Immunization with Bungarus candidus venom was performed in four rabbits at high dose (initial dose, 75 microg/kg) and low dose (initial dose, 50 microg/kg). Each dose group consisted of two rabbits; one rabbit received the venom subcutaneously (s.c.) and the other intradermally (i.d.). The venom was injected as emulsified solutions with the same volume of Freund's complete adjuvant until the 4th immunization, thereafter as plain solutions. By stepwise increments of the immunizing dose, the higher dose group received a dose of 200 microg/kg and the lower dose group 150 microg/kg after the 5th immunization, respectively. Thereafter, seven additional immunizations were performed within six months. All rabbits were sacrificed two weeks after the last immunization (12th). Antilethal activity of the immunized antisera thus obtained was determined not only with the homologous venom but also with two heterologous venoms from Bungarus fasciatus and Bungarus flaviceps. Immunodiffusion analysis was also performed with these venoms. The results obtained in this pilot trial provided useful information for production of Malayan krait antivenom at Queen Saovabha Memorial Institute.
Bungarus fasciatus is one of three species of krait found in Malaysia. Envenoming by B. fasciatus results in neurotoxicity due to the presence of presynaptic and postsynaptic neurotoxins. Antivenom, either monovalent or polyvalent, is the treatment of choice in systemically envenomed patients. In this study, we have isolated a postsynaptic neurotoxin which we named α-elapitoxin-Bf1b. This toxin has an approximate molecular weight of 6.9 kDa, with LCMS/MS data showing that it is highly homologous with Neurotoxin 3FTx-RI, a toxin identified in the Bungarus fasciatus venom gland transcriptome. α-Elapitoxin-Bf1b also shared similarity with short-chain neurotoxins from Laticauda colubrina and Pseudechis australis. α-Elapitoxin-Bf1b produced concentration- and time-dependent neurotoxicity in the indirectly-stimulated chick biventer cervicis muscle preparation, an effect partially reversible by repetitive washing of the preparation. The pA2 value for α-elapitoxin-Bf1b of 9.17 ± 0.64, determined by examining the effects of the toxin on cumulative carbacol concentration-response curves, indicated that the toxin is more potent than tubocurarine and α-bungarotoxin. Pre-incubation of Bungarus fasciatus monovalent and neuro polyvalent antivenom failed to prevent the neurotoxic effects of α-elapitoxin-Bf1b in the chick biventer cervicis muscle preparation. In conclusion, the isolation of a postsynaptic neurotoxin that cannot be neutralized by either monovalent and polyvalent antivenoms may indicate the presence of isoforms of postsynaptic neurotoxins in Malaysian B. fasciatus venom.
Malayan krait (Bungarus candidus) is a medically important snake species found in Southeast Asia. The neurotoxic effects of envenoming present as flaccid paralysis of skeletal muscles. It is unclear whether geographical variation in venom composition plays a significant role in the degree of clinical neurotoxicity. In this study, the effects of geographical variation on neurotoxicity and venom composition of B. candidus venoms from Indonesia, Malaysia and Thailand were examined. In the chick biventer cervicis nerve-muscle preparation, all venoms abolished indirect twitches and attenuated contractile responses to nicotinic receptor agonists, with venom from Indonesia displaying the most rapid neurotoxicity. A proteomic analysis indicated that three finger toxins (3FTx), phospholipase A2 (PLA2) and Kunitz-type serine protease inhibitors were common toxin groups in the venoms. In addition, venom from Thailand contained L-amino acid oxidase (LAAO), cysteine rich secretory protein (CRISP), thrombin-like enzyme (TLE) and snake venom metalloproteinase (SVMP). Short-chain post-synaptic neurotoxins were not detected in any of the venoms. The largest quantity of long-chain post-synaptic neurotoxins and non-conventional toxins was found in the venom from Thailand. Analysis of PLA2 activity did not show any correlation between the amount of PLA2 and the degree of neurotoxicity of the venoms. Our study shows that variation in venom composition is not limited to the degree of neurotoxicity. This investigation provides additional insights into the geographical differences in venom composition and provides information that could be used to improve the management of Malayan krait envenoming in Southeast Asia.