Neutralization of Micrurus distans distans venom by antivenin (Micrurus fulvius)
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1 Journal of Wilderness Medicine 3, (1992) ORIGINAL ARTICLE Neutralization of Micrurus distans distans venom by antivenin (Micrurus fulvius) R.e. DART, MD, PhD l, 2, P.e. O'BRIEN, Pharm D2, R.A. GARCIA, BS l, J.e. JARCHOW, DVM and J. McNALLY, Pharm D2 I Section ofemergency Medicine and 2Arizona Poison and Drug Information Center, University ofarizona, Tucson AZ, 85724, USA Neutralization of Micrurus distans distans venom by antivenin (Micrurus fulvius) was investigated. Pooled, lyophilized venom from a single specimen was used in all experiments. Micro-Ouchterlony demonstrated multiple precipitin lines between venom and M. fulvius antivenin (AV). The efficacy of M. fulvius AV in neutralizing and M. fulvius venoms in vivo was tested using CD-l mice. All mice received venom plus saline or AV. Survival times were improved in all animals receiving AV. Clinical observation in both groups showed that modes of death were similar. Antivenin (M. fulvius) Wyeth may be useful in the treatment of M. distans distans envenomation. Key words: snake venom, antivenin, envenomation Introduction The West Mexican coral snake (Micrurus distans distans) is found predominantly in the Mexican state of Sinaloa, but is also found in the southern portion of the Sonoran desert [1]. Although this species is rare, the management of patients bitten by the snake occasionally involves poison centers along the US-Mexico border. is considered a medically important snake in Mexico [2]. Antivenin (M. fulvius) Wyeth is effective against the venom of the Eastern coral snake (Micrurus fulvius fulvius) [3], but its ability to alter the lethality of has not been studied. The planned exhibition of an specimen prompted an investigation of the use of antivenin (M. fulvius) Wyeth in the treatment of M. distans distans envenomation. Material and methods Venom Pooled, lyophilized venom was obtained from a single specimen of. The specimen was 122 em long and had been collected in southern Sonora, Mexico. It was identified by a herpetologist at the University of Arizona. M. fulvius fulvius venom was purchased from Sigma Chemical Co, St. Louis, MO Chapman & Hall
2 378 Dart, O'Brien, Garcia, Jarchow and McNally Immunodiffusion The ability of M. fulvius antivenin (Wyeth, Marietta PA) to produce precipitin lines was determined by modified micro-ouchterlony double diffusion in 1% agarose gel [4]. The first test was 20 mg ml- 1 of venom against 100, 50, 25, 12.5, 6.25, and mg ml- 1 of antivenin (M. fulvius). A second test of antivenin (M. fulvius) 25 mg ml- 1 against 40,20, 10,5,2.5, and 1.25 mg ml- 1 was performed. To determine if the precipitation was caused by an antigen-antibody interaction, horse serum was tested against M. fulvius antivenin, venom and venom. All venom and antivenin dilutions were in normal saline. Antivenin efficacy The modified LDso was used to determine biological activity [5]. Six groups of CD-1 mice, g, (n = 5 per group) were administered venom, 0.175, 0.35, 0.7, 1.05, 2.5, or 3.75 Ilg g-l body weight. Doses were given by intraperitoneal injection in a total volume of 200 III using normal saline as diluent. Mice were observed for 9 h. Time and mode of death and were recorded ± 7.5 min. The efficacy of M. fulvius antivenin in neutralizing venom was tested in vivo. Four groups of five mice were administered a dose of venom that was 2.25 times the LDsO' Each dose was administered in a total volume of 200 III by intraperitoneal injection. Group 1 received only saline as a diluent; groups 2, 3, and 4 received the equivalent (on a g/kg basis) of 5, 10, and 20 vials of M. fulvius antivenin, respectively. The efficacy of M. fulvius antivenin in neutralizing M. fulvius fulvius venom was also tested. Two groups of five mice received 2.25 times the LD so of M. fulvius fulvius venom. One group received saline while the other group received the equivalent of 20 vials of antivenin. Results Immunodiffusion As expected, precipitin lines formed between M. fulvius fulvius venom and M. fulvius antivenin. Similarly, sharp precipitin lines were found using a range of concentrations of M. fulvius antivenin against 20 mg mil? venom (Fig. 1a). This observation is particularly evident between the venom and the 100 mg ml- 1 concentration of antivenin, and becomes less distinct with decreasing concentration. A second test with decreasing concentration of venom against M. fulvius antivenin demonstrated precipitin bands, particularly at the lower venom concentrations (Fig. 1b). No precipitin lines formed between horse serum and M. fulvius antivenin, M. fulvius fulvius venom or venom. Antivenin efficacy The modified LDso for was 0.71 Ilg g-l body weight. The modified LDso of M. fulvius fulvius venom was found to be 2.01 Ilg g-l body weight. Both laboratory animal groups quickly became unsteady and then prostrate. This was soon followed by decreased and irregular respiration, followed by death at periods ranging from a few minutes to hours. M. fulvius antivenin was effective in neutralizing M. distans distans venom in vivo. Analysis of variance, followed by Duncan's multiple range test,
3 and antivenin M. fulvius 379 Fig. 1. Immunodiffusion analysis of venom and antivenom (M. fulvius) Wyeth. (Left) The center wen contained venom (20 mg ml- 1 ). Wens 1 to 6 contained antivenin (M. fulvius), 100, 50, 25, 12.5, 6.25, mg ml- 1, respectively. (Right) The center wen contained antivenin (M. fulvius) 25 mg ml-'. Wens 1 to 6 contained venom, 40, 20, 10, 5, 2.5, and 1.25 mg ml- 1, respectively. revealed a statistical difference between control and each treatment group (p = 0.042) (Table 1). Testing of M. fulvius fulvius venom also demonstrated a significant extension of survival time (p = 0.023) (Table 1). Discussion The modified LD so of 2.01!lg g-i for M. fulvius fulvius venom determined in our study was higher than a previous report of 0.77!lg g-i using a traditional LD so The modified LD so of venom was lower than that of M. fulvius fulvius determined in this experiment but is nearly identical to a previous report [6]. The combination of precipitin line development and efficacy in the mouse model suggests that antivenin (M. fulvius) Wyeth could be useful in envenomation. The formation of precipitin lines on Ouchterlony does not alone assure efficacy of an antivenin because the conditions are non-physiologic and the antigens recognized by the antibody are not necessarily those that will neutralize the activity of the venom. The mouse has traditionally been used to determine the efficacy of antivenins and has proved to be a reliable indicator of efficacy, at least in terms of survival. This model may be less accurate in modelling tissue injury. Fortunately, local injury is not a problem in coral snake envenomation. Human envenomation by does occur, but the frequency is unknown. We know of one documented case of human envenomation. We have received sporadic reports of envenomation from southern Sonora, Mexico, of patients envenomed by a coral snake while handling the snake. Victims reportedly mistook their specimens as milksnakes (Lampropeltis sp.). Although these reports were second hand, they did feature primarily neurological findings similar to those of M. fulvius fulvius. The primary coral snake in southern Sonora appears to be. The death of an elderly
4 380 Dart, O'Brien, Garcia, Jarchow and McNally Table 1. Biological activity of M. fulvius antivenin against venom Venom M. fulvius fulvius. M. fulvius fulvius Treatment 1- Saline 2 - AV (5 vial equivalent) 3 - AV (10 vial equivalent) 4 - AV (20 vial equivalent) 5 - Saline 6 - AV (20 vial equivalent) n Mean survival SD time (min) * * * * *Significantly different (p < 0.05) from saline alone. man in Sonora who mistook the coral snake for a milksnake has come to our attention. We were contacted when the patient developed progressive respiratory failure. Antivenin (M. fulvius) was flown in, but arrived after the patient had died. Pettigrew and Glass [7] reported similar symptoms in a patient bitten by M. laticollaris. Coral snakes in the United States and in northern parts of Mexico such as Sonora can be identified by their banding pattern. In the coral snakes from these areas, the red and yellow rings touch, while the nonvenomous species, the yellow rings are separated on each side by a black ring [8]. This has led to a rhyme: Red on Yellow, Kill a Fellow Red on Black, Good for Jack Although this is true for coral snakes of the United States, this does not hold true for all coral species. Many coral snake species in South America, Central America and southern North American have red and yellow rings separated by black. Campbell and Lamar [1] have summarized the distribution of these snakes. The decision to use antivenin for snake envenomation is always difficult, the more so when the antivenin is not specific to the offending snake. Recent evidence suggests that cross-neutralization between subspecies and species may occur [9, 10]. Since there is no specific antivenin to venom, it may be reasonable, particularly in potentially serious envenomations, to utilize antivenin (M. fulvius) as part of the treatment. Kitchens and Van Mierop [3] have published recommendations for the management of M. fulvius fulvius envenomations which can be applied to other coral snake envenomations. References 1. Campbell, J.A. and Lamar, W.W. The Venomous Reptiles of Latin America Ithaca, NY: Cornell University Press, Bolanos, R., Cerdas, L. and Abalos, J.W. Venoms of coral snakes (Micrurus spp.): report on a multivalent antivenin for the Americas. Bull Pan Am Health Organ 1978; 12, Kitchens, C.S. and Van Mierop, L.H.S. Envenomation by the eastern coral snake (Micrurus fulvius fulvius). lama 1987; 258, Ouchterlony, O. and Nisson, L.A. Immunodiffusion and immunoelectrophoresis. In: Weir, D., ed. Handbook ofexperimental Immunology. 2nd ed. Oxford: Blackwell, 1973: Meier, J. and Theakston, R.D.G. Aproximate LD so determination of snake venoms using eight to ten experimental animals. Toxicon 1986; 24,
5 and antivenin M. fulvius Cohen, P. and Seligmann, E.B. Immunologic studies of coral snake venom. Mem Inst Butantan 1966; 33, Pettigrew, L.c. and Glass, J.P. Neurologic complications of a coral snake bite. Neurology 1985; 35, Russell, F.E. Snake venom poisoning. Great Neck, NY: Scholium International Inc., Mebs, D., Pohlman, S. and Von Tenspolde, W. Snake venom hemorrhagins; neutralization by commercial antivenoms. Toxicon 1988; 26, Bolanos, R., Cerdas, L. and Taylor, R. The production and characteristics of a coral snake (Micrurus mipartitus hertwigi) antivenin. Toxicon 1975; 13,
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