Article Figures & Data
Figures
- FIG 1
Effects of gamma irradiation on B. thuringiensis spore viability and Cry5B bioactivity. (A) Effect of 15 kGy of gamma irradiation on spore counts for two different Cry-deficient B. thuringiensis strains (4.D.8 and 4.D.9) transformed with a Cry5B-expressing plasmid. (B) Comparison of Cry5B efficacy expressed in 4.D.8 and 4.D.9 on C. elegans viability at 100 µg/ml before and after 15-kGy irradiation. Labels without “15 kGy” indicate non-gamma-irradiated samples.
- FIG 2
BaCC (bacteria with cytosolic crystals) and IBaCC (Inactivated bacteria with cytosolic crystals). (A) spo0A-deficient B. thuringiensis cells expressing Cry5B from a vegetative promoter before (BaCC) and after (IBaCC) treatment with essential oil. Cry5B bipyramidal crystals (dark) are evident inside the bacteria pre- and posttreatment. Scale bars, 5 µm. (B) Protein gel showing Cry5B protein expressed in spo0A-deficient cells before and after essential oil treatment. (C) Spore counts from spo0A-deficient cells expressing Cry5B before and after essential oil treatment along with standard deviation (actual spore counts, 2.1 × 107 CFU/ml in BaCC and 0 CFU/ml in IBaCC).
- FIG 3
Efficacy of IBaCC in vitro against nematodes. (A) C. elegans. (Left) Photos of C. elegans N2 exposed to various conditions (spore crystal lysate [SCL] and IBaCC; both 40 µg/ml Cry5B). Scale bar, 200 µm. C. elegans was treated with azide to immobilize them just prior to imaging. (Right) Viability of C. elegans glp-4(bn2) L4 hermaphrodites under various conditions (IBaCC, 29 µg/ml Cry5B). P value for comparison is from a one-tailed t test. (B) Hookworm larval development. Plotted are the numbers of L3i larvae that developed from 60 hookworm eggs within 7 days (A. ceylanicum, left; N. americanus, right). x axes indicate the concentrations of Cry5B for IBaCC (IBa, 0 ng/ml for all).
- FIG 4
Impact of Cry5B IBaCC on adult hookworms in vitro. Adult hookworm motility over time at 5, 50, and 200 µg/ml Cry5B for A. ceylanicum adults (A) and N. americanus adults (B) in vitro averaged over three independent trials. In all figures, plots show averages and standard errors. (C) (Left) SDS-PAGE gel showing IBaCC before (IBaCC) and after (rhod-IBaCC) labeling with rhodamine. (Right) UV fluorescence image of same gel, showing predominant labeling of full-length Cry5B band for rhod-IBaCC. (D) Uptake of rhod-IBaCC. (Left) Bright-field image of IBaCC and rhodamine fluorescence image of adult hookworm fed IBaCC for 4 h. (Right) Bright-field image of rhod-IBaCC and rhodamine fluorescence image of adult hookworm fed rhod-IBaCC for 4 h. Uptake of rhodamine-labeled Cry5B crystals is evident.
- FIG 5
Efficacy of IBaCC produced in the laboratory in vivo against both human hookworm genera. (A) Shown are mean A. ceylanicum hookworm burdens (left) and fecal egg counts (right) in infected hamsters treated with water control, IBa, or IBaCC containing Cry5B. Here and in subsequent figures, error bars are standard errors. EPG, eggs per gram of feces. (B) Mean N. americanus hookworm burdens (left) and fecal egg counts (right) in infected hamsters treated with water control or IBaCC containing Cry5B.
- FIG 6
Efficacy of production facility IBaCC in vivo against A. ceylanicum hookworms. (A) Dose response of A. ceylanicum burdens (left) and fecal egg counts (right) in infected hamsters treated with water control or CMO-produced IBaCC containing Cry5B. (B) A. ceylanicum burdens (left) and fecal egg counts (right) in infected hamsters treated with water or CMO-produced 15 mg/kg Cry5B in IBaCC. EPG, eggs per gram of feces. P values for relevant comparisons are given.
- FIG 7
Fit-for-purpose formulation studies for Cry5B IBaCC. (A) Mean A. ceylanicum hookworm burdens (left) and fecal egg counts (right) in infected hamsters treated with water control, IBaCC, or IBaCC mixed with sodium bicarbonate. No cimetidine pregavage was used in these experiments. (B) Mean A. ceylanicum hookworm burdens (left) and fecal egg counts (right) in infected hamsters treated with water control, IBaCC, or the same batch of IBaCC freeze-dried (FD). P values for relevant comparisons are given.
- FIG 8
Confirmation of fit-for-purpose Cry5B IBaCC efficacy against a different, luminal feeding parasite in a second host. Mean H. polygyrus bakeri burdens (left) and fecal egg counts (right) in infected mice treated with water, sodium bicarbonate, or single-dose freeze-dried (FD) Cry5B IBaCC mixed with sodium bicarbonate. The FD-IBaCC used was an independent batch from that used for the data shown in Fig. 7.
Additional Files
Supplemental material
- Supplemental file 1 -
Supplemental Figures S1 and S2 and Tables S1 to S3
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