Fig 1.
Meta-analysis of filarial adult parasitological yields in gerbils, CB.17 SCID or BALB/c IL-4Rα-/-IL-5-/- mice.
(A) Total, (B) female or (C) male B. malayi recovered from the peritonea of gerbils, CB.17 SCID or BALB/c IL-4Rα-/-IL-5-/- mice. Data are number of worms as a % of initial larval inoculation. (D) Total peritoneal mf yields recovered from gerbils, CB.17 SCID or BALB/c IL-4Rα-/-IL-5-/- mice adjusted per female B. malayi recovered. (E-H) data stratified into infections of <25 & >25 weeks. Each point represents an individual animal, red horizontal lines represent median and inter-quartile range values. Significance is indicated as ****P<0.0001, ***P<0.001, **P<0.01, *P<0.05 (Kruskal-Wallis with Dunn’s multiple comparison’s tests comparing between rodent models or Mann-whitney tests comparing between time points).
Table 1.
Summary of B. malayi adult parasitology in gerbils, CB.17 SCID mice and BALB/c IL-4Rα-/-IL-5-/- mice.
Fig 2.
female and male B. malayi survival, motility and metabolic activity during culture with different cell and media conditions.
(A) Kaplein-Meier survival or (B) average motility of female B. malayi over 14 days. (C) Individual female B. malayi motility scores or (D) MTT reductase activity of cultured female B. malayi at day 14, normalized to median MTT reductase activity in a sample five freshly isolated female B. malayi (median optical density = 0.48). (E) Kaplein-Meier survival or (F) average motility of male B. malayi over 14 days. (G) Individual male B. malayi motility scores or (H) MTT reductase activity of cultured male B. malayi at day 14, normalized to median MTT reductase activity in a sample five freshly isolated male B. malayi (median optical density = 0.32). Data is combined from 24 culture wells initially containing 48 viable filariae (A,E) or between 6–12 culture wells initially containing 12–24 viable filariae (B-D and F-H). Horizontal bars represent the median with interquartile range. Significance is indicated ***P<0.001, **P<0.01, *P<0.05 (Kruskal-Wallis with Dunn’s multiple comparison’s tests).
Fig 3.
21 day trans-well adult B. malayi female co-culture system with macrophage leukocytes or LEC bilayers.
(A) schematic of 2D trans-well co-culture system (B) Kaplein-Meyer survival curves or (C) average motility scores over 21-day culture period. (D) individual motility scores or (E) MTT reductase activity at day 21 normalized to median MTT reductase activity in a sample of five freshly isolated female B. malayi (median optical density = 0.48). Data is combined from 6 culture wells initially containing 12 viable female B. malayi. Horizontal bars represent the median with interquartile range. Significance is indicated ***P<0.001, **P<0.01, *P<0.05 (Mantel-Cox log-rank test or Kruskal-Wallis with Dunn’s multiple comparison’s tests).
Fig 4.
Comparison of Wolbachia titres over culture period in relation to in vivo recovered parasites.
(A) Microfilariae release / female B. malayi at days 3, 7 and 14 after culturing in LEC bilayers (B) Wolbachia titres / B. malayi female removed from IL-4Rα-/-IL-5-/- mice or (week 0) or maintained in LEC bilayer co-cultures for 1 or 2 weeks (C) schematic of experimental groups: group 1; female B. malayi freshly isolated from in vivo infection (CB.17 SCID mice), group 2; female B. malayi 2-weeks after single sex implantation into CB.17 SCID mice, group 3; female B. malayi 2-weeks after culture in LCE bilayers. (D) MTT reductase activity and (E) Wolbachia titres / B. malayi female derived from groups 1–3. Horizontal bars represent medians with interquartile range. Data is derived from a single experiment of 6 cultures (A) combined of three experiments of 6 cultures (B) or a single experiment of 12 cultures (D,E). Statistical significance determined by Kruskal-Wallis one-way ANOVA with Dunn’s post-hoc tests are indicated ns; not significant, *P<0.05, **P<0.01 and ***P<0.001.
Fig 5.
Validation of B. malayi female culture system as an anti-Wolbachia drug screen.
(A) Schematic of anti-Wolbachia drug screen (B) Kaplein-Meier survival or (C) average motility of female B. malayi over 14 days to indicated exposures of doxycycline (DOX), TylAMac or vehicle (1% DMSO), (D) Anti-Wolbachia efficacy following 7 days or (E) 14 days of indicated exposures to DOX, TylAMac or vehicle. Data plotted is wsp copy numbers/Bm female determined by qPCR (D-E). Data is combined from two experiments with 6 culture wells initially containing 12 viable female B. malayi (for vehicle & DOX groups) or a single experiment of 6 culture wells for Flubentylosin groups. Horizontal bars represent median with interquartile range. Significance is indicated ***P<0.001, **P<0.01, *P<0.05 (Kruskal-Wallis with Dunn’s multiple comparison’s tests).
Fig 6.
Validation of culture model as a direct-acting macrofilaricide drug model.
(A) Schematic of macrofilaricidal drug screen (B) Kaplein-Meyer survival or (C) average motility scores of female B. malayi over 14 days during exposure to 10μM of flubendazole (FBZ), suramin (SUR) or vehicle (1% DMSO), (D) individual motility scores or (E) MTT reductase activity following 14 days of 10μM FBZ, SUR or 1% DMSO vehicle. Data is combined from two experiments with 6 culture wells initially containing 12 viable female B. malayi. Horizontal bars represent median with interquartile range. Significance is indicated ***P<0.001, **P<0.01, *P<0.05 (Kruskal-Wallis with Dunn’s multiple comparison’s tests.