Journal of Wilderness Medicine 3,377-381 (1992)

ORIGINAL ARTICLE

Neutralization of Micrurus distans distans venomby 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 of Emergency Medicine and2Arizona 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-Ouchterlonydemonstrated multiple precipitin lines between M. distans distans venom and M. fulvius antivenin(AV). The efficacy of M. fulvius AV in neutralizing M. distans distans and M. fulvius venoms invivo was tested using CD-l mice. All mice received venom plus saline or AV. Survival times wereimproved in all animals receiving AV. Clinical observation in both groups showed that modes ofdeath were similar. Antivenin (M. fulvius) Wyeth may be useful in the treatment of M. distansdistans envenomation.

Key words: snake venom, antivenin, envenomation

Introduction

The West Mexican coral snake (Micrurus distans distans) is found predominantly in theMexican 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 snakeoccasionally involves poison centers along the US-Mexico border. M. distans distans isconsidered 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 M. distans distans hasnot been studied. The planned exhibition of an M. distans distans specimen prompted aninvestigation of the use of antivenin (M. fulvius) Wyeth in the treatment of M. distansdistans envenomation.

Material and methods

Venom

Pooled, lyophilized venom was obtained from a single specimen of M. distans distans.The specimen was 122 em long and had been collected in southern Sonora, Mexico. Itwas identified by a herpetologist at the University of Arizona. M. fulvius fulvius venomwas purchased from Sigma Chemical Co, St. Louis, MO.

0953-9859 © 1992 Chapman & Hall

378 Dart, O'Brien, Garcia, Jarchow and McNally

Immunodiffusion

The ability of M. fulvius antivenin (Wyeth, Marietta PA) to produce precipitin lines wasdetermined by modified micro-Ouchterlony double diffusion in 1% agarose gel [4]. Thefirst test was 20 mg ml-1 of M. distans distans venom against 100, 50, 25, 12.5, 6.25, and3.125 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 ifthe precipitation was caused by an antigen-antibody interaction, horse serum was testedagainst M. fulvius antivenin, venom and M. distans distans 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-1mice, 25-30 g, (n = 5 per group) were administered M. distans distans venom, 0.175,0.35, 0.7, 1.05, 2.5, or 3.75 Ilg g-l body weight. Doses were given by intraperitonealinjection in a total volume of 200 III using normal saline as diluent. Mice wereobserved for 9 h. Time and mode of death and were recorded ± 7.5 min.

The efficacy of M. fulvius antivenin in neutralizing M. distans distans venom wastested in vivo. Four groups of five mice were administered a dose of M. distans distansvenom that was 2.25 times the LDsO' Each dose was administered in a total volume of200 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. fulviusantivenin, respectively.

The efficacy of M. fulvius antivenin in neutralizing M. fulvius fulvius venom was alsotested. Two groups of five mice received 2.25 times the LDso of M. fulvius fulvius venom.One group received saline while the other group received the equivalent of 20 vials ofantivenin.

Results

Immunodiffusion

As expected, precipitin lines formed between M. fulvius fulvius venom and M. fulviusantivenin. Similarly, sharp precipitin lines were found using a range of concentrations ofM. fulvius antivenin against 20 mg mIl? M. distans distans venom (Fig. 1a). This obser­vation is particularly evident between the venom and the 100 mg ml-1 concentration ofantivenin, and becomes less distinct with decreasing concentration. A second test withdecreasing concentration of M. distans distans venom against M. fulvius antivenindemonstrated precipitin bands, particularly at the lower venom concentrations (Fig. 1b).No precipitin lines formed between horse serum and M. fulvius antivenin, M. fulviusfulvius venom or M. distans distans venom.

Antivenin efficacy

The modified LDso for M. distans distans was 0.71 Ilg g-l body weight. The modifiedLDso of M. fulvius fulvius venom was found to be 2.01 Ilg g-l body weight. Bothlaboratory animal groups quickly became unsteady and then prostrate. This was soonfollowed by decreased and irregular respiration, followed by death at periods rangingfrom a few minutes to hours. M. fulvius antivenin was effective in neutralizing M. distansdistans venom in vivo. Analysis of variance, followed by Duncan's multiple range test,

M. distans distans and antivenin M. fulvius 379

Fig. 1. Immunodiffusion analysis of M. distans distans venom and antivenom (M. fulvius) Wyeth.(Left) The center wen contained M. distans distansvenom (20 mg ml- 1). Wens 1 to 6 contained anti­venin (M. fulvius), 100, 50, 25, 12.5, 6.25, 3.125 mg ml- 1, respectively. (Right) The center wencontained antivenin (M. fulvius) 25 mg ml- '. Wens 1 to 6 contained M. distans distansvenom, 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 extensionof survival time (p = 0.023) (Table 1).

Discussion

The modified LDso of 2.01 !lg g-I for M. fulvius fulvius venom determined in our studywas higher than a previous report of 0.77 !lg g-I using a traditional LDso• The modifiedLDso of M. distans distans venom was lower than that of M. fulvius fulvius determined inthis experiment but is nearly identical to a previous report [6].

The combination of precipitin line development and efficacy in the mouse modelsuggests that antivenin (M. fulvius) Wyeth could be useful in M. distans distansenvenomation. The formation of precipitin lines on Ouchterlony does not alone assureefficacy of an antivenin because the conditions are non-physiologic and the antigensrecognized by the antibody are not necessarily those that will neutralize the activity of thevenom.

The mouse has traditionally been used to determine the efficacy of antivenins and hasproved to be a reliable indicator of efficacy, at least in terms of survival. This model maybe less accurate in modelling tissue injury. Fortunately, local injury is not a problem incoral snake envenomation.

Human envenomation by M. distans distans does occur, but the frequency isunknown. We know of one documented case of human envenomation. We have receivedsporadic reports of envenomation from southern Sonora, Mexico, of patients envenomedby a coral snake while handling the snake. Victims reportedly mistook their specimens asmilksnakes (Lampropeltis sp.). Although these reports were second hand, they didfeature primarily neurological findings similar to those of M. fulvius fulvius. The primarycoral snake in southern Sonora appears to be M. distans distans. The death of an elderly

380 Dart, O'Brien, Garcia, Jarchow and McNally

Table 1. Biological activity of M. fulvius antivenin against M. distans distans venom

Venom

M. distans distansM. distans distansM. distans distansM. distans distansM. fulvius fulvius.M. fulvius fulvius

Treatment

1- Saline2 - AV (5 vial equivalent)3 - AV (10 vial equivalent)4 - AV (20 vial equivalent)5 - Saline6 - AV (20 vial equivalent)

n Mean survival SD

time (min)

5 203 1315 245 37*5 326 53*5 334 47*5 56 275 156 75*

*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 canbe identified by their banding pattern. In the coral snakes from these areas, the red andyellow rings touch, while the nonvenomous species, the yellow rings are separated oneach side by a black ring [8]. This has led to a rhyme:

Red on Yellow, Kill a FellowRed on Black, Good for Jack

Although this is true for coral snakes of the United States, this does not hold true for allcoral species. Many coral snake species in South America, Central America and southernNorth 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 sowhen the antivenin is not specific to the offending snake. Recent evidence suggests thatcross-neutralization between subspecies and species may occur [9, 10]. Since there is nospecific antivenin to M. distans distans venom, it may be reasonable, particularly inpotentially 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 snakeenvenomations.

References

1. Campbell, J.A. and Lamar, W.W. The Venomous Reptiles of Latin America Ithaca, NY:Cornell University Press, 1989.

2. Bolanos, R., Cerdas, L. and Abalos, J.W. Venoms of coral snakes (Micrurus spp.): report on amultivalent antivenin for the Americas. Bull Pan Am Health Organ 1978; 12, 23-7.

3. Kitchens, C.S. and Van Mierop, L.H.S. Envenomation by the eastern coral snake (Micrurusfulvius fulvius). lAMA 1987; 258, 1615-18.

4. Ouchterlony, O. and Nisson, L.A. Immunodiffusion and immunoelectrophoresis. In: Weir, D.,ed. Handbook of Experimental Immunology. 2nd ed. Oxford: Blackwell, 1973: 10-12.

5. Meier, J. and Theakston, R.D.G. Aproximate LDso determination of snake venoms using eightto ten experimental animals. Toxicon 1986; 24, 395-401.

M. distans distans and antivenin M. fulvius 381

6. Cohen, P. and Seligmann, E.B. Immunologic studies of coral snake venom. Mem Inst Butantan1966; 33, 339-45.

7. Pettigrew, L.c. and Glass, J.P. Neurologic complications of a coral snake bite. Neurology1985; 35, 589-92.

8. Russell, F.E. Snake venom poisoning. Great Neck, NY: Scholium International Inc., 1983.9. Mebs, D., Pohlman, S. and Von Tenspolde, W. Snake venom hemorrhagins; neutralization by

commercial antivenoms. Toxicon 1988; 26,453-8.10. Bolanos, R., Cerdas, L. and Taylor, R. The production and characteristics of a coral snake

(Micrurus mipartitus hertwigi) antivenin. Toxicon 1975; 13, 139-42.