C57BL/6 mice were vaccinated with a bacterially expressed and purified
polyhistidine-tagged full-length version of the microneme
protein NcMIC3 (recNcMIC3) emulsified in
Ribi Adjuvant System (RAS). Subsequently, they were challenged by intraperitoneal inoculation of 2 x 10(6) live Neospora caninum tachyzoites. As controls, groups of mice received
phosphate-buffered saline (PBS)-RAS alone (adjuvant control) or were treated with PBS before
infection (infection control). The protective effect of vaccination was assessed by Neospora-specific polymerase chain reaction (PCR), immunohistochemical investigation of brain tissue, and serological means (
enzyme-linked
immunosorbent assay). Assessment by PCR performed on
DNA from different organs revealed that in all treatment groups parasite
DNA could only be detected in brain tissue. According to the PCR results. the recNcMIC3
vaccine conferred protection to 75% of mice (n = 16 in 2 independent experiments), whereas application of PBS-RAS and of PBS alone resulted in protection of 12.5% and 0% of mice, respectively (n = 16 as above). Mice in the PBS-treated infection control group were affected by evident clinical signs of neosporosis starting on day 6 postinfection (p.i.). Conversely, none of the animals treated with either PBS-RAS or recNcMIC3 exhibited any symptoms until day 21 p.i. Immunohistochemical staining of
paraffin-embedded brain tissue sections confirmed the protective effect of recNcMIC3 vaccination. Quantitative Neospora-specific real-time PCR revealed that
infection intensities were lower in the brain tissues of recNcMIC3-vaccinated mice compared with PBS-RAS-treated adjuvant control mice. Serological analysis showed that the protective effect observed in recNcMIC3-vaccinated mice was associated with a Th2-type
IgG1 antibody response directed against native NcMIC3 and a mixed IgG1-IgG2a antibody response directed against the recombinant
antigen itself. Taken together, these results demonstrated that recombinant NcMIC3
vaccine confers a significant protectivity against experimentally induced cerebral neosporosis in mice.