BS96hT2
Eighteen, 6-to-7-week-old, Toxocara-naive Beagle puppies were assigned to the study. Information about the rearing environment of puppies, absence of prior infection, and detection of experimental T. canis infection has been described in previous studies [12, 13, 14]. Briefly, 18 puppies were randomly assigned to 3 groups, each representing a time point of larval development in the host: 24 hpi, 96 hpi and 36 dpi. Each group of six dogs included three infected and three control dogs. For the infected groups, all puppies were orally inoculated with 300 embryonated T. canis eggs in 1 ml of sterile saline, while the puppies in the control groups were inoculated with 1 ml of saline, without eggs. Puppies were euthanized by intracardiac injection of potassium chloride (KCl) solution, under general anesthesia using Zoletil 50 (Virbac, Carros, France). This procedure was performed by a trained veterinarian with knowledge of anesthetic techniques to ensure the suitability of the anesthetic depth prior to administration of KCl. After washing the surface of the spleen with saline solution, ~12 g of splenic tissue was dissected out and stored in liquid nitrogen for extraction of the total RNA. None of the puppies exhibited any adverse health effects or died before they were euthanized.