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 × 106 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.

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