A new approach of immunotherapy against Crotalus snakes envenoming: ostrich (Struthio camelus) egg yolk antibodies (IgY-technology).

Un nuevo enfoque de inmunoterapia contra el envenenamiento de serpientes Crotalus: anticuerpos de yema de huevo de avestruz (Struthio camelus) (tecnología IgY).

• Carlos Bello Universidad Central de Venezuela, Caracas, República Bolivariana de Venezuela. https://orcid.org/0000-0002-5689-9284
• Fátima Torrico Universidad Central de Venezuela, Caracas, República Bolivariana de Venezuela. https://orcid.org/0000-0003-3388-8299
• Juan C. Jiménez Universidad Central de Venezuela, Caracas, República Bolivariana de Venezuela. https://orcid.org/0000-0002-1554-4292
• Mariana V. Cepeda Universidad Central de Venezuela, Caracas, República Bolivariana de Venezuela. https://orcid.org/0000-0002-5396-8483
• Miguel A. López Universidad Central de Venezuela, Caracas, República Bolivariana de Venezuela. https://orcid.org/0000-0002-6162-711X
• Alexis Rodríguez-Acosta Universidad Central de Venezuela, Caracas, República Bolivariana de Venezuela https://orcid.org/0000-0003-1234-7522

DOI: https://doi.org/10.54817/IC.v63n1a05

Keywords: Crotalus snakes, ostrich egg yolk, IgY, Struthio camelus, venom

Abstract

Crotalid envenomation is a neglected collective health problem involving many countries in America, which need secure and inexpensive snake anti-venom treatments. Here, high antibody titers (IgY) were raised in the Ostrich (Struthio camelus) egg yolk by immunizing with the venom of Venezuelan venomous Crotalus snakes. Ostriches were immunized with a pool of venoms from common rattlesnake (Crotalus durissus cumanensis), Uracoan rattlesnake (Crotalus vegrandis), Guayana rattlesnake (Crotalus durissus ruruima) and black rattlesnake (Crotalus pifanorum). The anti-snake venom antibodies were prepared from egg yolk by the water dilution method, enriched by the addition of caprylic acid (CA) and precipitation with ammonium sulfate at 30% (W/V). The purity and molecular mass of the final product was satisfactory, yielding a single ∼ 175 kDa band in SDS-PAGE gels ran under non-reducing conditions. In the immunoblot analysis, specific binding of the antivenom was observed with most venom proteins. The LD 50 was 16.5 g/mouse (825 μg/kg body weight). High titers of IgY against Crot/pool venom were shown by ELISA. The median effective dose (ED 50) was 19.66 mg/2LD 50. IgY antibodies neutralized efficiently the Crot/pool venom lethality. As far as we know, this is the first anti-snake venom produced in ostriches, which could make this technology an affordable alternative for low-income countries, since it is likely to produce about 2-4 g of IgY per ostrich egg. Hence, almost 400 g of IgY can be purified from only one ostrich during a year. In addition, there are enormous differences in the cost of investment in the maintenance of horses, from the points of view of infrastructure, feeding and veterinary care, in which the cost can reach USD 100 per animal per day, compared to a maintenance cost of USD 146 per month per producing bird. These results are encouraging and could easily be extrapolated to the manufacturing of other antivenoms and antitoxins as well, as they could be applied to the manufacturing of potential diagnostic tools.

References

Rengifo C, Rodríguez-Acosta A. Serpientes, Veneno y Tratamiento Médico en Venezuela. Caracas: Universidad Central de Venezuela, 2019; 1-272.

Aguilar I, Sánchez EE, Girón ME, Estrella A, Guerrero B. Rodríguez-Acosta A. Coral snake antivenom produced in chickens (Gallus domesticus). Rev Inst Med Trop Sao Paulo 2014; 56: 61-66.

Almeida CM, Kanashiro MM, Rangel-Filho FB, Mata MF, Kipnis TL, da Silva WD. Development of snake antivenom antibodies in chickens and their purification from yolk. Vet Rec 1998; 143: 579–584.

NIH, Principles of laboratory animal care. National Institute of Health of United States of America, Pub. 85-23, Maryland; 1985; 1–112.

McLaren RD, Prosser CG, Grieve RC, Borissenko M. The use of caprylic acid for the extraction of the immunoglobulin fraction from egg yolk of chickens immunised with ovine alpha-lactalbumin. J Immunol Methods 1994; 177:175-184.

Towbin H, Stachelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl Acad Sci USA 1979; 76: 4350–4354.

Spearman- Kärber R. Alternative methods of analysis for quantal responses. In: Finney D, editor. Statistical method in biological assay. London, Charles Griffin; 1978; 1– 78.

Bagcchi S. Experts call for snakebite to be re-established as a neglected tropical disease. BMJ. 2015; 351: h5313.

Larsson A, Balow RM, Lindahl TL, Forsberg PO. Chicken antibodies: taking advantage of evolution. A Rev Poultry Sci 1993; 72: 1807-1812.

Adachi K, Handharyani E, Sari DK, Takama K, Fukuda K, Endo I, Yamamoto R, Sawa M, Tanaka M, Konishi I, Tsukamoto Y. Development of neutralization antibodies against highly pathogenic H5N1 avian influenza virus using ostrich (Struthio camelus) yolk. Mol Med Rep 2008;1:203–209.

Larsson A, Mellstedt H. Chicken antibodies: a tool to avoid interference by human anti-mouse antibodies in ELISA after in vivo treatment with murine monoclonal antibodies. Hybridoma 1992; 11: 33–39.

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;33:129-154.

Thallay BS, Carroll SB. Rattle snake and scorpion antivenoms from the egg yolks of immunized hens. Biotechniques (NY) 1990; 8: 934–938.

Huang T, Zhang M, Wei Z, Wang P, Sun Y, Hu X, Ren L, Meng Q, Zhang R, Guo Y, Hammarstrom L, Li N, Zhao Y. Analysis of immunoglobulin transcripts in the ostrich (Struthio camelus), a primitive avian species. PLoS ONE 2012; 7: e34346.

Morimoto J, Sarkar M, Kenrick S, Kodadek T. Dextran as a generally applicable multivalent scaffold for improving immunoglobulin-binding affinities of peptide and peptidomimetic ligands. Bioconjug Chem 2014; 25:1479‐1491.

Parrilla P, Navarrete LF, Girón M.E, Aguilar I, Rodríguez-Acosta A. Use of chicken egg yolk-derived immunoglobulin against Scolopendra venom as an alternative to treat scolopendrism. Rev Cient FCV-LUZ 2008; 18: 385-392.

Leiva CL, Cangelosi A, Mariconda V, Farace M, Geoghegan P, Brero L, Fernández-Miyakawa M, Chacana P. IgY-based antivenom against Bothrops alternatus: Production and neutralization efficacy. Toxicon 2019; 163: 84-92.

Dos Santos MC, D’imperio-Lima MR, Furtado GC, Colletto GM, Kipnis TL, Dias da Silva W. Purification of F(ab’)2 anti-snake venom by caprylic acid: a fast method for obtaining IgG fragments with large neutralization activity, purity and yield. Toxicon 1989; 27: 297-303.

Rojas G, Jimenez JM, Gutierrez JM. Caprylic acid fractionation of hyper immune horse plasma: description of a simple procedure for antivenom production. Toxicon 1994; 32: 351-363.

Sutherland SK. Serum reactions. An analysis of commercial antivenoms and the possible role of anticomplementary activity in de-novo reactions to antivenoms and anti- toxins. Med J Aust. 1977; 1: 613–615.

Lee CH, Liu CI, Leu SJ, Lee YC, Chiang JR, Chiang LC, Mao YC, Tsai BY, Hung CS, Chen CC, Yang YY. Chicken antibodies against venom proteins of Trimeresurus stejnegeri in Taiwan. J Venom Anim Toxins Incl Trop Dis 2020; 26: e20200056.

Carbajo E. Produccion de avestruces: Una industria ya establecida. Rev Cien Vet 2007; 23: 3-5.