Genetic variability of <i>p29</i> within <i>E. granulosus</i> s.s. (G1) in Central Tunisia.

<p>(<b>A</b>) MAS-PCR: <i>p29</i> genotype profile of the <i>E. granulosus</i> s.s. (G1) by MAS-PCR. Two alleles encoding the P29 protein within <i>E. granulosus</i> s.s. (G1) were identified. Result of a MAS-PCR showed homozygotes A1/A1 (lanes 9 and 10), homozygotes A2/A2 (lanes 1–5) and heterozygotes (lanes 6–8), visualized on a 2% agarose gel. M: 100-bp DNA ladder (Promega). (<b>B</b>) Location of 34 patients used in this study for the <i>p29</i> multiplex allele specific (MAS)-PCR. The main area for human risk is located in Central Tunisia and includes Kairouan, Kasserine and Sidi Bouzid. All isolates were first genotyped as <i>E. granulosus</i> s.s. (G1). Samples identified as homozygote A1/A1 are represented by a black square, homozygotes A2/A2 are signified by a grey triangle and heterozygotes are showed by a white circle with black border.</p

Information of used primers.

<p>*Allele specific 3′-base in each primer.</p

Molecular characterization of <i>p29</i> genomic sequence within <i>Echinococcus</i> genus.

<p>*In total five clones were rejected because of the bad quality of their sequences (2 clones from sample 3, 2 from sample 5 and 1 clone from sample 7).</p

Alignment of amino acid sequences of the P29 <i>Echinococcus</i> protein.

<p>Two alleles within <i>E. granulosus</i> s.s. (G1) for the <i>p29</i> gene locus were identified. They code for the same protein and are 100% identical to the published <i>p29</i> sequence (GenBank accession no. <u>AF078931</u>). Deduced P29 protein homologs from <i>E. equinus</i> (G4), <i>E. ortleppi</i> (G5), <i>E. canadensis</i> (G6), <i>E. canadensis</i> (G7), <i>E. canadensis</i> (G10) and <i>E. multilocularis</i> (Four isolates from Switzerland, Germany, St. Lawrence Island and Canada) are aligned. The sequence alignment is numbered and the sequences are represented in blocks of 10 AAs. Identical residue sequences are presented in points, and substitutions are presented in letters. Numbers to the left of the sequence corresponds to the AA position at the start of each line.</p

Competitive Western blot analysis with recEg(G1)P29 expressed from <i>E. granulosus</i> s.s. (G1) and recEcnd(G6) expressed from <i>E. canadensis</i> (G6).

<p>(A) Shows a silver stained gel of expressed and purified recombinant antigens; recEg(G1)P29 (lane 1) and recEcnd(G6)P29 (lane 2). (<b>B</b>) Recombinant P29 proteins were separated by SDS-PAGE under reducing conditions and blotted onto a nitrocellulose membrane. Human serum from a CE patient either infected with <i>E. granulosus</i> s.s. (G1) or <i>E. canadensis</i> (G6) were added to recEg(G1)P29 (strip 1 and 2, respectively) or recEg(G6)P29 (strip 3 and 4, respectively). In a competition assay nitrocellulose strips loaded with recEcnd(G1)P29 were incubated with serum from CE patients either infected with <i>E. granulosus</i> s.s. (G1) or <i>E. canadensis</i> (G6). The sera were pre-incubated with the recombinant expressed competitor recEcnd(G6)P29 (strip 5 and 6) or as a control with recEg14-3-3 (strip 7 and 8). Immune sera were used at dilutions of 1∶100 and competitor/control at concentrations of 1 µg/mL.</p

Human <i>E. granulosus</i> s.s. (G1) allelic frequencies at <i>p29</i> loci genotyped with MAS-PCR.

<p>Human <i>E. granulosus</i> s.s. (G1) allelic frequencies at <i>p29</i> loci genotyped with MAS-PCR.</p

Determination of copy number and molecular structure of the <i>Echinococcus p29</i> gene.

<p>(<b>A</b>) Exon/intron structure analysis of the <i>Echinococcus p29</i> gene mapped to the alignments of full-length cDNA with the genomic DNA. The <i>p29</i> gene is of 1200 base pairs from the ATG start codon at position +1 to the TAG stop codon at positon +1200 and consists of 7 exons separated by six introns. At position P (−552) upstream of the start codon, we identified a TATA box and a eukaryotic transcriptional regulatory element. Additionally two possible polyadenylation sites were identified downstream of the TAG codon on P (+1365) and P (+1510) positions. (<b>B</b>) Graphical output of the BLAST analysis of <i>p29</i> cDNA (GenBank, accession no. AF078931) beween <i>E. granulosus</i> s.s. (G1) and <i>E. multilocularis</i> genomes performed at the <i>Echinococcus</i> blast server (available at: (available at: <a href="http://www.sanger.ac.uk/cgi-bin/blast/submitblast/Echinococcus" target="_blank">http://www.sanger.ac.uk/cgi-bin/blast/submitblast/Echinococcus</a>). The diagram shows the reads with significant BLAST scores to <i>p29</i> cDNA query (+hsps; high score probability). The reads cluster belongs to the same contig; pathogen_EgG_scaffold_0007 and pathogen EmW Chr 07 for <i>E. granulosus</i> and <i>E. multilocualris</i> respectively. Reads from the same cluster are contiguous and overlapping DNA fragments and when assembled resulted in a single and complete <i>p29</i> gene sequence.</p

Comparison of phylogenies of <i>Echinococcus</i> inferred by maximum likelihood (ML) analysis using gDNA data.

<p>(<b>A</b>) Maximum likelihood with molecular clock rooted cladogram from this study, based on <i>p29</i> gene sequence (exons and introns). (<b>B</b>) For comparison a published cladogram based on the DNA sequences of nuclear protein-coding genes is shown <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098357#pone.0098357-Saarma1" target="_blank">[38]</a>.</p

Serological analyses.

<p>Antibody responses of i.p. infected mice, vaccinated pre- or post-parasite challenge, were measured by ELISA using affinity-purified Em2 (G11) and rEmP29 antigen. Bars indicate SE. Immunization and then infection: mouse groups from experiment no. 2 were immunized with different preparations (Saline solution (NaCl), saponin (Sap), rEmP29 plus saponin (rEmP29-Sap) or with rNcMIC1 plus saponin (rNcMIC1-Sap)), and then challenged by infection with an <i>E</i>. <i>multilocularis</i> metacestode suspension. Infection and then immunization: mice from experiment no. 3 were infected with <i>E</i>. <i>multilocularis</i> metacestode suspension, and at 1 month p.i., animals were treated with different preparations (physiological saline solution (NaCl), saponin (Sap), rEmP29 plus saponin (rEmP29-Sap) or with rNcMIC1 plus saponin (rNcMIC1-Sap)).</p

Relative mRNA expression levels of IL-4 (A), IFN-γ (A), IL-2 (C) and IL-10 (D) in spleens from BALB/C mice, following immunization with rEmP29 (rEmP29-sap) or with adjuvant alone (sap).

<p>The IL-4/IFN-γ cytokine mRNA ratios were calculated from the three groups <b>(B)</b> and indicate a Th2-dominated response in rEmP29-sap and sap-alone-treated mice compared to none immunized mice.</p