Odile Bain (April 28, 1939–October 16, 2012): A Life Dedicated to Systematics and Biology of Filariae

<p>Odile Bain (April 28, 1939–October 16, 2012): A Life Dedicated to Systematics and Biology of Filariae</p

Photo courtesy of Kerstin Junker (Ile de France, 2008).

<p>Photo courtesy of Kerstin Junker (Ile de France, 2008).</p

Filarial nematodes used to assess the analytical specificity of the qPCR assay.

<p>Filarial nematodes used to assess the analytical specificity of the qPCR assay.</p

Immunization induces Mf-specific IgG1 and IgG2.

<p>Mice were immunized three times s.c. with 100,000 Mf in alum (Al-Mf/naïve, Al-Mf/Inf). Control mice received alum alone (Al/naïve, Al/Inf). <i>L. sigmodontis</i> challenge infection was performed one week after the last immunization (Al/Inf, Al-Mf/Inf) or left uninfected (Al/naïve, Al-Mf/naïve). Plasma levels of Mf-specific IgG1 (A) and IgG2a/b (B) were measured. Two-way ANOVA was used for statistical analysis, day 0 indicates day of challenge infection. Asterisks indicate significant differences between the immunized and infected, and the corresponding control group (*** <i>P</i><0.001) and pound signs between the immunized but uninfected, and the corresponding control group (^#<i>P</i><0.05, ^##<i>P</i><0.01, ^###<i>P</i><0.001). (C–F) Pleural space lavage was analyzed for specific IgG1 and IgG2a/b on days 22 (C, D) and 70 p.i. (E, F). Data analyzed with Welch-corrected t-test (mean, *** <i>P</i><0.001). Graphs show representatives of three independent experiments with eight to ten mice each group (additional experiments see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001558#pntd.0001558.s006" target="_blank">Figure S6A, B, E–J</a>).</p

Mice immunized with Mf in alum have reduced numbers of Mf.

<p>Mice were immunized three times s.c. with 100,000 Mf in alum. Control mice received alum alone. <i>L. sigmodontis</i> infection was performed one week after the last immunization. Microfilaraemia was monitored twice a week throughout patency. (A) Kinetics of Mf load of sham-treated (dashed line) and immunized (black line) mice in the peripheral blood. One representative of three independent experiments with ten mice per group is shown (2-way ANOVA, mean ± SEM), including both Mf⁻ and Mf⁺ mice. For additional experiments see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001558#pntd.0001558.s003" target="_blank">figure S3A</a>, B. (B) Percentage of Mf⁺ mice of three independent experiments was analyzed using Student's t-test. Each mouse with peripheral Mf at any given time point was defined as Mf⁺. (C, D) Mf burden in the pleural space days 70 (C) and 90 (D) p.i.. Graphs show one representative of three (C) and two (D) independent experiments (at least seven mice each group, see also <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001558#pntd.0001558.s003" target="_blank">Figure S3C</a>–E) and were analyzed with Welch-corrected t-test. Numbers below the symbols indicate the number of Mf⁺ mice (median, * <i>P</i><0.05, ** <i>P</i><0.005).</p

Skin samples tested for <i>Onchocerca lupi</i> by qPCR, divided (Groups 1–5) according to the parasitic load (mfs) microscopically detected.

<p>The mean, minimum, maximum and standard deviation (sd) values of the threshold cycle (Cq), parasite load (Starting Quantity (SQ) value, expressed as ng/μl of DNA for reaction) and microfilariae concentration, assessed by qPCR is reported.</p

Immunization inhibits embryogenesis in female worms.

<p>Mice were immunized three times s.c. with 100,000 Mf in alum. Control mice received alum alone. <i>L. sigmodontis</i> challenge infection was performed one week after the last immunization. Seventy days after infection female worms were analyzed for their embryonic stages. Representative pictures of oocyte (A; micron bar 10 µm), divided egg (B; 10 µm), pretzel stage (C; 15 µm) and stretched Mf (D; 30 µm) are shown. (E) Embryogram illustrating the composition of embryonic stages in female worms. If present, three female worms of each mouse were investigated (27 females in the control group, 28 females from the immunized group, additional experiments see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001558#pntd.0001558.s004" target="_blank">Figure S4</a>). Statistical analysis was performed with Mann-Whitney U-test (mean ± SEM, ** <i>P</i><0.01, *** <i>P</i><0.001).</p

Detection limit of the conventional PCR assay determined by 10-fold serial dilution of genomic DNA of microfilariae and adult of <i>Onchocerca lupi</i>.

<p>Lanes 1–4, from 3.6 ×10¹ pg/2μl to 3.6 ×10⁻³ pg/2μl of <i>O</i>. <i>lupi</i> mfs DNA (i.e., from 1 to 1×10⁻⁴ mfs); Lanes 5–15, from 8 ×10¹ ng/2μl to 8 x 10⁻³ fg/2μl of <i>O</i>. <i>lupi</i> adult DNA; Line 16, no-DNA control; M, 100 bp DNA marker.</p

A real-time PCR tool for the surveillance of zoonotic <i>Onchocerca lupi</i> in dogs, cats and potential vectors - Fig 2

<p>Standard curves generated from serial dilutions of (A) genomic DNA from adult (from 8 × 10⁴ to 8 × 10⁻³ fg/2μl of reaction) and microfilariae (B) (from 3.6 ×10⁻¹ ng/2μl to 3.6 ×10¹ fg/2μl of reaction) of <i>Onchocerca lupi</i>. Each point was tested in triplicate. Slope, efficacy and <i>R</i>² are reported on the bottom.</p

Assessment of the specificity of qPCR assay in the detection of <i>Onchocerca lupi</i> DNA.

<p>The amplification plot is represented by the fluorescent signal accordingly to relative fluorescence units (RFU) and threshold cycle.</p