Groups

of 20 adult horses (400–450 kg) are regularly used to produce anti-Crotalus antivenom. Six horses (two per group) were selected to be immunized with C. d. terrificus venom, purified crotoxin or PLA2 venom components. The animals, not previously immunized, were maintained in a special animal house at the São Joaquim Farm, Instituto Butantan, São Paulo, Brazil. Before immunization, the animals were vaccinated against the most common equine infectious diseases. Male Swiss out bred mice, 18–20 g, (four per group) were used in protocols to determine the lethality (LD50) of the venoms and the Adriamycin neutralizing potency (ED50) of the antivenoms. Mice were kept in standard conditions, with light between 7:00 a.m. and 6:00 p.m., a temperature of 22 ± 2 °C and laboratory food and water ad libitum. All animals used in this study were maintained and treated under strict ethical conditions in accordance with the “International Animal Welfare recommendations” ( Remfry, 1987) and the “Committee Members, International Society on Toxinology” (1992). This project was approved by the Ethics Committee of Animal Usage in Research (Protocol No: 790/11) of the Instituto Butantan. Horses selected to produce regular antivenom

received STI571 four inoculations of a mixture containing equal amounts of C. d. terrificus and C. d. collilineatus venoms. Horses were immunized as described in Guidolin et al. (2010). Horses were s.c. injected in the back with 2.0 mL of

incomplete MMT80 or complete MMT80 adjuvant mixtures, or with 0.15 M Niclosamide NaCl containing 2.5–5.0 mg of venom. Two vaccinations were performed, with two weeks between the vaccinations. Two weeks after the last immunization, the antisera antibody titers were evaluated: the horses were bled, and a volume of blood corresponding to one-twelfth of each animal’s body weight was collected in a sterile plastic bag containing anticoagulant. Plasma and cells were separated by gravity sedimentation, and the cells were re-infused into the corresponding horse through the jugular vein. Plasma from the same horse was pooled and stored at 4 °C. Before immunization, blood samples were drawn by jugular vein puncture, and sera was stored at −20 °C for use as negative controls in the antisera antibody determinations. Three months after bleeding, boosters with similar doses of venom in PBS were given, and blood was collected and processed as described previously ( Guidolin et al., 2010). This final procedure was generally repeated for the following two years. Horses included in the Experimental Groups received four inoculations of the following mixtures. Experimental Group 1 (n = 2): 500 μg/animal of crude C. d. terrificus venom; Experimental Group 2 (n = 2): 200 μg/animal of partially purified crotoxin; and Experimental Group 3 (n = 2): 100 μg/animal of partially purified PLA2. The first inoculation of every group was prepared in Complete MMT80 and the respective antigens.