Immunization of horses with Crotalus durissus Terrificus (South American Rattlesnake) venom: a comparision of four different procedures

RESUMO

A comparative study was carried out on horses immunized with Crotalus durissus terrificus venom using four different inoculation procedures, which included the use Freund's adjuvant, A1(OH)3 and liposomes as adjuvants. The antibody titer was assessed by enzyme linked immunosorbent assay (ELISA) and the neutralizing potency by the neutralizing median effective dose (ED50). The inoculation schedule used in horses to obtain antivenom serum consisted of scinjections of a 7.5 mg venom starting dose in 5.0ml sterile saline emulsified with an equal volume sterile saline at 2-dayintervals. This immunization procedure, based in low doses of antigen (37.5mg/horse) emulsified with Freund's adjuvant, proceduced a more protective andsustained immune response whencompared with other procedures using A1(OH)3 and 5.0 mg/horse in liposome) or high (870.0 mg/horse in A1(OH)3 and 20.0 mg/horse in liposome) antigen doses. The ED50 values evaluated at the end of the procedure were 15.4 *l serum/20 gmouse when antigen was emulsified with Freund's adjuvant; 21.7 * serum/20 g mouse when 870,0 mg antigen/horse was emulsified with A1(OH)3 and 30.0 *l serum/20 g mouse when 50.0 mg antigen/hors was emulsified with a1(OH)3.When antigen was emulsified with liposome, the immune serum was ineffective against the lethal effects of C.d terrificus venom. The inoculation schedule used in horses to obtain hyperimmune serum consisted of reimmunization with sc booster injections of 7.5 mg venom in 5.0 ml sterile saline emulsified with an equal volume of Freund's incomplete adjuvant. One week later, 2.5 mg venom in 12.0 ml sterile saline was inoculated at 2-day intervals.This reimmunization schedule,based on low doses of antigen (15.0 mg/horse) emulsified with Freund's incomplete adjuvant or with saline, produced a protective andsustained immune response, regardless of the initial immunization procedure. The ED50 evaluatedfor each of the animals five days after the reimmunization period was never more than 20 * serum/20 g mouse. The liposome inoculation method employed a membrane-stabilized reverse phase evaporation preparation of sphingomyelin/cholesterol 2.5/1 (w/w) liposomes. This procedure permits incorporation of 1.0 mg protein per mg of phospholipid. This liposome inoculation method , which stimulates a rapid, sustained and protective immune response in mice and rabbits inoculated with both C.d. collineatus and C.d. terrificus, was not effective when horses were immunized with C.