Fig. 4. 66NC protects C57BL/6 mice against intraperitoneal challenge with MAP K-10.

a Schematic diagram of the vaccination and MAP challenge timeline. C57BL/6 mice were subcutaneously injected two times (three weeks apart) with 50 μg 66NC fusion protein formulated in Montanide ISA 61 VG (61 VG) adjuvant. Control groups include mice immunized with Montanide ISA 61 VG adjuvant alone as a negative control or 74 F mixed MPL adjuvant as subunit vaccine control. Three weeks after the booster immunization, the mice were challenged with MAP K-10 by intraperitoneal injection. b Body weight was measured for 2, 4, 8, and 12 weeks after the MAP challenge (n = 6 per group). Monitor IgG (c) and IgM (d) antibody levels. Sera were collected and measured for specific anti-66NC or 74 F antibody response of both IgG and IgM at 2, 4, 6, 8, 10, 12, 14, 16, and 18 weeks after prime vaccination by ELISA. Bacterial load quantification was evaluated in the liver (e) and intestine (f) from mice challenged by intraperitoneal injection with MAP K-10 for 2, 4, and 8 weeks after vaccination. g Representative images of Ziehl-Neelsen acid-fast stain in the liver of vaccinated mice, two weeks after being challenged with MAP, are presented (scale bars, 100 μm (top) and 10 μm (bottom)). The bottom images are enlarged from the outlined areas of the top images. b, e, f Data indicate cumulative results from at least three to six independent replicates. Mean ± SEM and Two-way ANOVA were used to analyze the statistical significance, followed by Tukey’s multiple comparisons tests. ns, non-significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.