View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Toxicon 60 (2012) 1018–1021 Contents lists available at SciVerse ScienceDirect Toxicon journal homepage: www.elsevier.com/locate/toxicon Short communication Preclinical testing of Peruvian anti-bothropic anti-venom against Bothrops andianus snake venom Francisco S. Schneider a, Maria C. Starling a, Clara G. Duarte a, Ricardo Machado de Avila a, Evanguedes Kalapothakis a, Walter Silva Suarez b, Benigno Tintaya b, Karin Flores Garrido b, Silvia Seraylan Ormachea b, Armando Yarleque c, César Bonilla b, Carlos Chávez-Olórtegui a,* a Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Av. Antonio Carlos 6627, CP: 486, CEP: 31270-901, Belo Horizonte, Minas Gerais, Brazil b Instituto Nacional de Salud, Lima, Peru c Universidad Nacional Mayor de San Marcos, Lima, Peru article info abstract Article history: Bothrops andianus is a venomous snake found in the area of Machu Picchu (Peru). Its Received 3 April 2012 venom is not included in the antigenic pool used for production of the Peruvian anti- Received in revised form 20 June 2012 bothropic anti-venom. B. andianus venom can elicit many biological effects such as Accepted 28 June 2012 hemorrhage, hemolysis, proteolytic activity and lethality. The Peruvian anti-bothropic Available online 14 July 2012 anti-venom displays consistent cross-reactivity with B. andianus venom, by ELISA and Western Blotting and is also effective in neutralizing the venom’s toxic activities. Keywords: Ó 2012 Elsevier Ltd. Open access under the Elsevier OA license. Bothrops andianus Andean snake Venom Anti-venom Toxicity Snake bites represent an important health problem in involving Bothrops complex (Bothrops and Bothrocophias Peru, especially to the east of the Andes in the High Forest genera), is a whole IgG preparation produced from horse (600–3500 m altitude) and Tropical Rain Forest (<600 m plasma previously immunized with an antigenic pool altitude) (Ministério de Salúd Peru, 2004). These regions consisting of Bothrops atrox (50%), Bothrops pictus, Bothrops are known for containing the major Peruvian snake species barnetti, Bothrops brazili and Bothrocophias hyoprora (12.5% and most diversified ophidian population. The Instituto each) venoms (Laing et al., 2004; Rojas et al., 2005; Nacional de Salud (INS), located in Lima, Peru has been Theakston and Warrell, 1991). Besides neutralizing the producing commercial anti-venoms since 1978 (Ministério most severe toxic effects induced by envenomation de Salúd Peru, 2004). The Peruvian anti-bothropic poly- involving snakes from the antigenic pool, (Laing et al., valent anti-venom (PABA), used to treat envenomations 2004; Rojas et al., 2005) the preclinical assessment of anti-venom’sefficacy against venoms from other medically Abbreviations: INS, Instituto Nacional de Salud; PABA, Peruvian anti- important species would be useful in Latin America for bothropic polyvalent anti-venom; BSA, Bovine serum albumin; PBS, improving anti-venom production (Gutierrez et al., 2009). Phosphate buffered saline; LD50, Median lethal dose; MHD, Minimum This work describes the preclinical evaluation of the hemorrhagic dose; MPD, Minimum PLA2 Dose; DMC, Dimethylcasein; neutralizing capacity of PABA against lethality, hemor- ED , Median effective dose. 50 rhagic, proteolytic, and PLA effects of Bothrops andianus’ * Corresponding author. Tel.: þ55 31 3409 2625; fax: þ55 31 3441 2 5963. venom. B. andianus is a venomous snake found in the E-mail address: [email protected] (C. Chávez-Olórtegui). southern mountains of Peru and Bolivia and its venom is 0041-0101 Ó 2012 Elsevier Ltd. Open access under the Elsevier OA license. http://dx.doi.org/10.1016/j.toxicon.2012.06.018 F. S. Schneider et al. / Toxicon 60 (2012) 1018–1021 1019 not included in PABA production. In Peru, B. andianus is of PABA and incubating for 1 h at 37 C followed by i.p. found in the areas (departments) of Cuzco and Puno, at injection in 5 groups of 4 mice. Median effective dose elevations of 1800–3300 m (Ministério de Salúd Peru, (ED50) was calculated from the number of deaths within 2004). Its geographical distribution overlaps Machu Pic- 24 h of injection of the venom/anti-venom mixture using chu area, a UNESCO World Heritage Site (UNESCO, 2012), Probit analysis as described above. The ED50 was expressed which is an important touristic attraction and receives as ml anti-venom/mg of venom needed to prevent death in more than 600,000 tourists per year, increasing the risks of 50% of the injected mice. accidents involving this snake. In Peru, the snakes of genus To determine the neutralization of hemorrhagic activity, Bothrops are responsible for 80% of accidents and approx- PABA was incubated with either 3MHD or 5MHD for 30 min imately 6.5% of these accidents are registered in the Cuzco at 37 C according to manufacturer’s instructions (1 mLof and Puno Departments (Ministério de Salúd Peru, 2004). serum to 2.5 mg of venom) and inoculated in different For the experiments, male and female Swiss mice groups of 3 Swiss male mice (18–22 g) as described above. (18–22 g) were maintained in the Centro de Bioterismo of Positive and negative control groups, each consisting of 2 Instituto de Ciências Biológicas of Universidade Federal de mice were treated with venom alone (5MHD) or anti- Minas Gerais (UFMG), Brazil. All animals received water and venom, respectively. Two hours later, mice were eutha- food ad libitum under controlled environmental conditions. nized and the hemorrhage was measured (Kondo et al., The experimental protocols were approved by the Ethics 1960; Sanchez et al., 1992). Committee in Animal Experimentation (CETEA/UFMG). Inhibition of PLA2 activity of B. andianus venom by PABA PABA, crude venoms from B. andianus, and antigenic pool was conducted as described by Gutierrez et al. (1998). Two species were provided by INS. Venoms were kept at À20 C MPD of venom were incubated with 13, 6.5 and 3.25 mlof and anti-venom at 4 C temperature as indicated on their anti-venom for 30 min at 37 C, and 15 ml of each mixture prescription. The protein content in crude venoms and anti- added in triplicate to wells in agarose gels. Neutralization of venoms were determined according to Bradford’s method venom was checked by the absence of halos on the plate’s (1976) using BSA (Sigma Chemicals) as standard. surface. Lethality of B. andianus venom was assessed by the Inhibition of dimethylcasein hydrolysis by PABA was intra-peritoneal (i.p.) route. Groups of four mice were estimated by incubation (30 min at 37 C) of a fixed injected with increasing amounts of venom (34.6 mg–72 mg/ concentration of B. andianus venom with increasing mouse), dissolved in 0.5 ml of PBS–BSA 0.01% solution, pH amounts of anti-venom (ml). After incubation the mixtures 7.4. Twenty four hours later, deaths were counted and LD50 were tested as described before. The neutralizing ability of was calculated using Probit analysis (95% confidence) anti-venom is expressed as the quantity of anti-venom able (Finney, 1971). to neutralize 50% of proteolytic activity obtained by 1 mg of The hemorrhagic activity was assayed as described in venom. Kondo et al. (1960) and modified by Gutierrez et al. (1985). For the immunological assays (ELISA and Western Five different doses (3.72 mg; 5.2 mg; 7.29 mg; 10.2 mg; Blotting assays), Falcon flexible micro titration plates were 14.28 mg) of crude venom were inoculated subcutaneously used (Becton Dickinson France S.A). The plates were coated into dorsal shaved skin of mice in 0.1 ml NaCl 0.9%. Two overnight at 5 C with 100 mlofa5mg/ml solution of the hours later, mice were sacrificed by cervical dislocation and crude venoms (B. andianus, B. atrox, B. barnetti, B. brazili, B. back skin was totally removed in order to measure the area pictus and B. hyoprora) in 0.02 M sodium bicarbonate buffer, of the hemorrhagic lesion. MHD was defined by the dose pH 9.6. The assays were performed as described previously causing a lesion with a diameter of 10 mm. by Chávez-Olórtegui et al. (1991). Absorbance values were PLA2 activity was measured using an indirect hemolytic determined at 492 nm with a Biorad 680 Microplate assay (Gutierrez et al.,1988). Increasing concentrations of B. Reader. All measurements were made in triplicate and the andianus venom (from 0.004 mgupto10mg) were prepared results expressed as the median of two assays. For Western in a final volume of 15 ml in PBS and added to 2 mm wells in Blotting the venoms were subjected to electrophoresis agarose gel plates (0.8% in PBS, pH ¼ 8.1, containing 1.2% SDS-PAGE (15%) according to Laemmli (1970) in reducing sheep erythrocytes, 1.2% egg yolk and 100 mM CaCl2). Plates conditions. The proteins were transferred onto nitrocellu- were incubated at 37 C for 18 h and the diameters of the lose membranes (Towbin et al., 1979) and blocked with hemolytic haloes were measured. In controls, 15 ml of PABA PBS-Tween 0.3% containing 2% casein. The membranes was used. One unit (Minimum PLA2 Doses-MPD) corre- were incubated with PABA (1:10,000) for 1 h at room sponds to a minor concentration of venom which produced temperature. Immunoreactive proteins were detected a hemolytic halo of 10 mm diameter. Experiments were using anti-horse Sigma IgG conjugated with peroxidase conducted in triplicate. (1:3000). After washing three times for 5 min with PBS- Proteolytic activity was measured with dimethylcasein Tween 0.05%, blots were developed using DAB/chlor- (Sigma) as described in Lin et al. (1969) with the modifi- onaphthol according to the manufacturer’s instructions.