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.

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.

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.

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.

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.

Neutralization of Bothrops alternatus regional venom pools and individual venoms by antivenom: a systematic comparison.

In this study we report that variation in lethality, hemorrhagic potency and procoagulation between individual samples of Bothrops alternatus venom from a single region, and variation between regional pools at the national level are comparable in range. Furthermore, the range of relative neutralization potencies of individual venoms within a region overlaps, and sometimes exceeds, the range of neutralization of regional venom pools throughout the country. Thus, the potency of neutralization of a national venom pool is poorly predictive of the potencies of neutralization of its constituent regional venom pools and, furthermore, the potency of neutralization of a regional venom pool is poorly predictive of the potencies of neutralization of its individual venom constituents. The efficiencies of neutralization of each of these effects (lethality, hemorrhage and procoagulation) were not significantly related to each other and did not correlate to the corresponding toxic potency of each venom or venom pool. Some implications of these findings are discussed in the context of the distinction between experimental quantitation of antivenom potency and the amount of antivenom that might be actually required to successfully treat two apparently comparable B. alternatus envenomations.

Toxicity of Bothrops neuwiedi complex ("yarará chica") venom from different regions of Argentina (Serpentes, Viperidae).

We report a study of toxic and enzymatic activities of Bothrops neuwiedi complex venoms collected from specimens of different regions of Argentina and a pool of these same venoms. Were determined lethal, hemorrhagic and pro-coagulant (plasma and fibrinogen) doses and the neutralization of these activities by a bivalent antivenom. The electrophoretic pattern of different regions venom was studied by SDS-PAGE. All samples exhibited lethal potencies, hemorrhagic and coagulant (plasma and fibrinogen) activities with potencies concordant with previous studies. The only conspicuous difference in the toxicological pattern of Bothrops diporus venoms was the low-thrombin-like activity found in one sample. The antivenom used in this study could neutralize all the toxic activities tested and the neutralizing potency of the antivenom was comparable for all samples. Despite the wide distribution of B. neuwiedi complex throughout Argentina and the evident morphological variation between B. diporus (B. neuwiedi complex), this study establishes a remarkably similar toxicity profile throughout its range. This is the first systematic study on the regional variation of enzymatic and toxic activities of venom from species belonging to the B. neuwiedi complex, one of the snakes of highest sanitary importance in South America and their neutralization by the type of antivenom most commonly used in the South of South America.