Invest Clin 63(1): 57 - 69, 2022 https://doi.org/10.54817/IC.v63n1a05
Corresponding author: Alexis Rodríguez-Acosta. Instituto Anatómico, Ciudad Universitaria de Caracas, Universidad
Central de Venezuela, Caracas 1041. Email: rodriguezacosta1946@yahoo.es
A new approach of immunotherapy against
Crotalus snakes envenoming: ostrich
(Struthio camelus) egg yolk antibodies
(IgY-technology).
Carlos Bello1, Fátima Torrico1, Juan C. Jiménez1,2, Mariana V. Cepeda1,
Miguel A. López1 and Alexis Rodríguez-Acosta1,3
1Biotecfar C.A, Facultad de Farmacia, Universidad Central de Venezuela, Caracas,
República Bolivariana de Venezuela.
2Instituto de Inmunología, Facultad de Medicina, Universidad Central de Venezuela,
Caracas, República Bolivariana de Venezuela.
3Laboratorio de Inmunoquímica y Ultraestructura, Instituto Anatómico, Universidad
Central de Venezuela, Caracas, República Bolivariana de Venezuela.
Key words: antivenom; 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 Os-
trich (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 rattle-
snake (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 ad-
dition 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 LD50 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 (ED50) was 19.66 mg/2LD50. IgY antibodies neutral-
ized 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
58 Bello et al.
Investigación Clínica 63(1): 2022
Un nuevo enfoque de inmunoterapia contra el envenenamiento
de serpientes Crotalus: anticuerpos de yema de huevo de
avestruz (Struthio camelus) (tecnología IgY).
Invest Clin 2022; 63 (1): 57 – 69
Palabras clave: antiveneno; avestruz Crotalus; IgY; Struthio camelus; veneno.
Resumen. El envenenamiento por crotálidos es un problema de salud
colectiva desatendido, que involucra a muchos países del continente ameri-
cano, los cuales necesitan tratamientos seguros y económicos. En este tra-
bajo, se obtuvieron títulos altos de anticuerpos (IgY) producidos en yema
de huevo de avestruz (Struthio camelus) mediante la inmunización con
el veneno de serpientes venezolanas del genero Crotalus. Se inmunizaron
avestruces con una colección de veneno de serpientes de cascabel común
(Crotalus durissus cumanensis), cascabel de Uracoa (Crotalus vegrandis),
cascabel de Guayana (Crotalus durissus ruruima) y cascabel negra (Crota-
lus pifanorum). Los anticuerpos anti-veneno de serpiente se prepararon a
partir de yema de huevo por el método de dilución en agua, enriquecidos
mediante la adición de ácido caprílico (CA), seguido de una precipitación
con sulfato de amonio al 30% (P/V). La pureza y masa molecular de los
anticuerpos (IgY) se definieron mediante ensayos de SDS-PAGE nativos y
las masas moleculares se establecieron electroforéticamente, obteniéndose
una única banda de IgY de 175 kDa. El análisis de inmunotransferencia
mostró la unión específica del antiveneno con la mayoría de las proteínas
del veneno. La DL50 fue de 16,5 µg/ratón (825 µg / kg de peso corporal);
Se mostraron títulos altos de IgY contra el veneno de Crot / pool median-
te ELISA. La dosis mediana efectiva (DE50) fue de 19,66 mg/2 LD50. Los
anticuerpos IgY neutralizaron eficazmente la letalidad del veneno de Crot
/ pool. Hasta donde sabemos, se trata del primer antídoto de serpiente
producido en avestruces, lo que podría abaratar la producción de este tra-
tamiento en países del tercer mundo. Ya que es probable que se obtengan
alrededor de 2-4 g de IgY por huevo de avestruz. Por lo tanto, se podrían
purificar casi 400 g de IgY de un solo avestruz durante un año. Asimismo,
debido a las enormes diferencias en el costo de inversión en el manteni-
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 extrapo-
lated to the manufacturing of other antivenoms and antitoxins as well, as they
could be applied to the manufacturing of potential diagnostic tools.
Crotalus snakes envenoming treated with ostrich (Struthio camelus) IgY 59
Vol. 63(1): 57 - 69, 2022
INTRODUCTION
Venomous snakebite is a worldwide
problem, especially in tropical and subtropi-
cal geographical regions. There are more
than five million snakebite accidents annual-
ly worldwide; the members of the Viperidae
family 1 produce the most common snake-
bites that take place in the American conti-
nent (~ 98%).
Traditional antivenom production
is based on purified antibodies extracted
from hyperimmunized horses’ plasma 2. In
recent years, several authors report that
bird’s (mainly chicken) antibodies produced
against snake venoms, presented venom
effective neutralization activities 2,3. How-
ever, despite their potential considering
their body size and egg-laying advantages,
ostriches (Struthio camelus) have not been
previously tested for snake anti-venom pro-
duction. Clinical assays will be need to as-
sess their security as an antidote to human
victims of ophitoxemia and further experi-
mentation addressing IgY-based antivenoms
safety and effectiveness is required.
MATERIALS AND METHODS
Reagents
Polyvalent anti-ophidic serum (PAOS)
(Biotecfar C.A, Universidad Central de Vene-
zuela, Pharmacy Faculty, Caracas, República
Bolivariana de Venezuela). Sodium chloride,
sodium citrate, sodium azide, tris, hydro-
chloric acid, sodium hydroxide, Coomassie
blue, acrylamide, bis-acrylamide, ammoni-
um per-sulphate, glacial acetic acid, temed,
glycerol, sodium bicarbonate, caprylic acid
(Sigma-Aldrich, Missouri, USA). Saran wrap
(S.C. Johnson & Son, Inc, USA). Complete
Freund’s and incomplete Freund’s adjuvants
(GIBCO, USA). Antibody goat anti-avian (in-
cluded anti-ostrich) (Laboratories ABCAM,
USA). Peroxidase substrate (TMB)(Vector
Lab, USA). 30 kDa cassette filtration unit
(Vivaflow 50 R, Sartorius, Germany). Fil-
ter (Sartorious Laboratories, Germany).
Microtitration plates (Corning® ELISA mi-
croplates, USA). Automatic ELISA reader
(Bio-Tek Laboratories, USA). Mini-Protean
II system (Bio-Rad Laboratories, USA).
Trans Blot SD system (Bio-Rad Laboratories,
USA). Molecular mass standards for SDS-
PAGE were from Bio-Rad Laboratories Ltd
(California, USA).
Ostriches
Two female healthy adult ostriches
(Struthio camelus) were obtained from a lo-
cal ostrich farm (Villa de Cura town, Aragua
state, República Bolivariana de Venezuela).
They were housed under standard environ-
ment (humidity, lighting and temperature)
and fed ad libitum with standard ostrich diet
and potable water (Fig. 1).
Venom
A pool of venoms from common rat-
tlesnake (Crotalus durissus cumanensis)
(0.15 mg/mL), Uracoan rattlesnake (Crota-
lus vegrandis) (0.03 mg/mL), Guayana rat-
miento de los caballos desde el punto de vista de infraestructura, alimenta-
ción y atención veterinaria, en los que el costo puede llegar a los 100 USD
por día, frente a los 146 USD por mes de mantenimiento de la producción
de aves. Estos resultados abren un campo terapéutico, para la fabricación
de otros antivenenos contra un amplio espectro de toxinas y también como
probables herramientas de diagnóstico.
Received: 03-08-2021 Accepted: 04-11-2021