Histopathological changes of mice tissues in course of the envenomation by the Macrovipera lebetina obtusa venom and the neutralizing effect of the ovine-derived experimental antivenom.

Viper bites pose a significant public health issue in Armenia, even within urban areas, often resulting in clotting disorders, hypofibrinogenemia, and tissue necrosis in humans. This study investigates histopathological changes in various tissues during mice envenomation by West-Asian blunt-nosed viper (Macrovipera lebetina obtusa) venom, as well as the recovery process aided by experimental antivenom derived from sheep. The high venom dose caused substantial damage to the heart, lungs, liver, and kidneys in mice, indicating systemic harm. While antivenom administration can prevent mortality in mice envenomation, it may not fully mitigate histological damage in affected organs. Additionally, the study highlights the importance of timing antivenom administration, as the severity of tissue alterations can vary depending on the duration of envenomation. These findings shed light on antivenom's effects on viper envenomation and stress the need for further research to optimize its timing and dosage for minimizing histological damage and enhancing clinical outcomes.

Changes in attachment and metabolic activity of rat neonatal cardiomyocytes and nonmyocytes caused by Macrovipera lebetina obtusa venom.

The Caucasian viper Macrovipera lebetina obtusa (MLO) is one of the most prevalent and venomous snakes in the Caucasus and the surrounding regions, yet the effects of MLO venom on cardiac function remain largely unknown. We examined the influence of MLO venom (crude and with inhibited metalloproteinases and phospholipase A2) on attachment and metabolic activity of rat neonatal cardiomyocytes (CM) and nonmyocytes (nCM), assessed at 1 and 24 h. After exposing both CM and nCM to varying concentrations of MLO venom, we observed immediate cytotoxic effects at a concentration of 100 µg/ml, causing detachment from the culture substrate. At lower MLO venom concentrations both cell types detached in a dose-dependent manner. Inhibition of MLO venom metalloproteinases significantly improved CM and nCM attachment after 1-hour exposure. At 24-hour exposure to metalloproteinases inhibited venom statistically significant enhancement was observed only in nCM attachment. However, metabolic activity of CM and nCM did not decrease upon exposure to the lower dose of the venom. Moreover, we demonstrated that metalloproteinases and phospholipases A2 are not the components of the MLO venom that change metabolic activity of both CM and nCM. These results provide a valuable platform to study the impact of MLO venom on prey cardiac function. They also call for further exploration of individual venom components for pharmaceutical purposes.

Comparative Venom Proteomics of Iranian, Macrovipera lebetina cernovi, and Cypriot, Macrovipera lebetina lebetina, Giant Vipers.

Envenoming by Macrovipera lebetina subspecies causes severe life-threatening difficulties for people living in North Africa and the Middle East. To better understand the pathophysiology of envenoming and improve patient management, knowledge about the venom components of the subspecies is essential. Here, the venom proteomes of Macrovipera lebetina lebetina from Cyprus and Macrovipera lebetina cernovi from Iran were characterized using RP-HPLC separation of the crude venom proteins, SDS-PAGE of fractionated proteins, and LC-MS/MS of peptides obtained from in-gel tryptic digestion of protein bands. Moreover, we also used high-resolution shot-gun proteomics to gain more reliable identification, where the whole venom proteomes were subjected directly to in-solution digestion before LC-HR-MS/MS. The data revealed that both venoms consisted of at least 18 protein families, of which snake venom Zn2+-dependent metalloprotease (SVMP), serine protease, disintegrin, phospholipase A2, C-type lectin-like, and L-amino acid oxidase, together accounted for more than 80% of the venoms' protein contents. Although the two viper venoms shared mostly similar protein classes, the relative occurrences of these toxins were different in each snake subspecies. For instance, P-I class of SVMP toxins were found to be more abundant than P-III class in the venoms of M. l. cernovi compared to M. l. lebetina, which gives hints at a more potent myonecrotic effect and minor systemic hemorrhage following envenoming by M. l. cernovi than M. l. lebetina. Moreover, single-shot proteomics also revealed many proteins with low abundance (<1%) within the venoms, such as aminopeptidase, hyaluronidase, glutaminyl-peptide cyclotransferase, cystatin, phospholipase B, and vascular endothelial growth factor. Our study extends the in-depth understanding of the venom complexity of M. lebetina subspecies, particularly regarding toxin families associated with envenoming pathogenesis and those hard-detected protein classes expressed in trace amounts.

Diverse and Dynamic Alpha-Neurotoxicity Within Venoms from the Palearctic Viperid Snake Clade of Daboia, Macrovipera, Montivipera, and Vipera.

The targeting of specific prey by snake venom toxins is a fascinating aspect of molecular and ecological evolution. Neurotoxic targeting by elapid snakes dominates the literature in this regard; however, recent studies have revealed viper toxins also induce neurotoxic effect. While this effect is thought to primarily be driven by prey selectivity, no study has quantified the taxonomically specific neurotoxicity of the viper clade consisting of Daboia, Macrovipera, Montivipera, and Vipera genera. Here, we tested venom toxin binding from 28 species of vipers from the four genera on the alpha 1 neuronal nicotinic acetylcholine receptors (nAChRs) orthosteric sites of amphibian, avian, lizard, rodent, and human mimotopes (synthetic peptides) using the Octet HTX biolayer interferometry platform. Daboia siamensis and D. russelii had broad binding affinity towards all mimotopes, while D. palestinae had selectivity toward lizard. Macrovipera species, on the other hand, were observed to have a higher affinity for amphibian mimotopes except for M. schweizeri, which inclined more toward lizard mimotopes. All Montivipera and most Vipera species also had higher affinity toward lizard mimotopes. Vipera a. montandoni, V. latastei, V. nikolski, and V. transcaucasina had the least binding to any of the mimotopes of the study. While a wide range of affinity binding towards various mimotopes were observed within the clade, the lowest affinity occurred towards the human target. Daboia siamensis and Macrovipera lebetina exhibited the greatest affinity toward the human mimotope, albeit still the least targeted of the mimotopes within those species. Overlaying this toxin-targeting trait over phylogeny of this clade revealed multiple cases of amplification of this trait and several cases of secondary loss. Overall, our results reveal dynamic variation, amplification, and some secondary loss of the prey targeting trait by alpha-neurotoxins within the venoms of this clade, indicating evolutionary selection pressure shaping the basic biochemistry of these venoms. Our work illustrates the successful use of this biophysical assay to further research snake venom neurotoxins and emphasizes the risk of generalizing venom effects observed on laboratory animals to have similar effects on humans.

Development and characterization of human single chain antibody against Iranian Macrovipera lebetina snake venom.

Snakebite is an important public health problem in tropical and subtropical regions. Macrovipera lebetina is one of the most dangerous snakes in Iran. Envenoming by this snake can lead to respiratory distress, heart attack, bleeding, and death. The specific treatment available is immunized equine serum, which has several side effects like serum sickness. Nowadays, single-chain fragment variable antibodies (scFvs) are one of the fast growing classes of monoclonal antibodies, which are suggested for treatment of envenoming. This study aimed to achieve a fully human scFv antibody against M. lebetina venom from human non-immune library. In this study, scFvs against M. lebetina venom were isolated by phage display technique. Using three rounds of biopanning, two specific scFvs (C37 and C69) with the highest affinity were selected. The selected scFvs purified by nickel affinity chromatography. The specific binding of purified antibodies were confirmed by enzyme-linked immunosorbent assay. The LD50 as well as HD50 concentration of the crude venom were obtained to be 45 µg and 120 µg/ml, respectively. C69 neutralized 48% of the hemolysis activity of M. lebetina venom and C37 survived 66% of mice after 115 min of envenoming. Taken together, the results indicate the potential of human non-immune libraries for selection of functional antibodies against M. lebetina venom.

The development and evaluation of the efficacy of ovine-derived experimental antivenom immunoserum against Macrovipera lebetina obtusa (MLO) venom.

Here we describe the processing and development of animal-derived monovalent antibody serum against Macrovipera lebetina obtusa venom by purification and concentration of the immunoglobulins using caprylic acid. We demonstrate that this new viper venom antiserum is pre-clinically effective in neutralizing lethal toxicity and hemorrhagicity of the venom of the Armenian Levantine viper - a significant public health problem in Armenia and a wide region from south-east parts of Europe to south-west Asia. The developed product shows a high capacity to inhibit metalloproteinases and phospholipase activity of venom included in the study in comparison to current specific antivenoms, and following additional experimental approvals, it will be possible to derive the monovalent antivenom satisfying international standards, which will be much cheaper and accessible compared with the current market rivals.

Clinical implications of differential procoagulant toxicity of the palearctic viperid genus Macrovipera, and the relative neutralization efficacy of antivenoms and enzyme inhibitors.

Species within the viperid genus Macrovipera are some of the most dangerous snakes in the Eurasian region, injecting copious amounts of potent venom. Despite their medical importance, the pathophysiological actions of their venoms have been neglected. Particularly poorly known are the coagulotoxic effects and thus the underlying mechanisms of lethal coagulopathy. In order to fill this knowledge gap, we ascertained the effects of venom upon human plasma for Macrovipera lebetina cernovi, M. l. lebetina, M. l. obtusa, M. l. turanica, and M. schweizeri using diverse coagulation analysing protocols. All five were extremely potent in their ability to promote clotting but varied in their relative activation of Factor X, being equipotent in this study to the venom of the better studied, and lethal, species Daboia russelii. The Insoserp European viper antivenom was shown to be highly effective against all the Macrovipera venoms, but performed poorly against the D. russelii venom. Reciprocally, while Daboia antivenoms performed well against D. russelii venom, they failed against Macrovipera venom. Thus despite the two genera sharing a venom phenotype (Factor X activation) driven by the same toxin type (P-IIId snake venom metalloproteases), the surface biochemistries of the toxins differed significantly enough to impede antivenom cross- neutralization. The differences in venom biochemistry were reflected in coagulation co-factor dependence. While both genera were absolutely dependent upon calcium for the activation of Factor X, dependence upon phospholipid varied. The Macrovipera venoms had low levels of dependence upon phospholipid while the Daboia venom was three times more dependent upon phospholipid for the activation of Factor X. This suggests that the sites on the molecular surface responsible for phospholipid dependence, are the same differential sites that prevent inter-genera antivenom cross- neutralization. Due to cold-chain requirements, antivenoms may not be stocked in rural settings where the need is at the greatest. Thus we tested the efficacy of enzyme inhibitor Prinomastat as a field-deployable treatment to stabilise patients while being transported to antivenom stocks, and showed that it was extremely effective in blocking the Factor X activating pathophysiological actions. Marimastat however was less effective. These results thus not only shed light on the coagulopathic mechanisms of Macrovipera venoms, but also provide data critical for evidence-based design of snakebite management strategies.

Preclinical Assessment of a New Polyvalent Antivenom (Inoserp Europe) against Several Species of the Subfamily Viperinae.

The European continent is inhabited by medically important venomous Viperinae snakes. Vipera ammodytes, Vipera berus, and Vipera aspis cause the greatest public health problems in Europe, but there are other equally significant snakes in specific regions of the continent. Immunotherapy is indicated for patients with systemic envenoming, of which there are approximately 4000 annual cases in Europe, and was suggested as an indication for young children and pregnant women, even if they do not have systemic symptoms. In the present study, the safety and venom-neutralizing efficacy of Inoserp Europe-a new F(ab')2 polyvalent antivenom, designed to treat envenoming by snakes in the Eurasian region-were evaluated. In accordance with World Health Organization recommendations, several quality control parameters were applied to evaluate the safety of this antivenom. The venom-neutralizing efficacy of the antivenom was evaluated in mice and the results showed it had appropriate neutralizing potency against the venoms of several species of Vipera, Montivipera, and Macrovipera. Paraspecificity of the antivenom was demonstrated as well, since it neutralized venoms of species not included in the immunization schemes and contains satisfactory levels of total proteins and F(ab')2 fragment concentration. Therefore, this new polyvalent antivenom could be effective in the treatment of snake envenoming in Europe, including Western Russia and Turkey.

Biochemistry and pharmacology of proteins and peptides purified from the venoms of the snakes Macrovipera lebetina subspecies.

The isolation and characterization of individual snake venom components is important for a deeper understanding of the pathophysiology of envenomations, for improving the therapeutic procedures of patients, and it also opens possibilities for the discovery of novel toxins that might be useful as tools for understanding cellular and molecular processes. This review provides a summary of the different toxins that have been isolated and characterized from the venoms of Vipera lebetina (Macrovipera lebetina) subspecies Macrovipera lebetina cernovi, Macrovipera lebetina lebetina, Macrovipera lebetina obtusa, Macrovipera lebetina transmediterranea, Macrovipera lebetina turanica, the snake species causing the majority of human envenomings in Central Asia (Middle East) and North Africa. The venoms of these snakes contain proteins belonging to different families: Zn2+- metalloproteinases, serine proteinases, L-amino acid oxidase, 5'-nucleotidase, phosphodiesterase, phosphomonoesterase, nucleases, hyaluronidase, phospholipase A2, C-type lectin-like protein, disintegrin, DC-fragment, cystein-rich secretory protein, proteinase inhibitors, nerve growth factor (NGF), vascular endothelial growth factor (VEGF), low molecular weight peptides. Their main biochemical properties, toxic and pharmacological actions have been described. In this review we will provide an overview of the proteins and peptides from the venoms of M. lebetina subspecies, their biochemical, pharmacological and structural features and their role in snake venom toxinology. A lot of contributions have been made for better understandings of these venomous snakes, their venom, and their pharmacological effects. Many of these components are also fascinating models for drug design.

Vipers of the Middle East: A Rich Source of Bioactive Molecules.

Snake venom serves as a tool of defense against threat and helps in prey digestion. It consists of a mixture of enzymes, such as phospholipase A2, metalloproteases, and l-amino acid oxidase, and toxins, including neurotoxins and cytotoxins. Beside their toxicity, venom components possess many pharmacological effects and have been used to design drugs and as biomarkers of diseases. Viperidae is one family of venomous snakes that is found nearly worldwide. However, three main vipers exist in the Middle Eastern region: Montivipera bornmuelleri, Macrovipera lebetina, and Vipera (Daboia) palaestinae. The venoms of these vipers have been the subject of many studies and are considered as a promising source of bioactive molecules. In this review, we present an overview of these three vipers, with a special focus on their venom composition as well as their biological activities, and we discuss further frameworks for the exploration of each venom.

Anti-inflammatory properties of Centaurea calolepis Boiss. and cnicin against Macrovipera lebetina obtusa (Dwigubsky, 1832) and Montivipera xanthina (Gray, 1849) venoms in rat.

Macrovipera lebetina obtusa (Dwigubsky, 1832) and Montivipera xanthina (Gray, 1849) (Ottoman Viper) are viper snakes from Viperidae family and found in various locations in Anatolia. Both snakes are responsible for major snake bite cases in Turkey Their venoms cause necrosis, hemorrhage, pain and local edema. Centaurea L. (Asteraceae) species draw attention as potential anti-inflammatory sources due to their traditional uses and accomplished studies on this field. C. calolepis Boiss. is an endemic taxon distributed in Aegean and Antalya regions in Turkey. Chloroform extract of C. calolepis and its major compound cnicin, a sesquiterpene lactone, are reported to have strong anti-inflammatory activities in-vitro, by previous studies. In the present study, in-vivo anti-inflammatory activities of C. calolepis chloroform extract and the sesquiterpenoid cnicin against edema induced by Macrovipera lebetina obtusa and Montivipera xanthina venoms were evaluated in the rat model. Protein contents and induction doses of the venoms were determined. Carrageenan and snake venoms were used as inducing agents in paw edema tests. Extract demonstrated strong inhibition on edema at all doses and hours against M. xanthina venom and carrageenan. Inhibition ratio of extract at 25 mg/kg dose (84.13% inhibition) after 0.5 h M. xanthina venom injection was more than indomethacin's value (45.4% inhibition). The extract also showed significant effect also on inflammation caused by M. lebetina obtusa venom at all doses. However, 2.5 mg/kg cnicin was more effective than total extract of C. calolepis against rat paw edema induced by (27.31%) M. lebetina obtusa venom. This is the first study reported therapeutic potential of C. calolepis, an endemic plant of Turkey, in case of snake-bites cause inflammation by venomous species in natural fauna of Anatolia.

Snakebites in Lebanon: A Descriptive Study of Snakebite Victims Treated at a Tertiary Care Center in Beirut, Lebanon.

BACKGROUND: Snakebites lead to at least 421,000 envenomations and result in more than 20,000 deaths per year worldwide. Few reports exist in the Mediterranean region. This study describes demographic and clinical characteristics, treatment modalities, and outcomes of snakebites in Lebanon. MATERIALS AND METHODS: This was a retrospective chart review of patients who presented with snakebite complaint to the emergency department between January 2000 and September 2014. RESULTS: A total of 24 patients were included in this study. The mean age was 34.6 (±16.4) years and 58.3% were males. Local manifestations were documented in 15 (62.5%) patients, systemic effects in 10 (41.7%), hematologic abnormalities in 10 (41.7%), and neurologic effects in 4 (16.7%) patients. Nine patients (37.5%) received antivenom. The median amount of antivenom administered was 40 ml or 4 vials (range: 1-8 vials). About 50% of patients were admitted to the hospital with 75% to an Intensive Care Unit and 25% to a regular bed. All were discharged home with a median hospital length of stay of 4 (interquartile range 11) days. Among those admitted, seven patients (58.3%) had at least one documented complication (compartment syndrome, fasciotomy, intubation, deep vein thrombosis, coagulopathy, acute respiratory distress syndrome, sepsis, congestive heart failure, cellulitis, upper gastrointestinal bleeding, and vaginal bleeding). CONCLUSION: Victims of snakebites in Lebanon developed local, systemic, hematologic, or neurologic manifestations. Complications from snakebites were frequent despite antivenom administration. Larger studies are needed to assess the efficacy of available antivenom and to possibly create a local antivenom for the treatment of snakebites in Lebanon.

Next-generation sequencing yields the complete mitochondrial genome of the endangered Milos viper Macrovipera schweizeri (Reptilia, Viperidae).

The Milos viper, Macrovipera schweizeri, is an endangered viperid snake found on four Aegean islands (Greece). Its complete mitochondrial genome, the first reported for the genus Macrovipera, was assembled through next-generation sequencing. Its total length is 17,152 bp and includes 22 tRNAs, two ribosomal RNA genes, 13 protein-coding genes and two control regions, showing the typical gene-arrangement for Viperidae. Eight tRNAs and ND3 are encoded on the light strand, while all other genes are encoded on the heavy strand. A mitogenomic phylogeny that included Macrovipera schweizeri and 13 other viperid genera returned an unresolved relationship among the genera Macrovipera, Daboia and Vipera.

Lebein, a Snake Venom Disintegrin, Induces Apoptosis in Human Melanoma Cells.

Melanoma, the most threatening form of skin cancer, has a very poor prognosis and is characterized by its very invasive and chemoresistant properties. Despite the recent promising news from the field of immunotherapy, there is an urgent need for new therapeutic approaches that are free of resistance mechanisms and side effects. Anti-neoplasic properties have been highlighted for different disintegrins from snake venom including Lebein; however, the exact effect of Lebein on melanoma has not yet been defined. In this study, we showed that Lebein blocks melanoma cell proliferation and induces a more differentiated phenotype with inhibition of extracellular signal-regulated kinase (ERK) phosphorylation and microphthalmia-associated transcription factor (MITF) overexpression. Melanoma cells became detached but were less invasive with upregulation of E-cadherin after Lebein exposure. Lebein induced a caspase-independent apoptotic program with apoptosis inducing factor (AIF), BCL-2-associated X protein (BAX) and Bim overexpression together with downregulation of B-cell lymphoma-2 (BCL-2). It generated a distinct response in reactive oxygen species (ROS) generation and p53 levels depending on the p53 cell line status (wild type or mutant). Therefore, we propose Lebein as a new candidate for development of potential therapies for melanoma.

Haemostasis disorders caused by envenomation by Cerastes cerastes and Macrovipera mauritanica vipers.

Viper venoms are a real source of proteolytic enzymes causing clotting, bleeding, edema, necrosis, hemorrhage, pain at the bite site and systemic changes. This study was conducted to evaluate the changes induced in hematological and haemostatic parameters in rabbits after 1, 3, 6 and 24 h post-venom of subcutaneously administration of a sublethal dose of Cerastes cerastes and Macrovipera mauritanica venoms. Our results indicated that most hematological and haemostatic parameters showed significant changes 3 and 6 h after envenomation. The hemoglobin, hematocrit, red blood cells, platelets and prothrombin time were reduced significantly 3 h after envenomation. A very significant increase in the levels of white blood cells, lymphocytes, monocytes, activated thromboplastin time and fibrinogen were recorded 6 h following envenomation. However, no significant difference was found for the mean corpuscular volume, corpuscular hemoglobin content and mean corpuscular hemoglobin concentration throughout the whole duration of the experiment. These results suggest that severe hematological and haemostatic changes may be initiated during the early stages of envenomation leading to local and systemic hemorrhages and coagulopathies which are the main cause of death in case of vipers envenomation.

Paraspecificity of Vipera a. ammodytes-specific antivenom towards Montivipera raddei and Macrovipera lebetina obtusa venoms.

Antivenom raised against the venom of nose-horned viper, Vipera ammodytes (V. a.) ammodytes (European viper venom antiserum, Zagreb antivenom), contains neutralising equine F(ab')2 fragments that are clinically successful against homologous venom, but also against the venoms of several others medically important European snakes due to its paraspecific action. In this work we demonstrated that Zagreb antivenom is preclinically effective in neutralising lethal toxicity and hemorrhagicity of venoms of Armenian mountain snakes--Montivipera raddei and Macrovipera lebetina obtusa as well. In order to better understand the biochemical basis of the observed paraspecificity, the ability of anti-V. a. ammodytes serum to recognise and neutralise proteinases of the two venoms was also investigated. Anti-V. a. ammodytes serum showed surprisingly low capacity to inhibit metalloproteinases of both venoms included in the study, probably due to weak immunorecognition of their P-I representatives. Also, it completely failed to abolish enzymatic action of serine proteinases from Macrovipera lebetina obtusa venom. Relevance of such finding is yet to be established.

Determination of in vivo toxicity and in vitro cytotoxicity of venom from the Cypriot blunt-nosed viper Macrovipera lebetina lebetina and antivenom production

The venomous Levantine viper, Macrovipera lebetina lebetina is endemic to Cyprus. The objective of this study was to investigate in vitro cytotoxicity, in vivo lethality, and antivenom production followed by a re-immunization schedule in mice against Macrovipera lebetina lebetina venom. The LD50 value was estimated as 7.58 mg/kg within 24 hours by different venom doses administrated intraperitoneally in mice. Freund's complete and incomplete adjuvants were used for first and second immunization of mice in antivenom production. A cell-based assay was performed to determine the effects of Macrovipera lebetina lebetina venom and antivenom neutralizing potency on L929 cell viability. The snake venom toxicity and cytotoxicity were examined and comparison of results showed good correlation, the LD50 value was tenfold higher than the IC50 value. The IC50 value was 0.62 ± 0.18 mL after 48 hours treatment while the calculated value was 1.62 ± 0.25 mL for the culture media totally refreshed after two hours treatment with venom. The in vitro efficacy of antivenom against Macrovipera lebetina lebetina venom was found to be low. This is the first report that describes the in vivo and in vitro toxic effects of Macrovipera lebetina lebetina venom and antivenom production against this species.(AU)

General characterization of venom from the Moroccan snakes Macrovipera mauritanica and Cerastes cerastes

Ophidian envenomation accidents constitute a serious public health problem in many countries around the globe. Over 5 million such accident cases occur each year causing more than 100,000 deaths. In Africa, more than 20,000 deaths per year are registered while 400,000 envenomation victims retain severe and permanent functional sequelae. In Morocco, snakebites are frequent and of greater severity in children. They occur mostly in rural areas. The incidence of these bites remains poorly understood and vastly underestimated. The epidemiological data are not well known due to the absence of a national registry, whereas a significant proportion of envenomations receive only traditional treatment methods in non-medical intensive care. This prompted us to investigate the enzymatic and biological properties of venom biochemical constituents from two of the most dangerous snake venoms in Morocco: Cerastes cerastes (Cc) and Macrovipera mauritanica (Mm). Also, we studied the immune cross-reactivity of Cc and Mm venoms in comparison to that of another important dangerous Moroccan viper, Bitis arietans (Ba), to identify the best candidates (venom or a mixture of venoms) for producing the most efficient and protective antivenom. In the present study, we report a preliminary venom characterization of Cc and Mm and the cross-reactivity that may exist between their venoms and Ba. These venoms are known to be highly toxic and contain several proteins that differ by molecular weights. Interestingly, both Cc and Mm venoms are characterized by intense hemorrhagic and phospholipase A2 activities and their ability to degrade the α and γ chains of fibrinogen. They display very low proteolysis through the casein test. After injection into mice, Cc and Mm induce myonecrosis in skeletal muscles, which most likely reflects direct action of myotoxins and indirect action of hemorrhagic molecules present in these venoms. In mice, this myonecrosis diminishes serum creatine phosphokinase (CPK) levels. As expected, Cc venom is immunogenic and induces highly protective antivenom against Mm and Ba venom antigens. This protective capacity is similar to that of the antivenom produced against the Mm venom.(AU)