Immunization of horses with Crotalus durissus terrificus (South American rattlesnake) venom. A comparison of four different procedures.
Abstract: 1. A comparative study was carried out on horses immunized with Crotalus durissus terrificus venom using four different inoculation procedures, which included the use of 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). 2. The inoculation schedule used in horses to obtain antivenom serum consisted of sc injections of a 7.5 mg venom starting dose in 5.0 ml sterile saline emulsified with an equal volume of Freund's complete adjuvant. One week later, 7.5 mg venom in 5.0 ml sterile saline emulsified with an equal volume of Freund's incomplete adjuvant was injected. This was followed by three doses of 2.5 mg venom in 12.0 ml sterile saline at 2-day intervals. This immunization procedure, based on low doses of antigen (37.5 mg/horse) emulsified with Freund's adjuvant, produced a more protective and sustained immune response when compared with other procedures using A1(OH)3 or liposome emulsions with either low (50.0 mg/horse in 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 microliters serum/20 g mouse when antigen was emulsified with Freund's adjuvant; 21.7 microliters serum/20 g mouse when 870.0 mg antigen/horse was emulsified with A1(OH)3 and 30.0 microliters serum/20 g mouse when 50.0 mg antigen/horse 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. 3. 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 and sustained immune response, regardless of the initial immunization procedure. The ED50 evaluated for each of the animals five days after the reimmunization period was never more than 20 microliters serum/20 g mouse. 4. The liposome inoculation method employed a membrane-stabilized reverse phase evaporation preparation of sphingomyelin/cholesterol 2.5/l (w/w) liposomes. This procedure permits incorporation of 1.0 mg protein per mg of phospholipid.(ABSTRACT TRUNCATED AT 400 WORDS)
Publication Date: 1991-01-01 PubMed ID: 1823242
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- Comparative Study
- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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This research compares four procedures for immunizing horses against South American rattlesnake venom. Results indicate that the protocol using low doses of venom and Freund’s adjuvant produces a more protective and prolonged immune response than other methods.
Research Details and Procedure
- The study aimed to investigate and compare four different procedures for immunizing horses against the venom of South American rattlesnake, also known as Crotalus durissus terrificus.
- These procedures involved distinctive types of adjuvants – Freund’s adjuvant, A1(OH)3, and liposomes. Adjuvants are substances that enhance the body’s immune response to an antigen.
- Horses were injected with specific doses of venom in a series of injections – first using Freund’s complete adjuvant, followed by Freund’s incomplete adjuvant and then by sterile saline at regular intervals.
Results and Comparison
- The method involving low antigen doses (37.5mg/horse) with Freund’s adjuvant resulted in a better and lasting immune response as compared to other procedures that were tested during the study.
- Different antigen doses were trialled with the A1(OH)3 and liposome adjuvants, but these methods couldn’t match the efficacy achieved by Freund’s adjuvant.
- The immune potency against the venom was assessed by calculating the neutralizing median effective dose (ED50). This measures the amount of serum required to neutralize the venom in mice. Lower the ED50 value, higher the efficacy of immunity.
- Furthermore, the research found that horses immunized with the liposome adjuvant were not effectively protected from the lethal effects of the rattlesnake venom.
Re-immunization and Follow-up Study
- Later in the study, the horses were re-immunized using booster injections of the venom using Freund’s incomplete adjuvant or saline. This was done to obtain a hyperimmune serum response, which is a heightened or intense immune response.
- Interestingly, this reimmunization process also resulted in a protective and sustained immune reaction irrespective of the initial immunization protocol.
Liposome Inoculation Method
- This study further explored an inoculation procedure using liposomes, which are spherical vesicles that can incorporate protein into their structure. This method utilized a specific procedure for sturdying the membrane while preparing the liposome.
- Despite this innovative approach, the liposome-based procedure was not efficient in triggering an effective immune response against the venom.
Cite This Article
APA
Freitas TV, Fortes-Dias CL, Diniz CR, Velarde DT, Freitas CF.
(1991).
Immunization of horses with Crotalus durissus terrificus (South American rattlesnake) venom. A comparison of four different procedures.
Braz J Med Biol Res, 24(3), 281-290.
Publication
Researcher Affiliations
- Centro de Pesquisa e Desenvolvimento, Fundação Ezequiel Dias, Belo Horizonte, MG, Brasil.
MeSH Terms
- Animals
- Antibody Formation
- Antivenins / immunology
- Crotalid Venoms / immunology
- Enzyme-Linked Immunosorbent Assay
- Horses / immunology
- Immunization Schedule
- Immunization, Secondary
- Liposomes / immunology
- Mice
- Neutralization Tests
Citations
This article has been cited 3 times.- Arguedas M, Umaña D, Moscoso E, García A, Pereira C, Sánchez A, Durán G, Cordero D, Sánchez A, Segura Á, Vargas M, Herrera M, Villalta M, Gómez A, Salas C, Díaz C, María Gutiérrez J, León G. Comparison of adjuvant emulsions for their safety and ability to enhance the antibody response in horses immunized with African snake venoms. Vaccine X 2022 Dec;12:100233.
- Dias da Silva W, De Andrade SA, Megale ÂAA, De Souza DA, Sant'Anna OA, Magnoli FC, Guidolin FR, Godoi KS, Saladini LY, Spencer PJ, Portaro FCV. Antibodies as Snakebite Antivenoms: Past and Future. Toxins (Basel) 2022 Sep 1;14(9).
- Gilliam LL, Carmichael RC, Holbrook TC, Taylor JM, Ownby CL, McFarlane D, Payton ME. Antibody responses to natural rattlesnake envenomation and a rattlesnake toxoid vaccine in horses. Clin Vaccine Immunol 2013 May;20(5):732-7.
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