Additional file 1: of Efficient high-throughput molecular method to detect Ehrlichia ruminantium in ticks

Text. PCR protocol for the detection of E. ruminantium. Table S1. Primers and probes for pCS20 Sol1TqM, Sol1SG, Cow™ and 16SSG qPCRs. (DOC 48 kb

Additional file 2: of Efficient high-throughput molecular method to detect Ehrlichia ruminantium in ticks

Text. Protocol of extraction of tick DNA. (DOCX 12 kb

Schematic overview of metabolic pathways and membrane proteins found in ERG, based on genomic (KEGG database) and proteomic information obtained in this work.

<p>Nodes correspond to substrates and edges to enzymatic reactions and the 11 major metabolic pathways are color-coded (for example, light orange for amino acid metabolism). The proteins that were identified on both ERGvir and ERGatt are indicated in dark blue, the proteins and pathways found exclusively in ERGatt are in red and those detected only in ERGvir are highlighted in green. The dashed lines correspond to metabolic pathways found in both strains.</p

Venn diagram representing the number of non-redundant proteins identified in ERGvir and ERGatt and in both (core proteome) by 1DE-nanoLC-MALDI-TOF/TOF analysis.

<p>Three biological replicates per strain were independently used and the results analyzed with Peaks software (using using simultaneously Peaks DB, MASCOT and X!Tandem algorithms).</p

Proteins overexpressed (fold change ≥ 3) in ERGvir (A) and ERGatt (B) according to DIGE analyses and MALDI-TOF/TOF.

<p>Proteins overexpressed (fold change ≥ 3) in ERGvir (A) and ERGatt (B) according to DIGE analyses and MALDI-TOF/TOF.</p

Experimental workflow for <i>E</i>. <i>ruminantium</i> subcellular fractionation and proteome characterization.

<p><b>OM</b>, outer membrane; <b>I</b>, inner membrane; <b>C</b>, cytoplasm.</p

Evaluation of OM isolation quality.

<p>Transmission electron microscopy of (A) purified <i>E</i>. <i>ruminantium</i> and (B) the insoluble precipitate after 0.1% sarkosyl treatment; scale bar = 200 nm. (C) SDS-PAGE and (D) Western blot of <b>E</b> (elementary bodies), <b>S</b> (sarkosyl-soluble fraction), and <b>OM</b> (outer membrane fraction) using monoclonal antibodies against Map1. <u><b>Band 1</b></u>: Map1-14, X5HG56, GroEL; <u><b>Band 2</b></u>: Map1+1, Map1, Map1-6, VirB10, VirB4, GroEL, PyrE, Q5HAR6, X5HG56, 30S-S8; <u><b>Band 3</b></u>: Map1+1, Map1-6, Map2, GroEL, PyrE, Q5HAR6, X5HG56, Q5FGC2, Q5HBI2, Q5FHJ9; <u><b>Band 4</b></u>: Map1, Map1-6, VirB4, GroEL, DnaK, BamA, FusA, Pnp, Q5HAR6, X5HG56, Q5FH07, Q5HBS6; <u><b>Band 5</b></u>: VirB4, VirB10, VirB11, DnaK, HtpG, GroEL, FusA, 30S-S1, Q5FGV5, Q93FS2; <u><b>Band 6</b></u>: Map1, Map1-14, VirB10, PleD, GroEL, DnaK, FtsZ, 30S-S1, Q5HB83, Q5FGA7, Q5HBE1; <u><b>Band 7</b></u>: Map1-14, GroEL, DnaK, FtsZ, HtpG; <u><b>Band 8</b></u>: Map1-6, Map1, GroEL, DnaK, FtsZ, BamA; <u><b>Band 9</b></u>: Map1-6, Map1, Map1+1, Map1-14, GroEL, DnaK, BamA, Q5FFE6, Q5HAR6; <u><b>Band 10</b></u>: Map1-11, Map1-13, Map1, Map1+1, Map1-6, VirB10, VirB9, Q5FFE6, Q5HAR6, Q5HBI2, Q5HA95; <u><b>Band 11</b></u>: Map1, Map2, BamA, DnaK, GroEL, FusA, Def, 50S-L4, PyrE, X5HG56, Q5HBI2; <u><b>Band 12</b></u>: 30S-S18, 30S-S12, 50S-L7/L12, 50S-L18, 50S-L24, 50S-L28 X5HG56, Q5HBN6; <u><b>Band 13</b></u>: HupB, X5HG56; <u><b>Band 14</b></u>: 30S-S12, 50S-L7/L12, 50S-L18, GroEL, YajC, PyrE.</p