IN-VITRO HATCHING AND MAINTENANCE OF 2ND STAGE LARVAE OF TOXOCARA-VITULORUM

The tissue phase of Toxocara vitulorum cannot be diagnosed by conventi onal parasitological means. The present study was therefore aimed at d eveloping an appropriate immunodiagnostic test. For this T. vitulorum eggs, collected from the faeces of infected buffalo calves and termina l portion of the uterus of adult female worms, were embryonated in 0.5 % formalin at 28-degrees-C for 20-30 days. Thick outer coats of the em bryonated eggs were removed by treating with sodium hypochloride solut ion at 0.37-degrees-C for 4 hr. After removing the hypochlorite by 5 w ashings with sterile normal saline solution (NSS), decorticated eggs w ere vigorously shaken for 60 sec in a rubber-stoppered tube containing sterile sand and NSS. This process yielded more than 98% hatching of second stage larvae (L2). Larvae were cleaned of shell-debris and dead /injured larvae by Baemannisation for 8 hr in antibiotic-antimycotic a dded sterile Hank's Balanced Salt Solution with non-essential amino ac ids (HBSS-NEAA) using 3 layers of coarse cotton clotgh. Out of the 4 m edia used for maintaining the larvae in vitro, RPMI-1640 could maintai n more than 80% viable larvae for 28 days at 37-degrees-C and Eagle's Minimum Essential Medium with Hank's Salts (HMEM) for 21 days. In HBSS -NEAA and NSS all larvae were dead by 72 and 24 hr respectively. This technique could be used to obtain T. vitulorum L2 for antigen preparat ions, and RPMI-1640 and HMEM may be used as maintenance media for L2.