Extraction and Quantification of Cattle Nematode Larvae

This was done following a method we described previously [30 (link)], with slight modifications. Briefly, fresh pieces of nodule-containing umbilical cattle skin were purchased from a local slaughter house, washed repeatedly with tap water until all dirt was removed, and rinsed with distilled water. The skin was towel-dried and sterilized with 70% ethanol, allowed to dry in a laminar flow hood and firmly attached to an autoclaved cylindrical-shaped wooden block. Using sharp razor blades, criss-cross cuts were made into the skin and submerged in ICM (incomplete culture medium: RPMI-1640 containing penicillin, streptomycin and amphotericin B) for 4 hours, after which, the medium was centrifuged at 700xg for 20 minutes using an Eppendorf 5810R centrifuge (Eppendorf, Germany) to concentrate the mf. After viability check, the number of mf were counted and adjusted to the required number (4,000–22,725 in 200–1,000 μl of ICM) for experimental infection.