Toxicon 88 (2014) 34e37
Contents lists available at ScienceDirect
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, María Gutie rrez b, ngela Vargas b, Gladys Alvarez a, Jose Maria b ,* n Guillermo Leo a b
n en Salud Pública (INSPI), Guayaquil, Ecuador Instituto de Investigacio 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: Received 20 January 2014 Received in revised form 9 June 2014 Accepted 11 June 2014 Available online 17 June 2014
The toxicological profile of the venoms of the snakes Bothrops asper and Bothrops atrox from Ecuador was investigated, together with the venom of a population of B. asper formerly classified as ‘Bothrops xanthogrammus’. The three venoms exerted lethal, hemorrhagic, myotoxic, coagulant and defibrinogenating effects, in agreement with the characteristic toxicological profile of Bothrops sp venoms. A polyspecific antivenom (bothropic ecrotalicelachesic) manufactured in Costa Rica was assessed for its preclinical efficacy against the toxic activities of these Ecuadorian venoms. Antivenom was effective in the neutralization of the five activities tested in the three venoms. These observations are in agreement with previous reports on the extensive cross-reactivity and paraspecific neutralization of antivenoms manufactured in Latin America against the venoms of Bothrops sp snakes. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Bothrops asper Bothrops atrox Ecuador Antivenom Toxicity Neutralization
As occurs in the majority of Latin American countries, snakebite envenoming constitutes a significant public lezhealth hazard in Ecuador (Warrell, 2004; Gonza rrez, 2011). It has Andrade and Chippaux, 2010; Gutie 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 lez-Andrade and rate of 0.052 per 100,000 per year (Gonza 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: [email protected] (G. Leo http://dx.doi.org/10.1016/j.toxicon.2014.06.008 0041-0101/© 2014 Elsevier Ltd. All rights reserved.
there are 17 snake species of the family Viperidae, classified in the genera Bothrops, Bothriechis, Bothriopsis, Bothrocophias, Porthidium, and Lachesis, and 21 species of the family Elapidae, classified in the genera Micrurus and Leptomicrurus (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.
J. Laines et al. / Toxicon 88 (2014) 34e37
pico polivaA polyspecific antivenom (‘antiofídico botro lente’) has been manufactured in Ecuador by the former rez (http://apps.who.int/ Instituto Leopoldo Izquieta Pe bloodproducts/snakeantivenoms/database/) (Smalligan et al., 2004), later transformed into Instituto Nacional de n (INSPI). The effectiveness and Salud Pública e Investigacio 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 utilized (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 activities of venoms of B. asper and B. atrox from different lon et al., calities in Central and South America (Alape-Giro ~ ez et al., 2009; Gutie rrez et al., 2010; Calvete 2008; Nún 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 countries. 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 addition, the venom of a population of B. asper formerly classified as the separate species ‘Bothrops xanthogrammus’ was also analyzed, being hereby named as B. asper (population ‘xanthogrammus’). Venoms of B. asper, B. asper (population ‘xanthogrammus’) and B. atrox were collected from snakes kept in captivity in the serpentarium of the former Instituto Nacional de Higiene y Medicina Tropical “Dr Leopoldo Izquieta rez”, currently known as Instituto Nacional de Salud Pe n (INSPI). Venoms are pools obtained Pública e Investigacio 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 fractionated by caprylic acid precipitation (Rojas et al.,1994), and the product is constituted by whole IgG molecules. The antivenom 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 rrez et al. (1985). The Minimum Hemorrhagic Dose Gutie (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 et al. mined in human citrated plasma as described by Gene (1989). The Minimum Coagulant Dose (MCD) is the amount
35
of venom that induces clotting of plasma in 60 s. Defibrinogenating activity was determined in CD-1 mice et al. (1989). The Minimum (18e20 g), as described by Gene 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 injection, 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 protocols 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 rrez et al., 1985; Rojas et al., 2005). Briefly, mixtures (Gutie containing a constant amount of venom and various dilutions 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 mixtures, 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 et al. (1989). In as Effective Dose (ED), as defined by Gene 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 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 Hemorrhagic (MHD)b Coagulant (MCD)c Defibrinogenating (MDD)d Myotoxic (CK, U/L)e
91 (78e126) mg 1.1 ± 0.2 mgf 0.07 ± 0.02 mgf 2 mg 5516 ± 1350f
84 (73e126) mg 2.2 ± 0.3 mg₤ 0.10 ± 0.02 mgf 2 mg 2816 ± 1201₤
85 (33e107) mg 3.7 ± 1.0 mg₤ 2.90 ± 0.12 mg₤ 1 mg 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 activity between B. asper and B. atrox venoms (Segura et al., 2010). This experimental toxicological profile is compatible with the main clinical manifestations of envenomings by B. atrox and B. asper in Ecuador, characterized by prominent local tissue damage and systemic manifestations 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 preclinical 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 neutralization 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 antivenom/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 manufactured 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 ~ ez et al., 2009). venoms of B. atrox from Ecuador (Nún 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 antivenom of Instituto Clodomiro Picado (Costa Rica) is effective, 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 countries 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 2.9 (2.1e4.2) mg v/mL av 125 ± 4f 190 ± 1f 500
189 (116e238) mL av/mg v 5.3 (4.2e8.6) mg v/mL av 168 ± 9f 316 ± 3₤ 500
263 (154e385) mL av/mg v 3.8 (2.6e6.5) mg v/mL av 834 ± 96₤ 305 ± 5₤ 1000
Hemorrhagic (ED50)b Coagulant (ED)c Defibrinogenating (ED)d
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 performed 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 Laboratory 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 rrez, and Guillermo Leo n iangela Vargas, Jose work at Instituto Clodomiro Picado (Universidad de Costa Rica), where the antivenom tested in this study is manufactured.
References n, A., Sanz, L., Escolano, J., Flores-Díaz, M., Madrigal, M., Alape-Giro Sasa, M., Calvete, J.J., 2008. Snake venomics of the lancehead pitviper Bothrops asper: geographic, individual, and ontogenetic variations. J. Proteome Res. 7, 3556e3571. rrez, J.M., 1997. Clinical and laboratory alAngulo, Y., Estrada, R., Gutie terations in horses during immunization with snake venoms for the production of polyvalent (Crotalinae) antivenom. Toxicon 35, 81e90. Bogarín, G., Morais, J.F., Yamaguchi, I.K., Stephano, M.A., Marcelino, J.R., rrez, J.M., Nishikawa, A.K., Guidolin, R., Rojas, G., Higashi, H.G., Gutie 2000. Neutralization of crotaline snake venoms from Central and South America by antivenoms produced in Brazil and Costa Rica. Toxicon 38, 1429e1441. rez, A., Borges, A., Vargas, A.M., Lomonte, B., Calvete, J.J., Sanz, L., Pe rrez, J.M., Chalkidis, H.M., Moura ~o, R.H., Angulo, Y., Gutie Furtado, M.F.D., Moura-da-Silva, A.M., 2011. Snake population venomics and antivenomics of Bothrops atrox: paedomorphism along its transamazonian dispersal and implications of geographic venom variability on snakebite management. J. Proteomics 74, 510e527. Campbell, J.A., Lamar, W.W., 2004. The Venomous Reptiles of the Western Hemisphere. Cornell University Press, Ithaca, New York. de Roodt, A., Dolab, J.A., Fern andez, T., Segre, L., Haajos, S.E., 1998. Crossreactivity and heterologous neutralization of crotaline antivenoms used in Argentina. Toxicon 36, 1025e1038. Del Brutto, O.H., Del Brutto, V.J., 2012. Neurological complications of venomous snake bites: a review. Acta Neurol. Scand. 125, 363e372. Gaus, D.P., Herrera, D.F., Troya, C.J., Guevara, A.H., 2013. Management of snakebite and systemic envenomation in rural Ecuador using the 20minute whole blood clotting test. Wilderness Environ. Med. 24, 345e350. , J.A., Roy, A., Rojas, G., Gutie rrez, J.M., Cerdas, L., 1989. Comparative Gene study on coagulant, defibrinating, fibrinolytic and fibrinogenolytic activities of Costa Rican crotaline snake venoms and their neutralization by a polyvalent antivenom. Toxicon 27, 841e848. lez-Andrade, F., Chippaux, J.P., 2010. Snake bite envenomation in Gonza Ecuador. Trans. R. Soc. Trop. Med. Hyg. 104, 588e591.
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rrez, 2011. Envenenamientos por mordeduras de serpientes en Gutie rica Latina y el Caribe: una visio n integral de car Ame acter regional. Bol. Malariol. Salud Amb. LI, 1e16. rrez, J.M., Gene , J.A., Rojas, G., Cerdas, L., 1985. Neutralization of Gutie proteolytic and hemorrhagic activities of Costa Rican snake venoms by a polyvalent antivenom. Toxicon 23, 887e893. rrez, J.M., Sanz, L., Flores-Díaz, M., Figueroa, L., Madrigal, M., Gutie n, G., Estrada, R., Borges, A., AlapeHerrera, M., Villalta, M., Leo n, A., Calvete, J.J., 2010. Impact of regional variation in Bothrops Giro asper snake venom on the design of antivenoms: integrating antivenomics and neutralization approaches. J. Proteome Res. 9, 564e577. Kerrigan, K.R., 1991. Venomous snakebite in eastern Ecuador. Am. J. Trop. Med. Hyg. 44, 93e99. rrez, J.M., Freire, A., 1996. Biochemical and bioKuch, U., Mebs, D., Gutie logical characterization of Ecuadorian pitviper venoms (genera Bothriechis, Bothriopsis, Bothrops and Lachesis). Toxicon 34, 714e717. Larrick, J.W., Yost, J.A., Kaplan, J., 1978. Snake bite among the Waorani Indians of eastern Ecuador. Trans. R. Soc. Trop. Med. Hyg. 72, 542e543. ~ ez, V., Cid, P., Sanz, L., De La Torre, P., Angulo, Y., Lomonte, B., Nún rrez, J.M., Calvete, J.J., 2009. Snake venomics and antivenomics Gutie of Bothrops atrox venoms from Colombia and the Amazon regions of Brazil, Perú and Ecuador suggest the occurrence of geographic variation of venom phenotype by a trend towards paedomorphism. J. Proteomics 73, 57e78. ~ ez, V., Osorio, R.G., Gutie rrez, J.M., Giraldo, C.A., Posada, L.E., Otero, R., Nún 1995. Ability of six Latin American antivenoms to neutralize the equis (Bothrops atrox) from Antioquia and Choco venom of mapana (Colombia). Toxicon 33, 809e815. Parvin, R., Pande, S.V., Venkitasubramanian, A., 1965. On the colorimetric Biuret method for protein determination. Anal. Biochem. 12, 219e229. rrez, J.M., 2005. Rojas, E., Quesada, L., Arce, V., Lomonte, B., Rojas, G., Gutie Neutralization of four Peruvian Bothrops sp venoms by polyvalent antivenoms produced in Perú and Costa Rica: preclinical assessment. Acta Trop. 93, 85e95. nez, J.M., Gutie rrez, J.M., 1994. Caprylic acid fractionation of Rojas, G., Jime hyperimmune horse plasma: description of a simple procedure for antivenom production. Toxicon 32, 351e363. Castillo, M.C., Nún ~ ez, V., Yarleque , A., Gonçalves, L.R.C., Segura, A., ndez, M., Villalta, M., Bonilla, C., Herrera, M., Vargas, M., Ferna n, P., Tintaya, B., SanoYano, M.Y., Araújo, H.P., Boller, M.A., Leo mez, A., Fern Martins, I.S., Go andez, G.P., Geoghegan, P., Higashi, H.G., n, G., Gutie rrez, J.M., 2010. Preclinical assessment of the neutralLeo izing capacity of antivenoms produced in six Latin American countries against medically-relevant Bothrops snake venoms. Toxicon 56, 980e989. Smalligan, R., Cole, J., Brito, N., Laing, G.D., Merts, B.L., Manock, S., Maudlin, J., Quist, B., Holland, G., Nelson, S., Lalloo, D.G., Rivadeneira, G., Barragan, M.E., Dolley, D., Eddleston, M., Warrell, D.A., Theakston, R.D.G., 2004. Crotaline snake bite in the Ecuadorian Amazon: randomized double blind comparative trial of three South American polyspecific antivenoms. BMJ 329, 1129. Theakston, R.D.G., Laing, G.D., Fielding, C.M., Lascano, A.F., Touzet, J.M., Vallejo, F., Guderian, R.H., Nelson, S.J., Wüster, W., Richards, A.M., Rumbea Guzm an, J., Warrell, D.A., 1995. Treatment of snake bites by Bothrops species and Lachesis muta in Ecuador: laboratory screening of candidate antivenoms. Trans. R. Soc. Trop. Med. Hyg. 89, 550e554. Theakston, R.D.G., Reid, H.A., Larrick, J.W., Kaplan, J., Yost, J.A., 1981. Snake venom antibodies in Ecuadorian Indians. J. Trop. Med. Hyg. 84, 199e202. Warrell, D.A., 2004. Snakebites in Central and South America: epidemiology, clinical features, and clinical management. In: Campbell, J.A., Lamar, W.W. (Eds.), The Venomous Reptiles of the Western Hemisphere, vol. I. Cornell University Press, Ithaca, New York, pp. 709e761. World Health Organization, 1981. Progress in the Characterization of Venoms and Standardisation of Antivenoms. WHO Offset Publication No. 58. World Health Organization, Geneva.
Contents lists available at ScienceDirect
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, María Gutie rrez b, ngela Vargas b, Gladys Alvarez a, Jose Maria b ,* n Guillermo Leo a b
n en Salud Pública (INSPI), Guayaquil, Ecuador Instituto de Investigacio 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: Received 20 January 2014 Received in revised form 9 June 2014 Accepted 11 June 2014 Available online 17 June 2014
The toxicological profile of the venoms of the snakes Bothrops asper and Bothrops atrox from Ecuador was investigated, together with the venom of a population of B. asper formerly classified as ‘Bothrops xanthogrammus’. The three venoms exerted lethal, hemorrhagic, myotoxic, coagulant and defibrinogenating effects, in agreement with the characteristic toxicological profile of Bothrops sp venoms. A polyspecific antivenom (bothropic ecrotalicelachesic) manufactured in Costa Rica was assessed for its preclinical efficacy against the toxic activities of these Ecuadorian venoms. Antivenom was effective in the neutralization of the five activities tested in the three venoms. These observations are in agreement with previous reports on the extensive cross-reactivity and paraspecific neutralization of antivenoms manufactured in Latin America against the venoms of Bothrops sp snakes. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Bothrops asper Bothrops atrox Ecuador Antivenom Toxicity Neutralization
As occurs in the majority of Latin American countries, snakebite envenoming constitutes a significant public lezhealth hazard in Ecuador (Warrell, 2004; Gonza rrez, 2011). It has Andrade and Chippaux, 2010; Gutie 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 lez-Andrade and rate of 0.052 per 100,000 per year (Gonza 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: [email protected] (G. Leo http://dx.doi.org/10.1016/j.toxicon.2014.06.008 0041-0101/© 2014 Elsevier Ltd. All rights reserved.
there are 17 snake species of the family Viperidae, classified in the genera Bothrops, Bothriechis, Bothriopsis, Bothrocophias, Porthidium, and Lachesis, and 21 species of the family Elapidae, classified in the genera Micrurus and Leptomicrurus (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.
J. Laines et al. / Toxicon 88 (2014) 34e37
pico polivaA polyspecific antivenom (‘antiofídico botro lente’) has been manufactured in Ecuador by the former rez (http://apps.who.int/ Instituto Leopoldo Izquieta Pe bloodproducts/snakeantivenoms/database/) (Smalligan et al., 2004), later transformed into Instituto Nacional de n (INSPI). The effectiveness and Salud Pública e Investigacio 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 utilized (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 activities of venoms of B. asper and B. atrox from different lon et al., calities in Central and South America (Alape-Giro ~ ez et al., 2009; Gutie rrez et al., 2010; Calvete 2008; Nún 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 countries. 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 addition, the venom of a population of B. asper formerly classified as the separate species ‘Bothrops xanthogrammus’ was also analyzed, being hereby named as B. asper (population ‘xanthogrammus’). Venoms of B. asper, B. asper (population ‘xanthogrammus’) and B. atrox were collected from snakes kept in captivity in the serpentarium of the former Instituto Nacional de Higiene y Medicina Tropical “Dr Leopoldo Izquieta rez”, currently known as Instituto Nacional de Salud Pe n (INSPI). Venoms are pools obtained Pública e Investigacio 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 fractionated by caprylic acid precipitation (Rojas et al.,1994), and the product is constituted by whole IgG molecules. The antivenom 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 rrez et al. (1985). The Minimum Hemorrhagic Dose Gutie (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 et al. mined in human citrated plasma as described by Gene (1989). The Minimum Coagulant Dose (MCD) is the amount
35
of venom that induces clotting of plasma in 60 s. Defibrinogenating activity was determined in CD-1 mice et al. (1989). The Minimum (18e20 g), as described by Gene 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 injection, 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 protocols 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 rrez et al., 1985; Rojas et al., 2005). Briefly, mixtures (Gutie containing a constant amount of venom and various dilutions 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 mixtures, 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 et al. (1989). In as Effective Dose (ED), as defined by Gene 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 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 Hemorrhagic (MHD)b Coagulant (MCD)c Defibrinogenating (MDD)d Myotoxic (CK, U/L)e
91 (78e126) mg 1.1 ± 0.2 mgf 0.07 ± 0.02 mgf 2 mg 5516 ± 1350f
84 (73e126) mg 2.2 ± 0.3 mg₤ 0.10 ± 0.02 mgf 2 mg 2816 ± 1201₤
85 (33e107) mg 3.7 ± 1.0 mg₤ 2.90 ± 0.12 mg₤ 1 mg 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 activity between B. asper and B. atrox venoms (Segura et al., 2010). This experimental toxicological profile is compatible with the main clinical manifestations of envenomings by B. atrox and B. asper in Ecuador, characterized by prominent local tissue damage and systemic manifestations 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 preclinical 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 neutralization 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 antivenom/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 manufactured 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 ~ ez et al., 2009). venoms of B. atrox from Ecuador (Nún 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 antivenom of Instituto Clodomiro Picado (Costa Rica) is effective, 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 countries 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 2.9 (2.1e4.2) mg v/mL av 125 ± 4f 190 ± 1f 500
189 (116e238) mL av/mg v 5.3 (4.2e8.6) mg v/mL av 168 ± 9f 316 ± 3₤ 500
263 (154e385) mL av/mg v 3.8 (2.6e6.5) mg v/mL av 834 ± 96₤ 305 ± 5₤ 1000
Hemorrhagic (ED50)b Coagulant (ED)c Defibrinogenating (ED)d
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 performed 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 Laboratory 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 rrez, and Guillermo Leo n iangela Vargas, Jose work at Instituto Clodomiro Picado (Universidad de Costa Rica), where the antivenom tested in this study is manufactured.
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