Neutralizing capacity of a new monovalent anti-Bothrops atrox antivenom : comparison with two commercial antivenoms

Three horse-derived antivenoms were tested for their ability to neutralize lethal, hemorrhagic, edema-forming, defibrinating and myotoxic activities induced by the venom of Bothrops atrox from Antioquia and Chocó (Colombia). The following antivenoms were used: a) polyvalent (crotaline) antivenom produced by Instituto Clodomiro Picado (Costa Rica), b) monovalent antibothropic antivenom produced by Instituto Nacional de Salud-INS (Bogotá), and c) a new monovalent anti-B. atrox antivenom produced with the venom of B. atrox from Antioquia and Chocó. The three antivenoms neutralized all toxic activities tested albeit with different potencies. The new monovalent anti-B. atrox antivenom showed the highest neutralizing ability against edema-forming and defibrinating effects of B. atrox venom (41 ± 2 and 100 ± 32 μl antivenom/mg venom, respectively), suggesting that it should be useful in the treatment of B. atrox envenomation in Antioquia and Chocó. Correspondence


Introduction
Antioquia and Chocó are located in the northwest region of Colombia, a zone with large reserves of tropical rain forest that have allowed the development of an important biodiversity, including 104 snake species (1).These regions and the Amazonas have the highest incidence of snake bites in Colombia, most of them caused by the abundant species Bothrops atrox, with a death rate of 5% and sequelae in 6% of the patients (2,3).This is mainly the result of late arrival at the hospital or, in some cases, of an insufficient antivenom supply or the high cost of lyophilized products.The venom of B. atrox has proteolytic, defibrinating, hemorrhagic, myotoxic, edema-forming and indirect hemolytic activities, with regional differences in venom activities (4).
These facts prompted us to produce a monovalent anti-B.atrox antivenom specific for the regions of Antioquia and Chocó, and to compare its neutralizing activity against homologous venom with two other crotaline antivenoms available in this region.

Animals, venom and antivenoms
Webster white mice (18-20 g) were used for the in vivo experiments.The venom of B. atrox was a pool obtained from 40 specimens collected in Antioquia and Chocó, Colombia, lyophilized and stored at -20 o C until the time for use.One adult horse was immunized at Instituto Clodomiro Picado (Costa Rica) with B. atrox venom from Antioquia and Chocó, using the immunization protocol shown in Table 1.At the end of the immunization schedule, the horse was bled and plasma was fractionated by ammonium sulfate precipitation (5).
For comparative purposes in the neutralization experiments, the following two commercially available antivenoms were used: a) the polyvalent (crotaline) antivenom of Instituto Clodomiro Picado (ICP), San José, Costa Rica, batch 2460294 LQ, produced in horses by the same immunization schedule as described above, using a mixture of equal weights of solid venoms of B. asper, Lachesis muta and Crotalus durissus durissus from Costa Rica and, b) the monovalent antibothropic antivenom of Instituto Nacional de Salud (INS), Santafé de Bogotá, batch 140992, produced in horses immunized with B. atrox venom from various regions of Colombia.These two antivenoms were also produced using similar fractionation protocols based on ammonium sulfate precipitation of globulins, without pepsin digestion (5).Phenol (0.25%) was added as a preservative and the antivenoms were tested before their expiration dates.

Neutralization assays
The neutralization assays were perfomed by incubating a constant amount of solid venom with various dilutions of antivenom, in order to obtain several antivenom/venom ratios.Incubations were carried out at 37 o C for 30 min and the mixtures were then tested in the corresponding assay systems for each pharmacological activity.The doses of venom selected to test each effect were the following: a) edema: 6 MED = 9 µg; b) hemorrhage: 10 MHD = 16 µg; c) defibrination: 2 MDD = 2.3 µg; d) lethality: 4 LD 50 = 265 µg; e) myonecrosis: 50 µg.Neutralizing ability is reported as effective dose 50% (ED 50 ), defined as the µl antivenom/mg venom ratio that reduces by 50% the activity of venom alone.In the case of neutralization of defibrinating activity, results are reported as effective dose 100% (ED 100 ), defined as the µl antivenom/mg venom ratio at which the effect of the venom was completely neutralized.All experiments were repeated on three different days.

Statistical analysis
Data were analyzed statistically by oneway analysis of variance (ANOVA).When the values were significantly different (P<0.05), the differences between pairs of means were analyzed by the Tukey test.

Neutralization of venom activities
Table 2 shows that the three antivenoms neutralized all pharmacological activities of B. atrox venom studied, albeit with different potencies.Monovalent antibothropic (INS) and monovalent anti-B.atrox antivenoms were equally potent in the neutralization of lethality and hemorrhage, whereas the polyvalent (ICP) antivenom was less efficient in the neutralization of these effects.Regarding edema and defibrination, the monovalent anti-B.atrox antivenom showed the highest neutralizing ability.No significant differences were observed between the three antivenoms concerning neutralization of myonecrosis (Table 2).

Discussion
The parenteral administration of horseor sheep-derived antivenoms constitutes the cornerstone in the treatment of snakebite envenomation (16).Since venoms present Neutralization is reported as effective dose 50% for lethal, hemorrhagic, edema-forming and myonecrotic effects and as effective dose 100% for the defibrinating effect (see Material and Methods).For lethality, results are reported as mean and 95% confidence limits (given in parentheses).For the other effects, results are reported as mean ± SD (N = 3).Values with different superscripts (a,b,c) are significantly different (P<0.05)(Tukey test).+ In the neutralization of lethality, results are also reported as mg venom neutralized per ml antivenom.

Antivenom
Neutralization Our results indicate that the three antivenoms were effective in neutralizing the most relevant toxic effects of B. atrox venom.This suggests that there is a significant immunological cross-reactivity between the venoms of B. atrox from Antioquia and Chocó, B. atrox from other regions of Colombia and B. asper from Costa Rica.Crossneutralization of several antivenoms produced in Latin America was demonstrated when they were tested against the venom of B. atrox from Antioquia and Chocó (20).
On the other hand, when the effective doses 50% of these antivenoms were compared, the monovalent anti-B.atrox antivenom produced with venoms from Antioquia and Chocó showed the highest neutralizing ability against the venom of B. atrox from these regions.
Local tissue damage (myonecrosis, hemorrhage and edema) is one of the most typical and dangerous consequences of B. atrox envenomation in Colombia (2).These effects result in severe lesions and, in a number of cases, in sequelae (2,3).The rapid development of these local effects makes efficient neutralization by antivenoms difficult (9)(10)(11).Thus, it is necessary to have antivenoms with high neutralizing capacity against the toxins responsible for these effects in order to reduce the local tissue damage.
Our results indicate that the three antivenoms were similarly efficient in neutralizing myonecrosis, whereas monovalent anti-B.atrox antivenom had the highest neutralizing ability against the edema-forming effect.Regarding hemorrhage, monovalent anti-B.atrox and monovalent antibothropic (INS) antivenoms were more potent than polyvalent antivenom, although all three neutralized this effect at a low antivenom/venom ratio.
In conclusion, the three antivenoms tested are efficient in neutralizing the most relevant toxic effects induced by the venom of B. atrox from Antioquia and Chocó.The monovalent antivenom produced with venoms from specimens collected in these regions in Colombia showed the highest neutralizing potency.We suggest that this antivenom may be of high value in the treatment of B. atrox envenomation in Antioquia and Chocó.A randomized clinical trial is currently under way in Colombia in order to test this hypothesis.

Table 1 -
Immunization schedule for the production of B. atrox monovalent and crotaline polyvalent antivenoms at Instituto Clodomiro Picado.

Table 2 -
Neutralization of pharmacological activities of B. atrox venom from Antioquia and Chocó by the three antivenoms.