Toxicon 88 (2014) 34e37

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Toxicon
journal homepage: www.elsevier.com/locate/toxicon

Short communication

Toxicity of Bothrops sp snake venoms from Ecuador and

preclinical assessment of the neutralizing efficacy of a
polyspecific antivenom from Costa Rica


Johana Laines a, Alvaro Segura b, Mauren Villalta b, María Herrera b,
Maria
María Gutie

ngela Vargas b, Gladys Alvarez a, Jose
rrez b,
,*
Guillermo Leo
n b

a
n en Salud Pública (INSPI), Guayaquil, Ecuador

Instituto de Investigacio
b
Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San Jos

e, Costa Rica

a r t i c l e i n f o a b s t r a c t

Article history: The toxicological profile of the venoms of the snakes Bothrops asper and Bothrops atrox
Received 20 January 2014 from Ecuador was investigated, together with the venom of a population of B. asper
Received in revised form 9 June 2014 formerly classified as ‘Bothrops xanthogrammus’. The three venoms exerted lethal, hem-
Accepted 11 June 2014
orrhagic, myotoxic, coagulant and defibrinogenating effects, in agreement with the char-
Available online 17 June 2014
acteristic toxicological profile of Bothrops sp venoms. A polyspecific antivenom (bothropic
ecrotalicelachesic) manufactured in Costa Rica was assessed for its preclinical efficacy
Keywords:
against the toxic activities of these Ecuadorian venoms. Antivenom was effective in the
Bothrops asper
Bothrops atrox
neutralization of the five activities tested in the three venoms. These observations are in
Ecuador agreement with previous reports on the extensive cross-reactivity and paraspecific
Antivenom neutralization of antivenoms manufactured in Latin America against the venoms of
Toxicity Bothrops sp snakes.
Neutralization © 2014 Elsevier Ltd. All rights reserved.

As occurs in the majority of Latin American countries,
snakebite envenoming constitutes a significant public
health hazard in Ecuador (Warrell, 2004; Gonza
lez-
Andrade and Chippaux, 2010; Gutie
rrez, 2011). It has
been estimated that between 1400 and 1600 snakebite
cases occur every year in this country, with an incidence of
11.15 cases per 100,000 population per year, and a mortality
rate of 0.052 per 100,000 per year (Gonza
lez-Andrade and
Chippaux, 2010), although it is likely that these data, based
mostly on hospital statistics, represent an underreport of
the actual situation. The incidence is likely to be higher in
specific localities, such as in indigenous communities
(Larrick et al., 1978; Theakston et al., 1981). In Ecuador,
* Corresponding author. Tel.: þ506 2511 7877; fax: þ506 2292 0485.

n).
E-mail address: guillermo.leon@ucr.ac.cr (G. Leo
there are 17 snake species of the family Viperidae, classified
in the genera Bothrops, Bothriechis, Bothriopsis, Bothroco-
phias, Porthidium, and Lachesis, and 21 species of the family
Elapidae, classified in the genera Micrurus and Lep-
tomicrurus (Campbell and Lamar, 2004). The vast majority
of accidents, and the most severe ones, are induced by
species of Bothrops, especially Bothrops asper and Bothrops
atrox (Smalligan et al., 2004). The former inhabits the
western lowland rainforests of the country, whereas the
latter is distributed in the rainforests of the Amazonian
region, in the eastern part of Ecuador (Campbell and Lamar,
2004). Other species, most notably Bothropsis bilineata,
inflict a significant number of cases in the Amazonian basin,
although they usually are of less severity (Smalligan et al.,
2004).
The treatment of snakebite envenomings in Ecuador is
based on the administration of equine-derived antivenoms.

http://dx.doi.org/10.1016/j.toxicon.2014.06.008
0041-0101/© 2014 Elsevier Ltd. All rights reserved.
 J. Laines et al. / Toxicon 88 (2014) 34e37
A polyspecific antivenom (‘antiofídico botro
pico poliva-
lente’) has been manufactured in Ecuador by the former
Instituto Leopoldo Izquieta Pe
rez (http://apps.who.int/
bloodproducts/snakeantivenoms/database/) (Smalligan
et al., 2004), later transformed into Instituto Nacional de
Salud Pública e Investigacio
n (INSPI). The effectiveness and
safety of this formulation was previously demonstrated at
preclinical and clinical levels (Theakston et al., 1995;
Smalligan et al., 2004). In addition, polyspecific bothropic
antivenoms produced in Brazil and Colombia are also uti-
lized (Smalligan et al., 2004). Recently, the polyspecific
viperid antivenom of Instituto Clodomiro Picado, Costa
Rica, has been registered and is being used in Ecuador for
the treatment of envenomings by viperid species.
Previous studies have demonstrated the existence of
intraspecies variations in the proteomics and toxic activ-
ities of venoms of B. asper and B. atrox from different lo-
calities in Central and South America (Alape-Giro
n et al.,
2008; Nún ~ ez et al., 2009; Gutie
rrez et al., 2010; Calvete
et al., 2011). Therefore, in the light of this phenomenon, it
is relevant to evaluate whether polyspecific antivenoms
manufactured in some countries are effective in the
neutralization of toxic effects of venoms from other coun-
tries. In this communication, a preclinical assessment of the
neutralizing ability of this antivenom, against the venoms
of B. asper and B. atrox from Ecuador, is reported. In addi-
tion, the venom of a population of B. asper formerly clas-
sified as the separate species ‘Bothrops xanthogrammus’
was also analyzed, being hereby named as B. asper (popu-
lation ‘xanthogrammus’).
Venoms of B. asper, B. asper (population ‘xanthog-
rammus’) and B. atrox were collected from snakes kept in
captivity in the serpentarium of the former Instituto Nacio-
nal de Higiene y Medicina Tropical “Dr Leopoldo Izquieta
Pe
rez”, currently known as Instituto Nacional de Salud
Pública e Investigacio
n (INSPI). Venoms are pools obtained
from more than ten adult specimens. Once collected,
venoms were dried in a desiccator for stabilization, and
stored at 20  C. The lyophilized polyspecific antivenom
(‘suero antiofìdico polivalente’, batch 5240413POLF, expiry
date: April 2018), manufactured by Instituto Clodomiro
Picado, Universidad de Costa Rica, was used. It was prepared
by immunizing horses with a mixture of the venoms of B.
asper, Crotalus simus and Lachesis stenophrys from Costa Rica
(Angulo et al., 1997). Hyperimmune horse plasma was frac-
tionated by caprylic acid precipitation (Rojas et al.,1994), and
the product is constituted by whole IgG molecules. The an-
tivenom has a total protein concentration of 53.7 ± 0.3 g/L, as
determined by the Biuret method (Parvin et al., 1965).
Lethality of venoms was assessed in CD-1 mice
(16e18 g) by the intraperitoneal route, using eight mice per
experimental group. Deaths occurring within 48 h were
recorded and the Median Lethal Dose (LD50) was estimated
by SpearmaneKarber (WHO, 1981). Hemorrhagic activity
was determined by the mouse skin test, as described by
Gutie
rrez et al. (1985). The Minimum Hemorrhagic Dose
(MHD) corresponded to the venom dose that induced a
hemorrhagic lesion of 10 mm diameter in the inner side of
the skin 2 h after injection. Coagulant activity was deter-
mined in human citrated plasma as described by Gene
et al.
(1989). The Minimum Coagulant Dose (MCD) is the amount
35
of venom that induces clotting of plasma in 60 s. Defib-
rinogenating activity was determined in CD-1 mice
(18e20 g), as described by Gene
et al. (1989). The Minimum
Defibrinogenating Dose (MDD) is the amount of venom
that induced blood incoagulability in all mice after 1 h of
injection. Myotoxic activity was determined by injecting
50 mg venom, dissolved in 50 mL of 0.14 M NaCl, 0.04 M
phosphate, pH 7.2 (PBS), intramuscularly in the right
gastrocnemius of CD-1 mice (18e20 g). Three hr after in-
jection, mice were bled from the tail; blood was collected in
heparinized capillary tubes and plasma was separated.
Plasma creatine kinase (CK) activity was determined using
a commercial kit (CK LIQUI-UV, Stanbio Lab., Texas, USA).
CK activity was expressed as units/L. Experimental pro-
tocols involving the use of mice were approved by the
Institutional Committee for the Care and Use of Laboratory
Animals (CICUA) of the University of Costa Rica.
For assessing neutralization by the antivenom, the
protocols described in previous publications were followed
(Gutie
rrez et al., 1985; Rojas et al., 2005). Briefly, mixtures
containing a constant amount of venom and various di-
lutions of antivenom were prepared and incubated at 37  C
for 30 min. Controls included venom solutions incubated
with PBS instead of antivenom. Then, aliquots of the mix-
tures, containing a predefined ‘challenge dose’ of venom,
were tested in the experimental systems described above.
The challenge doses utilized for each effect were: For
lethality, 4 LD50s; for hemorrhagic activity, 5 MHDs; for
coagulant activity, 2 MCDs; for defibrinogenating activity, 2
MDD; and for myotoxic activity, 50 mg. For lethal and
hemorrhagic activities, neutralizing ability was expressed
as the Median Effective Dose (ED50), defined as the ratio mL
antivenom/mg venom at which the activity of venom was
inhibited by 50%. In the case of lethality, ED50 was also
expressed as mg venom neutralized per mL antivenom, as
this is a typical way of expressing neutralization in quality
control laboratories in Latin America. For coagulant and
defibrinogenating activities, neutralization was expressed
as Effective Dose (ED), as defined by Gene
et al. (1989). In
the case of myotoxic activity, only one antivenom/venom
ratio (1000 mL antivenom per mg venom) was tested. The
non-parametric KruskaleWallis test, followed by Dunn's
post-hoc test, were used to determine the significance of
the differences between mean values of experimental
groups; a p value <0.05 was considered significant.
The three venoms exerted lethal, hemorrhagic, coagu-
lant, defibrinogenating, and myotoxic activities (Table 1), in
agreement with a previous study describing the effects of
Ecuadorian snake venoms (Kuch et al., 1996). No significant
difference was observed in lethal activity between venoms,
whereas hemorrhagic activity was highest in the venom of
B. asper, followed by B. asper (population ‘xanthogrammus’)
and B. atrox. The two venoms of B. asper had higher in vitro
coagulant activity than the venom of B. atrox. The order of
myotoxic activity in these venoms was: B. asper > B. asper
(population ‘xanthogrammus’) > B. atrox (Table 1). Overall,
the general toxicological profiles of these venoms show
similarities with those described previously for B. asper and
B. atrox venoms from Ecuador and other countries in Latin
America (Otero et al., 1995; Theakston et al., 1995; Kuch
et al., 1996; Rojas et al., 2005; Segura et al., 2010),
36 J. Laines et al. / Toxicon 88 (2014) 34e37

Table 1
Toxic activities of Bothrops sp venoms from Ecuador.

Activity B. asper B. asper (population ‘xanthogrammus’) B. atrox

Lethal (LD50)a 91 (78e126) mg 84 (73e126) mg 85 (33e107) mg

Hemorrhagic (MHD)b 1.1 ± 0.2 mgf 2.2 ± 0.3 mg₤ 3.7 ± 1.0 mg₤
Coagulant (MCD)c 0.07 ± 0.02 mgf 0.10 ± 0.02 mgf 2.90 ± 0.12 mg₤
Defibrinogenating (MDD)d 2 mg 2 mg 1 mg
Myotoxic (CK, U/L)e 5516 ± 1350f 2816 ± 1201₤ 785 ± 181#

Values with different superscripts (f, ₤, #) are significantly different between them (p < 0.05), whereas values with the same superscript do not differ
significantly between them (p > 0.05).
a
LD50: Median Lethal Dose: Dose of venom that induces death in 50% of injected mice (16e18 g); 95% confidence limits are depicted in parentheses.
b
MHD: Minimum Hemorrhagic Dose: Dose of venom that induces a hemorrhagic lesion of 10 mm diameter in mice 2 h after injection. Results are
presented as mean ± S.D. (n ¼ 3).
c
MCD: Minimum Coagulant Dose: Dose of venom that induces clotting of citrated human plasma in 60 s. Results are presented as mean ± S.D. (n ¼ 3).
d
MDD: Minimum Defibrinogenating Dose: Dose of venom that induces blood incoagulability 1 h after i.v. injection in all mice tested (n ¼ 3).
e
Plasma CK activity of mice 3 h after intramuscular injection in the gastrocnemius of 50 mg venom (in 50 mL PBS). Control mice injected with PBS alone had
a CK activity of 220 ± 40 U/L. Results are presented as mean ± S.D. (n ¼ 5).

including the notorious difference in direct myotoxic ac-
tivity between B. asper and B. atrox venoms (Segura et al.,
2010). This experimental toxicological profile is compat-
ible with the main clinical manifestations of envenomings
by B. atrox and B. asper in Ecuador, characterized by
prominent local tissue damage and systemic manifesta-
tions associated with bleeding and coagulopathy (Kerrigan,
1991; Warrell, 2004; Smalligan et al., 2004; Gaus et al.,
2013). Severe cases are associated with cardiovascular
shock and cerebrovascular accidents (Kerrigan, 1991;
Warrell, 2004; Del Brutto and Del Brutto, 2012).
The polyspecific antivenom was effective at the pre-
clinical level in the neutralization of all the activities tested
for the three venoms studied (Table 2). Antivenom
neutralized lethal activity of the three venoms with similar
efficacy, as the 95% confidence limits of ED50s overlap.
Neutralizing ability against hemorrhagic activity was
higher with the venoms of the two samples of B. asper than
with the venom of B. atrox, whereas the order of neutrali-
zation of in vitro coagulant activity was B. asper > B. atrox
and B. asper (population ‘xanthogrammus’). Antivenom was
also effective in the neutralization of myotoxic activity
induced by the venoms of B. asper and B. atrox. At an an-
tivenom/venom ratio of 1000 mL antivenom/mg venom, the
plasma CK values were: For B. atrox venom: venom alone:
785 ± 181 U/L; venom plus antivenom: 248 ± 63 U/L. For B.
asper venom: venom alone: 5516 ± 1350 U/L; venom plus
antivenom: 505 ± 321 U/L; in both cases neutralization was
significant (p < 0.05). Plasma CK activity in control mice
injected with PBS was 220 ± 40 U/L. Our observations are in
agreement with previous studies performed in Latin
America on the paraspecific neutralization of Bothrops sp
venoms by antivenoms produced in various countries using
different venoms in the immunizing mixture (see for
example Theakston et al., 1995; Otero et al., 1995; de Roodt
et al., 1998; Bogarín et al., 2000; Rojas et al., 2005; Segura
et al., 2010). In the case of Ecuador, Theakston et al.
(1995) had demonstrated the ability of antivenoms man-
ufactured in Ecuador, Brazil and Colombia to neutralize
toxic activities of Bothrops sp venoms. Our results also
agree with ‘antivenomic’ observations which highlighted
the ability of the polyspecific antivenom tested in this study
to immunorecognize the majority of components of the
venoms of B. atrox from Ecuador (Nún ~ ez et al., 2009).
In conclusion, the venoms of B. asper and B. atrox from
Ecuador, and of a B. asper population previously classified
as ‘B. xanthogrammus’, present a toxicological profile
characterized by lethal, hemorrhagic, myotoxic, coagulant,
and defibrinogenating activities. The polyspecific anti-
venom of Instituto Clodomiro Picado (Costa Rica) is effec-
tive, at the preclinical level, in the neutralization of these
toxic activities induced by the venoms, thus extending
previous observations on the large extent of paraspecific
efficacy of polyspecific antivenoms raised in various coun-
tries in Latin America against the venoms of Bothrops sp
species.

Table 2
Neutralization of toxic activities of Ecuadoran Bothrops sp venoms by the polyspecific antivenom.

Activity B. asper B. asper (population ‘xanthogrammus’) B. atrox

Lethal (ED50)a 345 (238e476) mL av/mg v 189 (116e238) mL av/mg v 263 (154e385) mL av/mg v
2.9 (2.1e4.2) mg v/mL av 5.3 (4.2e8.6) mg v/mL av 3.8 (2.6e6.5) mg v/mL av
Hemorrhagic (ED50)b 125 ± 4f 168 ± 9f 834 ± 96₤
Coagulant (ED)c 190 ± 1f 316 ± 3₤ 305 ± 5₤
Defibrinogenating (ED)d 500 500 1000

Values with different superscripts (f, ₤) are significantly different between them (p < 0.05), whereas values with the same superscript do not differ
significantly between them (p > 0.05).
a
ED50: Ratio of mL antivenom/mg venom or mg venom/mL antivenom at which 50% of injected mice were protected; 95% confidence limits are depicted in
parentheses.
b
ED50: Ratio of mL antivenom/mg venom at which the diameter of hemorrhagic spots was reduced by 50%. Results are expressed as mean ± S.D. (n ¼ 5).
c
ED: Ratio of mL antivenom/mg venom at which the clotting time of plasma is increased three times as compared with the clotting time of plasma
incubated with venom alone, without antivenom. Results are expressed as mean ± S.D. (n ¼ 3).
d
ED: Ratio of mL antivenom/mg venom at which blood was clottable in all injected mice.
 J. Laines et al. / Toxicon 88 (2014) 34e37
Ethical statement
This manuscript presents an experimental study per-
formed following the standard procedure of scientific
ethics. All procedures used in this study were approved by
the Institutional Committee for the Care and Use of Labo-
ratory Animals (CICUA) of Universidad de Costa Rica.
Acknowledgments
The collaboration of Daniela Solano in the laboratory
work is acknowledged. This study was supported by Vice-

n, Universidad de Costa Rica
rrectoría de Investigacio
(project 741-B2-091).
Conflict of interest statement


Alvaro Segura, Mauren Villalta, María Herrera, Mar-


María Gutie
iangela Vargas, Jose
rrez, and Guillermo Leo

n
work at Instituto Clodomiro Picado (Universidad de Costa
Rica), where the antivenom tested in this study is
manufactured.
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