In vitro toxic effects of puff adder (Bitis arietans) venom, and their neutralization by antivenom

Abstract

This study investigated the in vitro toxic effects of Bitis arietans venom and the ability of antivenom produced by the South African Institute of Medical Research (SAIMR) to neutralize these effects. The venom (50 µg/mL) reduced nerve-mediated twitches of the chick biventer muscle to 19% ± 2% of initial magnitude (n = 4) within 2 h. This inhibitory effect of the venom was significantly attenuated by prior incubation of tissues with SAIMR antivenom (0.864 µg/µL; 67% ± 4%; P < 0.05; n = 3-5, unpaired t-test). Addition of antivenom at t50 failed to prevent further inhibition or reverse the inhibition of twitches and responses to agonists. The myotoxic action of the venom (50 µg/mL) was evidenced by a decrease in direct twitches (30% ± 6% of the initial twitch magnitude) and increase in baseline tension (by 0.7 ± 0.3 g within 3 h) of the chick biventer. Antivenom failed to block these effects. Antivenom however prevented the venom induced cytotoxic effects on L6 skeletal muscle cells. Venom induced a marginal but significant reduction in plasma clotting times at concentrations above 7.8 µg/100 µL of plasma, indicating poor procoagulant effects. In addition, the results of western immunoblotting indicate strong immunoreactivity with venom proteins, thus warranting further detailed studies on the neutralization of the effects of individual venom toxins by antivenom.

Figure 1

Neutralisation of in vitro neurotoxic effects by antivenom. Effect of venom (50µg/mL) alone and in the presence of South African Institute of Medical Research (SAIMR) polyvalent antivenom (0.864µg/µL) on the (a) nerve mediated twitches and (b) contractile responses to ACh, CCh or KCl in the chick biventer cervicis nerve muscle preparation. * Significantly different compared with B. arietans venom alone or ** venom + antivenom; P < 0.05; unpaired t-test; n = 3–5.

Figure 2

Reversal of neurotoxicity by antivenom. Effect of adding antivenom (0.864µg/µL) after the addition of venom (i.e., at t50 indicated by arrow) on (a) the nerve mediated twitches and (b) contractile responses to exogenous agonists of the chick biventer cervicis nerve muscle preparation n = 4; paired t-test; n = 4; * significantly different compared to antivenom alone.

Figure 3

Myotoxic effects of B. arietans venom. The effect of venom (50 µg/mL) alone or in the presence of SAIMR antivenom (0.864 µg/mL) on (a) the direct twitches and (b) baseline tension of the chick biventer cervicis preparation (n = 4).

Figure 4

Neutralisation of in vitro cytotoxic effects by antivenom.Effect of venom (10 µg/mL) alone, or in the presence of antivenom (0.11 µg/µL) on the viability of L6 cells. * P < 0.05; significantly different compared with control (Cells + media), one-way ANOVA, n = 4.

Figure 5

Analysis of venom proteins by 2D gel electrophoresis and western blotting. (a) B. arietans venom proteins (40 µg) were subjected to iso-electric focussing (pI indicated at the top) and resolved on 12% polyacrylamide gel; (b) Venom proteins resolved by 2D gel electrophoresis were detected by western blotting using SAIMR polyvalent antivenom (1:500 in 5% skim milk) and appropriate secondary antibodies. Molecular weight markers are indicated at the centre.