| Summary: | In terms of its veterinary importance, vaccine development against <i>Ehrlichia canis</i> is needed. However, the effect of developing vaccines on humoral immune response against <i>E. canis</i> infection is still unknown. Novel GP19<sub>4-43</sub> was synthesized according to <i>E. canis</i> GP19 epitope prediction. To restrict any loss and/or illness in the host animal, rabbits were used in this study to produce GP19<sub>4-43</sub> hyperimmune sera. The effect of GP19<sub>4-43</sub> hyperimmune sera on neutralization was examined in vitro by determining the inhibition of <i>E. canis</i> infection of the macrophage-like cell line (DH82) in the presence of the sera. Four groups of DH82 cells received differing treatments. These included <i>E. canis</i> experimentally infected DH82 cells, <i>E. canis</i>-infected DH82 cells with control rabbit serum (untreated group), <i>E. canis</i>-infected DH82 cells with GP19<sub>4-43</sub> rabbit antiserum (treated group) and uninfected cells (negative control group), respectively. The treated group developed a decrease (<i>p</i> < 0.01) in the percentage of <i>E. canis</i> infected cells after 3 days post-infection at 48.57 ± 1.28. In addition, real-time PCR analyses of cytokine mRNA expression involved with the macrophage, humoral, and cellular immune responses were conducted. The findings revealed an upregulated expression of <i>IFNG</i> in the treated group during the infection. This study demonstrated neutralization in the GP19<sub>4-43</sub> peptide hyperimmune sera of immunized rabbits. Notably, IFN-γ production could be effectively promoted in canine macrophages in relation to the activation of macrophages and adaptive immune responses. The results of this study indicate the potential for the use of this immunogen in further investigations involving immunized and infected dogs as <i>E. canis</i> host species.
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