(117) Assay Card

General Information
Summary A range of concentrations of kB1 was tested and scoring was conducted after 24, 48 and 72 h. After 24 h, motility scores of 0 were recorded at the two highest concentrations tested and the intermediate concentrations responded in a dose-dependent manner. After 48 h, there was a significant reduction in motility and, after 72 h, motility scores of 0 were recorded for all concentrations tested except the lowest two (<25 g/mL). It was also noted that while in the control assay tubes, a large proportion of laid eggs hatched to L1 larvae after 24 h, very few eggs hatched in the kB1 treated assay tubes above 400 g/mL and nearly all newly hatched larvae were dead above kB1 concentrations of 50 g/mL.
Condition The effects of cyclotides on the adult stages of H. contortus were assessed by observing the degree of motility shown by worms over a period of exposure to the peptides in vitro. Briefly, adult worms were recovered from sheep abomasa (approximately 6?10 weeks post infection) by manual picking from the gut contents. They were placed in culture medium (RPMI-1640, HEPES buffer, glucose, bovine serum, antibiotics, and fungicide) for several hours, and then groups of six were placed in separate tubes in 0.5 mL of culture medium. The only significant change from the previously described method was the inclusion of 20% newborn bovine serum in the culture medium, and the subsequent maintenance of the worms in an atmosphere of 20% CO2, 5% O2, and 75% N2 (compared to 5% CO2 in air for the earlier study). The cyclotides were added to the tubes at various concentrations and the tubes were kept at 37 C. Each compound was tested in triplicate. At 24 h intervals, the worms were observed and their degree of motility was scored. The assay tubes were placed onto a warm tray and tubes were held individually near a light for assessment of motility. Each tube was swirled to thoroughly disturb the nematodes and was scored according to the degree of motility shown by the worms using a scoring system. Briefly, the worms were scored as the following: 3, most individuals showing significant smooth sinusoidal motion, similar to motion at the start of the culture period; 2, significant movement shown by a small number of individuals, at least one individual able to move in a normal sinusoidal fashion; 1, only very limited movement in a small number of individuals, no sinusoidal motion; and 0, no movement.
Result A range of concentrations of kB1 was tested and scoring was conducted after 24, 48 and 72 h. After 24 h, motility scores of 0 were recorded at the two highest concentrations tested and the intermediate concentrations responded in a dose-dependent manner. After 48 h, there was a significant reduction in motility and, after 72 h, motility scores of 0 were recorded for all concentrations tested except the lowest two (<25 g/mL). It was also noted that while in the control assay tubes, a large proportion of laid eggs hatched to L1 larvae after 24 h, very few eggs hatched in the kB1 treated assay tubes above 400 g/mL and nearly all newly hatched larvae were dead above kB1 concentrations of 50 g/mL.
AssayType Nematocidal

References
Colgrave ML, Kotze AC, Huang YH, O'Grady J, Simonsen SM, Craik DJ. (2008) Cyclotides: natural, circular plant peptides that possess significant activity against gastrointestinal nematode parasites of sheep. Biochemistry 47:5581-5589

Cross-references
Proteins Assayed kalata B1