Neutralization Of The Hemorrhagic And Edemaâforming Activities ...
Rev. Vet. 10/11, 1 y 2, 1999–2000
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Neutralization of the hemorrhagic and edema–forming activities
from venom of Bothrops alternatus offsprings from Argentina
Zeinsteger, P.A.; Maruñak, S.L.; Acosta de Pérez, O.
Cátedra de Patología Médica, Facultad de Ciencias Veterinarias, UNNE,
Sargento Cabral 2139, Corrientes (3400), Argentina, Tel– Fax: 54–03783–425753,
E–mail: patmed@vet.unne.edu.ar
Abstract
Zeinsteger, P.A.; Maruñak, S.L.; Acosta de Pérez, O.: Neutralization of the hemor-
rhagic and edema–forming activities from venom of Bothrops alternatus offsprings from
Argentina. Tests of neutralization of hemorrhagic and edema–forming activities from the
venom of Bothrops alternatus (víbora de la cruz) one month–old offsprings were performed
using a bivalent antivenom, produced by Laboratorio Central de Salud Pública in La Plata,
Buenos Aires, Argentina. We determined that 172 µl of the antivenom neutralize 50 % of
the hemorrhagic activity, induced by 9 µg of the venom (challenge dose). Neutralization of
edema–forming activity was more effective when the antivenom was administered previous to
the venom. We conclude that the antivenom neutralizes the studied toxic activities, taking into
account that for its production horses are immunized using venom from adult snakes.
Key words: Bothrops alternatus, neutralization, hemorrhagic activity, edema–forming activity.
INTRODUCTION
The purpose of the present study was to evaluate
the neutralizing activity of a bivalent antivenom on the
In Argentina there are snakes from many species of hemorrhagic and edema–forming activities induced by
the Viperidae family. Bothrops alternatus and Bothrops venom from Bothrops alternatus offsprings, taking into
neuwiedii diporus are widespread all along the country. account that this venom in particular is not used for
The northeast region presents a subtropical climate; B. the immunization of the horses destined to antivenom
jararacussu, B. jararaca, B. cotiara and B. moojeni and production.
the two species mentioned above are common there. B.
ammodytoides inhabits the south area, which is arid MATERIAL AND METHODS
and cold. In this country, 90% of the envenomings are
caused by Bothrops genus 5.
Venom. A venom mixture from one month–old
Previous studies demonstrated that Bothrops al- offsprings Bothrops alternatus was used. After the
ternatus venom from the northeast of Argentina has a extraction of the venoms, the pool was homogenized,
prominent local effect, characterized by intense pain, dehydrated and maintained at –20 ºC.
edema, ecchymosis, hemorrhagic phlyctema and ne-
Antivenom. A bivalent hyperimmune antivenom ob-
crosis of the muscular and connective tissues 2, 3. The tained by horse immunization was used. It neutralizes
systemic effects lead to hepatic and renal alterations 3, 25 mg of Bothrops alternatus venom, and 15 mg of B.
as well as coagulation abnormalities 1.
neuwiedii venom (Lote Nº 021, Laboratorio Central de
The coordinated activity of the different compo- Salud Pública, La Plata, Buenos Aires, Argentina).
nents of the venom causes a complex envenomation that
Neutralization of the hemorrhagic activity (ED ).
50
must be treated using a specific antivenom, commonly Our laboratory previously determined the hemor-
obtained from horse immunization.
rhagic activity from the venom of Bothrops alternatus
Several studies state that there are considerable differ- offsprings (9 µg), expressed as minimum hemorrhagic
ences in the composition of venoms from the same spe- dose (MHD) 12. For this determination, we applied the
cies of snakes that inhabit different geographic areas 8, 9.
Kondo et al. 1960 method 10, modified by Gutiérrez et
A satisfactory effect has been obtained with the use of al., 1985 7. For the neutralization of the hemorrhagic
antivenoms in cases where the involved species were not activity, a challenge dose equivalent to 10 times MHD
used for horse immunization 11. An example of this would was used. Different proportion mixtures of antivenom
be the neutralization activity of a polyvalent antivenom and venom were incubated for 30 minutes at 37ºC, and
from South–African snakes which neutralizes the toxic from each of them 0.1 ml were injected to mice. Ani-
activities from venoms of North–American species 13.
mals were grouped in 5 individuals (weight varied from
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18 to 20 g). Each inoculation had 9 µg of the venom in rest of the body to determine the increment of weight
0.1 ml phosphate buffer solution (PBS) and was inject- due to edema.
ed intradermically. Two hours later, animals were eutha-
nized with ether and skin was removed to measure the RESULTS
hemorrhagic area. Controls were injected with 0.1 PBS.
The effective dose 50% (ED ) is the antivenom/venom
The antivenom satisfactorily neutralized the hemor-
50
reason that reduces 50% of the hemorrhagic effect, ac- rhagic and edema–forming activities induced by venom
cording to Gutiérrez et al., 1981 method 6.
from Bothrops alternatus offsprings, in spite of the fact
Neutralization of the edema–forming activity. Pre- that for the preparation of the antigenic booster for
vious experiences have established the minimum ede- horses only venom from adult snakes was used.
ma–forming dose (MED) from venom of B. alternatus
ED was calculated by linear regression. The dose
50
offsprings in 0.38 µg 12. This data was used for the de- of injected venom was constant (9 µg), and the conse-
termination of the neutralization capacity of the studied quent hemorrhagic halo showed an inverse proportion
antivenom on this effect. Mice from 18 to 20 g were ad- between µg of the venom and the different dilutions of
ministered intraperitoneally with 0.5 ml of antivenom, the antivenom. These haloes were similar when whole
and 2.28 µg venom in 0.05 ml PBS in the left plantar antivenom and dilutions up to 1/4 were used, while
pad. Groups of 5 individuals received the antivenom 1/8 and 1/16 dilutions had a lower neutralizing activity
30 minutes before, simultaneously and 30 minutes after (Figure 1). Statistical analysis showed r2 = 0.860; the
the injection of the venom. Animals were anesthetized calculated ED was 172 µl antivenom/venom.
50
and euthanized 60 minutes after the administration of
When the antivenom was injected prior to venom,
the antivenom and both limbs were separated from the the edema–forming activity was best neutralized, while
in the simultaneous injection a lower neutralizing activ-
ity was evident. Late administration of the antivenom
(30 minutes after venom injection) did not show signifi-
cant differences with those animals which received the
venom only (Figure 2).
DISCUSSION
Bothrops alternatus venom was barely studied in
the past. Our study showed that the hemorrhagic ac-
tivity from venom of offsprings was four times more
powerful than in adults (MHD 3.6 µg). This effect could
also be observed with the edema–forming activity as
it showed 10 times the power of the adult venom (4.4
Figure 1. Neutralization of the hemorrhagic activ-
µg) 12. This is remarkable as both offspring and adult
ity from venom of Bothrops alternatus offsprings.
snakes inhabit the same geographical area.
Graphic shows the neutralizing capacity of the whole
Despite the significant differences in the intensity
antivenom and different dilutions of it (X axis). Y axis
of the hemorrhagic and edema–forming activities from
represents mean diameter values (mm) of the hemor-
venoms of adults and offsprings, the neutralizing capac-
rhagic halo.
ity of the antivenom was efficient for both of them 4.
Compared to adults, offsprings venom had a greater
vasculotoxic and edema–forming activity. Nevertheless,
the antivenom was capable to neutralize both effects
when it was administered in time. This antivenom is
the same used in previous experiences to neutralize the
same toxic activities of adult venom, but from a differ-
ent lot.
It is important to study the neutralizing activity of
the bivalent antivenom in the neutralization of the toxic
activities of offsprings venom, considering that it is not
used in the preparation of the antigenic booster for the
immunization of horses. Paradoxically, compared to
adults a lower antivenom dose is needed to neutralize
Figure 2. Neutralization of the edema–forming activity the hemorrhagic activity of offsprings venom (172 vs.
from venom of Bothrops alternatus offsprings.
282.95 µl).
Mice were injected with 2.28 µg of the venom (chal-
Results may confirm the existence of a total im-
lenge dose) and 500 µl of the antivenom 30 minutes
munologic identity between offspringss and adults.
before, simultaneously and after it.
Differences might be attributable to variations in the
24
concentration of some enzymes in comparison to others,
de serpientes del género Bothrops y Crotalus de Argentina.
being this related to the ontogeny of the animals.
Toxicon 36: 1165–1172.
4. Acosta de Pérez O. 1999. Caracterización bioquímica y
Resumen
farmacológica del veneno de Bothrops alternatus. Tesis
Zeinsteger, P.A.; Maruñak, S.L.; Acosta de Pérez,
doctoral, Universidad Nacional de Buenos Aires (UBA),
O.: Neutralización de las actividades hemorrágica y
Argentina, p. 88.
edematizante de veneno de viboreznos de Bothrops al- 5. Esteso SC. 1985. Ofidismo en la República Argentina, Arpon,
ternatus. Se efectuaron pruebas de neutralización de las
Córdoba, p. 82–94.
actividades hemorrágica y edematizante causadas por 6. Gutiérrez JM, Chaves F, Bolaños R, Cerdas L, Rojas E,
veneno de viboreznos de Bothrops alternatus (víbora
Arroyo O, Portilla E. 1981. Neutralización de los efectos
de la cruz) de un mes de vida, utilizando un antiveneno
locales del veneno de Bothrops asper por un veneno poliva-
bivalente de origen equino, producido en el Laboratorio
lente. Toxicon 19: 493–500.
Central de Salud Pública de La Plata, Provincia de Bue- 7. Gutiérrez JM, Gené JA, Rojas G, Cerdas L. 1985.
nos Aires, Argentina. Se determinó que 172 µl de anti-
Neutralization of proteolytic and hemorrhagic activities
veneno neutralizan el 50% de la actividad hemorrágica
of costa rican snake venoms by a polyvalent antivenom.
inducida por 9 µg de veneno (dosis de reto). La neutra-
Toxicon 23: 887–893.
lización de la actividad edematizante fue más efectiva 8. Jimenez–Porras JM, Chaves F, Bolaños R. 1964. Venom
cuando se administró el antiveneno previo al veneno.
proteins of the fer–de–Lance Bothrops atrox, from Costa
Se concluye que el antiveneno utilizado neutraliza satis-
Rica. Toxicon 2: 155–166.
factoriamente las actividades tóxicas estudiadas, a pesar 9. Jimenez–Porras JM. 1964. Intraspecific variation in the
que para la obtención del mismo, se inmunizan equinos
composition of the jumping viper, Bothrops nummifer.
con veneno de ejemplares adultos.
Toxicon 2: 187–195.
10. Kondo H, Kondo S, Ibezawa I, Murata R, Osaka A. 1960.
Palabras clave: Bothrops alternatus, neutralización,
Studies on the quantitative method for determination of the
actividad hemorrágica, actividad edematizante.
hemorrhagic activity of Habu snake venom. Jpn. J. Med.
Sci. Biol. 13: 43–51.
11. Lomonte B, Furtado MF, Rovira ME, Carmona E, Rojas
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