Estimated O-glycan compositions of SRf1 from mass spectrometric data.

*<p>δmass = [observed m/z] – [theoretical m/z]. Abbreviations: Hex, hexose (e.g., mannose, galactose); HexNAc, N-acetylhexosamine (e.g., GlcNAc, GalNAc); NeuAc, N-acetylneuraminic acid; Sulph, sulfated glycan; Pent, pentose.</p

Localization of SRf1 in the larval and adult stages of <i>E. multilocularis</i>.

<p>Immunostaining was performed using polyclonal mouse anti-SRf1 antiserum. Panels A (40×) and B (100×) show adult worms harbored in the epithelium of the small intestine of infected dogs at 23 days postinfection. Panels C (100×) and D (200×) show protoscoleces in cysts derived from infected cotton rats. The brown color indicates specific antibody reactivity; the blue color indicates hematoxylin staining of nuclei. Anti-SRf1 antibodies were detected on the surface, including suckers, rostella, and hooks in both stages of worm development.</p

Identification and purification of vaccine antigen (SRf1) from <i>E. multilocularis</i>.

<p>Crude extracts were prepared from protoscoleces (PCE) and adult worms (ACE). A total of 120 µg protein was applied to 2D-PAGE. The proteins were blotted onto a PVDF membrane, and reactivity between proteins and intestinal IgA from dogs experimentally infected with <i>E. multilocularis</i> was examined. Panels A and B: PCE and ACE, respectively; panels 1 and 5: CBB-stained gels; panels 2 and 6: tested with intestinal IgA from dogs infected 5 times; panels 3 and 7: tested with intestinal IgA from dogs infected 3 times; and panels 4 and 8: tested with intestinal IgA from dogs infected once. Molecular size markers are indicated on the left (in kDa). Panel C: a gel filtration chromatogram of the vaccine antigen (SRf1) and 2D-western blot analysis; panels 9 and 11: CBB-stained gels of SRf1 and SRf2; panel 10: 2D-western blotting for SRf1 using intestinal swabs from dogs infected 5 times; panel 12: 2D-western blotting for SRf2 using sera from dogs infected 5 times. Panel D: SDS-PAGE analysis of SRf1; lanes 1 and 2: glycoprotein stained-gel of molecular size markers and SRf1; 3 and 4: CBB-stained gel with molecular size markers and SRf1. Glycoprotein detection was performed with a Pro-Q Emerald 300 gel stain kit and CandyCane glycoprotein molecular weight standards.</p

Tolerance of SRf1 against gastric protease digestion.

<p>SRf1 was digested in the presence of pepsin (1 mg/mL) at pH 2 or in the presence of trypsin (0.4 mg/mL) and chymotrypsin (1.7 mg/mL) at pH 7.4. After 1 or 4 h digestion, the reaction mixture was applied to a Superose 6 gel filtration column, and peak areas were compared to those of the control. Panel B: 1D-western blot analysis of digested SRf1s detected using sera from dogs infected 5 times. Lanes: M, molecular marker; C, controls (no proteases); 1 and 2, pepsin digestion for 1 and 4 h, respectively; 3 and 4, tryptic digestion for 1 and 4 h, respectively.</p

Phylogenetic analyses of diagnostic antigen gp50 amino acid sequences.

<p>The neighbor-joining tree was constructed by MEGA 6.0 (<a href="http://www.megasoftware.net" target="_blank">www.megasoftware.net</a>); bootstrap values were obtained from 1,000 replicates. Nonc: RPKM>100 at stages of non-activated oncosphere; Aonc: RPKM>100 at stages of activated oncosphere; P_gravid: FPKM>100 at stage of pre-gravid [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004634#pntd.0004634.ref023" target="_blank">23</a>]; Gravid: FPKM>100 at stage of gravid [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004634#pntd.0004634.ref023" target="_blank">23</a>].</p

Analysis on Gene Expression Profile in Oncospheres and Early Stage Metacestodes from <i>Echinococcus multilocularis</i>

<div><p>Alveolar echinococcosis is a worldwide zoonosis of great public health concern. Analysis of genome data for <i>Echinococcus multilocularis</i> has identified antigen families that can be used in diagnostic assays and vaccine development. However, little gene expression data is available for antigens of the egg and early larval stages. To address this information gap, we used a Next-Generation Sequencing approach to investigate three different stages (non-activated and activated oncospheres, and early stage metacestodes) of <i>E</i>. <i>multilocularis</i> (Nemuro strain). Transcriptome data analysis revealed that some diagnostic antigen gp50 isoforms and the antigen Eg95 family dominated in activated oncospheres, and the antigen B family dominated in early stage metacestodes. Furthermore, heat shock proteins and antigen II/3 are constantly expressed in the three stages. The expression pattern of various known antigens in <i>E</i>. <i>multilocularis</i> may give fundamental information for choosing candidate genes used in diagnosis and vaccine development.</p></div

Overview of the sequencing reads.

<p>Overview of the sequencing reads.</p

Morphology of different life cycle stages of <i>E</i>. <i>multilocularis</i>.

<p>A: Egg; B: Oncospheres (non-activated); C: Oncospheres (activating) with the hooks dispersive and body swelling; D: Oncospheres (activated) with the hooks aggregation in the smaller lobe after 24 hours activation; E: 4-week metacestodes miniature vesicles; F: Metacestodes small vesicles cultivated in vitro; Bar: 10μm (A-E), 1mm (F); Arrowhead: Miniature vesicles.</p

Primers for real-time PCR.

<p>Primers for real-time PCR.</p

Analyses of differentially expressed genes (DEGs) among non-activated oncophere (Nonc), activated oncosphere (Aonc) and early stage of metacestode (Emet).

<p>Analyses of differentially expressed genes (DEGs) among non-activated oncophere (Nonc), activated oncosphere (Aonc) and early stage of metacestode (Emet).</p