Login Register Cart 0
Generic placeholder image

Journal of Current Toxicology and Venomics

Editor-in-Chief

ISSN (Print): 2950-5704
ISSN (Online): 2950-5712

Back
Journal Home About Journal Editorial Board Current Issue Volumes/Issues
Subscribe
Research Article

Pre-clinical Efficacy and Immunogenicity of IgY Antibodies Directed against Crotalus durissus cumanensis Venom

Author(s): Yuyibeth Montero, Noraida Zerpa, Yudith Neves, Oriana Zerpa, Aurora Álvarez and Caridad Malavé*

Volume 4, 2024

Published on: 28 May, 2024

Article ID: e280524230401 Pages: 11

DOI: 10.2174/0126661217296708240506074324

Price: $65

Abstract

Background: Crotalus durissus cumanensis (C.d.c.) is the most widely distributed snake in Venezuela, causing the majority of snakebite envenoming.

Objective: The purpose of this study was to produce IgY antibodies against a C.d.c. venom pool from different Venezuelan regions and evaluate their neutralization capacity on various venom toxic activities.

Methods: Anti-C.d.c. venom antibodies are purified from chicken egg yolks by precipitation with polyethylene glycol and further analyzed by Multiple Antigen Blot Assay, indirect ELISA, Western blot, and Inhibition assays. In addition, we evaluate the phospholipase, edematogenic, and hemorrhagic activities. In addition, a new envenoming simulation study using anti-C.d.c. venom IgY in mice is presented.

Results: In this study, we show that anti-C.d.c. venom IgY is capable of neutralizing 4 LD50 doses of the Cdc venom (i.e., 1.76 mg of IgY neutralized 14 μg of C.d.c. venom) and effectively neutralizing the phospholipase, edematogenic and hemorrhagic activities. Additionally, the anti C.d.c. venom IgY specifically recognizes polypeptide bands with apparent molecular masses of ~ 54.55, 30.39, 24.1, 14.02, and 9.44 kDa by western blot. The IgY specificity is demonstrated by a dose-dependent inhibition, in which antibodies pre-adsorbed with the C.d.c. venom does not recognize the proteins contained in the venom. Furthermore, in the simulation study of envenoming, the mice inoculated with IgY showed no response.

Conclusion: Our results support the use of anti-venom IgY as an alternative to traditional equine therapy in animals and, eventually, in human patients bitten by C.d.c snakes.

[1]
Parra L, Peña J, Parra AR, Echezuria L, Rodriguez-Morales A. Trends in fatal snakebites in Venezuela, 2003–2007. Int J Infect Dis 2010; 14: e138.
[http://dx.doi.org/10.1016/j.ijid.2010.02.1789]
[2]
De Sousa L, Bastouri-Carrasco J, Matos M, et al. Epidemiology of ophidism in Venezuela (1996-2004). Invest Clin 2013; 54(2): 123-37.
[PMID: 23947002]
[3]
Aguilar I, Guerrero B, Maria Salazar A, et al. Individual venom variability in the South American rattlesnake Crotalus durissus cumanensis. Toxicon 2007; 50(2): 214-24.
[http://dx.doi.org/10.1016/j.toxicon.2007.03.012] [PMID: 17482229]
[4]
Hernández M, Scannone H, Finol HJ, et al. Alterations in the ultrastructure of cardiac autonomic nervous system triggered by crotoxin from rattlesnake (Crotalus durissus cumanensis) venom. Exp Toxicol Pathol 2007; 59(2): 129-37.
[http://dx.doi.org/10.1016/j.etp.2007.04.002] [PMID: 17616380]
[5]
Montilla J, Alvares de Montilla M, Diaz E. Hiperinmunizacion de ovinos contra veneno de Crotalus durisus cumanensis del Estado Zulia, Venezuela. Revista Científica 1999; IX: 388-94.
[6]
Theakston RDG, Warrell DA, Griffiths E. Report of a WHO workshop on the standardization and control of antivenoms. Toxicon 2003; 41(5): 541-57.
[http://dx.doi.org/10.1016/S0041-0101(02)00393-8] [PMID: 12676433]
[7]
Rodríguez-Acosta A. Los venenos y el síndrome de envenenamiento ofídico. 2001. Available From: https://dialnet.unirioja.es/servlet/articulo?codigo=1227970
[8]
Thalley BS, Carroll SB. Rattlesnake and scorpion antivenoms from the egg yolks of immunized hens. Biotechnology (N Y) 1990; 8(10): 934-8.
[PMID: 1366776]
[9]
Maya Devi C, Vasantha Bai M, Vijayan Lal A, Umashankar PR, Krishnan LK. An improved method for isolation of anti-viper venom antibodies from chicken egg yolk. J Biochem Biophys Methods 2002; 51(2): 129-38.
[http://dx.doi.org/10.1016/S0165-022X(02)00002-7] [PMID: 12062112]
[10]
Karlson-Stiber C, Persson H. Antivenom treatment in Vipera berus envenoming—report of 30 cases. J Intern Med 1994; 235(1): 57-61.
[http://dx.doi.org/10.1111/j.1365-2796.1994.tb01032.x] [PMID: 8283161]
[11]
Araújo AS, Lobato ZIP, Chávez-Olórtegui C, Velarde DT. Brazilian IgY-Bothrops antivenom: Studies on the development of a process in chicken egg yolk. Toxicon 2010; 55(4): 739-44.
[http://dx.doi.org/10.1016/j.toxicon.2009.11.004] [PMID: 19925817]
[12]
Paul K, Manjula J, Deepa EP, Selvanayagam ZE, Ganesh KA, Subba Rao PV. Anti-Echis carinatus venom antibodies from chicken egg yolk: Isolation, purification and neutralization efficacy. Toxicon 2007; 50(7): 893-900.
[http://dx.doi.org/10.1016/j.toxicon.2007.06.017] [PMID: 17681579]
[13]
Almeida CMC, Kanashiro MM, Rangel Filho FB, Mata MFR, Kipnis TL, Dias da Silva W. Development of snake antivenom antibodies in chickens and their purification from yolk. Vet Rec 1998; 143(21): 579-84.
[http://dx.doi.org/10.1136/vr.143.21.579] [PMID: 9854769]
[14]
Lee CH, Lee YC, Liang MH, et al. Antibodies against venom of the snake Deinagkistrodon acutus. Appl Environ Microbiol 2016; 82(1): 71-80.
[http://dx.doi.org/10.1128/AEM.02608-15] [PMID: 26475102]
[15]
Duan H, He Q, Zhou B, et al. Anti-Trimeresurus albolabris venom IgY antibodies: Preparation, purification and neutralization efficacy. J Venom Anim Toxins Incl Trop Dis 2016; 22(1): 23.
[http://dx.doi.org/10.1186/s40409-016-0078-3] [PMID: 27563307]
[16]
Liu J, He Q, Wang W, et al. Preparation and neutralization efficacy of IgY antibodies raised against Deinagkistrodon acutus venom. J Venom Anim Toxins Incl Trop Dis 2017; 23(1): 22.
[http://dx.doi.org/10.1186/s40409-017-0112-0] [PMID: 28396683]
[17]
Korah MC, Hima SP. v SR, Anil A, Harikrishnan VS, Krishnan LK. Pharmacokinetics and pharmacodynamics of avian egg-yolk derived pure anti-snake venom in healthy and disease animal-model. J Pharm Sci 2022; 111(6): 1565-76.
[http://dx.doi.org/10.1016/j.xphs.2022.02.008] [PMID: 35196538]
[18]
de Andrade FG, Eto SF, Navarro dos Santos Ferraro AC, et al. The production and characterization of anti-bothropic and anti-crotalic IgY antibodies in laying hens: A long term experiment. Toxicon 2013; 66: 18-24.
[http://dx.doi.org/10.1016/j.toxicon.2013.01.018] [PMID: 23416799]
[19]
da Rocha DG, Fernandez JH, de Almeida CMC, et al. Development of IgY antibodies against anti-snake toxins endowed with highly lethal neutralizing activity. Eur J Pharm Sci 2017; 106: 404-12.
[http://dx.doi.org/10.1016/j.ejps.2017.05.069] [PMID: 28595875]
[20]
Leiva CL, Cangelosi A, Mariconda V, et al. IgY-based antivenom against Bothrops alternatus: Production and neutralization efficacy. Toxicon 2019; 163: 84-92.
[http://dx.doi.org/10.1016/j.toxicon.2019.03.020] [PMID: 30914282]
[21]
Klemperer F. Ueber natürliche Immunität und ihre Verwerthung für die Immunisirungstherapie. Naunyn Schmiedebergs Arch Pharmacol 1893; 31(4-5): 356-82.
[http://dx.doi.org/10.1007/BF01832882]
[22]
Schade R, Calzado EG, Sarmiento R, Chacana PA, Porankiewicz-Asplund J, Terzolo HR. Chicken egg yolk antibodies (IgY-technology): A review of progress in production and use in research and human and veterinary medicine. Altern Lab Anim 2005; 33(2): 129-54.
[http://dx.doi.org/10.1177/026119290503300208] [PMID: 16180988]
[23]
Tini M, Jewell UR, Camenisch G, Chilov D, Gassmann M. Generation and application of chicken egg-yolk antibodies. Comp Biochem Physiol A Mol Integr Physiol 2002; 131(3): 569-74.
[http://dx.doi.org/10.1016/S1095-6433(01)00508-6] [PMID: 11867282]
[24]
Alarcón CE, Hurtado H, Castellanos JE. Anticuerpos aviares: Alternativa en producción y diagnóstico. Biomédica 2000; 20(4): 338-43.
[http://dx.doi.org/10.7705/biomedica.v20i4.1077]
[25]
Gassmann M. Efficient production of chicken egg yolk antibodies against a conserved mammalian protein. Faseb J 1990; 4(8): 2528-32.
[http://dx.doi.org/10.1096/fasebj.4.8.1970792]
[26]
Mine Y, Kovacs-Nolan J. Chicken egg yolk antibodies as therapeutics in enteric infectious disease: A review. J Med Food 2002; 5(3): 159-69.
[http://dx.doi.org/10.1089/10966200260398198] [PMID: 12495588]
[27]
Sharma JM. Introduction to poultry vaccines and immunity. Adv Vet Med 1999; 41: 481-94.
[http://dx.doi.org/10.1016/S0065-3519(99)80036-6]
[28]
Alvarez A, Montero Y, Jimenez E, Zerpa N, Parrilla P, Malavé C. IgY antibodies anti-Tityus caripitensis venom: Purification and neutralization efficacy. Toxicon 2013; 74: 208-14.
[http://dx.doi.org/10.1016/j.toxicon.2013.08.058] [PMID: 23994592]
[29]
Polson A, Coetzer T, Kruger J, von Maltzahn E, van der Merwe KJ. Improvements in the isolation of IGY from the yolks of eggs laid by immunized hens. Immunol Invest 1985; 14(4): 323-7.
[http://dx.doi.org/10.3109/08820138509022667] [PMID: 4065934]
[30]
Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227(5259): 680-5.
[http://dx.doi.org/10.1038/227680a0] [PMID: 5432063]
[31]
Voller A, Bidwell D, Huldt G, Engvall E. A microplate method of enzyme-linked immunosorbent assay and its application to malaria. Bull World Health Organ 1974; 51(2): 209-11.
[PMID: 4377238]
[32]
Walker JM. Methods in Molecular Biology. Berlin, Heidelberg: Springer 2024.
[33]
Noya O, Alarcón de Noya B. The multiple antigen blot assay (MABA): A simple immunoenzymatic technique for simultaneous screening of multiple antigens. Immunol Lett 1998; 63(1): 53-6.
[http://dx.doi.org/10.1016/S0165-2478(98)00055-8] [PMID: 9719439]
[34]
Towbin H, Staehelint T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc Natl Acad Sci USA 1979; 76(9): 4350-4.
[35]
Zerpa NC, Wide A, Noda J, Bermúdez H, Pabón R, Noya OO. Immunogenicity of synthetic peptides derived from Plasmodium falciparum proteins. Exp Parasitol 2006; 113(4): 227-34.
[http://dx.doi.org/10.1016/j.exppara.2006.01.007] [PMID: 16513113]
[36]
Finney DJ. Statistical Method in Biological Assay. (3rd ed.). London: Charles and Griffin and Co Ltd 1978; p. 508.
[37]
Gutiérrez J, Gené J, Rojas G, Cerdas L. Neutralization of proteolytic and hemorrhagic activities of Costa Rican snake venoms by a polyvalent antivenom. Toxicon 1985; 23(6): 887-93.
[http://dx.doi.org/10.1016/0041-0101(85)90380-0] [PMID: 3913055]
[38]
Yamakawa M, Nozaki M, Hokama Z. Fractionation of sakishima habu (Trimeresurus elegans) venom and lethal hemorrhagic, and edema forming activities of fractions – ScienceOpen Animal. Plant Microbial Toxins 1976; 4: 97-109.
[39]
Gutiérrez J, Rojas G, Lomonte B, Gené J, Cerdas L. Comparative study of the edema-forming activity of Costa Rican snake venoms and its neutralization by a polyvalent antivenom. Comp Biochem Physiol C Comp Pharmacol 1986; 85(1): 171-5.
[http://dx.doi.org/10.1016/0742-8413(86)90069-1] [PMID: 2877785]
[40]
León G, Monge M, Rojas E. Comparison between IgG and F(ab’)(2) polyvalent antivenoms: Neutralization of systemic effects induced by Bothrops asper venom in mice, extravasation to muscle tissue, and potential for induction of adverse reactions. Toxicon 2001; 39(6): 793-801.
[41]
Romito M, Viljoen GJ, Du Plessis DH. Eliciting antigen-specific egg-yolk IgY with naked DNA. Biotechniques 2001; 31(3) 670-675, 672, 674-675.
[http://dx.doi.org/10.2144/01313dd05] [PMID: 11570510]
[42]
Gutiérrez JM, Sanz L, Flores-Díaz M, et al. Impact of regional variation in Bothrops asper snake venom on the design of antivenoms: Integrating antivenomics and neutralization approaches. J Proteome Res 2010; 9(1): 564-77.
[http://dx.doi.org/10.1021/pr9009518] [PMID: 19911849]
[43]
Ledsgaard L, Jenkins T, Davidsen K, et al. Antibody cross-reactivity in antivenom research. Toxins (Basel) 2018; 10(10): 393.
[http://dx.doi.org/10.3390/toxins10100393] [PMID: 30261694]
[44]
Baudou FG, Rodriguez JP, Fusco L, de Roodt AR, De Marzi MC, Leiva L. South American snake venoms with abundant neurotoxic components. Composition and toxicological properties. A literature review. Acta Trop 2021; 224: 106119.
[http://dx.doi.org/10.1016/j.actatropica.2021.106119] [PMID: 34481791]
[45]
Calvete JJ, Sanz L, Cid P, et al. Snake venomics of the Central American rattlesnake Crotalus simus and the South American Crotalus durissus complex points to neurotoxicity as an adaptive paedomorphic trend along Crotalus dispersal in South America. J Proteome Res 2010; 9(1): 528-44.
[http://dx.doi.org/10.1021/pr9008749] [PMID: 19863078]
[46]
Rodríguez-Vargas A, Vega N, Reyes-Montaño E, et al. Intraspecific differences in the Venom of Crotalus durissus cumanensis from Colombia. Toxins (Basel) 2022; 14(8): 532.
[http://dx.doi.org/10.3390/toxins14080532] [PMID: 36006194]
[47]
Meléndez-Martínez D, Plenge-Tellechea LF, Gatica-Colima A, Cruz-Pérez MS, Aguilar-Yáñez JM, Licona-Cassani C. Functional mining of the Crotalus spp. venom protease repertoire reveals potential for chronic wound therapeutics. Molecules 2020; 25(15): 3401.
[http://dx.doi.org/10.3390/molecules25153401] [PMID: 32731325]
[48]
Gutiérrez J, Escalante T, Rucavado A, Herrera C, Fox J. A comprehensive view of the structural and functional alterations of extracellular matrix by snake venom metalloproteinases (SVMPs): Novel perspectives on the pathophysiology of envenoming. Toxins (Basel) 2016; 8(10): 304.
[http://dx.doi.org/10.3390/toxins8100304] [PMID: 27782073]
[49]
Saravia-Otten P, Arce V, Velasquez R. Geographic and ontogenic variability in the venom of the neotropical rattlesnake Crotalus durissus: Pathophysiological and therapeutic implications. Rev Biol Trop 2002; 50(1): 337-46.
[50]
Solano Godoy JA, Molano Cardona ED, Bernal Bautista MH, Murillo Arango W. Actividad Fosfolipasa, Hemolítica y Bactericida preliminar del Veneno de la Serpiente de cascabel del Tolima. Cienc Desarro 2020; 11(1): 119-27.
[http://dx.doi.org/10.19053/01217488.v11.n1.2020.9869]
[51]
de Roodt AR, Estévez-Ramírez J, Paniagua-Solís JF, et al. Toxicidad de venenos de serpientes de importancia médica en México. Gac Med Mex 2005; 141(1): 13-21.
[PMID: 15754746]
[52]
Aguiar AS, Melgarejo AR, Alves CR, Giovanni-De-Simone S. Single-step purification of crotapotin and crotactine from Crotalus durissus terrificus venom using preparative isoelectric focusing. Braz J Med Biol Res 1997; 30(1): 25-8.
[http://dx.doi.org/10.1590/S0100-879X1997000100004] [PMID: 9222399]
[53]
Rangel-Santos A, Dos-Santos EC, Lopes-Ferreira M, Lima C, Cardoso DF, Mota I. A comparative study of biological activities of crotoxin and CB fraction of venoms from Crotalus durissus terrificus, Crotalus durissus cascavella and Crotalus durissus collilineatus. Toxicon 2004; 43(7): 801-10.
[http://dx.doi.org/10.1016/j.toxicon.2004.03.011] [PMID: 15284014]
[54]
Rangel-Santos AC, Mota I. Effect of heating on the toxic, immunogenic and immunosuppressive activities of Crotalus durissus terrificus venom. Toxicon 2000; 38(10): 1451-7.
[http://dx.doi.org/10.1016/S0041-0101(99)00238-X] [PMID: 10758279]
[55]
Grillo Rodríguez O, Scannone HR. Fractionation of Crotalus durissus cumanensis venom by gel filtration. Toxicon 1976; 14(5): 400-3.
[http://dx.doi.org/10.1016/0041-0101(76)90090-8] [PMID: 982480]
[56]
Salazar AM, Aguilar I, Guerrero B, et al. Intraspecies differences in hemostatic venom activities of the South American rattlesnakes, Crotalus durissus cumanensis, as revealed by a range of protease inhibitors. Blood Coagul Fibrinolysis 2008; 19(6): 525-30.
[http://dx.doi.org/10.1097/MBC.0b013e328304e02e] [PMID: 18685436]
[57]
Rodriguez OG, Scannone HR, Parra ND. Enzymatic activities and other characteristics of Crotalus durissus cumanensis venom. Toxicon 1974; 12(3): 297-302.
[http://dx.doi.org/10.1016/0041-0101(74)90073-7] [PMID: 4376285]
[58]
Cavalcante WLG, Ponce-Soto LA, Marangoni S, Gallacci M. Neuromuscular effects of venoms and crotoxin-like proteins from Crotalus durissus ruruima and Crotalus durissus cumanensis. Toxicon 2015; 96: 46-9.
[http://dx.doi.org/10.1016/j.toxicon.2015.01.006] [PMID: 25598498]
[59]
Aguilar I, Girón ME, Rodríguez-Acosta A. Purification and characterisation of a haemorrhagic fraction from the venom of the Uracoan rattlesnake Crotalus vegrandis. Biochim Biophys Acta Protein Struct Mol Enzymol 2001; 1548(1): 57-65.
[http://dx.doi.org/10.1016/S0167-4838(01)00217-5] [PMID: 11451438]
[60]
Rodriguez-Acosta A, Aguilar I, Giron M. Haemorrhagic activity of neotropical rattlesnake (Crotalus vegrandis Klauber, 1941) venom. Nat Toxins 1998; 6(1): 15-8.
[http://dx.doi.org/10.1002/(SICI)1522-7189(199802)6:1<15:AID-NT2>3.0.CO;2-S]
[61]
Camey KU, Velarde DT, Sanchez EF. Pharmacological characterization and neutralization of the venoms used in the production of Bothropic antivenom in Brazil. Toxicon 2002; 40(5): 501-9.
[http://dx.doi.org/10.1016/S0041-0101(01)00245-8] [PMID: 11821121]
[62]
Meenatchisundaram S, Parameswari G, Michael A, Ramalingam S. Neutralization of the pharmacological effects of Cobra and Krait venoms by chicken egg yolk antibodies. Toxicon 2008; 52(2): 221-7.
[http://dx.doi.org/10.1016/j.toxicon.2008.04.179] [PMID: 18590753]
[63]
Teixeira CFP, Landucci ECT, Antunes E, Chacur M, Cury Y. Inflammatory effects of snake venom myotoxic phospholipases A2. Toxicon 2003; 42(8): 947-62.
[http://dx.doi.org/10.1016/j.toxicon.2003.11.006] [PMID: 15019493]
[64]
Mora J, Mora R, Lomonte B, Gutiérrez JM. Effects of Bothrops asper snake venom on lymphatic vessels: Insights into a hidden aspect of envenomation. PLoS Negl Trop Dis 2008; 2(10): e318.
[http://dx.doi.org/10.1371/journal.pntd.0000318] [PMID: 18923712]
[65]
Pereira EPV, van Tilburg MF, Florean EOPT, Guedes MIF. Egg yolk antibodies (IgY) and their applications in human and veterinary health: A review. Int Immunopharmacol 2019; 73: 293-303.
[http://dx.doi.org/10.1016/j.intimp.2019.05.015] [PMID: 31128529]

Rights & Permissions Print Cite
© 2025 Bentham Science Publishers | Privacy Policy