Annotation of the Complete Wolbachia Genome

<p>(Figure from [<a href="http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.0020110#pmed-0020110-b18" target="_blank">18</a>])</p

Cross-Section of a Female Worm from an Extirpated Nodule from a Patient with Onchocerciasis

<div><p>Wolbachia, endosymbiotic bacteria of filarial nematodes important for embryo development, are targets for antifilarial treatment. The endobacteria cause inflammation and contribute to the pathology of lymphatic filariasis and onchocerciasis. The bacteria here were stained with antibodies against Yersinia Hsp60. The bacteria are located in the hypodermis and oocytes of the worm (arrows). h, hypodermis; i, intestine; m, muscle; u, uterine tube.</p> <p>(Photo: D.W. Büttner, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany)</p></div

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

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

Predicted minimum remaining program duration required until elimination of onchocerciasis, assuming ivermectin efficacy as in assumption set 1 and low inter-individual variation in exposure to fly bites.

<p>Panels illustrate the minimum remaining program duration (y-axis) required for 99% probability of elimination (absence of infection 50 years after the mass last treatment), given the number of annual mass treatment rounds already completed (x-axis), as predicted by ONCHOSIM (1,000 simulations per scenario). Each panel compares four strategies: continuing annual mass treatment at same coverage (solid black line), switching to 6-monthly mass treatment at same coverage (dashed black line), switching to 3-monthly mass treatment at same coverage (dotted black line), or continuing annual treatment at increased coverage (+15 percentage points; solid blue line; only for past mass treatment coverage of 50% and 65%). Different panels pertain to increasing pre-control infection levels (top to bottom), and increasing values of past mass treatment coverage (left to right). Grey lines represent smoothed and where relevant extrapolated trendline of simulated outcomes, fitted such that they intersect with the x-axis at the same point as graph lines for annual mass treatment (black solid lines). Values in the corner of each panel represent reductions in remaining program duration (pooled over scenarios for different numbers of past treatment rounds), when increasing coverage (a), switching to 6-monthly mass treatment (b), or switching to 3-monthly mass treatment (c), compared to continuing annual treatment at the same coverage. Panels marked with an asterisk (*) pertain to simulations that did not result in 99% probability of elimination within 20 future treatment rounds, and hence contain no graph lines.</p

Sensitivity analysis for assumptions about density dependence in transmission and permanent effects of ivermectin on adult worms.

<p>The results presented here are based on a setting where the pre-control community microfilarial load is 30 microfilariae per skin snip.</p><p>* Numbers in parentheses represent differences relative to the strategy of continuing mass treatment annually at 65% coverage.</p><p>** The probability of elimination was less than 99% within the scope of the simulations (maximum 20 future treatment rounds).</p><p>*** Permanent effects of ivermectin on adult worms were assumed to be either a factor 2/3 lower or a factor 3/2 higher (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115886#pone-0115886-t002" target="_blank">Table 2</a> for details).</p><p>Sensitivity analysis for assumptions about density dependence in transmission and permanent effects of ivermectin on adult worms.</p

Two sets of assumptions about ivermectin efficacy in ONCHOSIM.

<p>Assumption set 1 was quantified such that ONCHOSIM could reproduce trends in skin mf levels as observed in a community trial that encompassed five consecutive annual ivermectin treatments <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115886#pone.0115886-Plaisier2" target="_blank">[14]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115886#pone.0115886-Alley1" target="_blank">[19]</a>. Assumption set 2 was quantified such that ONCHOSIM could reproduce trends in worm survival during three years of 3-monthly and 6-monthly mass treatment, as estimated from nodulectomy data <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115886#pone.0115886-Cupp3" target="_blank">[23]</a>, and trends in skin mf levels up to two years after a single dose of ivermectin as reported in a published meta-analysis <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115886#pone.0115886-Basez2" target="_blank">[31]</a>. Parameter values were fitted to the data with maximum likelihood, using the mean output of 100 repeated ONCHOSIM simulations as expected values (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115886#pone.0115886.s007" target="_blank"><b>S1 Text</b></a> for details).</p>a<p>Excess mortality has been reported for both female <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115886#pone.0115886-Cupp3" target="_blank">[23]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115886#pone.0115886-Klger2" target="_blank">[30]</a> and male worms <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115886#pone.0115886-Duke2" target="_blank">[25]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115886#pone.0115886-Duke3" target="_blank">[26]</a>. In the current study, excess mortality due to ivermectin was allowed to differ between male and female worms, reflecting the relative absence of male worms from subcutaneous nodules after repeated ivermectin treatment <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115886#pone.0115886-Cupp2" target="_blank">[22]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115886#pone.0115886-Klger2" target="_blank">[30]</a>. The macrofilaricidal effects of ivermectin were allowed to vary per treatment; however, this variation could not be estimated due to the aggregated nature of the Guatemalan data <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115886#pone.0115886-Cupp3" target="_blank">[23]</a>. Instead, we arbitrarily assumed beta distributions with sample size 50 and mean 6% for males (2.5% and 97.5% percentiles 1.3%–14.0%) and 12% (3.9%–19.0%) for females, with the macrofilaricidal effects on male and female worms being perfectly correlated. Macrofilaricidal effects were assumed to be independent of earlier exposure to ivermectin and worm age, and hence reproductive capacity of the worm. In the sensitivity analysis, we set the average macrofilaricidal effects to either 4% and 8% (for males and females), or 9% and 18% (difference of factor 2/3 or 3/2) while keeping the sample size of the beta distribution at 50.</p>b<p>This treatment effect was assumed to vary per worm and treatment; 2.5% and 97.5% percentiles 2–24 months.</p>c<p>This assumption represents the notion that ivermectin causes temporary congestion of female worm uteri with dead mf, effectively preventing insemination and release of microfilariae <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115886#pone.0115886-Chavasse1" target="_blank">[20]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115886#pone.0115886-Klger1" target="_blank">[21]</a>. Time until recovery was assumed to vary per worm and treatment, and to follow an exponential distribution with mean 3.5 years (fitted to data <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115886#pone.0115886-Basez2" target="_blank">[31]</a>). This implies that 5% of adult female worms can be inseminated and release microfilariae within two months after exposure to ivermectin. Likewise, congestion resolves in 25%, 50%, 75%, and 95% of adult female worms within 1, 2.5, 5, and 10.5 years after exposure to ivermectin, respectively.</p>d<p>To account for variation in treatment efficacy between persons and treatments, for every simulated person and treatment, the average reduction was multiplied with a random value drawn from a Weibull distribution with mean 1 and shape 2 (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115886#pone.0115886.s007" target="_blank">S1 Text</a>). In the sensitivity analysis, the average reduction was set to 23% or 52% (difference of factor 2/3 or 3/2).</p><p>Two sets of assumptions about ivermectin efficacy in ONCHOSIM.</p

Immunization reduces adult worm burden, but does not affect their development.

<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. Numbers of worms on days 15 (A), 56 (B), 70 (C) and 90 (D) p.i. (additional experiments see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001558#pntd.0001558.s005" target="_blank">Figure S5A</a>–C), gender balance (E) (individual experiments see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001558#pntd.0001558.s005" target="_blank">Figure S5D</a>, E), as well as length of males (F) and females (G) at day 90 p.i. (10/90 percentile, outliers are indicated, individual experiments see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001558#pntd.0001558.s005" target="_blank">Figure S5F</a>–I) were analyzed with Student's t-test (** <i>P</i><0.01, *** <i>P</i><0.001).</p

Predicted trends in probability of elimination over time for settings with different history of control.

<p>The three panels represent predictions for different histories of control in terms of number of past treatment rounds (14 or 8) and mass treatment coverage (65% or 80%). Black lines represent the probability of elimination (y-axis) if mass treatment were to be suspended at a certain point in time (x-axis). Trends until now (time 0) are displayed against a shaded background, while expected future trends are shown against a white background. Different line types pertain to different future mass treatment frequencies (annual, 6-monthly, or 3-monthly). Red lines highlight the predicted minimum remaining program duration required to achieve 99% probability of elimination (based on 1,000 repeated simulations). The three panels are equal with respect to assumed transmission conditions (pre-control community microfilarial load of about 30 mf per skin snip, low variation between individuals in relative exposure to fly bites) and ivermectin efficacy (assumption set 1). Elimination was defined as absence of infection 50 years after suspension of mass treatment.</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