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.

Venomous Landmines: Clinical Implications of Extreme Coagulotoxic Diversification and Differential Neutralization by Antivenom of Venoms within the Viperid Snake Genus Bitis

“…Venoms which act on the coagulation cascade are particularly evident in species known to prey on mammals, as is the case for the giant Bitis species [30,31,32,33]. Comparatively the diets of the dwarf Bitis species are less known and therefore predictions about their venom effects cannot be made in an evidence-based manner.…”

“…The most well studied species ( B. arietans , B. gabonica and B. rhinoceros ) have been studied in-depth due to the potent anticoagulant and hemorrhagic effects of their venom, being responsible for a significant proportion of the mortality and morbidity of snakebites in Africa [4,5,30,33,34]. The venom of both lineages of giant Bitis species causes a variety of effects including spontaneous and persistent bleeding, incoagulable blood, hemorrhage, hypotension and rapid defibrinogenation [30,31,32,33]. Hemorrhage and oligemic shock caused by extravazation is the reported cause of death in many clinical accounts from envenomations by the giant Bitis species [4,5,33].…”

The effect of physiological levels of South African puff adder (Bitis arietans) snake venom on blood cells: an in vitro model

“…The clinical consensus in practice is to observe patient closely for signs and symptoms of envenomation, which usually manifest between 15 minutes and up to two hours after the snakebite, occurred. If none of the typical signs or symptoms of envenomation have been noted after two hours of clinical observation, the possibility of a dry bite (mechanical bite with no venom injected) is embraced by most clinicians 16 . Anti-venom dosage is based on the clinical response of the patient, where up to 6 to 8 vials of polyvalent anti-venom from the South African Vaccine Producers (SAVP) are administered as a typical baseline dosage in cases of puff adder envenomation.…”

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“…All the later tests that were performed were done with a concentration of the plant extracts below the cytotoxicity level. 16,17 Inhibition of procoagulant activity: The assay for procoagulant activity was performed according to the method described by Theakston and Reid, various concentrations of venom (100µl) were added to 200μl of human citrated plasma at 37 0 C 18,19 . Coagulation time was recorded and the minimum coagulant dose (MCD) was determined as the venom concentration which induced clotting of plasma within 60 seconds.…”