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. 2017 Aug 9;7:360. doi: 10.3389/fcimb.2017.00360

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Copyright © 2017 Contreras, Alberdi, Fernández De Mera, Krull, Nijhof, Villar and De La Fuente.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Selection of candidate tick protective antigens. Purified rabbit IgG against recombinant candidate tick protective antigens were incubated with I. scapularis ISE6 and I. ricinus IRE/CTVM20 tick cells before infection with A. phagocytophilum. (A) Characterization of the effect of rabbit IgG antibodies on ISE6 tick cells apoptosis. (B) Characterization of the effect of rabbit IgG antibodies on pathogen infection of ISE6 tick cells. (C) Sequence identity between I. scapularis and I. ricinus homologs. The accession numbers are shown together with corresponding percent identity for nucleotide (nt) and amino acid (aa) sequences and E-values. The selected I. scapularis sequences were blasted against the I. ricinus database using the Blastp tool from BLAST, and the sequences with the lowest E-value were selected. (D) Characterization of the effect of rabbit IgG antibodies on IRE/CTVM20 tick cells apoptosis. (E) Characterization of the effect of rabbit IgG antibodies on pathogen infection of IRE/CTVM20 tick cells. The percentage of apoptotic cells was determined by flow cytometry after Annexin V-FITC and PI labeling. A. phagocytophilum DNA levels were determined by msp4 real-time PCR normalizing against tick rpS4. Control cells were incubated with rabbit pre-immune IgG (negative control, Control -) or rabbit anti-ISE6 IgG (positive control, Control +). Results were presented as average + S.D. normalized Ct-values and compared between each treatment and negative control by Student's t-test with unequal variance (P ≤ 0.05; N = 4). The selected candidate protective antigens are shown with arrows.