Effectiveness of two common antivenoms for North, Central, and South American Micrurus envenomations.

1 Journal of Toxicology
2 CLINICAL TOXICOLOGY
3 Vol. 42, No. 0, pp. 1–8, 2004
4 Effectiveness of Two Common Antivenoms for North, Central,
5 and South American Micrurus Envenomations
6 Adolfo R. de Roodt, D.V.M., Ph.D.,1,*
7 Jorge F. Paniagua-Solis, Pharm.D., Ph.D.,2 Jorge A. Dolab, B.Sc.,3
8 Judith Este´vez-Ramire´z, M.Sc.,4 Blanca Ramos-Cerrillo, B.Sc.,5
9 Silvana Litwin, B.Sc.,1 J. C. EQ1Dokmetjian, B.Sc.,1
10 and Alejandro Alago´n, M.D., Ph.D.5
1123
14 1A´rea de Investigacio´n y Desarrollo/Serpentario, Instituto Nacional de Produccio´n
15 de Biolo´gicos—A.N.L.I.S. ‘‘Dr. Carlos G. Malbra´n’’, Buenos Aires, Argentina
16 2Direccio´n de Investigacio´n y Desarrollo, Laboratorios Silanes S.A. de C.V.,
17 Col. Del Valle, Me´xico DF, Me´xico
18 3Departamento de Vacunas y Sueros, Instituto Nacional de Produccio´n de
19 Biolo´gicos—A.N.L.I.S. ‘‘Dr. Carlos G. Malbra´n’’, Buenos Aires, Argentina
20 4Laboratorio Investigacio´n y Desarrollo, Instituto Bioclo´n,
21 Col. Toriello Guerra, Me´xico DF, Me´xico
22 5Departamento de Medicina Molecular y Bioprocesos, Instituto de Biotecnologı´a
23 de la UNAM, Cuernavaca, Morelos, Me´xico
24
25 ABSTRACT
26
27 Micrurus snakes (coral snakes) may produce severe envenomation that can lead to
28 death by peripheral respiratory paralysis. Only few laboratories produce specific
29 antivenoms, and despite the cross-reactivity found in some Micrurus species venoms,
30 the treatment is not always effective. To test two therapeutic antivenoms against the
31 venom of four species of Micrurus from Southern America, North of South America,
32 Central America, and North America, the determination of the lethal potency of the
33 venoms, the study of some biochemical and immunochemical characteristics, and the
34 determination of the neutralizing activity of both antivenoms were studied. North
35 American and South American antivenoms neutralized well venoms from Micrurus
36 species of the corresponding hemisphere but displayed lower effectiveness against
*Correspondence: Adolfo R. de Roodt, D.V.M., Ph.D., A´rea de Investigacio´n y Desarrollo/Serpentario, Instituto Nacional
de Produccio´n de Biolo´gicos—A.N.L.I.S. ‘‘Dr. Carlos G. Malbra´n’’, Av. Ve´lez Sarsfield 563, CP 1281, Buenos Aires, Argentina;
Fax: +54-11-4303-2492; E-mail: aderoodt@uolsinectis.com.
1
DOI: 10.1081/CLT-120030943 0731-3810 (Print); 1097-9875 (Online)
Copyright D 2004 by Marcel Dekker, Inc. www.dekker.com
120030943_CLT_42_00_R2_032904

37 venoms of species from different hemispheres. It was concluded that the neutralization
38 of Micrurus venoms by regional antivenoms could be useful to treat the envenomation
39 by some Micrurus snakes but is necessary to evaluate carefully the antivenoms to be
40 used with the venoms from the snakes of the region. Also considering the difficulties
41 for coral snake antivenom production, the development of a polyvalent antivenom
42 useful to treat the envenomation by coral snakes from different regions is necessary.
43 Key Words: Micrurus; Antivenoms; Venoms; Severe envenomation; Snakes;
44 Therapeutics.
45
46 INTRODUCTION
47 Accidents by venomous snakes represent a health
48 problem worldwide that varies in severity in different
49 regions, ranging from a sanitary problem of scarce
50 significance in the North of Europe to a very important
51 sanitary problem in the whole of Africa (1).
52 In America the snakes responsible for the highest
53 number of envenomations in humans belong to the
54 Viperidae Family (pit vipers, lance-headed vipers).
55 Information from Argentina, Brazil, Colombia, Costa
56 Rica, and Mexico indicates that these snakes are
57 responsible for over 95% of the accidents (2–8).
58 The remaining small percentage of accidents by
59 venomous snakes is due to the American Elapids,
60 represented by the Genus Micrurus, Micruroides, and
61 Leptomicrurus. Most of the accidents are due to
62 Micrurus species, owing mostly to their distribution
63 range (9), from Patagonia (M. pyrrhocryptus) to the
64 United States (M. fulvius). The American Elapids are
65 named coral snakes because of their bright red color
66 that contrasts with the black, white, or yellow bands
67 along their body.
68 Coral snake venoms are extremely toxic and the
69 bite of Micrurus constitutes a medical emergency
70 because of high risk of death due to their high
71 neurotoxicity. These venoms produce loss of muscle
72 strength and, in general, death by respiratory paralysis
73 of peripheral origin in animals and humans. The
74 neurotoxicity of these venoms can be produced by a
75 postsynaptic action (alpha neurotoxins, e.g., M. fronta-
76 lis), block of the end-plate receptors (alpha neuro-
77 toxins), and inhibition of evoked acetylcholine release
78 by the motor nerve endings (presynaptic-like action
79 e.g., M. corallinus), or, venoms that block endplate
80 receptors (alpha neurotoxins) and depolarize the muscle
81 fiber membrane (cardiotoxins or myotoxic phospholi-
82 pases A2, i.e., M. nigrocinctus and M. fulvius (10).
83 However, envenomation by these snakes is not
84 frequent, and the accidents are uncommon for several
85 reasons, among which we can summarize the follow-
86 ing: Elapid venomous apparatus (proterogliphous) is
87not as efficient as the viper’s (solenogliphous) in
88venom delivery; the small size of the snake’s mouth
89and the inability to maintain a large aperture of the
90jaws hinders utility to bite a human; there is a need for
91some time to inject a high amount of venom by their
92unsealed venomous conducts; coral snakes are not
93aggressive, in fact they are rather shy snakes; and they
94live mostly underground. For these reasons, accidents
95by Micrurus are most common in snake handlers in
96serpentariums or in children in the forest, who take the
97snakes to play because of their nonaggressive character
98and very attractive colors. In the North of Argentina it
99is not unusual for children from the communities of the
100forest to play with coral snakes.
101However, even if not frequent, accidents by
102Micrurus do happen and do constitute a medical
103emergency. As has been mentioned, venoms from
104Micrurus spp. are rich in a-neurotoxins that bind to
105alpha subunits of acetylcholine receptors at the myoneu-
106ral plate, leading to muscular and respiratory paralysis
107and death (5,6,10,11). Although experimentally it has
108been documented that some Micrurus venoms may
109produce myotoxicity and local lesions (12–15), the
110paramount feature of the coral snake toxicity is the
111neurotoxicity, and all the cases are considered serious
112because of the high risk of death.
113The only specific treatment for Micrurus enveno-
114mation is the application of the specific antivenom.
115Although Micrurus antivenoms are produced by several
116laboratories like Wyeth (United States), Bioclo´n
117(Me´xico), Clodomiro Picado Institute (Costa Rica),
118Butantan Insitute (Brazil), and the Instituto Nacional de
119Produccio´n de Biolo´gicos (Argentina) (3,5,6,16–18),
120they are not always available for treatment, even for
121use in the same country, and sometimes its production
122is discontinued. This lack of regular availability is due
123to various technical causes, among which are the
124difficulty in the obtention of coral snakes for venom
125extraction (in some regions of South America it is
126necessary to hunt the snakes in the jungle); the
127demands of maintaining Micrurus snakes in captivity
128in good state of health and feeding (19); and the small
2 de Roodt et al.
120030943_CLT_42_00_R2_032904

129 amount of venom that can be extracted per snake
130 (20). These facts make the production of Micrurus
131 antivenom more difficult than the production of
132 viper antivenom.
133 If a-neurotoxins are responsible for the toxicity of
134 these venoms and all coral snakes have this type of
135 toxins (11) and considering that cross-reactivity among
136 several venoms of Micrurus has been reported (21–24),
137 it may be hypothesized that the use of a given Micrurus
138 antivenom could be used to treat envenomation caused
139 by other species of Micrurus. However such statement
140 seems not to be entirely true, since lack of neutraliza-
141 tion of venoms from Micrurus by antivenom produced
142 with venom from snakes of the same country has been
143 also reported (25).
144 In spite of the above, and as a first step to the
145 obtention of an effective polyvalent anti-Micrurus
146 antivenom to be used in several regions of America,
147 we studied the neutralizing capacity of two antivenoms
148 of therapeutic use against the venoms of Micrurus
149 snakes of different regions.
150 One of the antivenoms was from North America
151 and another from South America. Both antivenoms
152 were tested against venoms from Micrurus species
153 from North America (M. fulvius), Central America
154 (M. nigrocinctus), and South America (M. surinamensis
155 and M. pyrrhocryptus) in order to evaluate their neu-
156 tralizing capacity.
157 MATERIALS AND METHODS
158 Venoms
159 Venoms from healthy specimens of M. fulvius (La
160 Florida), M. nigrocinctus (Costa Rica), and M. surina-
161 mensis (Leticia, Colombia) were provided from the
162 Bank of Venoms of Bioclo´n Institute, Me´xico DF.
163 Venom from M. pyrrhocryptus (Argentina) was provid-
164 ed from the Centro Zootoxicolo´gico de Misiones,
165 Obera´, Misiones, Argentina. All the venoms were ob-
166 tained by manual extraction, and immediately frozen at
167 �20�C and lyophilized. The venoms were aliquoted and
168 stored at �20�C until use.
169 Determination of Lethal Potency
170 Mice (CF-1 strain, 18–22 g, 5 to 8 animals per
171 dose level) were injected by i.p. route with different
172 amounts of venom in 0.15 M NaCl. From the number of
173 surviving animals 48 h after the injection, the LD50 was
174 calculated by non-linear regression using the combined
175Prism and Stat-Mate softwares (GraphPad, Inc., San
176Diego, CA). It was defined as the amount of venom that
177produce the death of 50% of the challenged mice
178(26,27).
179Electrophoretic Study
180Samples prepared under non-reducing conditions
181were separated on a vertical slab of 12.5% acrylamide
182gel using the discontinuous buffer system described by
183Laemmli (28). For molecular weight estimation of
184venom proteins, a kit of molecular weight markers
185(BioRad Broad Range) was run in the same gel. Gels
186were stained with Coomasie Brilliant Blue R (Sigma).
187Double Immunodiffusion
188Double immunodiffusions were performed in Petri
189dishes (10 cm) containing 1% Agarose (Sigma) in PBS
190pH 7.4 as described by Siles Villarroel (29). Wells
191(0.3 cm) were punched and filled with 10 ml of the
192different Micrurus venoms (concentration 1 mg/ml).
193The venoms were confronted against serial dilutions
194of the different antivenoms. After 48 h, Petri dishes
195were washed with 0.15 M NaCl, dried at 37�C,
196and immunocomplexes were stained with Amido
197Black (Sigma).
198Antivenoms
199The antivenoms used were Suero Anti-Micrurus,
200from the Instituto Nacional de Produccio´n de Bio-
201lo´gicos—A.N.L.I.S. ‘‘Dr. Carlos G. Malbra´n,’’ Buenos
202Aires, Argentina (batch 111, expiration date June 20,
2031999) with a protein content of 55 ± 2.5 mg/ml; and
204Coralmyn, from Instituto Bioclo´n, Mexico DF, Mexico
205(batch B-2D-06, expiration date October 16, 2004). The
206pharmaceutical presentation is lyophilized to be recon-
207stituted in 5 ml of diluent, with a final protein content
208of 40 ± 1.5 mg/ml. Both antivenoms were F(ab’)2
209fragments of equine immunoglobulins with similar
210degree of purity.
211Neutralization Assay
212This assay was performed as suggested by the
213World Health Organization (26,27). CF-1 mice (18–
21420 g) were injected i.p. with 3.0 LD50 of each venom
215preincubated for 30 min at 37�C with different doses of
216each antivenom (six animals per dose) in a final volume
217of 0.5 ml. After 48 h deaths were recorded and the data
EQ2 Effectiveness of Two Common Antivenoms for Micrurus Envenomations 3
120030943_CLT_42_00_R2_032904

218 analyzed by nonlinear regression using the software
219 Prism (GraphPad Inc., CA). The neutralizing capacity
220 was expressed in microliters as the effective dose 50%
221 (ED50), that is, the antivenom dose which protects half
222 of the injected mice (26,27) or as the milligram of
223 venom neutralized per vial of antivenom (30).
224 RESULTS
225 The electrophoretic profile showed differences
226 between the venoms. The venom from M. surinamensis
227 showed few proteins over 20 kDa with strong stained
228 bands under 14 kDa whereas the venoms from M.
229 nigrocinctus and M. fulvius showed strong stained
230 bands around 6.4–50 kDa with differences in intensity
231 and mobility between both venoms (Fig. 1).
232 Double immunodiffusion showed that Coralmyn
233 strongly recognized the venom from M. nigrocinctus
234 and M. fulvius and did not recognize the venom of M.
235 surinamensis (Fig. 2) or M. pyrrhocryptus (data not
236 shown) venoms. On the other hand, Anti-Micrurus
237 antivenom recognized the venom of M. surinamensis
238 and weakly the venoms of M. nigrocinctus and M.
239 fulvius (Fig. 2).
240 The lethal doses found for the different venoms
241 were 0.85 mg/kg (confidence interval, CI, 0.75 to 1.11
242 mg) for M. nigrocinctus venom, 0.48 mg/kg (CI 0.45 to
Figure 1. SDS-PAGE of venoms from different species of
Micrurus. It was performed in a 12.5% Acrilamyde/Bisacri-
lamyde gel in not reducing conditions. (1) M. surinamensis
venom (from left to right: 10, 20 and 30 mg); (2) M.
nigrocinctus venom (20, 35 and 50 mg) and (3) M. fulvius
(same as in M. nigrocinctus). The migration of the molecular
weight markers is expressed in kDa in the right (Molecular
weight markers BioRad Broad Range).
Figure 2. Double immunoprecipitation (Ouchterlony meth-
od) in 1% agarose of the venoms of M. fulvius, M. nigrocinctus
and M. surinamensis against Coralmyn (left side of the gels)
and Anti-Micrurus (right side of the gels) antivenoms. The
central wells were filled with 10 ml of a solution of 1 mg/ml
of M. fulvius (Mf, a), M. nigrocinctus (Mn, b), or M. suri-
namensis (Ms, c) venoms. The peripheral wells were filled
with 10 ml of dilutions of the antivenoms (1/1, 1/2, 1/4 and 1/8 in
NaCl 0.15 M). After 48 h the gels were dried and stained with
Amido Schwarz.
4 de Roodt et al.
120030943_CLT_42_00_R2_032904

243 0.50) for M. fulvius venom, 0.38 mg/kg (CI 0.25 to
244 0.95) for M. surinamensis and 1.3 mg/kg (CI 0.6 to 1.9)
245 for M. pyrrhocryptus venom.
246 The antivenoms showed different neutralizing ca-
247 pacity against the different venoms (Table 1). Coralmyn
248 was effective against venoms from M. fulvius and M.
249 nigrocinctus (ED50s of 39 ml and 63 ml, respectively) but
250 did not neutralize the venom from the South American
251 Micrurus since the ED50s were over 500 ml of antivenom.
252 On the other hand, Anti-Micrurus antivenom was
253 effective in the neutralization of M. surinamensis and
254 M. pyrrhocryptus venom (ED50 of 36 ml and 88 ml,
255 respectively), but it showed lower neutralizing capacity
256 against M. fulvius venom (385 ml) and M. nigrocinctus
257 venom (123 ml), being necessary to duplicate the dose to
258 neutralize M. nigrocinctus venom or to use tenfold the
259 dose to neutralize M. fulvius venom when compared with
260 the ED50 determined for these venoms with Coralmyn
261 (Table 1).
262 The calculated neutralizing potency per vial of an-
263 tivenom (30) for the Anti-Micrurus were 2.95 mg for M.
264 pyrrhocryptus, 2.11 mg for M. surinamensis, 0.25 mg
265 for M. fulvius, and 1.4 mg for M. nigrocinctus venoms.
266 For Coralmyn the potency per vial was 2.46 mg for M.
267 fulvius venom and 2.69 mg for M. nigrocinctus venom.
268 DISCUSSION
269 The venoms showed differences in lethal potency
270 and in electrophoretic pattern. A differential immuno-
271 chemical reactivity of the venoms with the different
272 antivenoms was also observed, which was consistent
273 with the data obtained from the experiments of sero-
274 neutralization of lethality.
275 The lethal potencies of the venoms found in this
276 study are close to those reported for other Micrurus
277 species (31), and the electrophoretic profiles of these
278 venoms in general have the major characteristics
279described for Micrurus venoms (31–34) with most of
280the Coomassie-stained material under 20 kDa.
281The immunochemical assay showed that the
282antivenoms show greater reactivity toward venoms
283from geographically related snakes. The neutralization
284assays showed similar results. Coralmyn, an antivenom
285widely used in North America and Central America,
286showed a good neutralization capacity against the ven-
287om of M. nigrocinctus (the venom used as immunogen)
288from Costa Rica and M. fulvius from La Florida
289(United States) but was not effective against venom
290of M. surinamensis (Colombia) or M. pyrrhocryptus
291(Argentina). On the other hand, Anti-Micrurus, an
292antivenom used in Argentina, was effective against the
293venoms of M. surinamensis and M. pyrrhocryptus (the
294latter used as immunogen), but it confers very
295low protection against venom from M. nigrocinctus
296and M. fulvius.
297With the exception of M. corallinus venom, that
298possesses presynaptic neurotoxins in addition to the
299alpha neurotoxins (10,35,36), the principal toxic com-
300ponents in Micrurus venoms are the latter (10,24,
30137–40). For this reason, in Brazil an antivenom raised
302against venom of M. frontalis and M. corallinus is used
303(6,23,41). However, M. corallinus venom seems to be
304effectively neutralized by antivenoms raised against
305other venoms (22).
306An important cross-reactivity among Micrurus ven-
307oms has been well described (22–24,43–46). Bolan˜os
308(22) proposed four antigenically related groups: 1) M.
309fulvius, M. nigrocinctus, and M. carinicauda, 2) M.
310corallinus, M. frontalis, and M. spixii, 3) M. halleni and
311M. mipartitus and 4) M. surinamensis venom as a
312separate group. In addition, Alape´-Giro´n (42) described,
313by means of monoclonal antibodies, three antigenically
314related groups: 1) M. nigrocinctus, M. fulvius, M.
315dumerilii, and M. albicinctus, 2) M. frontalis and M.
316brasiliensis, 3) M. alleni and M. spixii that present
317features of groups 1) and 2) but with particular char-
318acteristics. Venoms from M. surinamensis, M. corallinus,
Table 1. Neutralizing capacity of the antivenoms on the lethal potency of the different Micrurus venoms.T1.1
Antivenoms Coralmyn Anti-MicrurusT1.2
Venoms ED50 Potency per vial (5 ml) ED50 Potency per vial (5 ml)T1.3
M. fulvius 39 ml (25–58) 2.46 mg 385 ml (360–412) 0.25 mgT1.4
M. nigrocinctus 63 ml (50–78) 2.69 mg 123 ml (114–131) 1.4 mgT1.5
M. surinamensis > 500 ml < 0.15 mg 36 ml (29–44) 2.11 mgT1.6
M. pyrrhocryptus > 500 ml < 0.52 mg 88 ml (82–95) 2.95 mgT1.7
The ED50 (Effective Dose 50%) indicate the amount of antivenom required to protect 50% of mice challenged with 3.0 i.p. doses of
each venom and it is expressed in microliters. The 95% intervals of confidence are indicated into brackets. The potency per vial
expresses the amount of venom (in milligrams) neutralized by one vial of antivenom.
Effectiveness of Two Common Antivenoms for Micrurus Envenomations 5
120030943_CLT_42_00_R2_032904