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Maintaining Coral Snakes (Micrurus nigrocinctus, Serpentes: Elapidae) for venom production on an alternative fish-based diet
Toxicon 60 (2012) 249–253
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Toxicon journal homepage: www.elsevier.com/locate/toxicon
Maintaining Coral Snakes (Micrurus nigrocinctus, Serpentes: Elapidae) for venom production on an alternative fish-based diet Danilo Chacón 1, Santos Rodríguez 1, Jazmín Arias 1, Gabriela Solano 1, Fabián Bonilla 1, Aarón Gómez* Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San José, Costa Rica
a r t i c l e i n f o
a b s t r a c t
Article history: Received 3 January 2012 Received in revised form 29 March 2012 Accepted 3 April 2012 Available online 19 April 2012
American Elapid snakes (Coral Snakes) comprise the genera Leptomicrurus, Micruroides and Micrurus, which form a vast taxonomic assembly of 330 species distributed from the South of United States to the southern region of South America. In order to obtain venom for animal immunizations aimed at antivenom production, Coral Snakes must be kept in captivity and submitted periodically to venom extraction procedures. Thus, to maintain a snake colony in good health for this purpose, a complete alternative diet utilizing an easily obtained prey animal is desirable. The development of a diet based on fish is compared to the wild diet based on colubrid snakes, and assessed in terms of gain in body weight rate (g/week), longevity (weeks), venom yield (mg/individual), venom median lethal dose (LD50) and venom chromatographic profiles. The animals fed with the fish-based diet gained more weight, lived longer, and produced similar amount of venom whose biological and biochemical characteristics were similar to those of venom collected from specimens fed with the wild diet. This fish-based diet appears to be suitable (and preferable to the wild diet) to supply the nutritional requirements of a Micrurus nigrocinctus snake collection for the production of antivenom. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Fish-based diet Snake development Coral Snake Micrurus nigrocinctus Venom production
1. Introduction Snakebite envenomation is an important public health problem around the world (Kasturiratne et al., 2008). In the Americas, snakes of the family Viperidae produce most of the human envenomations (Warrell, 2004). Nevertheless, accidents involving snakes of the family Elapidae can also be severe or lethal (Bucaretchi et al., 2006; Norris et al., 2009). American elapid snakes (Coral Snakes) comprise the genera Leptomicrurus, Micruroides and Micrurus, which form a vast taxonomic assembly of 330 species distributed from the South of United States to the southern region of South
* Corresponding author. Tel.: þ506 25114962, þ506 25114934; fax: þ506 22920485. E-mail addresses: [email protected], [email protected] (A. Gómez). 1 Tel.: þ506 25114934; fax: þ506 22920485. 0041-0101/$ – see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.toxicon.2012.04.332
America (Campbell and Lamar, 1989; Roze, 1996; Greene, 1997; Stafford, 2000). According to Bolaños et al. (1978), the most important Coral Snakes, from a medical point of view, belong to the genus Micrurus, including Micrurus fulvius (United States), Micrurus diastema, Micrurus distans and Micrurus laticoralis (México and Guatemala); Micrurus nigrocinctus and Micrurus alleni (Central America); and Micrurus mipartitus, Micrurus corallinus, Micrurus frontalis, Micrurus spixii, Micrurus dumerilli carnicauda, Micrurus surinamensis and Micrurus isozonus (South America). Micrurus snake venoms are composed of several dozens of different molecules including predominantly phospholipases A2 (PLA2) and three-finger toxins (3FTXs) (such as a-neurotoxins), in addition to L-amino-acid oxidases (LAAOs), metalloproteinases and other components (Olamendi-Portugal et al., 2008; Fernández et al., 2011; Rey-Suárez et al., 2011). The 3FTXs in particular confer to the venom the ability to block the neuromuscular junctions
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and to induce paralysis of intercostal muscles and diaphragm. In severe envenomations, death is due to respiratory arrest (Bucaretchi et al., 2006). Such neurotoxic activity is due to the action of a-neurotoxins, although some venoms also display presynaptic neurotoxicity due to the action of PLA2s. Thus, these venoms induce signs and symptoms such as palpebral ptosis, ophtalmoplegia, paralysis of jaw, larynx and pharynx muscles, sialorrhea and paralysis of neck and limb muscles. Some Micrurus sp. venoms also show myotoxicity and cardiotoxicity when injected intravenously (Da Silva and Aird, 2001; Cecchini et al., 2005). Cardiotoxic, myotoxic, hemolytic, hemorrhagic and edematogenic manifestations upon envenoming have been also described in animal models as well as in patients bitten by different species of Coral Snakes (Corrêa-Netto et al., 2011). In order to counteract pathophysiological effects, medical treatment of snakebite envenomation is based on the parenteral administration of animal-derived antivenoms. Snake antivenoms are formulations of immunoglobulins or their F(ab’)2 or Fab fragments, purified from plasma of animals immunized with snake venoms (WHO, 2010; Gutiérrez et al., 2011). Because snake venom composition often varies significantly between species, antivenoms are able to neutralize only venoms antigenically related with those used in the antivenom preparation. In the case of Micrurus venoms, limited cross neutralization has been demonstrated in several studies (Keegan et al., 1961; Bolaños et al., 1973, 1975; Tanaka et al., 2010); nevertheless, protective action can be conferred by the use of different antivenoms produced from other species of Micrurus sp. or even other genera, to the particular case of murine models (Winiewski et al., 2003; Sánchez et al., 2008). In order to obtain venom for immunization, Coral Snakes must be kept in captivity and submitted periodically to venom extraction procedures. Each Micrurus specimen produces around 5 mg venom/extraction (Minton, 1957, 1974; Russell, 1967; Bolaños, 1972). Therefore, snake collections used to provide venom for industrial antivenom production must consist of numerous specimens. The development of a colony of snakes for venom production purposes has been reported for many institutions worldwide (Ashley and Burchfield, 1968). Usually, Coral Snakes feed only on natural prey, such as colubrid snakes belonging to the genera Geophis and Ninia (Da Silva et al., 2001; Urdaneta et al., 2004). In captivity, maintenance of a long-term collection is affected by a number of considerations including the quality of food and type provided (Kirkwood and Gili, 1994). Most Coral Snakes do not feed readily, and they generally die a few months after being captured (Serapicos and Merusse, 2002b). Although some herpetologists use a forced-feeding strategy (Fix and Minton, 1976), the survival rate of these specimens rarely exceeds one year. Thus, feeding Coral Snakes is a major problem to overcome in order to maintain a well-suitable collection to yield the necessary amount of venom to produce antivenom. Several studies have reported prey animals and stomach contents of Coral Snakes, which include caecilians, amphisbaenians, lizards and the fresh-water fish Synbranchus marmoratus (Schmidt, 1932; Cunha and Nascimento, 1978; Roze, 1982; Greene, 1984; Sazima and Abe, 1991).
Because of the impracticality of supplying the required quantities of these foods and the low survival rate of Coral Snakes that prompted us to search for a diet that is readily accepted by the snakes and that provides all the nutritional requirements to keep a well sustained colony. In this work, the suitability of a fish-based diet to supply the nutritional requirements to a M. nigrocinctus snake collection is described, as applied to a snake collection aimed at generating venom for the production of antivenom. 2. Materials and methods 2.1. Natural prey and feeding techniques Colubrid snakes of the genera Ninia sp. and Geophis sp. were kept at the Serpentarium of the Instituto Clodomiro Picado, until their use. These snakes were euthanized by CO2, and placed into plastic bags filled with water in order to freeze them. The bags were identified with the genus and the quantity of snakes and kept at 10 C until they were used. When Coral Snakes were fed, Ninia sp. and Geophis sp. snakes were thawed with water at room temperature; then, they were placed into a container with water in order to maintain turgor. After this, natural prey specimens were selected and offered to Coral Snakes according to their body weight and length. In detail, the natural prey was held with forceps and brought near the Coral Snake, moving it in a lifelike fashion in order to stimulate feeding behavior. Then, simulating snake-like movements, the natural prey was placed near the Coral Snake so that it could bite and hold it. 2.2. Fish-based diet preparation and feeding techniques Fillets of tilapia (Oreochromis sp.) fish were used; selecting those with proper texture, integrity and length. Basically, around 2 kg of tilapia fillets were placed and separated on a plastic dish and frozen for 24 h at 10 C; after this, the fillets were taken out and thawed at room temperature for about 20 min in order to cut them into longitudinal sections. The size of the sections depended on the weight and size of the specimens of Coral Snakes to be fed. The fillet sections were placed in a container with water at room temperature in order to completely defrost the meat. Lastly, in order to avoid some deficiencies in terms of calcium and vitamin D supply, we added a calcium supplement (RepCalÒ) to the fillets before offering them to the Coral Snakes. The elongate pieces of fish were grasped with forceps and brought near the Coral Snakes, simulating snake-like movements in order to stimulated the snakes; then, fish was left as close to the Coral Snake as possible. 2.3. Nutritional analysis The nutritional and compositional analyses were performed by Laboratorio de Servicios Analíticos of the School of Chemistry, University of Costa Rica (report No. 355-07). For these analyses, 50 g of both ground Geophis and Ninia snakes or 50 g of ground tilapia fillet were used. The samples were analyzed by gravimetry, volumetry and atomic absorption spectrometry for the quantification of protein and fat content, as well as for calcium and
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potassium, in addition to dry weight (ash) and moisture content (Skoog and Leary, 1994; Skoog et al., 1997). 2.4. Diet assessment The specimens of M. nigrocinctus used in this study were collected, mainly, from Santo Domingo, Heredia, and Coronado, San José, both localities in the Central Valley of Costa Rica. Likewise, the sample size studied was 92 individuals, separated in two groups: those assigned to the natural prey diet (n ¼ 33), and those corresponding to the fish-based diet (n ¼ 59). The snakes were fed every 15 days with a prey item according to the groups assigned. Thus, the gain/loss body weight rate (expressed as g/week) was defined as the difference between the initial and the final body weight recorded, divided by the period of time maintained in the study. The body weight was determined every 15 days, before the Coral Snakes were fed with either diet. Then, the gain/loss body weight rate was evaluated for both experimental groups, as described. Additionally, the longevity of the Coral Snakes was defined as the number of days that the snakes survived under both diets. Lastly, the venom yield was defined as the total amount of venom obtained from individual specimens assigned to these diets. 2.5. Biological and biochemical characterization of the venom Pools of venom were obtained from M. nigrocinctus snakes fed on either diet. The venoms were extracted every four months interval. These venoms were lyophilized and stored at 20 C until used. For the reverse-phase (RP) HPLC separation, 2 mg of venom were dissolved in 200 mL of 5% acetonitrile containing 0.1% trifluoroacetic acid, centrifuged for 5 min at 13,000 rpm, and loaded onto an Agilent Zorbax Eclipse Plus C18 Analytical column (4.6 250 mm, 5-Micron), using an Agilent 1100 Series chromatographer. Elution was performed at 1 mL/min as described by Fernández et al. (2011), and the absorbance at 215 nm was monitored. The Median Lethal Dose (LD50) test was conducted using venoms from the two experimental groups of Coral Snakes. Groups of six mice (16–18 g body weight, CD-1 strain) were injected with 500 mL of sterilized saline solution containing the venom by the intraperitoneal (i.p.) route. Assays were performed using six levels (3.5; 4.9; 6.86; 9.6; 13.44; 18.82 mg) with a dilution factor of 1/1.2 and deaths were recorded after 72 h (Bolaños et al., 1978). The results were calculated by Probits. Experiments were performed five times each venom and results expressed as mean SD. 2.6. Statistical analysis All the statistical tests were performed using SPSS 17.0 for Windows, and all P 0.05 values were considered significant. The LD50 values were obtained using the Probitos software. 3. Results and discussion 3.1. Nutritional analysis Chemical analyses revealed differences in calcium and protein concentrations between the two diets (Table 1).
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Table 1 Nutritional analysis of tilapia fillets and the natural prey of M. nigrocinctus. Fifty grams of fish or colubrid snakes (Ninia sp. and Geophis sp.), were used in the analysis. Analysis
Natural prey
Fat content (% m/m) Protein (% m/m) Calcium (mg/100 g) Potassium (mg/100 g) Ash (% m/m) Moisture content (% m/m)
7.5 2.0 1095.3 27.5 4.3 75.6
0.1 0.1 0.1a 0.1 0.1 0.5
Tilapia fillets 6.9 12.6 668.7 18.8 0.2 83.5
0.1 0.1a 0.5 0.1 0.1 0.5
a Indicates where the diets differed most regarding the compounds analyzed. All the values are expressed as mean standard deviation.
The natural prey had a higher calcium concentration than the fish fillets, whereas the latter had higher protein content. Considering that a fresh-water fish has been reported as part of the Coral Snakes’ diet, it makes sense to assume that an alternative diet based on fish will supply most of the nutrients needed by Coral Snakes. Potential deficiencies can be circumvented by the addition of an external source of calcium plus vitamin D3. 3.2. Diet assessment The assessment of the diets was based on the comparison and analysis of body weight gain, longevity and venom yielded by the snakes fed with either diet. The body weight gain for snakes fed on natural prey was 0.0778 0.5893 g/week, whereas that of snakes fed on fish-based diet was 0.264 0.6288 g/week. These results were not significantly different (Table 2) (F ¼ 0.038; df ¼ 1, 90; P ¼ 0.845). The body weight gain is a major factor to be considered, since the body mass affects the survival rate and the longevity (Kirkwood and Gili, 1994; Serapicos and Merusse, 2002a). On the other hand, the longevity was affected by the type of diet. Coral Snakes fed on natural prey survived 32 3 weeks, whereas those fed with fish diet survived 168 21 weeks (F ¼ 23.493; df ¼ 1, 90; P < 0.001, n ¼ 92). Thus, our observations on body weight gain and longevity support the view that a fish-based diet is highly successful as well as an useful strategy to maintain a large collection of M. nigrocinctus aimed at obtaining venom for antivenom production, without having to use colubrid snakes for feeding. Since the venom yield of Coral Snakes is rather low, the extension in survival time in captivity has a highly significant impact in venom production. Concerning the venom yielded by Coral Snakes when they were fed on either fish-based diet or natural prey diet, the former had a venom yield of 5 3 mg/individual (total protein content ¼ 1.8 0.1 g/dL), whereas the Coral Table 2 Effects of the two different diets on various parameters of M. nigrocinctus in captivity. Test
Natural prey diet
Fish-based diet
Body weight gain Venom yield Longevity LD50
0.0778 0.5893 g/week
0.264 0.6288 g/week
3 1 mg/individual 32 3 weeks 0.41 0.08 mg/g mouse
5 3 mg/individual 168 21 weeks 0.55 0.09 mg/g mouse
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3.3. Biological and biochemical venom characterization
Fig. 1. Coral Snake (Micrurus nigrocinctus) feeding on a tilapia fillet as an alternative diet. Notice that the snake bites toward the thinner end of the tilapia fillet.
Snakes fed on their natural prey had a venom yield of 3 1 mg/individual (total protein content ¼ 1.8 0.9 g/dL). Fish-fed snakes averaged higher venom yields, but these differences were not significantly different (t ¼ 1.363; df ¼ 7; P ¼ 0.215). These yields are comparable to previously reported yields for M. nigrocinctus and M. fulvius (Bolaños, 1972; Minton, 1957, 1974; Russell, 1967; Fix and Minton, 1976); however, these studies did not consider effect of diet on venom yields. Together with increased longevity effects, maintaining a colony of Coral Snakes on a fish-based diet guarantees an increased supply of venom for antivenom production (Fig. 1).
The elution profile of the venoms from Coral Snakes fed on either diet and separated by RP-HPLC revealed nearly 50 peaks (Fig. 2). The chromatographic profiles of both venoms were very similar to each other and to the pattern previously described for this species’ venom by Fernández et al. (2011). Thus, variations in diet do not seem to modify the overall venom composition. In agreement with previous results of Bolaños (1972), the median lethal dose (LD50) obtained for the venom from snakes fed on natural prey was 0.41 0.08 mg/g mouse, whereas the LD50 of the venom from snakes fed on a fishbased diet was 0.55 0.09 mg/g mouse (Table 2); this difference was not statistically significant (t ¼ 2.15, df ¼ 6, P ¼ 0.075). Likewise, these values of LD50 do not differ from those previously reported by León et al. (1999), and from those regularly obtained at the Quality Control Laboratory of Instituto Clodomiro Picado (data not shown). These findings indicate that overall venom toxicity is not affected by the shift in diet. In conclusion, our observations on two groups of M. nigrocinctus fed with different diets, one based on their natural prey (colubrid snakes) and the other based on fish, indicate that these two groups do not differ significantly in terms of body weight gain, venom yield, venom HPLC pattern and LD50; further, snakes fed with fish showed a significantly higher survival time in captivity. These results support the use of a fish-based diet for the feeding of a collection of M. nigrocinctus. The prolonged life span of these specimens in captivity and the stable characteristics of the venom indicate that this fish-based diet is an important innovation for maintaining Coral Snakes in
Fig. 2. Chromatographic profiles of Micrurus nigrocinctus venom proteins by RP-HPLC. Two milligrams of pooled venoms were applied to a C18 column, as described in Materials and Methods (gradient line was omitted for clarity). A) Elution profile of M. nigrocinctus venom proteins fed on its natural prey. B) Elution profile of M. nigrocinctus venom proteins fed on the fish-based diet.
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captivity for venom production used in antivenommanufacturing laboratories. Acknowledgments This study was supported by Vicerrectoría de Investigación, Universidad de Costa Rica (project 741-A09-003). The authors thank our colleagues at Instituto Clodomiro Picado for their collaboration. Conflict of interest There are no conflicts of interest. References Ashley, B.D., Burchfield, P.M., 1968. Maintenance of a snake colony for the purpose of venom extraction. Toxicon 5, 267–275. Bolaños, R., 1972. Toxicity of Costa Rican snake venoms for the white mouse. Am. J. Trop. Med. Hyg. 21, 360–361. Bolaños, R., Cerdas, L., Taylor, R., 1973. Estudios inmunológicos de los venenos de las principales Micrurus de Norte América, Centro América, Panamá y Colombia. In: Trabajo presentado en el 3er Congreso Latinoamericano de Parasitología y 3er Congreso Colombiano de Parasitología y Medicina Tropical. Medellín, Colombia, 9–12 de diciembre. Bolaños, R., Cerdas, L., Taylor, R., 1975. The production and characteristics of a Coral snake (Micrurus mipartitus hertwigi) antivenin. Toxicon 13, 139–142. Bolaños, R., Cerdas, L., Abalos, J.W., 1978. Venenos de serpientes coral (Micrurus spp.): informe sobre un antiveneno polivalente polivalente para las Américas. Bol Of. Sanit Panam 84 (2), 128–133. Bucaretchi, F., Hyslop, S., Vieira, R.J., Toldedo, A.S., Madureira, P.R., de Capitani, E.M., 2006. Bites by coral snakes (Micrurus spp.) in Campinas, state of São Paulo, Southeastern Brazil. Rev. Inst. Med. Trop. S. Paulo 48, 141–145. Campbell, J.A., Lamar, W.W., 1989. The Venomous Reptiles of Latin America. Cornell University Press, Ithaca and New York. Cecchini, A.L., Marcussi, S., Silveira, L.B., Borja-Oliveira, C.R., RodriguesSimoni, L., Amara, S., Stábeli, R.G., Giglio, J.R., Arantes, E.C., Soares, A. M., 2005. Biological and enzymatic activities of Micrurus (Coral) snake venoms. Comp. Biochem. Physiol. Part A 140, 125–134. Corrêa-Netto, C., Junqueira-de-Azevedo, I.L.M., Silva, D.A., Leitáo-deAraújo, A., Alves, M.L.M., Sanz, L., Foguel, D., Zingali, R.B., Calvete, J.J., 2011. Snake venomics and venom gland transcriptomic analysis of Brazilian coral snakes Micrurus altirostris and M. corallinus. J. Prot. 74 (9), 1795–1809. Cunha, O.R., Nascimento, F.P., 1978. Ofidios de Amazônia. X. As cobras de região leste do Pará, Belem. Mus. Par. Emilio Goeldi Publ. Avuls 31, 1–128. Da Silva, N.J., Aird, S.D., 2001. Prey specifity, comparative lethality and compositional differences of coral snakes venoms. Comp. Biochem. Physiol. Part C 128, 425–456. Da Silva, A.R., Prieto, Yamagushi, I.K., Morais, J.F., Higashi, H.G., Raw, I., Ho, P.L., de Oliveira, J.S., 2001. Cross reactivity of different specific Micrurus antivenom sera with homologous and heterologous snakes venoms. Toxicon 34, 949–953. Fernández, J., Alape-Girón Alberto, Angulo, Y., Sanz Libia, Gutiérrez, J.M., Calvete, J.J., Lomonte, B., 2011. Venomic and antivenomic analyses of the Central America Coral Snake, Micrurus nigrocinctus (Elapidae). J. Prot. Res. 10, 1816–1827. Fix, J.D., Minton, S.A., 1976. Venom extraction and yields from the North American coral snake, Micrurus fulvius. Toxicon 14, 143–145. Greene, H.W., 1984. Feeding behavior and diet of the Eastern coral snake, Micrurus fulvius. In: Seigel, R.A., Hunt, L.E., Knight, J.L., Malaret, L., Zuschlag, N.L. (Eds.), Vertebrate Ecology and Systematics – A Tribute to Henry S. Fitch. Museum of Natural History, University of Kansas, Lawrence, pp. 147–161. Greene, H.W., 1997. Snakes: The Evolution of Mystery in Nature. University of California Press, Berkeley, California. Gutiérrez, J.M., León, G., Lomonte, B., Angulo, Y., 2011. Antivenoms for snake envenomings. Inflamm. Allergy Drug Targets 10, 369–380.
253
Kasturiratne, A., Wickremasinghe, A.R., de Silva, N., Gunawardena, N.K., Pathmeswaran, A., Premaratna, R., Saviolo, L., Lalloo, D.G., de Silva, H.J., 2008. The global burden of snakebite: a literature analysis and modelling based on regional estimates of envenoming and deaths. PLoS Med. 5, e218. Keegan, H.L., Whittemore, F.W., Flanigan, J.F., 1961. Heterologous antivenin in neutralization of North American coral snake venom. Public Health Rep., Wash 76, 540. Kirkwood, J.K., Gili, C., 1994. Food consumption in relation to bodyweight in captive snakes. Res. Vet. Sci. 57, 35–38. León, G., Stiles, B., Alape, A., Rojas, G., Gutiérrez, J.M., 1999. Comparative study on the ability of IgG and F(ab’)2 antivenoms to neutralize lethal and myotoxic effects Induced by Micrurus nigrocinctus (coral snake) venom. Am. J. Trop. Med. Hyg. 6 (2), 266–271. Minton, S.A., 1957. An immunological investigation of rattlesnake venoms by the agar diffusion method. Am. J. Trop. Med. Hyg. 6 Minton, S.A., 1974. Venom Diseases. Charles C. Thomas, Springfield, 1–235 pp. Norris, R.L., Pfalzgraf, R.R., Laing, G., 2009. Death following coral snake bite in the United States: first documented case (with ELISA confirmation of envenomation) in over 40 years. Toxicon 53, 693–697. Olamendi-Portugal, T., Batista, C.V.F., Restano-Cassulini, R.R., Pando, V., Villa-Hernandez, O., Zavaleta-Martínez, A., Salas-Arruz, M.C., Rodríguez, R.C., Becerril, B., Posan, L.D., 2008. Proteomic analysis of the venoms from the fish eating coral snake Micrurus surinamensis: novel toxins, their function and phylogeny. Proteomics 8, 1919–1932. Rey-Suárez, P., Núñez, V., Gutiérrez, J.M., Lomonte, B., 2011. Proteomic and biological characterization of the venom of the redtail coral snake, Micrurus mipartitus (Elapidae), from Colombia and Costa Rica. J. Proteomics 75 (2), 655–667. Roze, A.J., 1982. New World coral snakes (Elapidae): a taxonomic and biological summary. Mem. Inst. Butantan 46, 305–338. Roze, A.J., 1996. Coral Snakes of the America: Biology, Identification and Venoms. Krieger Publ. Comp. Malabar, Florida. Russell, F.E., 1967. Bites by the sonoran coral snake Micruroides euryxanthus. Toxicon 5 (1), 39–42. Sánchez, E.E., Lopez-Johnston, J.C., Rodríguez-Acosta, A., Pérez, J.C., 2008. Neutralization of two North American coral snake venoms with United States and Mexican antivenoms. Toxicon 51, 297–303. Sazima, I., Abe, A.S., 1991. Habits of five Brazilian snakes with coral-snakes pattern, including a summary of defensive tactics. Stud. Neotrop. Fauna Environ. 26, 159–164. Schmidt, K.P., 1932. Stomach contents of some American coral snakes, with the description of a new species of Geophis. Copeia 1, 6–9. Serapicos, E.O., Merusse, J.L.B., 2002a. Variação de peso e sobrevida de Micrurus corallinus sobre diferentes condições de alimentação em biotério (Serpentes, Elapidae). Iheringia, Sér. Zool., Porto Alegre 92 (4), 105–109. Serapicos, E.O., Merusse, J.L.B., 2002b. Análise comparativa entre tipos de alimentação de Micrurus corallinus (Serpentes, Elapidae) em cautiverio. Iheringa, Sér. Zool., Porto Alegre. 92 (4), 99–103. Skoog, D.A., Leary, J.J., 1994. Análisis Instrumental, fourth ed. Editorial MacGraw-Hill, Madrid. Skoog, D.A., West, D.M., Holler, F.J., 1997. Fundamentos de Química Analítica, 4ta ed., Tomo I. Editorial Reverté, Barcelona. Stafford, P.J., 2000. On the status of the coral snake Micrurus nigrocinctus (Serpentes, Elapidae) in Belize, and its Northernmost distribution in Atlantic Middle America. Herpetological Review 31 (2), 78–82. Tanaka, G.D., Furtado, M.F.D., Portaro, F.V.C., Sant’Anna, O.A., Tambourgi, D.V., 2010. Diversity of Micrurus snake species related to their venom toxic effects and the prospective of antivenom neutralization. PLos Negl. Trop. Dis. 4 (3), e622. Urdaneta, A.H., Bolaños, F., Gutiérrez, J.M., 2004. Feeding behavior and venoms toxicity of coral snake Micrurus nigrocinctus (Serpentes: Elapidae) on its natural prey in captivity. Comp. Biochem. Physiol. 138, 485–492. Warrell, D.A., 2004. Epidemiology, clinical features and management of snake bites in Central and South America. In: Campbell, J., Lamar, W.W. (Eds.), Venomous Reptiles of the Western Hemisphere. Cornell University Press, Ithaca, pp. 709–761. WHO, 2010. Guidelines for the Production Control and Regulation of Snake Antivenom Immunoglobulins. Geneva, Switzerland. Winiewski, M.S., Hill, R.E., Havey, J.M., Bogdan, G.M., Dart, R.C., 2003. Australian tiger snake (Notechis scutatus) and Mexican coral snake (Micrurus species) antivenoms prevent death from United States coral snake (Micrurus fulvius fulvius) venom in a mouse model. J. Toxicol. Clin. Toxicol. 41 (1), 7–10.
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Toxicon journal homepage: www.elsevier.com/locate/toxicon
Maintaining Coral Snakes (Micrurus nigrocinctus, Serpentes: Elapidae) for venom production on an alternative fish-based diet Danilo Chacón 1, Santos Rodríguez 1, Jazmín Arias 1, Gabriela Solano 1, Fabián Bonilla 1, Aarón Gómez* Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San José, Costa Rica
a r t i c l e i n f o
a b s t r a c t
Article history: Received 3 January 2012 Received in revised form 29 March 2012 Accepted 3 April 2012 Available online 19 April 2012
American Elapid snakes (Coral Snakes) comprise the genera Leptomicrurus, Micruroides and Micrurus, which form a vast taxonomic assembly of 330 species distributed from the South of United States to the southern region of South America. In order to obtain venom for animal immunizations aimed at antivenom production, Coral Snakes must be kept in captivity and submitted periodically to venom extraction procedures. Thus, to maintain a snake colony in good health for this purpose, a complete alternative diet utilizing an easily obtained prey animal is desirable. The development of a diet based on fish is compared to the wild diet based on colubrid snakes, and assessed in terms of gain in body weight rate (g/week), longevity (weeks), venom yield (mg/individual), venom median lethal dose (LD50) and venom chromatographic profiles. The animals fed with the fish-based diet gained more weight, lived longer, and produced similar amount of venom whose biological and biochemical characteristics were similar to those of venom collected from specimens fed with the wild diet. This fish-based diet appears to be suitable (and preferable to the wild diet) to supply the nutritional requirements of a Micrurus nigrocinctus snake collection for the production of antivenom. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Fish-based diet Snake development Coral Snake Micrurus nigrocinctus Venom production
1. Introduction Snakebite envenomation is an important public health problem around the world (Kasturiratne et al., 2008). In the Americas, snakes of the family Viperidae produce most of the human envenomations (Warrell, 2004). Nevertheless, accidents involving snakes of the family Elapidae can also be severe or lethal (Bucaretchi et al., 2006; Norris et al., 2009). American elapid snakes (Coral Snakes) comprise the genera Leptomicrurus, Micruroides and Micrurus, which form a vast taxonomic assembly of 330 species distributed from the South of United States to the southern region of South
* Corresponding author. Tel.: þ506 25114962, þ506 25114934; fax: þ506 22920485. E-mail addresses: [email protected], [email protected] (A. Gómez). 1 Tel.: þ506 25114934; fax: þ506 22920485. 0041-0101/$ – see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.toxicon.2012.04.332
America (Campbell and Lamar, 1989; Roze, 1996; Greene, 1997; Stafford, 2000). According to Bolaños et al. (1978), the most important Coral Snakes, from a medical point of view, belong to the genus Micrurus, including Micrurus fulvius (United States), Micrurus diastema, Micrurus distans and Micrurus laticoralis (México and Guatemala); Micrurus nigrocinctus and Micrurus alleni (Central America); and Micrurus mipartitus, Micrurus corallinus, Micrurus frontalis, Micrurus spixii, Micrurus dumerilli carnicauda, Micrurus surinamensis and Micrurus isozonus (South America). Micrurus snake venoms are composed of several dozens of different molecules including predominantly phospholipases A2 (PLA2) and three-finger toxins (3FTXs) (such as a-neurotoxins), in addition to L-amino-acid oxidases (LAAOs), metalloproteinases and other components (Olamendi-Portugal et al., 2008; Fernández et al., 2011; Rey-Suárez et al., 2011). The 3FTXs in particular confer to the venom the ability to block the neuromuscular junctions
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and to induce paralysis of intercostal muscles and diaphragm. In severe envenomations, death is due to respiratory arrest (Bucaretchi et al., 2006). Such neurotoxic activity is due to the action of a-neurotoxins, although some venoms also display presynaptic neurotoxicity due to the action of PLA2s. Thus, these venoms induce signs and symptoms such as palpebral ptosis, ophtalmoplegia, paralysis of jaw, larynx and pharynx muscles, sialorrhea and paralysis of neck and limb muscles. Some Micrurus sp. venoms also show myotoxicity and cardiotoxicity when injected intravenously (Da Silva and Aird, 2001; Cecchini et al., 2005). Cardiotoxic, myotoxic, hemolytic, hemorrhagic and edematogenic manifestations upon envenoming have been also described in animal models as well as in patients bitten by different species of Coral Snakes (Corrêa-Netto et al., 2011). In order to counteract pathophysiological effects, medical treatment of snakebite envenomation is based on the parenteral administration of animal-derived antivenoms. Snake antivenoms are formulations of immunoglobulins or their F(ab’)2 or Fab fragments, purified from plasma of animals immunized with snake venoms (WHO, 2010; Gutiérrez et al., 2011). Because snake venom composition often varies significantly between species, antivenoms are able to neutralize only venoms antigenically related with those used in the antivenom preparation. In the case of Micrurus venoms, limited cross neutralization has been demonstrated in several studies (Keegan et al., 1961; Bolaños et al., 1973, 1975; Tanaka et al., 2010); nevertheless, protective action can be conferred by the use of different antivenoms produced from other species of Micrurus sp. or even other genera, to the particular case of murine models (Winiewski et al., 2003; Sánchez et al., 2008). In order to obtain venom for immunization, Coral Snakes must be kept in captivity and submitted periodically to venom extraction procedures. Each Micrurus specimen produces around 5 mg venom/extraction (Minton, 1957, 1974; Russell, 1967; Bolaños, 1972). Therefore, snake collections used to provide venom for industrial antivenom production must consist of numerous specimens. The development of a colony of snakes for venom production purposes has been reported for many institutions worldwide (Ashley and Burchfield, 1968). Usually, Coral Snakes feed only on natural prey, such as colubrid snakes belonging to the genera Geophis and Ninia (Da Silva et al., 2001; Urdaneta et al., 2004). In captivity, maintenance of a long-term collection is affected by a number of considerations including the quality of food and type provided (Kirkwood and Gili, 1994). Most Coral Snakes do not feed readily, and they generally die a few months after being captured (Serapicos and Merusse, 2002b). Although some herpetologists use a forced-feeding strategy (Fix and Minton, 1976), the survival rate of these specimens rarely exceeds one year. Thus, feeding Coral Snakes is a major problem to overcome in order to maintain a well-suitable collection to yield the necessary amount of venom to produce antivenom. Several studies have reported prey animals and stomach contents of Coral Snakes, which include caecilians, amphisbaenians, lizards and the fresh-water fish Synbranchus marmoratus (Schmidt, 1932; Cunha and Nascimento, 1978; Roze, 1982; Greene, 1984; Sazima and Abe, 1991).
Because of the impracticality of supplying the required quantities of these foods and the low survival rate of Coral Snakes that prompted us to search for a diet that is readily accepted by the snakes and that provides all the nutritional requirements to keep a well sustained colony. In this work, the suitability of a fish-based diet to supply the nutritional requirements to a M. nigrocinctus snake collection is described, as applied to a snake collection aimed at generating venom for the production of antivenom. 2. Materials and methods 2.1. Natural prey and feeding techniques Colubrid snakes of the genera Ninia sp. and Geophis sp. were kept at the Serpentarium of the Instituto Clodomiro Picado, until their use. These snakes were euthanized by CO2, and placed into plastic bags filled with water in order to freeze them. The bags were identified with the genus and the quantity of snakes and kept at 10 C until they were used. When Coral Snakes were fed, Ninia sp. and Geophis sp. snakes were thawed with water at room temperature; then, they were placed into a container with water in order to maintain turgor. After this, natural prey specimens were selected and offered to Coral Snakes according to their body weight and length. In detail, the natural prey was held with forceps and brought near the Coral Snake, moving it in a lifelike fashion in order to stimulate feeding behavior. Then, simulating snake-like movements, the natural prey was placed near the Coral Snake so that it could bite and hold it. 2.2. Fish-based diet preparation and feeding techniques Fillets of tilapia (Oreochromis sp.) fish were used; selecting those with proper texture, integrity and length. Basically, around 2 kg of tilapia fillets were placed and separated on a plastic dish and frozen for 24 h at 10 C; after this, the fillets were taken out and thawed at room temperature for about 20 min in order to cut them into longitudinal sections. The size of the sections depended on the weight and size of the specimens of Coral Snakes to be fed. The fillet sections were placed in a container with water at room temperature in order to completely defrost the meat. Lastly, in order to avoid some deficiencies in terms of calcium and vitamin D supply, we added a calcium supplement (RepCalÒ) to the fillets before offering them to the Coral Snakes. The elongate pieces of fish were grasped with forceps and brought near the Coral Snakes, simulating snake-like movements in order to stimulated the snakes; then, fish was left as close to the Coral Snake as possible. 2.3. Nutritional analysis The nutritional and compositional analyses were performed by Laboratorio de Servicios Analíticos of the School of Chemistry, University of Costa Rica (report No. 355-07). For these analyses, 50 g of both ground Geophis and Ninia snakes or 50 g of ground tilapia fillet were used. The samples were analyzed by gravimetry, volumetry and atomic absorption spectrometry for the quantification of protein and fat content, as well as for calcium and
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potassium, in addition to dry weight (ash) and moisture content (Skoog and Leary, 1994; Skoog et al., 1997). 2.4. Diet assessment The specimens of M. nigrocinctus used in this study were collected, mainly, from Santo Domingo, Heredia, and Coronado, San José, both localities in the Central Valley of Costa Rica. Likewise, the sample size studied was 92 individuals, separated in two groups: those assigned to the natural prey diet (n ¼ 33), and those corresponding to the fish-based diet (n ¼ 59). The snakes were fed every 15 days with a prey item according to the groups assigned. Thus, the gain/loss body weight rate (expressed as g/week) was defined as the difference between the initial and the final body weight recorded, divided by the period of time maintained in the study. The body weight was determined every 15 days, before the Coral Snakes were fed with either diet. Then, the gain/loss body weight rate was evaluated for both experimental groups, as described. Additionally, the longevity of the Coral Snakes was defined as the number of days that the snakes survived under both diets. Lastly, the venom yield was defined as the total amount of venom obtained from individual specimens assigned to these diets. 2.5. Biological and biochemical characterization of the venom Pools of venom were obtained from M. nigrocinctus snakes fed on either diet. The venoms were extracted every four months interval. These venoms were lyophilized and stored at 20 C until used. For the reverse-phase (RP) HPLC separation, 2 mg of venom were dissolved in 200 mL of 5% acetonitrile containing 0.1% trifluoroacetic acid, centrifuged for 5 min at 13,000 rpm, and loaded onto an Agilent Zorbax Eclipse Plus C18 Analytical column (4.6 250 mm, 5-Micron), using an Agilent 1100 Series chromatographer. Elution was performed at 1 mL/min as described by Fernández et al. (2011), and the absorbance at 215 nm was monitored. The Median Lethal Dose (LD50) test was conducted using venoms from the two experimental groups of Coral Snakes. Groups of six mice (16–18 g body weight, CD-1 strain) were injected with 500 mL of sterilized saline solution containing the venom by the intraperitoneal (i.p.) route. Assays were performed using six levels (3.5; 4.9; 6.86; 9.6; 13.44; 18.82 mg) with a dilution factor of 1/1.2 and deaths were recorded after 72 h (Bolaños et al., 1978). The results were calculated by Probits. Experiments were performed five times each venom and results expressed as mean SD. 2.6. Statistical analysis All the statistical tests were performed using SPSS 17.0 for Windows, and all P 0.05 values were considered significant. The LD50 values were obtained using the Probitos software. 3. Results and discussion 3.1. Nutritional analysis Chemical analyses revealed differences in calcium and protein concentrations between the two diets (Table 1).
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Table 1 Nutritional analysis of tilapia fillets and the natural prey of M. nigrocinctus. Fifty grams of fish or colubrid snakes (Ninia sp. and Geophis sp.), were used in the analysis. Analysis
Natural prey
Fat content (% m/m) Protein (% m/m) Calcium (mg/100 g) Potassium (mg/100 g) Ash (% m/m) Moisture content (% m/m)
7.5 2.0 1095.3 27.5 4.3 75.6
0.1 0.1 0.1a 0.1 0.1 0.5
Tilapia fillets 6.9 12.6 668.7 18.8 0.2 83.5
0.1 0.1a 0.5 0.1 0.1 0.5
a Indicates where the diets differed most regarding the compounds analyzed. All the values are expressed as mean standard deviation.
The natural prey had a higher calcium concentration than the fish fillets, whereas the latter had higher protein content. Considering that a fresh-water fish has been reported as part of the Coral Snakes’ diet, it makes sense to assume that an alternative diet based on fish will supply most of the nutrients needed by Coral Snakes. Potential deficiencies can be circumvented by the addition of an external source of calcium plus vitamin D3. 3.2. Diet assessment The assessment of the diets was based on the comparison and analysis of body weight gain, longevity and venom yielded by the snakes fed with either diet. The body weight gain for snakes fed on natural prey was 0.0778 0.5893 g/week, whereas that of snakes fed on fish-based diet was 0.264 0.6288 g/week. These results were not significantly different (Table 2) (F ¼ 0.038; df ¼ 1, 90; P ¼ 0.845). The body weight gain is a major factor to be considered, since the body mass affects the survival rate and the longevity (Kirkwood and Gili, 1994; Serapicos and Merusse, 2002a). On the other hand, the longevity was affected by the type of diet. Coral Snakes fed on natural prey survived 32 3 weeks, whereas those fed with fish diet survived 168 21 weeks (F ¼ 23.493; df ¼ 1, 90; P < 0.001, n ¼ 92). Thus, our observations on body weight gain and longevity support the view that a fish-based diet is highly successful as well as an useful strategy to maintain a large collection of M. nigrocinctus aimed at obtaining venom for antivenom production, without having to use colubrid snakes for feeding. Since the venom yield of Coral Snakes is rather low, the extension in survival time in captivity has a highly significant impact in venom production. Concerning the venom yielded by Coral Snakes when they were fed on either fish-based diet or natural prey diet, the former had a venom yield of 5 3 mg/individual (total protein content ¼ 1.8 0.1 g/dL), whereas the Coral Table 2 Effects of the two different diets on various parameters of M. nigrocinctus in captivity. Test
Natural prey diet
Fish-based diet
Body weight gain Venom yield Longevity LD50
0.0778 0.5893 g/week
0.264 0.6288 g/week
3 1 mg/individual 32 3 weeks 0.41 0.08 mg/g mouse
5 3 mg/individual 168 21 weeks 0.55 0.09 mg/g mouse
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3.3. Biological and biochemical venom characterization
Fig. 1. Coral Snake (Micrurus nigrocinctus) feeding on a tilapia fillet as an alternative diet. Notice that the snake bites toward the thinner end of the tilapia fillet.
Snakes fed on their natural prey had a venom yield of 3 1 mg/individual (total protein content ¼ 1.8 0.9 g/dL). Fish-fed snakes averaged higher venom yields, but these differences were not significantly different (t ¼ 1.363; df ¼ 7; P ¼ 0.215). These yields are comparable to previously reported yields for M. nigrocinctus and M. fulvius (Bolaños, 1972; Minton, 1957, 1974; Russell, 1967; Fix and Minton, 1976); however, these studies did not consider effect of diet on venom yields. Together with increased longevity effects, maintaining a colony of Coral Snakes on a fish-based diet guarantees an increased supply of venom for antivenom production (Fig. 1).
The elution profile of the venoms from Coral Snakes fed on either diet and separated by RP-HPLC revealed nearly 50 peaks (Fig. 2). The chromatographic profiles of both venoms were very similar to each other and to the pattern previously described for this species’ venom by Fernández et al. (2011). Thus, variations in diet do not seem to modify the overall venom composition. In agreement with previous results of Bolaños (1972), the median lethal dose (LD50) obtained for the venom from snakes fed on natural prey was 0.41 0.08 mg/g mouse, whereas the LD50 of the venom from snakes fed on a fishbased diet was 0.55 0.09 mg/g mouse (Table 2); this difference was not statistically significant (t ¼ 2.15, df ¼ 6, P ¼ 0.075). Likewise, these values of LD50 do not differ from those previously reported by León et al. (1999), and from those regularly obtained at the Quality Control Laboratory of Instituto Clodomiro Picado (data not shown). These findings indicate that overall venom toxicity is not affected by the shift in diet. In conclusion, our observations on two groups of M. nigrocinctus fed with different diets, one based on their natural prey (colubrid snakes) and the other based on fish, indicate that these two groups do not differ significantly in terms of body weight gain, venom yield, venom HPLC pattern and LD50; further, snakes fed with fish showed a significantly higher survival time in captivity. These results support the use of a fish-based diet for the feeding of a collection of M. nigrocinctus. The prolonged life span of these specimens in captivity and the stable characteristics of the venom indicate that this fish-based diet is an important innovation for maintaining Coral Snakes in
Fig. 2. Chromatographic profiles of Micrurus nigrocinctus venom proteins by RP-HPLC. Two milligrams of pooled venoms were applied to a C18 column, as described in Materials and Methods (gradient line was omitted for clarity). A) Elution profile of M. nigrocinctus venom proteins fed on its natural prey. B) Elution profile of M. nigrocinctus venom proteins fed on the fish-based diet.
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captivity for venom production used in antivenommanufacturing laboratories. Acknowledgments This study was supported by Vicerrectoría de Investigación, Universidad de Costa Rica (project 741-A09-003). The authors thank our colleagues at Instituto Clodomiro Picado for their collaboration. Conflict of interest There are no conflicts of interest. References Ashley, B.D., Burchfield, P.M., 1968. Maintenance of a snake colony for the purpose of venom extraction. Toxicon 5, 267–275. Bolaños, R., 1972. Toxicity of Costa Rican snake venoms for the white mouse. Am. J. Trop. Med. Hyg. 21, 360–361. Bolaños, R., Cerdas, L., Taylor, R., 1973. Estudios inmunológicos de los venenos de las principales Micrurus de Norte América, Centro América, Panamá y Colombia. In: Trabajo presentado en el 3er Congreso Latinoamericano de Parasitología y 3er Congreso Colombiano de Parasitología y Medicina Tropical. Medellín, Colombia, 9–12 de diciembre. Bolaños, R., Cerdas, L., Taylor, R., 1975. The production and characteristics of a Coral snake (Micrurus mipartitus hertwigi) antivenin. Toxicon 13, 139–142. Bolaños, R., Cerdas, L., Abalos, J.W., 1978. Venenos de serpientes coral (Micrurus spp.): informe sobre un antiveneno polivalente polivalente para las Américas. Bol Of. Sanit Panam 84 (2), 128–133. Bucaretchi, F., Hyslop, S., Vieira, R.J., Toldedo, A.S., Madureira, P.R., de Capitani, E.M., 2006. Bites by coral snakes (Micrurus spp.) in Campinas, state of São Paulo, Southeastern Brazil. Rev. Inst. Med. Trop. S. Paulo 48, 141–145. Campbell, J.A., Lamar, W.W., 1989. The Venomous Reptiles of Latin America. Cornell University Press, Ithaca and New York. Cecchini, A.L., Marcussi, S., Silveira, L.B., Borja-Oliveira, C.R., RodriguesSimoni, L., Amara, S., Stábeli, R.G., Giglio, J.R., Arantes, E.C., Soares, A. M., 2005. Biological and enzymatic activities of Micrurus (Coral) snake venoms. Comp. Biochem. Physiol. Part A 140, 125–134. Corrêa-Netto, C., Junqueira-de-Azevedo, I.L.M., Silva, D.A., Leitáo-deAraújo, A., Alves, M.L.M., Sanz, L., Foguel, D., Zingali, R.B., Calvete, J.J., 2011. Snake venomics and venom gland transcriptomic analysis of Brazilian coral snakes Micrurus altirostris and M. corallinus. J. Prot. 74 (9), 1795–1809. Cunha, O.R., Nascimento, F.P., 1978. Ofidios de Amazônia. X. As cobras de região leste do Pará, Belem. Mus. Par. Emilio Goeldi Publ. Avuls 31, 1–128. Da Silva, N.J., Aird, S.D., 2001. Prey specifity, comparative lethality and compositional differences of coral snakes venoms. Comp. Biochem. Physiol. Part C 128, 425–456. Da Silva, A.R., Prieto, Yamagushi, I.K., Morais, J.F., Higashi, H.G., Raw, I., Ho, P.L., de Oliveira, J.S., 2001. Cross reactivity of different specific Micrurus antivenom sera with homologous and heterologous snakes venoms. Toxicon 34, 949–953. Fernández, J., Alape-Girón Alberto, Angulo, Y., Sanz Libia, Gutiérrez, J.M., Calvete, J.J., Lomonte, B., 2011. Venomic and antivenomic analyses of the Central America Coral Snake, Micrurus nigrocinctus (Elapidae). J. Prot. Res. 10, 1816–1827. Fix, J.D., Minton, S.A., 1976. Venom extraction and yields from the North American coral snake, Micrurus fulvius. Toxicon 14, 143–145. Greene, H.W., 1984. Feeding behavior and diet of the Eastern coral snake, Micrurus fulvius. In: Seigel, R.A., Hunt, L.E., Knight, J.L., Malaret, L., Zuschlag, N.L. (Eds.), Vertebrate Ecology and Systematics – A Tribute to Henry S. Fitch. Museum of Natural History, University of Kansas, Lawrence, pp. 147–161. Greene, H.W., 1997. Snakes: The Evolution of Mystery in Nature. University of California Press, Berkeley, California. Gutiérrez, J.M., León, G., Lomonte, B., Angulo, Y., 2011. Antivenoms for snake envenomings. Inflamm. Allergy Drug Targets 10, 369–380.
253
Kasturiratne, A., Wickremasinghe, A.R., de Silva, N., Gunawardena, N.K., Pathmeswaran, A., Premaratna, R., Saviolo, L., Lalloo, D.G., de Silva, H.J., 2008. The global burden of snakebite: a literature analysis and modelling based on regional estimates of envenoming and deaths. PLoS Med. 5, e218. Keegan, H.L., Whittemore, F.W., Flanigan, J.F., 1961. Heterologous antivenin in neutralization of North American coral snake venom. Public Health Rep., Wash 76, 540. Kirkwood, J.K., Gili, C., 1994. Food consumption in relation to bodyweight in captive snakes. Res. Vet. Sci. 57, 35–38. León, G., Stiles, B., Alape, A., Rojas, G., Gutiérrez, J.M., 1999. Comparative study on the ability of IgG and F(ab’)2 antivenoms to neutralize lethal and myotoxic effects Induced by Micrurus nigrocinctus (coral snake) venom. Am. J. Trop. Med. Hyg. 6 (2), 266–271. Minton, S.A., 1957. An immunological investigation of rattlesnake venoms by the agar diffusion method. Am. J. Trop. Med. Hyg. 6 Minton, S.A., 1974. Venom Diseases. Charles C. Thomas, Springfield, 1–235 pp. Norris, R.L., Pfalzgraf, R.R., Laing, G., 2009. Death following coral snake bite in the United States: first documented case (with ELISA confirmation of envenomation) in over 40 years. Toxicon 53, 693–697. Olamendi-Portugal, T., Batista, C.V.F., Restano-Cassulini, R.R., Pando, V., Villa-Hernandez, O., Zavaleta-Martínez, A., Salas-Arruz, M.C., Rodríguez, R.C., Becerril, B., Posan, L.D., 2008. Proteomic analysis of the venoms from the fish eating coral snake Micrurus surinamensis: novel toxins, their function and phylogeny. Proteomics 8, 1919–1932. Rey-Suárez, P., Núñez, V., Gutiérrez, J.M., Lomonte, B., 2011. Proteomic and biological characterization of the venom of the redtail coral snake, Micrurus mipartitus (Elapidae), from Colombia and Costa Rica. J. Proteomics 75 (2), 655–667. Roze, A.J., 1982. New World coral snakes (Elapidae): a taxonomic and biological summary. Mem. Inst. Butantan 46, 305–338. Roze, A.J., 1996. Coral Snakes of the America: Biology, Identification and Venoms. Krieger Publ. Comp. Malabar, Florida. Russell, F.E., 1967. Bites by the sonoran coral snake Micruroides euryxanthus. Toxicon 5 (1), 39–42. Sánchez, E.E., Lopez-Johnston, J.C., Rodríguez-Acosta, A., Pérez, J.C., 2008. Neutralization of two North American coral snake venoms with United States and Mexican antivenoms. Toxicon 51, 297–303. Sazima, I., Abe, A.S., 1991. Habits of five Brazilian snakes with coral-snakes pattern, including a summary of defensive tactics. Stud. Neotrop. Fauna Environ. 26, 159–164. Schmidt, K.P., 1932. Stomach contents of some American coral snakes, with the description of a new species of Geophis. Copeia 1, 6–9. Serapicos, E.O., Merusse, J.L.B., 2002a. Variação de peso e sobrevida de Micrurus corallinus sobre diferentes condições de alimentação em biotério (Serpentes, Elapidae). Iheringia, Sér. Zool., Porto Alegre 92 (4), 105–109. Serapicos, E.O., Merusse, J.L.B., 2002b. Análise comparativa entre tipos de alimentação de Micrurus corallinus (Serpentes, Elapidae) em cautiverio. Iheringa, Sér. Zool., Porto Alegre. 92 (4), 99–103. Skoog, D.A., Leary, J.J., 1994. Análisis Instrumental, fourth ed. Editorial MacGraw-Hill, Madrid. Skoog, D.A., West, D.M., Holler, F.J., 1997. Fundamentos de Química Analítica, 4ta ed., Tomo I. Editorial Reverté, Barcelona. Stafford, P.J., 2000. On the status of the coral snake Micrurus nigrocinctus (Serpentes, Elapidae) in Belize, and its Northernmost distribution in Atlantic Middle America. Herpetological Review 31 (2), 78–82. Tanaka, G.D., Furtado, M.F.D., Portaro, F.V.C., Sant’Anna, O.A., Tambourgi, D.V., 2010. Diversity of Micrurus snake species related to their venom toxic effects and the prospective of antivenom neutralization. PLos Negl. Trop. Dis. 4 (3), e622. Urdaneta, A.H., Bolaños, F., Gutiérrez, J.M., 2004. Feeding behavior and venoms toxicity of coral snake Micrurus nigrocinctus (Serpentes: Elapidae) on its natural prey in captivity. Comp. Biochem. Physiol. 138, 485–492. Warrell, D.A., 2004. Epidemiology, clinical features and management of snake bites in Central and South America. In: Campbell, J., Lamar, W.W. (Eds.), Venomous Reptiles of the Western Hemisphere. Cornell University Press, Ithaca, pp. 709–761. WHO, 2010. Guidelines for the Production Control and Regulation of Snake Antivenom Immunoglobulins. Geneva, Switzerland. Winiewski, M.S., Hill, R.E., Havey, J.M., Bogdan, G.M., Dart, R.C., 2003. Australian tiger snake (Notechis scutatus) and Mexican coral snake (Micrurus species) antivenoms prevent death from United States coral snake (Micrurus fulvius fulvius) venom in a mouse model. J. Toxicol. Clin. Toxicol. 41 (1), 7–10.