Toxicon 39 (2001) 401405

www.elsevier.com/locate/toxicon

Short Communication

The venom of Bothrops asper from Guatemala: toxic

activities and neutralization by antivenoms
Patricia Saravia a, Ermila Rojas b, Teresa Escalante b, Viviana Arce b,
Esteban Chaves c, Ruben Velasquez a, Bruno Lomonte b, Gustavo Rojas b,
Jose Mar a Gutierrez b,*
a

Departamento de Bioqumica, Facultad de Ciencias Qumicas y Farmacia, Universidad de San Carlos de Guatemala, Guatemala
b
Instituto Clodomiro Picado, Facultad de Microbiologa, Universidad de Costa Rica, San Jose, Costa Rica
c
Departamento de Microbiologa e Inmunologa, Facultad de Microbiologa, Universidad de Costa Rica, San Jose, Costa Rica
Received 18 January 2000; accepted 7 March 2000

Abstract
Bothrops asper is responsible for approximately half of the snakebite envenomations in Central America. Despite
its medical relevance, only the venom of Costa Rican populations of this species has been studied to some detail,
and there is very little information on intraspecies variability in venom composition and toxicity. Venom of B. asper
from Guatemala was analyzed by sodium dodecyl sulphatepolyacrylamide gel electrophoresis and two-dimensional
gel electrophoresis, and its basic pharmacological activities were investigated with standard laboratory assays.
Venom has lethal, hemorrhagic, myotoxic, edema-forming, coagulant, debrinating and phospholipase A2 activities,
showing a similar toxicological prole to the one previously described for B. asper from Costa Rica. In addition,
polyvalent antivenoms produced in Mexico and Costa Rica, and currently used in Guatemala, were tested for their
ability to neutralize venom's toxic activities. Both antivenoms were eective against all eects studied, although the
Costa Rican product showed higher potency against most activities tested and higher antibody titer against venom
components, as determined by enzyme immunoassay. It is suggested that dierent dosage regimes should be
considered when using these antivenoms in B. asper envenomations in Guatemala. 7 2000 Elsevier Science Ltd. All
rights reserved.
Keywords: Venom; Bothrops asper; Antivenom; Neutralization; Guatemala

Bothrops asper, popularly known as ``terciopelo'',

``barba amarilla'' and ``nauyaca'', constitutes the most
dangerous poisonous snake in Southern Mexico and
Central America (Campbell and Lamar, 1989), being

* Corresponding author. Tel.: +1-506-2290344; fax: +1506-2920485.

E-mail address: jgutierr@cariari.ucr.ac.cr (J.M. Gutierrez).

responsible for approximately half of the snakebite

accidents in this region (Gutierrez, 1995). These envenomations are characterized by conspicuous local tissue damage and, in severe cases, by systemic bleeding,
coagulopathies, cardiovascular shock and renal disturbances (Hardy, 1992; Gutierrez, 1995; Arroyo et al.,
1999). The venom of B. asper from Costa Rica has
been characterized pharmacologically and biochemically (see reviews by Gutierrez and Lomonte, 1989,

0041-0101/00/$ - see front matter 7 2000 Elsevier Science Ltd. All rights reserved.
PII: S 0 0 4 1 - 0 1 0 1 ( 0 0 ) 0 0 1 2 2 - 7

402

P. Saravia et al. / Toxicon 39 (2001) 401405

1997), and its neutralization by commercially available

antivenoms has been assessed (Bogar n et al., 1995).
However, despite the medical relevance of this species
in the region, few studies have been performed on the
characteristics of its venom in other Central American
countries (Rojas et al., 1987). This work describes a
toxicological characterization of B. asper venom from
Guatemala and evaluates the neutralizing ability of
two polyvalent antivenoms commercially available in
this country.
Venom was a pool obtained from four adult specimens of B. asper collected in the Atlantic versant of
Guatemala and maintained in captivity at Museo de
Historia Natural, Escuela de Biolog a, Universidad de
San Carlos, Guatemala, and Museo Nacional de Historia Natural, Guatemala. Venom was lyophilized and
kept at 208C. Venom was analyzed by sodium dodecyl sulphatepolyacrylamide gel electrophoresis (SDS
PAGE), run under reducing conditions in 12% acrylamide gels (Laemmli, 1970), and by two-dimensional
electrophoresis (Cortes-Bratti et al., 1999). Briey,
samples were treated with acetone and separated by
isoelectric focusing in 11 cm Immobiline dry strips (pH
310; Pharmacia Biotech AB, Sweden). Then, proteins
were separated in the second dimension by 10% SDS
PAGE and visualized after silver staining. Lethality
was assessed by the intraperitoneal route in 1618 g
SwissWebster mice (Bolanos, 1972). Median Lethal
Dose (LD50) was estimated by SpearmanKarber
(WHO, 1981). Hemorrhagic activity was determined
by intradermal injection in mice (Gutierrez et al.,
1985), and edema-forming activity was quantitated as
the increment in footpad weight 1 h after venom injection (Yamakawa et al., 1976). Coagulant activity was
assessed in human citrated plasma (Theakston and
Reid, 1983; Gene et al., 1989) and debrination was
determined in mice (Theakston and Reid, 1983; Gene
et al., 1989). Myotoxicity was evaluated by determining the plasma creatine kinase (CK) activity 3 h after
intramuscular injection of venom in the right gastrocnemius muscle of 1820 g mice (Gutierrez et al.,
1980). Phospholipase A2 activity was quantitated by
incubating venom with egg yolk phospholipids (Gutierrez et al., 1986). Released fatty acids were extracted
and titrated according to Dole (1956). Indirect hemolytic activity was evaluated in agaroseegg yolkerythrocyte gels (Gutierrez et al., 1988).
Neutralization studies were performed with the
liquid polyvalent antivenom from Instituto Clodomiro
Picado, Costa Rica (batch 2941297LQ, expiration date
December 2000) and the MYN lyophilized polyvalent
antivenom (Bioclon S.A. de C.V., Mexico, batch B6A-06, expiration date January 2001). Neutralization
experiments involved assays in which venom and antivenom were incubated for 30 min at 378C before testing in the corresponding pharmacological systems

(Gutierrez et al., 1990; Bogar n et al., 1995). In all

cases, a constant amount of venom, corresponding to
a ``challenge dose'' selected on the basis of dose-response studies, was incubated with various dilutions of
antivenom (Gutierrez et al., 1990; Bogar n et al.,
1995). The challenge doses for the dierent eects were
the following: (a) Lethal: 4 LD50s; (b) hemorrhagic: 10
Minimum Hemorrhagic Doses (MHD); (c) myotoxic:
Four Minimum Myotoxic Doses (MMD); (d) coagulant: Two Minimum Coagulant Doses (MCD); (e) debrinating: Two Minimum Debrinating Doses; (f)
edema-forming: Four Minimum Edema-forming Doses
(MED); (g) indirect hemolytic: One Minimum Hemolytic Dose (MHeD). Neutralization was expressed as
Eective Dose 50% (ED50), dened as the ratio ml
antivenom/mg venom at which the eect was neutralized by 50% (Gutierrez et al., 1990; Bogar n et al.,
1995). In the cases of in vitro coagulant and in vivo
debrinating activities, neutralization was expressed as
Eective Dose (Gene et al., 1989). In addition, the
antibody titer against B. asper venom was assessed by
enzyme immunoassay (ELISA) (Lomonte et al., 1991;
Bogar n et al., 1995). Briey, microplates were coated
using 1 mg of venom/well, and various dilutions of
either ICP antivenom or MYN antivenom were
assayed. Bound antibodies were detected by an antiequine immunoglobulin/peroxidase conjugate. Normal
equine serum was included as a control. The signicance of the dierences between the mean values of
two experimental groups was determined by the Student's t-test.
Electrophoretic prole of B. asper venom from Guatemala in SDSPAGE showed the presence of several
protein bands ranging from 16 to 90 kDa (results not
shown). Two-dimensional electrophoresis evidenced a
complex pattern of approximately 140 protein spots,
ranging from acidic to highly basic components
(Fig. 1). The venom from Guatemala induced lethality,
hemorrhage, edema, myonecrosis, coagulation in vitro
and debrination, also presenting phospholipase A2 activity evidenced by titrimetric and indirect hemolytic
assays (Table 1). Both antivenoms were eective in the
neutralization of toxic and enzymatic activities of the
venom, although antivenom from Instituto Clodomiro
Picado was more eective than MYN antivenom in
the neutralization of hemorrhagic, edema-forming, coagulant, debrinating, phospholipase A2 and indirect
hemolytic activities, whereas no signicant dierences
were observed in the neutralization of myotoxic activity (Table 1). Regarding lethality, ICP antivenom
showed higher potency, although 95% condence
limits slightly overlap (Table 1). ELISA evidenced a
signicantly higher antibody titer against B. asper
venom antigens in ICP antivenom than in MYN antivenom (Fig. 2).
Intraspecic venom variation is a common phenom-

P. Saravia et al. / Toxicon 39 (2001) 401405

403

Fig. 1. Two-dimensional gel electrophoresis of B. asper venom from Guatemala. Samples were treated with acetone and initially
separated by isoelectric focusing on Immobiline dry strips (pH 310). Then, proteins were separated in the second dimension by
10% SDSPAGE. Proteins were visualized by silver staining. The molecular weights of several markers (in kDa) are shown.

enon in snakes (Chippaux et al., 1991), with evident

clinical and therapeutic implications, particularly in
species having wide distribution ranges. B. asper is distributed from southern Mexico to the northern regions

of South America (Campbell and Lamar, 1989). B.

asper from Middle America can be classied into two
major groups on the basis of scutellation and color
pattern characters: those from Mexico and Nuclear

Table 1
Toxic activities induced by B. asper venom from Guatemala and their neutralization by two antivenomsa
Eect

Lethal (LD50; mg/mouse)c

Hemorrhagic (MHD; mg)d
Myotoxic (MMD; mg)e
Coagulant (MCD; mg)f
Debrinating (MDD; mg)g
Edema-forming (MED; mg)h
Phospholipase A2(mEq/mg.min)i
Indirect hemolytic (MHeD; mg)j

Activity

56.8 (44.572.4)
1123
1323
3.920.08
2.5
120.1
2422
7.620.01

Neutralization (Eective Dose 50%), ml antivenom/mg venomb

ICP antivenom

MYN antivenom

342 (224465)
175237
5552159
13328
1000
14822120
12682182
1427250

934 (4481053)
272238
4582117
723214
2000
18372152
23522225
24722102

Except for lethality, results are presented as mean2SD n 4).  p < 0:05 when the two antivenoms are compared.
Eective Dose 50%, dened as the ratio ml antivenom/mg venom at which the eect is reduced by 50%. In the case of coagulant and debrinating activities, neutralization is expressed as Eective Dose (Gene et al., 1989).
c
Lethality was determined in 1618 g mice by the intraperitoneal route; 95% condence limits are included.
d
MHD (Minimum Hemorrhagic Dose): amount of venom inducing a hemorrhagic area of 10 mm diameter, 2 h after intradermal
injection in mice.
e
MMD (Minimum Myotoxic Dose): amount of venom that, 3 h after i.m. injection, induces an increment in plasma CK activity
corresponding to four times the CK activity of mice injected with PBS.
f
MCD (Minimum Coagulant Dose): amount of venom which induces coagulation of citrated human plasma in 60 s.
g
MDD (Minimum Debrinating Dose): minimum amount of venom that renders blood unclottable 1 h after i.v. injection in
mice.
h
MED (Minimum Edema-forming Dose): amount of venom inducing 30% edema 1 h after s.c. injection in mice.
i
Phospholipase A2 activity, expressed as mEq fatty acid released per mg protein per min.
j
MHeD (Minimum Hemolytic Dose): amount of venom that induces a hemolytic halo of 20 mm diameter in agaroseerythrocyteegg yolk gels after 20 h of incubation at 378C.
a

404

P. Saravia et al. / Toxicon 39 (2001) 401405

Central America and those from Isthmian Central

America (Sasa, 1996). Since the venom from Costa
Rican populations has been widely studied, it is relevant to characterize B. asper venom of populations
from the northern Central American countries. The
present investigation shows, for Guatemalan B. asper
venom, a qualitatively similar toxicological prole to
those previously described for B. asper from Costa
Rica (Bogar n et al., 1999) and Honduras (Rojas et al.,
1987), despite the observation of quantitative variations in these activities. Thus, no conspicuous dierences seem to exist in the pharmacological proles of
the venoms of various populations of this species in
Central America. Moreover, an antivenom produced
using B. asper venom from Costa Rica in the immunization mixture was highly eective in the neutralization
of Guatemalan venom, evidencing their close immunological relationship. Two-dimensional electrophoresis
revealed a complex protein pattern in B. asper venom.
This technique represents a useful tool to characterize
snake venoms and to detect ontogenetic and geographic variations in venom protein composition
(Rioux et al., 1998).
Although no systematic clinical investigations have
been published regarding B. asper envenomations in
Guatemala, unpublished observations in the northern
region of this country have shown that their pathophysiology resembles that of bites inicted by this species
in Costa Rica (Bolanos, 1982; de Franco et al., 1983;

Arroyo et al., 1999) and in other Central American

countries (Hardy, 1994). Since parenteral administration of antivenoms constitutes the central therapy in
snakebite envenomations (Warrell, 1992), evaluation of
the neutralizing ability of commercially available antivenoms in dierent countries is a relevant and urgent
task. These analyses should be performed using
venoms from specimens collected in the country where
the antivenom will be utilized. Moreover, antivenoms
should be tested not only against lethal activity of
venoms, but also against other relevant pharmacological eects (WHO, 1981; Gutierrez et al., 1990). In the
case of crotaline snake venoms, neutralization of
hemorrhagic, myotoxic, edema-forming, coagulant and
debrinating eects is necessary. Our data indicate
that both tested antivenoms neutralize these activities
of B. asper venom from Guatemala, although ICP
antivenom has a higher neutralizing ecacy. On the
basis of the observed variations in neutralizing
potency, it is suggested that dierent dosage regimes
should be considered when using these antivenoms in
Guatemala, a hypothesis that needs to be addressed in
randomized clinical trials.
Acknowledgements
The authors thank Mahmood Sasa for his comments
on the variations in B. asper populations, as well as
the sta of Instituto Clodomiro Picado, Programa de
Investigacion en Enfermedades Tropicales (Escuela de
Medicina Veterinaria, Universidad Nacional, Costa
Rica), Escuela de Biolog a and Departamento de Bioqu mica, Universidad de San Carlos de Guatemala.
The collaboration of Museo Nacional de Historia
Natural, Guatemala, is gratefully acknowledged. This
investigation was supported by NeTropica, by Vicerrectora de Investigacion, Universidad de Costa Rica
(projects 741-89-057 and 731-99-526) and by CONCYT
of Guatemala (project Facyt MS-058-1).
References

Fig. 2. Titration of antibodies to B. asper venom from Guatemala by enzyme immunoassay. Microplates were coated using
1 mg of venom/well, and various dilutions of either ICP antivenom (w) or MYN antivenom (q) were assayed. Bound
antibodies were detected by an anti-equine immunoglobulin/
peroxidase conjugate. Normal equine serum (r) was included
as a control. Each point represents mean 2 SD of duplicate
well readings.

Arroyo,
O.,
Rojas,
G.,
Gutierrez,
J.M.,
1999.
Envenenamiento por mordedura de serpiente en Costa
Rica en 1996: epidemiologa y consideraciones cl nicas.
Acta Medica Costarricense 41, 2329.
Bogar n, G., Romero, M., Rojas, G., Lutsch, C.,
Casadamont, M., Lang, J., Otero, R., Gutierrez, J.M.,
1999. Neutralization, by a monospecic Bothrops lanceolatus antivenom, of toxic activities induced by homologous
and heterologous Bothrops snake venoms. Toxicon 37,
551557.
Bogar n, G., Segura, E., Duran, G., Lomonte, B., Rojas, G.,
Gutierrez, J.M., 1995. Evaluacion de la capacidad de cuatro antivenenos comerciales para neutralizar el veneno de

P. Saravia et al. / Toxicon 39 (2001) 401405

la serpiente Bothrops asper (terciopelo) de Costa Rica.
Toxicon 33, 12421245.
Bolanos, R., 1972. Toxicity of Costa Rican snake venoms for
the white mouse. Am. J. Trop. Med. Hyg. 21, 360363.
Campbell, J.A., Lamar, W.W., 1989. The Venomous Reptiles
of Latin America. Cornell University Press, Ithaca, p. 425.
Chippaux, J.P., Williams, V., White, J., 1991. Snake venom
variability: methods of study, results and interpretation.
Toxicon 29, 12791303.
Cortes-Bratti, X., Chaves-Olarte, E., Lagergard, T.,
Thelestam, M., 1999. The cytolethal distending toxin from
the chancroid bacterium Haemophilus ducreyi induces cellcycle arrest in the G2 phase. J. Clin. Invest. 103, 107115.
de Franco, D., Alvarez, I., Mora, L.A., 1983. Mordedura de
odios venenosos en ninos en la region del Pacco Sur.
Analisis de ciento sesenta casos. Acta Medica
Costarricense 26, 6170.
Dole, V.P., 1956. Relation between monesteried fatty acids
and the metabolism of glucose. J. Clin. Invest. 35, 150
154.
Gene, J.A., Roy, A., Rojas, G., Gutierrez, J.M., Cerdas, L.,
1989. Comparative study on the coagulant, debrinating,
brinolytic and brinogenolytic activities of Costa Rican
crotaline snake venoms and their neutralization by a polyvalent antivenom. Toxicon 27, 841848.
Gutierrez, J.M., 1995. Clinical toxicology of snakebite in
Central America. In: Meier, J., White, J. (Eds.),
Handbook of Clinical Toxicology of Animal Venoms and
Poisons. CRC Press, , Boca Raton, FL, pp. 645665.
Gutierrez, J.M., Arroyo, O., Bolanos, R., 1980. Mionecrosis,
hemorragia y edema inducidos por el veneno de Bothrops
asper en raton blanco. Toxicon 18, 603610.
Gutierrez, J.M., Avila, C., Rojas, E., Cerdas, L., 1988. An
alternative in vitro method for testing the potency of the
polyvalent antivenom produced in Costa Rica. Toxicon
26, 411413.
Gutierrez, J.M., Gene, J.A., Rojas, G., Cerdas, L., 1985.
Neutralization of proteolytic and hemorrhagic activities of
Costa Rican snake venoms by a polyvalent antivenom.
Toxicon 23, 887893.
Gutierrez, J.M., Lomonte, B., 1989. Local tissue damage
induced by Bothrops snake venoms. A review. Mem. Inst.
Butantan 51, 211223.
Gutierrez, J.M., Lomonte, B., 1997. Phospholipase A2 myotoxins from Bothrops snake venoms. In: Kini, R.M. (Ed.),
Venom Phospholipase A2 Enzymes: Structure, Function
and Mechanism. Wiley, UK, pp. 321352.
Gutierrez, J.M., Lomonte, B., Chaves, F., Moreno, E.,

405

Cerdas, L., 1986. Pharmacological activities of a toxic

phospholipase A isolated from the venom of the snake
Bothrops asper. Comp. Biochem. Physiol. 84C, 159164.
Gutierrez, J.M., Rojas, G., Lomonte, B., Gene, J.A., Chaves,
F., Alvarado, J., Rojas, E., 1990. Standardization of
assays for testing the neutralizing ability of antivenoms.
Toxicon 28, 11271129.
Hardy, D.L., 1994. Snakebite and eld biologists in Mexico
and Central America: report of ten cases with recommendations for eld treatment. Herpetological Natural History
2, 6782.
Laemmli, U.K., 1970. Cleavage of structural proteins during
the assembly of the head of bacteriophage T4. Nature 227,
680685.
Lomonte, B., Gutierrez, J.M., Rojas, G., Calderon, L., 1991.
Quantitation by enzyme-immunoassay of antibodies
against Bothrops myotoxins in four commercially available
antivenoms. Toxicon 29, 695702.
Rioux, V., Gerbod, M.C., Boued, F., Menez, A., Galat, A.,
1998. Divergent and common groups of proteins in glands
of venomous snakes. Electrophoresis 19, 788796.
Rojas, G., Gutierrez, J.M., Gene, J.A., Gomez, M., Cerdas,
L., 1987. Neutralizacion de las actividades toxicas y enzimaticas de cuatro venenos de serpientes de Guatemala y
Honduras por el antiveneno polivalente producido en
Costa Rica. Rev. Biol. Trop. 35, 6769.
Sasa, M., 1996. Morphological variation in the lancehead
snake Bothrops asper (Garman) from Middle America.
M.Sc. Thesis, University of Texas at Arlington. Arlington,
TX.
Theakston, R.D.G., Reid, H.A., 1983. Development of simple
standard assay procedures for the characterization of
snake venoms. Bull. World Health Org. 61, 949956.
Warrell, D.A., 1992. The global problem of snake bite: its
prevention and treatment. In: Gopalakrishnakone, P.,
Tan, C.K. (Eds.), Recent Advances in Toxinology
Research, vol. 1. National University of Singapore,
Singapore, pp. 121153.
World Health Organization, 1981. Progress in the characterization of venoms and standardization of antivenoms. WHO
Oset Publication 58, Geneva, 44 p.
Yamakawa, M., Nozaki, M., Hokama, Z., 1976.
Fractionation of sakishima habu (Trimeresurus elegans )
venom and lethal, hemorrhagic, and edema-forming activities of the fractions. In: Ohsaka, A., Hayashi, K., Sawai,
Y. (Eds.), Animal, Plant and Microbial Toxins, vol. 1.
Plenum Press, New York, pp. 97109.