version ISSN 0104-7930
J. Venom. Anim. Toxins vol. 5 n. 1 Botucatu 1999
A.R. de ROODT, J. A. DOLAB, L. SEGRE, C. SIMONCINI,
S. E. HAJOS, T. FERNANDEZ, J. C. DOKMETJIAN, S. LITWIN,
1 Instituto Nacional de Producción de Biológicos ANLIS "Dr. Carlos G. Malbrán", Av. Vélez Sarsfield 563, 1281-Buenos Aires, Argentina, 2 Cátedra de Inmunología, Facultad de Farmacia y Bioquímica, University of Buenos Aires, 3 Area de Iologia, Museo de Ciencias Naturales "Bernardino Rivadavia," (CONICET), Buenos Aires, Argentina.
ABSTRACT: The immunochemical reactivity and neutralizing capacity of polyvalent Vipera antivenom (Vipera ammodytes, Vipera aspis, Vipera berus, Vipera lebetina, and Vipera xanthina) were tested on the enzymatic and biological activities of Crotalus durissus terrificus and the following Bothrops venoms from Argentina (Bothrops alternatus, Bothrops ammodytoides, Bothrops neuwiedii, Bothrops jararaca, Bothrops jararacussu, and Bothrops moojeni). The Vipera antivenom reacted weakly when tested by double immunoprecipitation (DIP) and reacted with all the venoms when tested by ELISA. Several components in all the venoms studied were recognized in Western blots. Vipera antivenom deactivated to different degrees in vitro procoagulant, (indirect) hemolytic, and proteolytic activities in all the venoms studied. Preincubation of Bothrops alternatus venom with Vipera antivenom neutralized a lethal potency of 4.5 LD50 in mice with an ED50 of 1.25 ± 0.25 ml per mg of venom, and with 1.0 ml/mg inhibited 54% of the hemorragic activity and 48% of necrotic activity. Vipera antivenom (2.0 ml per mg toxin) inhibited the phospholipase A2 activity of purified crotoxin and decreased its lethal potency by 60%, while the neutralizing capacity on the lethal potency of crude Crotalus durissus terrificus venom was poor even at a level of 5.0 ml/mg of venom.
KEY WORDS: antivenom, Vipera, Bothrops, Crotalus, cross-reactivity, cross-neutralization.
Cross-reactivity and cross-neutralization between antivenoms and venoms from snake species belonging to the same Genus or Family is frequently observed (8,9,12,22,27-30). This may be related to the high degree of similarity in the primary, secondary, and tertiary structures of homologous venom proteins, such as phospholipases A2 (3,33) and metalloproteinases (4), even from taxonomically unrelated species.
On the other hand, cross-reactivity and cross-neutralization studies have been used as taxonomic tools (2) to ascertain the level of contribution of some venom components to the physiopathology of envenomation (8) These studies could even be critically important for therapeutic use in emergencies, such as snakebites by foreign specimens in zoos, serpentariums, or for people who keep snakes as pets.
Immunochemical cross-reactivity between antivenoms and venoms has been reported to be relatively high for crotalids and viperids, lower for viperids and elapids, and very poor for crotalids and elapids (24). Thus, high amounts of commercial Vipera ammodytes antivenom from Behringwerke (Mardeburg, Germany) were reported to protect mice against B. alternatus, B. neuwiedii, and B. jararaca venoms, but were ineffective in neutralizing the lethal potency of C. d. terrificus venom (10). In contrast, polyvalent Behringwerke antivenom (raised against Bitis, Echis, Dendroaspis, and African Naja venoms) protected mice against the lethal potency of Crotalus atrox venom, while Wyeth polyvalent antivenom (raised against several Crotalus and Bothrops venoms) neutralized the hemorrhagic activity of Bitis arietans venom (20).
In this study, the immunochemical reactivity and neutralizing capacity of commercial Vipera antivenom were tested on the enzymatic and toxic activities present in the venoms of several crotalid species from Argentina.
MATERIALS AND METHODS
VENOMS: The venoms of C. d. terrificus, B. alternatus, B. jararaca, B. jararacussu, B. moojeni, and B. neuwiedii were obtained from healthy adult specimens kept at the serpentarium of the Instituto Nacional de Producción de Biológicos A. N. L. I. S. "Dr. Carlos G. Malbrán" (Buenos Aires, Argentina). The snakes were milked and the venoms were collected in cold sterile Petri dishes, desiccated under vacuum, and kept in tightly closed vials at 4ºC. B. ammodytoides venom was a gift from Dr. E. Gould, Fundación de Estudios Biológicos (Buenos Aires, Argentina). Venom solutions were prepared in 0.15 M NaCl just before use and sterilized by filtration.
Crotoxin complex was purified from C. d. terrificus venom as previously described (23). The purified preparation had a specific phospholipase A2 activity of 25 ± 3 units mg protein. SDS-PAGE under non-reducing conditions showed two bands with mobilities corresponding to molecular weights of 9.5 ± 0.3 kD (sub-unit A) and 14.5 ± 0.2 kDa (sub-unit B). The crotoxin preparation was equilibrated in 0.15 M NaCl by chromatography on Sephadex G-25 and sterilized by filtration.
ANTIVENOM: Horse "Immunoserum contra Venena Viperarum Europearum", Series 160 (Exp. 2/98) from Imunolsky zabod Laboratories (Zagreb, Croatia) was kindly provided by Prof. Dr. Franc Gubensek, Dept. of Biochemistry and Molecular Biology, Josef Stefan Institute (Ljubljana, Slovenia). According to the manufacturers, 1.0 ml of antivenom neutralizes 100 LD50 of V. ammodytes or V. aspis venom, and 50 LD50 of V. berus, V. lebetina, or V. xanthina venoms.
DOUBLE IMMUNOPRECIPITATION (DIP): This was performed in Petri dishes with a 3.0 mm thick layer of agarose (Ultrapure, Sigma Chemical Co.) with 10.0 ml of venom solutions (0.5 mg/ml) in the peripheral wells and 10.0 ml of Vipera antivenom in the central well. Diffusion was allowed to proceed for 72 h at 25ºC in a humid chamber (27,28). DIP was stained with amidoblack (Sigma Chemical Co.).
SDS-PAGE AND WESTERN BLOT: SDS-PAGE in non-reducing conditions was performed on an acrylamide 12.5/2.5 (T/C) gel, with a vertical unit Miniprotean II (Bio Rad), using the discontinuous buffer system described by Laemmli (16). After electrophoretic separation, the bands were stained with Coomassie Brilliant Blue R250 (Bio Rad), or transferred to a nitrocellulose membrane, as described by Towbin (32). The molecular weight markers used were those included in the Sigma VII-S kit.
ELISA: Each well of a 96-well Corning plate was sensitized with 100 ml of either venom or crotoxin solution (1.0 mg/ml in barbital buffer pH 9.0 for 12 h at 4ºC). Before all subsequent steps, the wells were washed 4 times with 0.15 M NaCl, 10 mM phosphate buffer pH 7.4, containing 0.05% Tween 20 (Sigma Chemical Co.). The final volume of each addition was 100 ml. The wells were treated sequentially: for 2 h at 37ºC with 10% defatted milk as a blocking agent, then for 40 min at 37ºC with 1/200 to 1/128000 dilutions of Vipera antivenom, and then for 40 min at 37ºC with a 1/10000 dilution of rabbit anti-horse IgG-peroxidase conjugate (Sigma Chemical Co.). The plates were washed and 100 ml of o-phenylene diamine, hydrogen peroxide at pH 5.7 were added to each well, incubated for 25 min in the dark, and the reaction was stopped by the addition of 3.0 N sulfuric acid. The absorbance at 490 nm was measured with an ELISA reader. Wells sensitized with normal horse serum or with venom, but with no antivenom, were used as negative controls.
LD50: Mice (CF1 strain, 18-22 g, n=6 per dose level) were injected intravenously with different concentrations of B. alternatus or C. d. terrificus venoms in 0.15 M NaCl. The LD50 value was calculated by regression using Prisma software (GraphPad Software, Inc. San Diego, CA). The LD50 for C. d. terrificus venom and crotoxin via intraperitoneal was determined by the procedure described by Meier and Theakston (21).
NEUTRALIZATION OF LETHAL POTENCY: Mice (CF1 strain, 18-22 g, n = 5) were injected intravenously with 25 mg (7.1 LD50) of C. d. terrificus venom or with 100 mg (4.5 LD50) of B. alternatus venom preincubated for 45 min at 37ºC in 300 ml of 0.15 M NaCl (positive controls). The other groups (n = 5 each) were injected with the same amounts of venoms preincubated for 45 min at 37ºC with Vipera antivenom at 0.5, 1.0, 2.0, 3.0, 4.0, and 5.0 ml per mg venom in 300 ml of 0.15 M NaCl. The ED50 was estimated from plotting the number of surviving animals against the volume (microliters) of antivenom.In another set of experiments, crude C. d. terrificus venom or crotoxin alone and preincubated with 2.0 and 4.0 ml of antivenom per mg of venom or toxin were intraperitonealy injected to mice at different dose levels. The comparison of the plots of dose versus dose/time of death, which according to Meier and Theakston (21), allowed estimation of the degree of protection.
HEMORRHAGIC AND NECROTIC ACTIVITY OF B. ALTERNATUS VENOM: Six mice (CF1 strain, 25 ± 2 g) were injected intradermically with 100 mg (hemorrhagic) or 50 mg (necrotic) of B. alternatus venom preincubated for 45 min at 37º C in 150 ml of 0.15 M NaCl (positive controls). Three groups of mice (n = 6 each) were injected intradermically with the same amounts of venom preincubated for 45 min at 37ºC with 25.0, 50.0, and 100.0 ml of antivenom in a final volume of 150 ml. The hemorrhagic areas were measured 3 h after injection, as described by Ferreira et al. (11,12). The necrotic areas were measured 72 hours after injection, as described by Theakston and Reid (31).
PROTEOLYTIC ACTIVITY ON GELATIN: Three ml of 20% gelatin in PBS pH 7.2 at 37ºC were mixed with 350 ml of 0.15 M NaCl containing 100 mg of venom or the same amount of venom preincubated with Vipera antivenom at 0.25, 0.5, 1.0, and 2.0 ml per mg of venom. The samples were incubated for 3 h at 37ºC and proteolysis was stopped by the addition of 100 ml of 2% EDTA. The samples were kept at 4ºC for 12 h and their physical state was compared to those of negative (gelatin plus 0.15 M NaCl or antivenom) and positive controls (gelatin plus venom). The proteolytic activity of the Bothrops venoms (positive controls) resulted in liquefaction of the gelatin samples. C. d. terrificus did not produce liquefaction of gelatin even with the addition of 1.0 mg of venom per sample.
INDIRECT HEMOLYSIS AND PHOSPHOLIPASE A2 ACTIVITY: Indirect hemolysis was determined using the procedure described by Al-Abdhula et al.(1). Mixtures containing 0.5 ml of egg yolk, 0.5 ml of washed and pelleted human erythrocytes, and 100 ml of 0.1 M CaCl2 in 10.0 ml of 0.15 M NaCl, 10 mM phosphate buffer pH 7.2 were incubated for 60 min at 37ºC with 0.15 M NaCl (negative controls), 1 % Triton X-100 (positive control), 100 or 200 mg of venom preincubated with either 0.15 M NaCl or Vipera antivenom at 0.5 and 1.0 ml per mg of venom in 0.15 M NaCl. The reaction was stopped with 1.0 ml of 10% EDTA and the mixtures were centrifuged for 30 min at 1500 r.p.m. The absorbances at 550 nm of the supernatants were recorded. The differences due to preincubation with antivenom were expressed as percentage of those obtained with 100 and 200 mg of venom.
Phospholipase A2 activity of the crotoxin complex before and after incubation with Vipera antivenom at 1.0 and 2.0 ml per mg of toxin was measured by automatic titration of the fatty acids released from a sample of egg yolk using a pH-Stat (Radiometer). One unit of phospholipase A2 activity was defined as the consumption of 1.0 mEquiv of NaOH (or the release of 1.0 mEquiv of fatty acids) per min at 25ºC.
STATISTICAL ANALYSIS: The results are presented as Mean ± S.D values. The Student t-test was used for the comparison between means and variances. The comparison between groups was made using the Student t-test and the Wilcoxons method.
Cross reactivity between Vipera antivenom and all the crotalid venoms tested was measured using DIP, in which several weak precipitation archs were observed. ELISA showed positive for all Bothrops venoms using dilutions of Vipera antivenom from 1:128000 (B. neuwiedii) up to 1:64000 (B. moojeni). The detection of C. d. terrificus venom and crotoxin required dilutions of the antivenom from 1: 64000 to 1: 32000 and from 1: 32000 to 1: 16000, respectively.
The Western blots of the different venoms used are shown in Figure 1. Using B. neuwiedii and B. jararacussu venoms, Vipera antivenom produced a band with molecular weight of about 67.4 kD. Except for B. jararaca and B. moojeni venoms, all the other bothropic venoms exhibited one to three strongly stained bands with molecular weights between 56.5 and 60.0 kD, as well as between 38.0 and 53.0 kD. Bands of about 34.0 and 38.0 kD were strongly stained with B. neuwiedii and B. jararaca venoms and less stained with B. alternatus venom. Except for B. jararacussu, all the Bothrops venoms showed one or more strongly stained bands of about 20-24 kD. Stained bands between 18.0 and 19.0 kD were observed with B. neuwiedii, B. jararaca, B. jararacussu, and B. moojeni. Using the venom of C. d. terrificus, strong stained bands of 57.3-59.7 kD, 45-46 kD and weakly stained bands were observed in the region with molecular weights of 39.0, 31.0, 27.0, 20.0, and 14.3 kD. Despite the different contents in phospholipase A2, only C. d. terrificus venom exhibited a band of 14.3 kD.
FIGURE 1. Western blot of crotalid venoms against Vipera (European) antivenom. Lanes 1 to 7: 1. Bothrops ammodytoides venom, 2. Bothrops alternatus venom, 3. Bothrops neuwiedii venom, 4. Bothrops jararaca venom, 5. Bothrops jararacussu venom, 6. Bothrops moojeni venom, and 7. Crotalus durissus terrificus venom. Numbers on the left represent the mobility of molecular weight markers in kD.
The LD50 of C. d. terrificus venom in mice was 0.175 ± 0.03 mg/g (intravenous) and 0.33 ± 0.1 mg/g (intraperitoneal). The intraperitoneal LD50 of crotoxin was 0.13 ± 0.09 mg/g. The intravenous LD50 of B. alternatus venom in mice was 1.1 ± 0.2 mg/g.
After the intravenous injection of 1.24 mg/g of C. d. terrificus venom (7.1 LD50), all the animals died within 20 min. When the same amount of venom preincubated with Vipera antivenom at 0.5, 1.0, 2.0, 3.0, 4.0, and 5.0 ml per mg was injected intravenously, all the animals died, although the survival time was longer (up to 6 h with 5.0 ml per mg venom).
A poor neutralizing effect of the antivenom was again observed after the intraperitoneal injection of 4.5 LD50 of C. d. terrificus venom. Only one in five mice survived after the injection of this amount of venom preincubated with Vipera antivenom at 5.0 ml per mg. According to Meier and Theakston (21), plots of the results obtained after intraperitoneal injection of different doses of C. d. terrificus venom preincubated with either 0.15 M NaCl or Vipera antivenom at 2.0 or 4.0 ml per mg suggest that the antivenom produces a decrease in the lethal potency of about 16% (p = 0.05).
According to Meier and Theakston, plots of results obtained after intraperitoneal injection of pure crotoxin preincubated with 0.15 M NaCl or Vipera antivenom (at 2.0 ml per mg toxin) are shown in Figure 2. The apparent LD50 of the antivenom-treated toxin (0.47 ± 0.16 mg/g) was higher than that of untreated toxin (0.13 ± 0.09mg/g), suggesting a significant decrease (about 60%, p<0.01) in lethal potency.
FIGURE 2. The effect of preincubation with Vipera antivenom on the lethal potency of crotoxin in mice. Samples containing 0.12 to 4.2 mg of crotoxin per g of body weight, or the same amounts of crotoxin preincubated with Vipera antivenom at 2.0 ml per mg of toxin were injected intraperitoneally into mice and the times of death (min after injection) were recorded. The doses were plotted against the ratios [Dose/Time of death] as described by Meier and Theakston (21). Data with crotoxin were fitted to a straight line with a slope 69.84 (± 5.09), intercept at 0.2308 (± 0.060) mg/g, and r= 0.965. Data with preincubated crotoxin were fitted to a straight line with a slope 46.34 (±3.88), intercept at 0.469 (± 0.162) mg/g, and r= 0.966. Dotted lines represent 95% confidence intervals.
Conversely, Vipera antivenom afforded effective protection against the lethal action of 4.5 LD50 of B. alternatus venom. The calculated ED50 was 1.25 ± 0.25 ml of antivenom per mg of venom, an amount only 4-5 times higher than that obtained with the homologous antivenom.
As shown in Table 1, preincubation of B. alternatus venom with Vipera antivenom resulted in neutralization of its hemorrhagic and necrotic activities.
TABLE 1. The inhibition of hemorrhagic and necrotic activities of B. alternatus venom by Vipera antivenom. Crude B. alternatus venom was preincubated for 45 min at 37ºC with similar volumes of either 0.15 M NaCl or Vipera antivenom as indicated in the first column. Hemorrhagic activity in CF-1 mice (n = 6 per group) was measured 3 h after intradermal injection of 100 mg (~5.8 MHD) venom. Necrotic activity in CF-1 mice (n = 6 per group) was measured 72 h after the intradermal injection of 50.0 µg ( ~ 1.8 MND) venom. Columns 2 and 4 show the average diameters (cm) of hemorrhagic (column 2) and necrotic (column 4) haloes. Inhibition (%) by incubation with Vipera antivenom is shown in columns 3 (hemorrhagic) and 5 (necrotic).
Preincubation of all the Bothrops venoms used in this study with 2.0 ml of Vipera antivenom per mg of venom resulted in complete inhibition of their proteolytic activity on gelatin.
The inhibitory effect of Vipera antivenom on the procoagulant activity of all the venoms studied is shown in Table 2. When compared to the crude venoms, preincubation with Vipera antivenom at 1.0 ml per mg of venom increased the time for clot formation 2-4 times with B. ammodytoides, B alternatus, B. neuwiedii, and B jararacussu, 6 times with B.moojeni, and more than 6 times with B. jararaca and C. d. terrificus venoms.
TABLE 2. The neutralization of procoagulant activity of crotalid venoms by Vipera antivenom. Horse plasma (500 ml) was treated with either 50.0 µg venom preincubated for 45 min at 37ºC with 50 ml of 0.15 M NaCl, or 50 µl of Vipera antivenom. The time required for clot formation was recorded. The last column shows the increase in clotting time produced by preincubation with Vipera antivenom as the ratio between the effects of pretreated/untreated venoms.
The results of indirect hemolysis assay are shown in Table 3. Preincubation with 0.5 ml of Vipera antivenom per mg of venom resulted in a poor inhibition of indirect hemolysis with B. ammodytoides and B. neuwiedii venoms, and inhibitions ranging between 25 and 50% with the other venoms. Preincubation with 1.0 ml of antivenom per mg of venom did not increase inhibition with B. ammodytoides and B. jararaca venoms, somewhat increased inhibition with B. moojeni, B. jararacussu, B. neuwiedii, and C. d. terrificus venoms, and produced complete inhibition with B. alternatus venom.
TABLE 3. The inhibition of indirect hemolytic activity of crotalid venoms by Vipera antivenom. Venoms (100 or 200 mg) were preincubated for 45 min at 37º C with 100 ml of either 0.15 M NaCl or Vipera antivenom. Samples were assayed for indirect hemolytic activity as (see Methods). The results are presented as percentage inhibition compared to the activity of 100 or 200 µg crude venom samples preincubated with 0.15 M NaCl.
Phospholipase A2 activity of crotoxin, as measured in the pH-Stat with 10.0, 20.0, and 40.0 mg of toxin increased linearly with the toxin concentration. The calculated specific activity was 25.0±3.0 mEquiv. minmg protein. Preincubation with Vipera antivenom at 1.0 and 2.0 ml per mg of crotoxin was 20% (p < 0.05) and 45% (p < 0.01) inhibition, respectively.
Both DIP and ELISA indicate a strong immunochemical cross-reactivity between Vipera antivenom and all the venoms used in this study. The concentrations of Vipera antivenom required for the detection of the crotalid venoms using ELISA were only 2-3 times higher than those using homologous antivenoms. These results confirm the high degree of immunochemical cross-reactivity between antivenoms raised against venoms of the Family Viperidae and venoms of the Family Crotalidae (10,20), suggesting that Vipera antivenom could inhibit some of the biological activities of the crotalid venoms.
Hemorrhagic, necrotic, and procoagulant activities play an important role in the physiopathology of envenomation by Bothrops species (14,15,17,25,26) and venoms from Vipera species have a procoagulant activity and also produce hemorrhage and necroses (19).
The hemorrhagic activity of both viperid and crotalid venoms is caused by metalloproteinases, known generically as "hemorrhagins" (4,18). Bjarnasson and Fox (4) have classified these metalloproteinases into four groups, and the hemorrhagins from viperid and crotalid venoms can be found in most of them: LEBETASE from V. lebetina, HF1 from B. jararaca, and MPA from B. moojeni in group I; HT-1, HT-2, and HT-3 from V. ammodytes, HMP from V. berus, HR1, HR2, and HR3 from V. palestinae, BOTHROPASINE from B. jararaca, NHFa and NHFb from B. neuwiedii venom, in group II; HT-1 from V. aspis, HF2, HF3, and JARARRHAGIN from B. jararaca venoms in group IV.
Hemorrhagic and necrotic activities of B. alternatus and B. ammodytoides venoms appear to be associated with fractions of molecular weights between 40 and 70 kD, while the procoagulant activity appears to be associated mainly with fractions of molecular weights between 20 and 40 kD (6). Western blots (Figure 1) showed a strong recognition of venom components in those range of molecular weights, and preincubation with Vipera antivenom inhibited the hemorrhagic, necrotic, and procoagulant activities of B. alternatus venom. This may explain the significant neutralizing capacity of Vipera antivenom against the lethal potency of B. alternatus venom with an estimated ED50 of 1.25 ± 0.25 ml per mg of venom (only 4 to 5 times higher than that of the homologous antivenom).
In all the bothropic venoms studied, the hemorragic and proteolytic activities on gelatin appear to be highly correlated (r= 0.6) (7), and preincubation with Vipera antivenom at 2.0 ml per mg of Bothrops venoms resulted in complete inhibition of the proteolytic activity on gelatin.
The indirect hemolysis assay (related to phospholipase A2 activity) showed that preincubation with 1.0 ml of Vipera antivenom per mg of venom produced complete inactivation of B. alternatus venom, 45% to 66% inactivation with B. moojeni and B. jararacussu venoms, and only a poor inhibition (14%) with B. ammodytoides and B. jararaca venoms (Table 3). Cross-inactivation of indirect hemolysis may be explained due to viperid and crotalid venoms contain Group II phospholipases A2 (5) and also due to a high degree of similarity between the in primary, secondary, and tertiary structures of this large family of homologous proteins (3).C. d. terrificus venom differs significantly from the Bothrops venoms in composition, as well as in the physiopathology of envenomation (2). In fact, envenomation by C. d. terrificus is characterized by neurotoxicity and paralysis more than by systemic hemorrhages and local signs. Neurotoxicity of this venom is attributed to the presence of crotoxin, a neurotoxic phospholipase A2 that forms about 50% of the venom protein. On a quantitative basis, 2.6 mg of whole C. d. terrificus venom (~ 1.0 LD50 I.V. for a 20 g mouse) contains about 1.3 mg crotoxin (1.0 LD50 I.V.), 0.25 mg of convulxin (0.001 LD50), and may contain 0.5 mg crotamin (0.016 LD50). Thus, crotoxin is responsible for the lethal potency of this venom, and the contribution of the other toxic components is usually neglected. When tested using ELISA, Vipera antivenom reacted with both whole C. d. terrificus venom or crotoxin, which confirms the known cross-reactivity between crotoxin and antibodies anti-ammodytoxin, (the neurotoxic phospholipase A2 from V. ammodytes venom(13). However, the results of neutralization experiments using Vipera antivenom and pure crotoxin (4.0 ml per mg of toxin) do not agree with those using 2.0 ml of antivenom per m g of whole C. d. terrificus venom. In fact, preincubation with Vipera antivenom resulted in the inhibition of phospholipase A2 activity of pure crotoxin (about 45% in the pH-Stat assay) accompanied by a decrease in a similar degree (about 60%) of the lethal potency, as mentioned above. In contrast, while preincubation of whole C. d. terrificus venom with Vipera antivenom also produced a significant inhibition of phospholipase A2 activity (about 60 % in the indirect hemolysis test), it produced only in a marginally significant decrease (about 16%) in the lethal potency. The results of crotoxin inactivation are compatible with the observation that all mice died after the intravenous injection of 7.1 LD50 (25 mg) of whole C. d. terrificus venom preincubated with the antivenom. This amount of whole venom contains about 10 mg crotoxin (7.1 LD50 I.V.), and preincubation with the antivenom would result in inactivation of about 6.5 mg. The remaining 3.5 mg of non-neutralized crotoxin represents 2.5 LD50, an amount sufficient to cause death. However, inactivation of crotoxin does not appear to be consistent with the fact that all animals died after the intraperitoneal injection of 4.5 LD50 of whole C. d. terrificus venom preincubated with Vipera antivenom, since a similar calculation indicates that the amount of non-neutralized crotoxin represents only 1.0 LD50. The poor neutralizing capacity of Vipera antivenom on the lethal potency of whole C. d. terrificus venom confirms the observation reported by Elliott (10). When this is considered along with the significant neutralizing capacity of the anivenom on the lethal potency of crotoxin, it suggests that other venom components, different from crotoxin and not neutralized by the Vipera antivenom, contribute significantly to the lethal potency of C. d. terrificus venom.
This work was partially supported by grants from the Fundación "FUNMECACET" and the Ministry of Health. The authors are indebted to Prof. Dr. F. Gubensek for his encouragement and suggestions. J. C. Vidal is Career Investigator of Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Argentina.
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