortion, parturition and infection in the sheep and goats after challenge with the virulent strain B. melitensis 16M.
Fig 5.
Index of infection for sheep and goats challenged with B. melitensis 16M at 113–120 days post-vaccination.
Pregnant sheep and goats in the group I (Flu-BA_Omp19-SOD) were immunized twice concurrently via the subcutaneous and conjunctival routes of administration at an interval of 21 days with vaccines generated from the influenza viral vectors (IVV) subtypes H5N1 (prime vaccination) and H1N1 (booster vaccination). The vaccination of animals of group II (Flu-BA_Omp19-SOD_TV) was carried out in the same way as in group I, but only the vaccine generated from IVV subtypes H5N1 was used, which was administered three times at 21 days intervals. Sheep and goats in the negative control group (IV) were vaccinated with 20% Montanide Gel01 adjuvant in PBS three times at 21 days intervals. Animals in the positive control group (III) were immunized once subcutaneously in the axillary region (right side) with commercial vaccine B. melitensis Rev.1 according to the manufacturer's instructions. Challenge with the virulent strain B. melitensis 16M was performed via the subcutaneous route (106 CFU/animal). The index of infection is the number of animals from which Brucella was isolated from the organs and lymph nodes. The data presented as mean ± standard error; * P <0.0001 vs. appropriate control group IV. Statistical analysis was performed using two-way ANOVA followed by Tukey's multiple comparisons test. P values < 0.05 were considered significant.
Table 2.
Colonization and incidence of recovery of B. melitensis in tissues after challenge with B. melitensis 16M.