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

Serological analysis of infected mice 14 weeks post-infection.

<p>Antibody responses of subcutaneously (sc) and intraperitoneally (ip) infected mice, either treated with albendazole (ABZ) or not (untreated) were measured by ELISA employing recombinant Em18 (EM18), Em2(G11)-antigen (EM2) or hydatid fluid. Error bars indicate standard deviations. There is no difference in the responses of treated versus respective untreated animals, and no significant difference can be seen between the two infection models.</p

Information of used primers.

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

Parasite burden and weight control.

<p><b>A</b>) Parasite burden of intraperitoneally (ip) and subcutaneously (sc) infected animal models. Albendazole treatment was initiated at 6 weeks post-infection. Each treatment group comprised 5 infected animals. scABZ and ipABZ groups received albendazole in honey/CMC, sc-untreated and ip-untreated groups received honey/CMC without compound. The <i>P</i> values indicate the scores obtained by student's t-test in comparison with the respective untreated groups. <b>B</b>) Weight control of mice throughout the study. Representation of average weight of different experimental groups at defined timepoints during the study. Treatments were initiated at day 42 p.i. The non-infected controls are not shown. The groups were divided into route of infection (subcutaneous = sc; intraperitoneal = ip), and in albendazole-treated (scABZ; ipABZ) or non-treated (sc-untreated; ip-untreated).</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

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