Protective efficacy of vaccination with NcMIC3 and NcMIC8 against <i>Neospora caninum</i> infection in mice

doi:10.15302/J-FASE-2019253

Front. Agr. Sci. Eng.

2019, Vol. 6

Issue (2) : 188-196 https://doi.org/10.15302/J-FASE-2019253

RESEARCH ARTICLE

Protective efficacy of vaccination with NcMIC3 and NcMIC8 against Neospora caninum infection in mice

Taotao ZHANG, Xiao ZHANG, Qun LIU, Jianhai XU, Jing LIU(

)

National Animal Protozoa Laboratory, College of Veterinary Medicine, China Agricultural University, Beijing 100193, China

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Abstract

Microneme proteins (MICs) are important for Apicomplexan parasite invasion due to their adhesion to host cells. Several studies have indicated that Neospora caninum MIC3 and MIC8 are important adhesion factors and potential vaccine candidates against neosporosis. In this study, we evaluated the protective efficacy of recombinant proteins and DNA vaccines of NcMIC3 and NcMIC8. BALB/c mice were immunized with rNcMIC3, rNcMIC8, pcDNA3.1-NcMIC3 and pcDNA3.1-NcMIC8 respectively, and challenged with N. caninum tachyzoites. The immune responses were evaluated through cytokine, antibody measurements and the parasite burden in the mice brain tissues. Serological analysis showed that recombinant protein vaccines induced higher levels of immunoglobulin G (IgG) than other groups. The percentage of IgG1 and IgG2a in the recombinant protein groups was higher than the other groups, and with a predominance of IgG1 over IgG2a, suggesting that recombinant protein vaccines elicited a Th2-type immune response, while DNA vaccines mainly produce a Th1-type immune response. In addition, mice immunized with rNcMIC3 and rNcMIC8 a had lower parasite burden in brain tissue compared with the other groups. These results demonstrate that rNcMIC3 and rNcMIC8 could induce humoral and Th2-type immune response, leading to a considerable level of resistance against neosporosis.

Keywords NcMIC3 NcMIC8 Neospora caninum vaccination

Corresponding Author(s): Jing LIU

Just Accepted Date: 31 January 2019 Online First Date: 12 March 2019 Issue Date: 22 May 2019

Cite this article:

Taotao ZHANG,Xiao ZHANG,Qun LIU, et al. Protective efficacy of vaccination with NcMIC3 and NcMIC8 against Neospora caninum infection in mice[J]. Front. Agr. Sci. Eng. , 2019, 6(2): 188-196.

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https://academic.hep.com.cn/fase/EN/10.15302/J-FASE-2019253
https://academic.hep.com.cn/fase/EN/Y2019/V6/I2/188

Fig.1 Construction of eukaryotic expression plasmids and expression in vitro. A: Amplification of NcMIC3 gene (672 bp, lane 1) and identification of the recombinant plasmid pcDNA3.1-NcMIC3 by double digestion (KpnI and EcoRV, lane 2) and PCR (lane 3); B: Amplification of NcMIC8 gene (927 bp, lane 1) and identification of the recombinant plasmid pcDNA3.1-NcMIC8 by double digestion (BamHI and XhoI, lane 2) and PCR (lane 3); C: Expression of NcMIC3 and NcMIC8 in HEK 293T cells by IFA. (a) HEK 293T cells transfected with pNcMIC3 react with the anti-NcMIC3 antibody; (b) HEK 293T cells transfected with pNcMIC8 react with the anti-NcMIC8 antibody; (c) negative control, HEK 293T cells transfected with empty pcDNA3.1 do not react with the anti-NcMIC3 or anti-NcMIC8 antibody. Bar= 20 mm

Fig.2 The purification of NcMIC3 and NcMIC8 recombinant proteins. A: Purified rNcMIC3 (about 32 kDa) was stained by Coomassie Brilliant Blue after SDS-PAGE; B: Purified rNcMIC8 (about 38 kDa) was stained by Coomassie Brilliant Blue after SDS-PAGE. M: Protein markers.

Fig.3 Neospora caninum-specific antibody levels in the sera of immunized BALB/c mice. A: Levels of IgG antibodies in sera of rNcMIC3, rNcMIC8, pNcMIC3, pNcMIC8, pcDNA3.1, and PBS groups were determined using indirect ELISA. Samples were collected preimmunization, after the third booster immunization and 30 days post-infection (n = 10); B: The levels of IgG1 and IgG2a subtypes in the sera 28 days after the last immunization were determined by ELISA; C: The IgG1/IgG2a ratio was calculated to determine whether the immunized mice induced a Th1- or a Th2-type immune responses. The results are expressed as the means±SD from three independent experiments. The asterisk designates statistically significant differences (P<0.05) between groups. * P<0.05, compared to control groups; ** P<0.01, compared to control groups.

Tab.1 Cytokines detected in sera from immunized mice

Fig.4 Neospora caninum burden in brain tissues of mice. The numbers on the y-axis correspond to the parasite number per mg of DNA, and each point represents individual animals. The results show that mice immunized with rNcMIC3 or rNcMIC8 and pcDNA3.1-NcMIC3 had reduced brain parasite loads compared to the control groups. In contrast, the pcDNA3.1-NcMIC8 group induced a higher cerebral parasite burden in mice. Values are shown as the means±SEM. P<0.05 (*), P<0.01 (**) and P<0.001 (***) were statistically significant as indicated.

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