Bull Pan Am Health Organ 12(l), 1918.

VENOMS OF CORAL SNAKES (MICRURUS SPP.): REPORT ON A MULTIVALENT ANTIVENIN FOR THE AMERICAS’

R. Boltis, L. Cerdas,s and J. W. Abalos4

A recently developed antivenin has Proved capable of neutral- iring venoms from nine coral snakes (Micrurus s#p.) of the Americas. The authors feel that this antivenin will prove effi- cacious against a very significant share of all coral snake bites in the area extending from the southern United States to Argentina.

According to Roze (1970) and Hoge and M. spixii, M. surinamensis, and M. Roman0 (1971) there are 111 kinds of coral isozonous-could be the most frequently snakes. These are classified in three involved in accidents or else the more genera-Leptomicrurus, Micruroides, and abundant and potentially important species Micrurus-containing a little over 50 (Figure 1). Unfortunately, not all their species. Available infbrmation on the venoms are available for study. medical importance of this group is very Cohen and his collaborators (1966, 1968, limited. The few papers published on this 1971) have reported important immuno- matter, our own experience, and a personal logical cross-reactions among Micrurus letter from Dr. J anis Roze suggest to us that venoms, and have attempted to design a the situation is as follows: Micrurus fulvius multivalent antivenin for use against the is the most important species in the United principal coral snakes of the Americas. States (Parrish, 1967), although some mild Work at our own laboratories has also bites have also been produced by Micru- produced data on the immunological char- roides euryxcznthus (Russell, 1967). Micru- acteristics of some Micrurus venoms. Some rus diastema, M. distans, and M. laticollaris of these data were presented at the Third seem to be the most important species in Latin American Congress of Parasitology Mexico and Guatemala, while Micrurus (Bolaiios, 1973) and some were previously nigrocinctus and M. alleni are the most reported in Toxicon (Bolafios, 1975). important in the rest of Central America Using all available data, we feel that we and Panama. In South America a wide can divide the venoms studied into three variety of Micrurus species-including serologically related groups, as shown in MiCTUTUS carinicaudus, M. mipartitus, M. Table 1. In the first two groups the immu- corallinus, M. frontalis, M. lemniscatus, nological relationship seems to be complete, at least in horses, because the specific anti- ‘Paper presented at the Fifth International Sympo- venins protect against all three venoms. The sium on Animal, Plant, and Microbial Toxins held at third group, however, seems to have only a San JOG, Costa Rica, in August 1976. Also appearing in Spanish in the BoletCz de la Oficina Sanitaria one-way relationship, because M. alleni Panamericana. antivenin does not offer protection against ZDirector, Clodomiro Picado Institute, and Profes- M. mi#artitus venom, but M. mipartitus sor of Immunology, University of Costa Rica. SChief, Production Division, Clodomiro Picado antivenin has been shown to protect mice Institute, and Associate Professor of Immunology, against challenges with M. alleni venom. University of Costa Rica. These findings, taken together, suggest 4Director, Applied Zoology Center, and Professor of Invertebrate Zoology, National University of CBrdoba, that an antivenin prepared by inoculating Argentina. horses with a mixture of venom from all

23 24 PAHO BULLETIN l vol. XII, no. 1, 1978

Figure 1. Medically important coral snake (Mi- Materials and Methods crurus) species of the Americas. The following three venoms were used for immunization: M. frontalis fiyrrhocryptus venom obtained from the Applied Zoology Center of the National University of C6r- M. diastema doba, Argentina: M. mipartitus hertwigi M. IaticoIlaris venom obtained from the Clodomiro Picado Institute of the University of Costa Rica; and M. nigrocinctus venom (a mixture of M. n. nigTocinctus and M. n. mosquitensis venoms) likewise obtained from the Clodo- miro Picado Institute. These three venoms were also employed for assay purposes, M. canhucaudus along with the following: M. alleni venom M. isozonus from the Clodomiro Picado Institute; M. fulvius fulvius and M. carinicaudus du- M. spivii M. Iemniscatus merilii venoms from the Division of Bio- M. corallinus logical Standards, U.S. National Institutes M. frontalis of Health; M. corallinus, M. spixii, and M. lemniscatus venoms purchased from the Miami Serpentarium Laboratories in Miami, Florida; and M. surinamensis venom obtained from a single specimen of this snake kindly donated by Dr. Juan Manuel Renjifo of the National Institute of Special Health Programs of Colombia. Table l-Groups of cross-reacting Micruru.s The immunization procedure was essen- venoms. tially the same used previously to prepare antivenin against Micrurus mipartitus (Bo- Venom Micrurus Habitat group species laiios, 1975), except that the antigen was an equal mixture of the M. frontalis, M. M. fulvius North America mipartitus, and M. nigrocinctus venoms. 1 M. nigTocinctus Central America M. carinicaudus South America Two healthy five-year-old horses were M. corallinus South America used. They were inoculated with nine pro- 2 M. fTontalis South America gressively larger amounts of venom mix- M. @xii South America ture, ranging from 1 mg initially to 75 mg 3 M. alleni Central and with the final injection. Sodium alginate M. mifiartitus South America was employed as an adjuvant depot with all the injections except the second to last, which was administered with Freund’s complete adjuvant. three groups might be effective against the Thirty liters of blood were withdrawn more common Micrurus in the Hemisphere. from each horse two weeks after the last The present article describes the production injection (over four successive days) with of such an antivenin and the in vitro assay reinjection of red blood cells. The material of its neutralizing potency against available was then fractionated with ammonium venoms. sulfate and concentrated to 10 per cent Boian’os et al. l CORAL SNAKE ANTIVENIN FOR THE AMERICAS 25

protein. The purified antivenin was bottled indicated. As can be seen, at least three I in 10 ml lots and liophilized. LD50 of venom were given in each case. Neutralization tests were performed with With the exception of M. surinamensis mice of both sexes weighing 16 to 18 grams. venom, all the venoms were neutralized to Aliquots of venom, containing 450 pg of M. some extent, suggesting the antivenin has mipartitus, M. frontalis, or M. corilanus efficacy in the event of coral snake bites. venom, or 300 pg of one of the other Considering the very small amounts of venoms, were prepared. These were mixed venom obtained by exhaustive milking of with progressively larger amounts of anti- coral snakes, it would seem that two or three venin, and the resulting mixtures were 10 ml vials of the antivenin should suffice to brought to a final volume of 6 ml. These neutralize the venom injected by a natural mixtures were then incubated in a water bite. This would appear true even if the bite bath at 37OC for 30 minutes and inoculated were administered by M. mipartitus or M. intraperitoneally (ip) into groups of five alleni, the two species whose venoms were mice (one ml per mouse). The neutraliza- neutralized at the lowest titers. Neverthe- tion titers, calculated in milligrams of less, the fact that M. mipartitus (and M. venom neutralized per ml of antivenin, alleni) venom was neutralized at a low titer were based on the smallest amount of anti- suggests a need for increasing the propor- venin which protected at least four of the tion of M. mipartitus venom in the im- five animals. The postinoculation observa- munization formula, or else modifying the tion period for the test animals was 72 immunization procedure so as to administer hours. more doses or larger amounts of venom. This is not possible at present because of Results and Discussion shortages of M. mipartitus specimens. How- ever, there is a fine opportunity for collect- Table 2 shows the neutralization titers ing specimens of this snake in Colombia’s obtained with the refined and concentrated coffee-growing regions. antivenin when tested against both homo- M. surinamensis venom does not seem to logous and heterologous venoms. The ip have a close immunologic relationship with LD50 for each venom and the challenge any of the other venoms tested. This is dose administered to each mouse are also important, because it leads to the hypothe-

Table 2-Neutralization titers of a multivalent Mkrurus antivenin tested against ten of the more important Micrurus venoms.

Observed neutralization Micrurus species Venom toxicity (one LDbO in Challenge venom Titer (mg of venom pg per mouse*) (pg per mouse*) per ml of serum)

M. fulvius 9 50 0.4-0.5 Ad. nigrocinctus 13 50 0.6-0.8 M. carinicaudus 17 50 1.0-1.5 M. frontalis 15-20 75 0.4-0.5 M. corallinus 20-25 75 1.5-2.0 M. spixiz’ 10-15 50 0.3-0.4 M. mipartitus 23 75 0.2-0.3 M. alleni 12 50 0.2-0.3 M. lemniscatus 5 50 0.6-0.7 M. surinamensis 5-10 50

*16-18 gram mice of both sexes. 26 PAHO BULLETIN l vol. XII, no. 1, 1978 sis that none of the various Micrurus enough venom could be stored) the inclu- , antivenins now on the market are effective sion of M. surinamensis venom in an immu- against this venom. nization formula for amultivalent antidote. If the multivalent antivenin just des- To date we have had no opportunity to cribed is included, we can say that there are test our antivenin against the more impor- now a total of six coral snake antivenins. tant coral snakes of Mexico and Guatemala, The other five are anti-M. fulvius anti- or against M. isozonous, which is important venin (USA), anti-M. nigrocinctus and M. in South America. Nevertheless, we believe alleni antivenin (Costa Rica), anti-M. that it will prove efficacious against a very mifiartitus antivenin (Costa Rica), and significant percentage of all coral snake anti-M. frontalis and M. corallinus anti- bites that occur in the Western Hemisphere. venin (Brazil and Argentina). All these This antivenin could be prepared easily homologous venoms were included in this through cooperative efforts by scientists in study: none appears to have immunological different countries, and could readily be cross reactions with M. surinamensis. We distributed through international agencies feel that this situation justifies the prepara- such as the Pan American Health Organi- tion of a monovalent antivenin or else (if zation.

ACKNOWLEDGMENTS

This investigation was supported by a re- Research, by the University of Costa Rica, search grant from Costa Rica’s National and by the National University of Cbrdoba, Council of Scientific and Technological Argentina,

SUMMARY

A multivalent coral snake antivenin was pre- talk, M. lemniscatus, M. mifiartitus, M. nigro- pared in horses immunized with a mixture of cinctus, and M. spixik The authors note that venoms from the species Micrurus nigrocinctus, appropriate cooperation by scientists in various M. mifiartitus, and M. frontalis, following countries would make production of this immunization procedures previously reported antivenin an easy matter, and that the product (2). Plasma from the horses was fractionated could conveniently be distributed via PAHO or with ammonium sulfate. other international agencies. They also note that The antivenin produced was then tested the antivenin showed no significant neutralizing against venoms from ten species. The neutraliza- effect against M. surinamensis venom. This tion titers obtained indicate it would be useful in situation would appear to justify preparing a treating bites received from most of the impor- monovalent antivenin against M. surinamensis, tant coral snake species in North and South or else including M. surinamensis venom in an America, namely: M. fuluius, M. alleni, M. immunization formula for a multivalent anti- carinicaudus dumerilii, M. corallinus, M. fron- dote.

REFERENCES

(I) Bolafios, R., L. Cerdas, and R. T. Taylor. Latin American Congress of Parasitology). Estudios inmunoldgicos de 10s venenos de las Antioquia Midica 23:518, 1973. principales Micrurus de Norte Am&rica, Centro (2) Bolafios, R., L. Cerdas, and R. T. Taylor. America, Panama y Colombia (Abstract, Third The production and characteristics of a coral Bolafios et al. 9 CORAL SNAKE ANTIVENIN FOR THE AMERICAS 27

snake (Micrurus mipartitus hertwigi) antivenin. snakes. In: W. Biicherl and E. Buckley (eds.), Toxicon 13:139, 1975. Venomous Animals and Their Venoms: Ven- (3) Cohen, P., and E. B. Seligmann, Jr. Im- omous Vertebrates (Volume 2). Academic Press, munology studies of coral snake venom. Mem New York, 1971, pp. 211-396. Znst Butantan 33:339, 1966. (7) Parrish, H. M. Bites by coral snakes: (4) Cohen, P., J. H. Dawson, and E. B. Reports of 11 representative cases. Am J Med Sci Seligmann, Jr. Cross neutralization of Micrurus 253:561, 1967. fuluius f&&s (coral snake) venom by anti- (8) Raze, J. A. Micrurus. In: J. A. Peters Micrurus carinicauda dumerilii serum. Am J and B. Orejas-Miranda (eds.), Catalogue of the Trap Med Hyg 17:308, 1968. Neotropical Squamata: Snakes (Part 1). Smith- (5) Cohen, P., W. H. Berkeley, and E. B. sonian Institution Press, Washington, D.C., Seligmann, Jr. Coral snake venoms: In vitro 1970. relation of neutralizing and precipitating anti- (9) Russell, F. E. Bites by the Sonoran coral bodies. Am J Trap Med Hyg 20:646, 1971. snake Micruroides euryxanthus. Toxicon 5:39, (6) Hoge, A. R., and S.A.R.W.D.L. Romano. 1967. Neotropical pit vipers, sea snakes, and coral