Immune Maturation Effects on Viral Neutralization and Avidity of Hyperimmunized Equine Anti-SARS-CoV-2 Sera.

Mass-vaccination against COVID-19 is still a distant goal for most low-to-middle income countries. The experience gained through decades producing polyclonal immunotherapeutics (such as antivenoms) in many of those countries is being redirected to develop similar products able to neutralize SARS-CoV-2 infection. In this study we analyzed the biological activity (viral neutralization or NtAb) and immunochemical properties of hyperimmune horses' sera (HHS) obtained during initial immunization (I) and posterior re-immunization (R) cycles using the RBD domain of the SARS-CoV-2 spike protein as antigen. HHS at the end of the R cycle showed higher NtAb titers when compared to those after the I cycle (35,585 vs. 7000 mean NtAb, respectively). Moreover, this increase paralleled an increase in avidity (95.2% to 65.2% mean avidity units, respectively). The results presented herein are relevant for manufacturers of these therapeutic tools against COVID-19.

Cross-reactivity of some Micrurus venoms against experimental and therapeutic anti-Micrurus antivenoms.

We developed experimental equine polyvalent and monovalent antivenoms against the venoms of Micrurus (M.) fulvius, M. nigrocinctus and M. surinamensis and studied their immunochemical reactivity on the venoms used as immunogens and on M. pyrrhocryptus, M altirostris and M. balyocoriphus venoms. Assessment of the neutralizing capacity of the polyvalent experimental antivenom was based on inhibition of lethality (preincubation and rescue assay experiments in mice) and indirect hemolytic and phospholipase activities. The immunochemical reactivity and neutralizing capacity were compared with those of two therapeutic antivenoms used for the treatment of coral snake envenomation in North America and in Argentina. In general, the experimental antivenom conferred a comparable level of neutralization against the venoms used as immunogens when compared to the therapeutic antivenoms and a certain level of cross-neutralization against the other venoms. The results suggest the need for additional venoms in the immunogenic mixture used, in order to obtain a broad spectrum anti-Micrurus antivenom with a good neutralizing potency. Paraspecific neutralization of South American coral snake venoms, although present at a higher level than the neutralization conferred by available nonspecific Micrurus therapeutic antivenoms, was rather low in relation to the specific neutralizing capacity.

Biotechnological potential of Phospholipase D for Loxosceles antivenom development.

Loxoscelism is one of the most important forms of araneism in South America. The Health Authorities from countries with the highest incidence and longer history in registering loxoscelism cases indicate that specific antivenom should be administered during the first hours after the accident, especially in the presence or at risk of the most severe clinical outcome. Current antivenoms are based on immunoglobulins or their fragments, obtained from plasma of hyperimmunized horses. Antivenom has been produced using the same traditional techniques for more than 120 years. Although the whole composition of the spider venom remains unknown, the discovery and biotechnological production of the phospholipase D enzymes represented a milestone for the knowledge of the physiopathology of envenomation and for the introduction of new innovative tools in antivenom production. The fact that this protein is a principal toxin of the venom opens the possibility of replacing the use of whole venom as an immunogen, an attractive alternative considering the laborious techniques and low yields associated with venom extraction. This challenge warrants technological innovation to facilitate production and obtain more effective antidotes. In this review, we compile the reported studies, examining the advances in the expression and application of phospholipase D as a new immunogen and how the new biotechnological tools have introduced some degree of innovation in this field.

Study on the obtaining of Tityus trivittatus venom in Argentina.

Envenomation by scorpions of the genus Tityus is an important public health problem in Argentina, involving near 8000 stings and 2 deaths each year. Treatment for envenomation is the use of specific antivenom and intensive hospital care. Antivenom is produced by the Ministry of Health and freely distributed throughout the country. For antivenom production it is necessary to collect scorpion venom, which is a difficult task because although scorpions can be found in Argentina, they are less abundant than in warmer latitudes. For this reason venom collection constitutes a bottleneck for antivenom production. Although in Argentina several species of Tityus can be found, most of the accidents are caused by Tityus trivittatus, and the venom of this scorpion has historically been the venom used for antivenom production. We analyzed retrospectively 26 pools of telson homogenates (6964 telsons) and 37 pools of milked venom obtained by electrical stimulation (equivalent to 6841 milkings). Lethal potencies of samples from different provinces were very similar, although venom from scorpions of Buenos Aires city showed the lowest potency. The venom obtained by milking (median LD50 12.3 µg), provided batches containing LD50s more potent when compared with the venom obtained from telson homogenates (p < 0.0001). Many batches of telson homogenates (30%) showed lower potencies than acceptable for antivenom production and control. In addition to the study of the venom yield, the records of immunization of horses, the potency of the batches and the protein content of each batch of anti-scorpion antivenom produced were analyzed, comparing those produced using milked venom with those using telson homogenates as immunogens. Batches produced using milked venom required a shorter period of immunization (p < 0.0001), rendered higher neutralizing titers (p 0.0350) and possessed lower protein content (p 0.0092). Results clearly showed that the milking of scorpions is a more efficient tool to obtain venom for antivenom production in comparison to the use of telson homogenates.

New insights into the proteomic characterization of the coral snake Micrurus pyrrhocryptus venom.

A proteomic analysis of the soluble venom of the coral snake Micrurus pyrrhocryptus is reported in this work. The whole soluble venom was separated by RP-HPLC and the molecular weights of its components (over 100) were determined by mass spectrometry. Three main sets of components were identified, corresponding to peptides with molecular masses from 5 to 8 kDa, proteins from 12 to 16 kDa and proteins from 20 to 30 kDa. Two components were fully sequenced: one α-neurotoxic peptide of 7210 Da with slight blocking activity of the nicotinic acetylcholine receptor (nAChR) and a phospholipase A2 (PLA2) with molecular weight 13517 Da and no effect on the nAChR. PLA2 activity was evaluated for all RP-HPLC components. In addition, N-terminal sequence was obtained for eleven components using Edman degradation. Among these, three were similar to known PLA2's, six to three-finger toxins (3FTx) and one to Kunitz-type serine protease inhibitors. Two-dimensional gel electrophoresis of the venom allowed the separation of about thirty spots with components of molecular weights from 25 to 70 kDa. Seventeen spots were recovered from the gel, digested with trypsin and the corresponding peptides (85) were sequenced by MS/MS allowing identification of amino acid sequences with similarities to snake venom metalloproteases (SVMP), PLA2's, L-amino acid oxidases (LAAO), acetylcholinesterases (AChE) and serine proteases (SP). In addition, LC-MS analysis of peptides obtained from tryptic digestion of whole soluble venom allowed the identification of 695 peptides, whose amino acid sequence could correspond to at least 355 components found in other snake venoms, where C-type lectins, vespryns, zinc finger proteins, and waprins were found, among others. These results show the complexity of the venom and provide important knowledge for future work on identification and activity determination of venom components from this coral snake.

Antivenom against Crotalus durissus terrificus venom: Immunochemical reactivity and experimental neutralizing capacity.

The venom of Crotalus durissus terrificus produces a neurotoxic and myotoxic syndrome that can lead to the death. Specific antivenom is the only treatment to neutralize the toxicity of the venom and the precocity in applying the antivenom is crucial for the efficiency of the treatment. We studied the variation of the immunochemical reactivity and neutralizing capacity of the specific antivenom on this venom in pre-incubation and rescue experiments, at different times. ELISA titers increased with longer venom-antivenom incubation times (p < 0.05) nevertheless incubation times had no effect on the neutralizing capacity of the antivenom. The antivenom dose necessary to rescue mice injected with 1.5 MMD (minimal mortal dose) 30 min after venom inoculation was over ten folds the dose of antivenom theoretically required to neutralize the same dose of venom according values obtained from pre-incubation experiments. Results showed that the in vitro immunochemical reactivity is not directly related to the neutralizing capacity. These observations underline the need for a rapid antivenom administration. Although preincubation experiments in mice are a powerful tool for the validation of the potency of the antivenoms in the productive process, it is clear that the nominal neutralization of the antivenoms must not be considered as a "stoichiometric" value regarding the venom to be neutralized in case of natural envenomation and emphasize the need of realization of clinical trials in order to evaluate the adequate doses of antivenom to be therapeutically used.

Accidentes y muertes por animales venenosos en Argentina durante el período 2000- 2011 / Accidents and deaths by venomous animals in Argentina during the period 2000-2011

En Argentina los accidentes causados por animales venenosos constituyen un problema de salud pública. En este estudio analizamos los accidentes y muertes por envenenamientos por mordedura o picadura de serpientes, escorpiones y arañas, y las muertes causadas por abejas- avispas-avispones, centípedos, animales marinos, otros artrópodos y otros animales venenosos en Argentina, recibidos por las autoridades sanitarias nacionales. Se analizó la información sobre accidentes y muertes producidas por este tipo de animales que fueron recibidas por el Ministerio de Salud de la Nación durante el período 2000-2011. Debido al tipo de sistema de notificación solo se pudieron calcular tasas de morbilidad y letalidad en los accidentes por arañas, escorpiones y serpientes. Contrariamente a lo que se suponía, no fueron estos los grupos que mayor cantidad de muertes causaron en el período de estudio, sino los himenópteros (abejas-avispas- avispones). El actual esquema de notificación de envenenamiento por animales venenosos provee información importante para el sistema sanitario. Sin embargo, deben realizarse modificaciones a fin de mejorar el conocimiento epidemiológico referente a envenenamientos por la mordedura o picadura de animales venenosos. De esta manera, se podrá progresar en la implementación de las medidas de prevención de estas patologías, su atención a nivel del primer nivel asistencial y la planificación de la producción de antivenenos.

In Argentina, accidents by venomous animals are a problem of public health. In this study we analyzed the envenomation caused by the bite or sting of snakes, scorpions and spiders and the deaths produced by the envenomation caused by this animals and the deaths caused by bees-wasp-hornet, centipedes, marine animals, other arthropods and other venomous animals in Argentina, recorded by the National Ministry of Health in the period 2000 ­ 2011. Most of the accidents were caused by scorpions nevertheless the highest lethality was observed in snakebites envenomation. Contrary to the general suppositions, hymenoptera (bee-wasp- hornet) were the responsible for the highest number of deaths. The present system of information of envenomation by venomous animals gives important information. However some modifications should be done to achieve a best knowledge of the epidemiological situation of the envenomation caused by bites or sting of the different venomous animals, in order to improve the prevention measures as well as the attention at the first level of sanitary assistance and the planning of antivenom production.

Toxicity of the venom of Latrodectus (Araneae: Theridiidae) spiders from different regions of Argentina and neutralization by therapeutic antivenoms.

"Black widow" spiders belong to the genus Latrodectus and are one of the few spiders in the world whose bite can cause severe envenomation in humans and domestic animals. In Argentina, these spiders are distributed throughout the country and are responsible for the highest number of bites by spiders of toxicological sanitary interest. Here, we studied the toxicity and some biochemical and immunochemical characteristics of eighteen venom samples from Latrodectus spiders from eight different provinces of Argentina, and the neutralization of some of these samples by two therapeutic antivenoms used in the country for the treatment of envenomation and by a anti-Latrodectus antivenom prepared against the venom of Latrodectus mactans from Mexico. We observed important toxicity in all the samples studied and a variation in the toxicity of samples, even in those from the same region and province and even in the same Latrodectus species from the same region. The therapeutic antivenoms efficiently neutralized all the venoms studied.

Lethality and histopathological alterations caused by Phoneutria nigriventer spider venom from Argentina: Neutralization of lethality by experimental and therapeutic antivenoms.

Although the spiders of the genus Phoneutria cause envenomation and their presence has been described in several provinces of the north of Argentina, they are not as common as other spiders of sanitary importance. In the present work, we studied the toxicity of samples of venom of Phoneutria spiders from the provinces of Misiones (where severe envenomation and deaths by Phoneutria have been recorded) and Jujuy (where no deaths have been recorded and severe envenomations are not frequent). To this end, we assessed the lethal potency in mice and guinea pigs and the histopathological alterations caused by both venoms, as well as the neutralization by the commonly used therapeutic antivenom produced by the Butantan Institute in Brazil and by an experimental antivenom developed with venom of P. nigriventer from Misiones. There were no differences in the lethality of the venoms of spiders from both regions. Post mortem examination showed that the heart and lungs were the most affected organs, while important pulmonary edema was seen macroscopically. Histological analysis showed edema, atelectasis, emphysema and cardiac lesion in both experimental models. The antivenoms assayed showed good neutralization of the venoms in the two experimental models. Despite the different geographic origins, the venoms showed similar toxicity and both the experimental antivenom and therapeutic antivenmos were able to neutralize the venoms of Argentinean P. nigriventer.

Cross neutralization of coral snake venoms by commercial Australian snake antivenoms.

CONTEXT: Although rare, coral snake envenomation is a serious health threat in Brazil, because of the highly neurotoxic venom and the scarcely available antivenom. The major bottleneck for antivenom production is the low availability of venom. Furthermore, the available serum is not effective against all coral snake species found in Brazil. An alternative to circumvent the lack of venom for serum production and the restricted protection of the actually available antivenom would be of great value. We compared the Brazilian coral snake and mono and polyvalent Australian antivenoms in terms of reactivity and protection. METHODS: The immunoreactivity of venoms from 9 coral snakes species were assayed by ELISA and western blot using the Brazilian Micrurus and the Australian pentavalent as well as monovalent anti-Notechis, Oxyuranus and Pseudechis antivenoms. Neutralization assays were performed in mice, using 3 LD50 of the venoms, incubated for 30 minutes with 100 µL of antivenom/animal. DISCUSSION: All the venoms reacted against the autologous and heterologous antivenoms. Nevertheless, the neutralization assays showed that the coral snake antivenom was only effective against M. corallinus, M. frontalis, M. fulvius, M. nigrocinctus and M. pyrrhocryptus venoms. On the other hand, the Australian pentavalent antivenom neutralized all venoms except the one from M. spixii. A combination of anti-Oxyuranus and Pseudechis monovalent sera, extended the protection to M. altirostris and, partially, to M. ibiboboca. By adding Notechis antivenom to this mixture, we obtained full protection against M. ibiboboca and partial neutralization against M. lemniscatus venoms. CONCLUSIONS: Our findings confirm the limited effectiveness of the Brazilian coral snake antivenom and indicate that antivenoms made from Australian snakes venoms are an effective alternative for coral snake bites in South America and also in the United States were coral snake antivenom production has been discontinued.

Venom yield and its relationship with body size and fang separation of pit vipers from Argentina.

The amount of venom that a snake can inject is related to its body size. The body size is related to head size and to the distance between fangs. To correlate snake body size, distance between fangs and distance between puncture wounds with the venom yield (and consequently with the venom dose potentially injected in a single snakebite), we studied these variables in two species of public health importance in South America, Bothrops (Rhinocerophis) alternatus, and Crotalus durissus terrificus. In all cases a positive correlation was observed between body length, fang separation distance, distance between puncture wounds and venom yield, with a regression coefficient over 0.5 for Bothrops alternatus and over 0.6 for Crotalus durissus terrificus in all cases, being the relation distance between punctures wounds and venom yield of 0.54 and 0.69 respectively. The difference between fang separation and puncture separation was never greater than 30%, with a mean difference around 13%. The strong relationships between body size, fang separation and venom yield may be useful for planning potential venom production in serpentariums. In addition, because puncture mark separation gives an approximate idea of the size of the snake, this provides a rough idea of the size of the snake that produced a bite and the potential amount of venom that could have been injected.

Veneno de escorpiones (alacranes) y envenenamiento / Scorpion (alacran) venom and envenomation / Veneno de escorpiÒo (alacran) e envenenamento

En las últimas décadas en Argentina se han incrementado de forma muy importante las notificaciones por picadura de escorpiones y de muertes debido a éstas. En el período 1993-1999 se comunicaron 3 muertes y en el período 2000-2006 se registraron 19, haciendo que el envenenamiento por estos arácnidos se haya transformado en uno de los emponzoñamientos de mayor impacto sanitario en Argentina. En esta breve revisión se describen algunos de los mecanismos tóxicos conocidos y propuestos para el envenenamiento por escorpiones, así como algunos de los componentes conocidos del veneno de Tityus trivittatus, el escorpión de mayor importancia médica de Argentina.(AU)

In the last decades the number of notifications by scorpion stings and deaths by scorpion envenoming increased very significantly in Argentina. In the period 1993-1999 three deaths were reported while in the period 2000-2006, nineteen deaths were registered, turning the envenomation by these arachnids in one of the envenoming of highest sanitary impact in Argentina. In this brief revision some of the known and proposed toxic mechanisms for scorpion envenomation as well as some of the venom discovered components in the venom of Tityus trivittatus the scorpion of highest medical importance in Argentina, are described.(AU)

Nas últimas décadas na Argentina aumentaram de forma muito significativa notificaþ§es de picadas de escorpiÒo e de mortes devido a elas. No período 1993- 1999 trÛs mortes foram registradas e no período 2000-2006 foram 19, fazendo com que o envenenamento por estes aracnídeos se tenha transformado num dos envenenamento de maior impacto sanitário na Argentina. Nesta breve revisÒo sÒo descritos alguns dos mecanismos tóxicos conhecidos e propostos para o evenenamento por escorpi§es, bem como alguns dos componentes conhecidos do veneno do Tityus trivittatus, o escorpiÒo de mayor importÔncia médica da Argentina.(AU)

Identificación de los escorpiones de importancia médica en la Argentina / Identification of scorpions with medical importance in Argentina

. Los escorpiones de importancia médica en el mundo pertenecen mayoritariamente a la familia Buthidae. En Argentina, los miembros de esta familia no son numerosos, siendo mucho mayor el número de especies de la familia Bothriuridae, cuyos miembros hasta el momento no revisten importancia médica. Dentro de los tres géneros de Buthidae que viven en el país, el género Tityus es el que posee especies cuya picadura e inoculación de veneno puede provocar accidentes severos. En general, la diferenciación entre Tityus trivittatus, el escorpión de mayor importancia médica de Argentina, respecto a otros escorpiones, es fácil en las provincias de la zona central del país. Sin embargo, las reglas a utilizar para la diferenciación entre escorpiones de importancia médica de aquellos que no la tienen, no son similares en todo el país. En esta actualización, se brindan elementos sencillos y claros para que el personal de salud pueda diferenciar entre las especies de Tityus de importancia médica de los escorpiones de la familia Bothriuridae, ampliamente distribuidos en la superficie del país. De esta forma, se espera que el diag­nóstico acertado sobre la especie de escorpión pueda mejorar la atención en el caso de la picadura por animales potencialmente peligrosos. Por otro lado, permitirá diferenciar a los escorpiones sin importancia médica lo que redundará en la mejor utilización de los recursos disponibles en los niveles de atención primaria, ante la emergencia que representa una picadura de alacrán.

The scorpions of medical importance in the world mostly belong to Buthidae family. In Argentina, members of this family are not numerous, being much most numerous the species from the Bothriuridae family, whose members so far are of no medical importance. Within the three genera of Buthidae that inhabit in the country, only species from Tityus genus can cause severe ac­cidents. The differentiation between the scorpions of highest medical importance in Argentina (Tityus trivittatus), regarding other scorpions, is easy in the provinces of the central region. However, the rules used for the differentiation of medically important scorpions regarding those not dangerous, is not similar through the country. In this update, we provide simple and clear tools for the health personnel to differentiate between species of Tityus with medical importance and the scorpions from Bothriuridae fam­ily, widely distributed in the country. Thus it is expected that the accurate diagnosis of the species of scorpions may improve the attention in the case of the sting of potentially dangerous scorpions. On the other hand, the differentiation of the not dangerous scorpions from those with medical importance will result in the best use of the available resources in primary care level, consider­ing the emergency that represents a scorpion sting.

Identificación de los escorpiones de importancia médica en la Argentina / Identification of scorpions with medical importance in Argentina

. Los escorpiones de importancia médica en el mundo pertenecen mayoritariamente a la familia Buthidae. En Argentina, los miembros de esta familia no son numerosos, siendo mucho mayor el número de especies de la familia Bothriuridae, cuyos miembros hasta el momento no revisten importancia médica. Dentro de los tres géneros de Buthidae que viven en el país, el género Tityus es el que posee especies cuya picadura e inoculación de veneno puede provocar accidentes severos. En general, la diferenciación entre Tityus trivittatus, el escorpión de mayor importancia médica de Argentina, respecto a otros escorpiones, es fácil en las provincias de la zona central del país. Sin embargo, las reglas a utilizar para la diferenciación entre escorpiones de importancia médica de aquellos que no la tienen, no son similares en todo el país. En esta actualización, se brindan elementos sencillos y claros para que el personal de salud pueda diferenciar entre las especies de Tityus de importancia médica de los escorpiones de la familia Bothriuridae, ampliamente distribuidos en la superficie del país. De esta forma, se espera que el diag¡nóstico acertado sobre la especie de escorpión pueda mejorar la atención en el caso de la picadura por animales potencialmente peligrosos. Por otro lado, permitirá diferenciar a los escorpiones sin importancia médica lo que redundará en la mejor utilización de los recursos disponibles en los niveles de atención primaria, ante la emergencia que representa una picadura de alacrán.(AU)

The scorpions of medical importance in the world mostly belong to Buthidae family. In Argentina, members of this family are not numerous, being much most numerous the species from the Bothriuridae family, whose members so far are of no medical importance. Within the three genera of Buthidae that inhabit in the country, only species from Tityus genus can cause severe ac¡cidents. The differentiation between the scorpions of highest medical importance in Argentina (Tityus trivittatus), regarding other scorpions, is easy in the provinces of the central region. However, the rules used for the differentiation of medically important scorpions regarding those not dangerous, is not similar through the country. In this update, we provide simple and clear tools for the health personnel to differentiate between species of Tityus with medical importance and the scorpions from Bothriuridae fam¡ily, widely distributed in the country. Thus it is expected that the accurate diagnosis of the species of scorpions may improve the attention in the case of the sting of potentially dangerous scorpions. On the other hand, the differentiation of the not dangerous scorpions from those with medical importance will result in the best use of the available resources in primary care level, consider¡ing the emergency that represents a scorpion sting.(AU)

Intraspecific differences in the immunochemical reactivity and neutralization of venom from Argentinean Bothrops (Rhinocerophis) alternatus by specific experimental antivenoms.

The venoms of Bothrops (Rhinocerophis) alternatus (B.a.) from different regions of Argentina have shown biochemical, toxicological and immunological variations. Considering these variations, we produced nine experimental antisera (rabbit, IgG) against venoms from snakes of nine different regions and a pool of venom, comprised of equal amounts of venoms from each region. The immunologic studies (ELISA, Westernblot) showed significant cross reactivity among all regional antivenoms with all regional venoms, with no significant differences regarding the specificity of the immunogens used for the production of antivenom. Neutralization of hemorrhage was variable (although all the antivenoms neutralized this activity in all venoms) and the neutralization of coagulant and phospholipase activities were evident in all cases. Some antivenoms neutralized toxic activities that were absent or very low in the venoms used as immunogen, on other non-homologous venoms (e.g. thrombin like activity). Despite the different toxic potencies of regional venoms, antivenoms developed using venoms of snakes from a particular region showed high immunochemical reactivity and cross-neutralizing capacity on snake venoms from different and distant regions, in occasions over those of the homologous antivenoms. These findings could be used to improve the generation of pools of venoms for the production of antivenoms.

Comments on Environmental and Sanitary Aspects of the Scorpionism by Tityus trivittatus in Buenos Aires City, Argentina.

Deaths by venomous animals are medical emergencies that can lead to death and thus constitute sanitary problems in some regions of the world. In the South of America, the accidents by these animals are a common sanitary problem especially in warm, tropical or subtropical regions, related with rural work in several countries. Argentina is located in the extreme South of South America and a minor part of the continental surface is in tropical or subtropical regions, where most of the accidents by venomous animals happen. However, in the big cities in the center and South of the country, with no relation to rural work, scorpionism, mostly due to the synanthropic and facultative parthenogenetic scorpion Tityus trivittatus, has become a sanitary problem in the last few decades. This scorpion is present in the biggest cities of Argentina and in the last decades has killed over 20 children in provinces of the center and north of the country, mostly in big cities. In addition, it seems that this species is growing and spreading in new regions of the cities. In this revision, some characteristics of this scorpion regarding its habitat, spreading in Buenos Aires city, combat measures and available treatments are discussed.

Epidemiology of snakebite and use of antivenom in Argentina.

BACKGROUND: The incidence and case fatality rate of snakebite in Argentina are poorly known. METHODS: The authors used questionnaires provided with antivenoms by the primary manufacturer of anti-venoms in Argentina. RESULTS: A total of 8083 completed questionnaires was collected between 1978 and 1998. The annual incidence of snakebite was 1.8 bites per 100,000 inhabitants, with a high geographical heterogeneity; in the northern provinces of the country, the incidence can exceed 150 snakebites per 100,000 people per year. Bothrops (pit viper) bites predominated, accounting for 96.6% (6720/6957) of envenomations, bites from Crotalus (rattlesnake) accounted for 2.8% (195/6957), and bites from Micrurus (coral snake) for 0.6% (42/6957). Most patients were young men, who were generally bitten during agricultural activities, i.e. while working in the fields. Most snakebites (78.9%, 5852/7419) were to the lower limb, including 58.3% (4322/7419) to the foot. The case fatality rate was <0.04% (3/8083). Most envenomations (90%, 7275/8083) were treated with specific antivenom during the first 4 h after the bite. The median dose of antivenom was two vials for viper bites (Bothrops and Crotalus) and three vials for Micrurus bites. CONCLUSION: These preliminary results should enable manufacturers to increase the availability of appropriate antivenom and health authorities to improve the management of snakebites where they are most common.

Envenoming by coral snakes (Micrurus) in Argentina, during the period between 1979-2003.

Envenomation by coral snakes (Micrurus sp.) is one of the most dangerous injuries in America and it is considered as a serious medical emergency, however bites by these snakes appear to be rare. We analyzed epidemiological data, clinical signs and antivenom use in Argentina during the period between 1979-2003. During this period of study 46 non-fatal Micrurus bites were reported. The majority of cases were men from 31 to 40 years old. Bites occurred primarily in spring and summer. Most cases were reported from the northeast and northwest provinces of the country. The bites were mostly located on hands or feet and occurred mostly during agricultural activities and so mainly involved farmers. Only four cases occurred as a result of handling snakes. The median time it took for antivenom to be administrated was 60 minutes after the bite, and the median number of vials applied was 2. Local pain was mentioned and edema was reported in 41% of patients. All patients recovered without sequelae. This study showed a low incidence of Micrurus bites and low severity of envenomation. However, although no deaths have been reported during the last 30 years, given the toxicity of the venom of Micrurus snakes, the risk of severe envenomation should be considered.

Myotoxicity and nephrotoxicity by Micrurus venoms in experimental envenomation.

Micrurus venoms are essentially neurotoxic but other activities, such as myotoxicity, may be apparent under experimental conditions. Although this myotoxicity has been occasionally reported, there are no studies addressing it systematically across the genus, particularly in its relationship to other systemic manifestations such as renal impairment. The lethal potency of Micrurus fulvius, Micrurus nigrocinctus, Micrurus surinamensis, Micrurus altirostris, Micrurus balyocoriphus and Micrurus pyrrhocryptus venoms determined by us were in the range described for the genus and all venoms exhibited phospholipase activity, albeit at significantly different levels. Intramuscular venom injection caused variable local inflammation-edema; myotoxicity (as determined by plasma creatine kinase levels and histopathology) was apparent only in those venoms with highest phospholipase activity, namely M. fulvius, M. nigrocinctus and M. pyrrhocryptus. Kidneys of animals injected with these strongly myotoxic venoms showed lesions consisting in extensive tubular necrosis with nuclear fragmentation, destruction of the brush border, rupture of basal membrane and epithelial exfoliation of tubular cells, granular cast and thickening of tubules. The histological characteristics of the lesions suggest an important role for indirect glomerular damage by myoglobin deposits. Phospholipase and myotoxic activities did not correlate significantly to the lethal potency; renal lesions were, however, evident only in those venoms that caused extensive muscular damage. Although kidney lesions have not been described in clinical cases of Micrurus envenomation, the potential for nephrotoxicity of some of these venoms should be considered in the overall toxicological picture, at least in experimental conditions.

The neuromuscular activity of Micrurus pyrrhocryptus venom and its neutralization by commercial and specific coral snake antivenoms.

The neuromuscular activity ofMicrurus pyrrochryptus venom was studied in chick biventer cervicis (BC) and mouse phrenic nerve-diaphragm (PND) preparations. The venom (0.5-50µg/ml) caused irreversible, time- and concentration-dependent blockade, with BC being more sensitive than PND (50% blockade with 10µg/ml in 22±;3min and 62±4min, respectively; mean±SEM, n=6; p<0.05). In BC preparations, venom (0.5µg/ml) progressively abolished ACh-induced contractures, whereas contractures to exogenous KCl and muscle twitches in curarized preparations were unaffected. The venom neither altered creatine kinase release (venom: 25.8±1.75IU/l vs control: 24.3±2.2IU/l, n=6, after 120min), nor it caused significant muscle damage (50µg of venom/ml vs control: 3.5±0.8% vs 1.1±0.7% for PND; 4.3±1.5% vs 1.2±0.5% for BC, n=5). The venom had low PLA(2) activity. Neurotoxicity was effectively neutralized by commercial Micrurus antivenom and specific antivenom. These findings indicate that M. pyrrhocryptus venom acts postsynaptically on nicotinic receptors, with no significant myotoxicity.