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In Colombia, Micrurus snakebites are classified as severe according to the national clinical care guidelines and must be treated with specific antivenoms. Unfortunately, these types of antivenoms are scarce in certain areas of the country and are currently reported as an unavailable vital medicine. To address this issue, La Universidad de Antioquia, through its spin-off Tech Life Saving, is leading a project to develop third-generation polyvalent freeze-dried antivenom. The goal is to ensure access to this therapy, especially in rural and dispersed areas. This project aims to evaluate the physicochemical and preclinical parameters (standard quality characteristics) of a lab-scale anti-elapid antivenom batch. The antivenom is challenged against the venoms of several Micrurus species, including M. mipartitus, M. dumerilii, M. ancoralis, M. dissoleucus, M. lemniscatus, M. medemi, M. spixii, M. surinamensis, and M. isozonus, following the standard quality characteristics set by the World Health Organization (WHO). The antivenom demonstrates an appearance consistent with standards, 100% solubility within 4 min and 25 s, an extractable volume of 10.39 mL, a pH of 6.04, an albumin concentration of 0.377 mg/mL (equivalent to 1.22% of total protein), and a protein concentration of 30.97 mg/mL. Importantly, it maintains full integrity of its F(ab')2 fragments and exhibits purity over 98.5%. Furthermore, in mice toxicity evaluations, doses up to 15 mg/mouse show no toxic effects. The antivenom also demonstrates a significant recognition pattern against Micrurus venoms rich in phospholipase A2 (PLA2) content, as observed in M. dumerilii, M. dissoleucus, and M. isozonus. The effective dose 50 (ED50) indicates that a single vial (10 mL) can neutralize 2.33 mg of M. mipartitus venom and 3.99 mg of M. dumerilii venom. This new anti-elapid third-generation polyvalent and freeze-dried antivenom meets the physicochemical parameters set by the WHO and the regulators in Colombia. It demonstrates significant efficacy in neutralizing the venom of the most epidemiologically important Micrurus species in Colombia. Additionally, it recognizes seven other species of Micrurus venom with a higher affinity for venoms exhibiting PLA2 toxins. Fulfilling these parameters represents the first step toward proposing a new pharmacological alternative for treating snakebites in Colombia, particularly in dispersed rural areas, given that this antivenom is formulated as a freeze-dried product.

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RESUMO

Snakes in the family Elapidae largely produce venoms rich in three-fnger toxins (3FTx) that bind to the α1 subunit of nicotinic acetylcholine receptors (nAChRs), impeding ion channel activity. These neurotoxins immobilize the prey by disrupting muscle contraction. Coral snakes of the genus Micrurus are specialist predators who produce many 3FTx, making them an interesting system for examining the coevolution of these toxins and their targets in prey animals. We used a bio-layer interferometry technique to measure the binding interaction between 15 Micrurus venoms and 12 taxon-specifc mimotopes designed to resemble the orthosteric binding region of the muscular nAChR subunit. We found that Micrurus venoms vary greatly in their potency on this assay and that this variation follows phylogenetic patterns rather than previously reported patterns of venom composition. The long-tailed Micrurus tend to have greater binding to nAChR orthosteric sites than their short-tailed relatives and we conclude this is the likely ancestral state. The repeated loss of this activity may be due to the evolution of 3FTx that bind to other regions of the nAChR. We also observed variations in the potency of the venoms depending on the taxon of the target mimotope. Rather than a pattern of prey-specifcity, we found that mimotopes modeled after snake nAChRs are less susceptible to Micrurus venoms and that this resistance is partly due to a characteristic tryptophan→serine mutation within the orthosteric site in all snake mimotopes. This resistance may be part of a Red Queen arms race between coral snakes and their prey.

RESUMO

Snakes in the family Elapidae largely produce venoms rich in three-fnger toxins (3FTx) that bind to the α1 subunit of nicotinic acetylcholine receptors (nAChRs), impeding ion channel activity. These neurotoxins immobilize the prey by disrupting muscle contraction. Coral snakes of the genus Micrurus are specialist predators who produce many 3FTx, making them an interesting system for examining the coevolution of these toxins and their targets in prey animals. We used a bio-layer interferometry technique to measure the binding interaction between 15 Micrurus venoms and 12 taxon-specifc mimotopes designed to resemble the orthosteric binding region of the muscular nAChR subunit. We found that Micrurus venoms vary greatly in their potency on this assay and that this variation follows phylogenetic patterns rather than previously reported patterns of venom composition. The long-tailed Micrurus tend to have greater binding to nAChR orthosteric sites than their short-tailed relatives and we conclude this is the likely ancestral state. The repeated loss of this activity may be due to the evolution of 3FTx that bind to other regions of the nAChR. We also observed variations in the potency of the venoms depending on the taxon of the target mimotope. Rather than a pattern of prey-specifcity, we found that mimotopes modeled after snake nAChRs are less susceptible to Micrurus venoms and that this resistance is partly due to a characteristic tryptophan→serine mutation within the orthosteric site in all snake mimotopes. This resistance may be part of a Red Queen arms race between coral snakes and their prey.

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The ex situ reproduction of wild animals still faces several obstacles, such as minimum reproductive rates or even reproductive inactivity, data that makes specialists worried considering the growing number of species threatened with extinction. Reproductive biotechniques are already well developed among farm animals and widely used in conservation programs, but studies are still needed to develop protocols adapted for snakes, regarding their wide variety of species and their particularities. Therefore, this work seeks to compare sperm survival over time in Oxyrhopus guibei when kept at 24°C and under refrigeration. For this, sperm analysis will be made by evaluating parameters such as motility, progressive motility, presence of morphological changes and mitochondrial activity in spermatozoa. Three males of O. guibei were used, belonging to the Ecology and Evolution Laboratory of the Butantan Institute, and semen was collected via digital stimulation without the use of local anesthesia. This study showed that it is possible to maintain O. guibei sperm cells in HAM-F10 cell culture medium for up to 8 hours at 24°C and for up to 48 hours under refrigeration at 4°C.

A reprodução de animais silvestres ex situ ainda enfrenta diversos obstáculos, como taxas reprodutivas mínimas ou até mesmo inatividade reprodutiva, dados que preocupam especialistas considerando o crescente número de espécies ameaçadas de extinção. As biotécnicas reprodutivas são bem desenvolvidas entre animais de produção e amplamente empregadas em programas de conservação, porém ainda carecem estudos para desenvolvimento de protocolos adaptados para serpentes, tendo em vista a grande variedade de espécies e suas particularidades. Dessa forma, este trabalho busca comparar a sobrevivência espermática ao longo do tempo em Oxyrhopus guibei quando mantidas a 24°C e sob refrigeração. Para isso, foi feita a análise espermática avaliando parâmetros como motilidade, motilidade progressiva, presença de alterações morfológicas e a atividade mitocondrial em células espermáticas. Foram utilizados 3 machos de O. guibei, pertencentes ao plantel do Laboratório de Ecologia e Evolução do Instituto Butantan e foi feita a colheita de sêmen via estimulação digital sem uso de anestesia local. Este estudo mostrou que é possível manter espermatozoides de O. guibei em meio HAM-F10 por até 8 horas a 24°C e por até 48h sob refrigeração, a 4°C.

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Micrurus dumerilii is a coral snake of clinic interest in Colombia. Its venom is mainly composed of phospholipases A2 being MdumPLA2 the most abundant protein. Nevertheless, Micrurus species produce a low quantity of venom, which makes it difficult to produce anticoral antivenoms. Therefore, in this work, we present the recombinant expression of MdumPLA2 to evaluate its biological activities and its immunogenic potential to produce antivenoms. For this, a genetic construct rMdumPLA2 was cloned into the pET28a vector and expressed heterologously in bacteria. His-rMdumPLA2 was extracted from inclusion bodies, refolded in vitro, and isolated using affinity and RP-HPLC chromatography. His-rMdumPLA2 was shown to have phospholipase A2 activity, a weak anticoagulant effect, and induced myonecrosis and edema. The anti-His-rMdumPLA2 antibodies produced in rabbits recognized native PLA2, the complete venom of M. dumerilii, and a phospholipase from another species of the Micrurus genus. Antibodies neutralized 100% of the in vitro phospholipase activity of the recombinant toxin and a moderate percentage of the myotoxic activity of M. dumerilii venom in mice. These results indicate that His-rMdumPLA2 could be used as an immunogen to improve anticoral antivenoms development. This work is the first report of an M. dumerilii functional recombinant PLA2.

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Micrurus is a medically relevant genus of venomous snakes composed of 85 species. Bites caused by coral snakes are rare, but they are usually associated with very severe and life-threatening clinical manifestations. Ecuador is a highly biodiverse country with a complex natural environment, which is home to approximately 20% of identified Micrurus species. Additionally, it is on the list of Latin American countries with the highest number of snakebites. However, there is no local antivenom available against the Ecuadorian snake venoms, and the biochemistry of these venoms has been poorly explored. Only a limited number of samples collected in the country from the Viperidae family were recently characterised. Therefore, this study addressed the compositional patterns of two coral snake venoms from Ecuador, M. helleri and M. mipartitus, using venomics strategies, integrating sample fractionation, gel electrophoresis, and mass spectrometry. Chromatographic and electrophoretic profiles of these snake venoms revealed interspecific variability, which was ascertained by mass spectrometry. The two venoms followed the recently recognised dichotomic toxin expression trends displayed by Micrurus species: M. helleri venom contains a high proportion (72%) of phospholipase A2, whereas M. mipartitus venom is dominated by three-finger toxins (63%). A few additional protein families were also detected in these venoms. Overall, these results provide the first comprehensive views on the composition of two Ecuadorian coral snake venoms and expand the knowledge of Micrurus venom phenotypes. These findings open novel perspectives to further research the functional aspects of these biological cocktails of PLA2s and 3FTxs and stress the need for the preclinical evaluation of the currently used antivenoms for therapeutic purposes in Ecuador.

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In Colombia, the genus Micrurus includes 30 species, of which M. mipartitus and M. dumerilii are the most widely distributed. Micrurus causes less than 3% of the approximately 5000 cases of snakebite per year. The elapid envenomation caused by the snakes from the Micrurus genus, are characterized by the severity of their clinical manifestations, due to the venom neurotoxic components such as three-finger toxins (3FTx) and phospholipases (PLA2). The treatment for snakebites is the administration of specific antivenoms, however, some of them have limitations in their neutralizing ability. A strategy proposed to improve antivenoms is to produce antibodies against the main components of the venom. The aim of this work was to produce an antivenom, using an immunization protocol including the main 3FTx and PLA2 responsible for M. mipartitus lethality. The antibody titers were determined by ELISA in rabbits' serum. The immunized animals elicited a response against toxins and whole venom. The Immunoglobulin G (IgGs) obtained were able to neutralize the lethal effect of their homologous toxins. A combination of antivenom from M. mipartitus with antitoxins improved their neutralizing ability. In the same way, a mixture of anti 3FTx and PLA2 protected the mice from a 1.5 median lethal dose (LD50) of M. mipartitus venom. The results showed that this might be a way to improve antibody titers specificity against the relevant toxins in M. mipartitus venom and indicated that there is a possibility to develop and use recombinant 3FTx and PLA2 toxins as immunogens to produce antivenoms. Additionally, this represents an alternative to reduce the amount of venom used in anti-coral antivenom production.

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Snakebite is a neglected tropical disease with a massive global burden of injury and death. The best current treatments, antivenoms, are plagued by a number of logistical issues that limit supply and access in remote or poor regions. We explore the anticoagulant properties of venoms from the genus Micrurus (coral snakes), which have been largely unstudied, as well as the effectiveness of antivenom and a small-molecule phospholipase inhibitor—varespladib—at counteracting these effects. Our in vitro results suggest that these venoms likely interfere with the formation or function of the prothrombinase complex. We find that the anticoagulant potency varies widely across the genus and is especially pronounced in M. laticollaris. This variation does not appear to correspond to previously described patterns regarding the relative expression of the three-finger toxin and phospholipase A2 (PLA2) toxin families within the venoms of this genus. The coral snake antivenom Coralmyn, is largely unable to ameliorate these effects except for M. ibiboboca. Varespladib on the other hand completely abolished the anticoagulant activity of every venom. This is consistent with the growing body of results showing that varespladib may be an effective treatment for a wide range of toxicity caused by PLA2 toxins from many different snake species. Varespladib is a particularly attractive candidate to help alleviate the burden of snakebite because it is an approved drug that possesses several logistical advantages over antivenom including temperature stability and oral availability.

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People who live in rural or forested areas are more likely to be affected by snakebites, due to their presence in the natural habitat of snakes and due to activities such as extractivism and agriculture. To conduct an ethnobiological study regarding the knowledge related to venomous snakes, snakebites and the attitudes of people who frequent areas of floodplain forests in the Alto Juruá (Brazilian Amazon), and correlate this information with data on snakebites in the region and the ecology of the ophiofauna, 100 residents, who are actively involved in extractivism, fishing, or hunting in the forests of the region were interviewed. Boards with photographs of venomous snakes from the region were used to ask questions about their experiences. The sampling of snakes was carried on trails in a forest used by residents of the region in their extractivism activities. Four venomous species (Bothrops atrox, B. bilineatus smaragdinus, Micrurus lemniscatus and M. surinamensis) were recorded. Among the interviewees, 31% claimed that they had already suffered at least one snakebite. The B. atrox snake is the species that the inhabitants encounter most and the one that is most associated with snakebites. Bothrops b. smaragdinus was the most common snake found during the search. Regarding accident prevention, 60% of them reported wearing boots when walking in the forest. In relation to practices adopted after a bite, the majority stated that they would seek medical attention; however, many reported using first aid measures that would not be medically effective for the victim's recovery. Despite B. b. smaragdinus being the most common snake found by researchers, it is responsible for very few snakebites. This is probably due to its arboreal habits, since it is found at a higher than average height (6.3 m) which is much higher than the height of a human being. The surveyed population demonstrated knowledge of the main preventive measures against snakebites, although some inadequate and/or ineffective first aid measures are still adopted.

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