Sequence analysis of the cDNA encoding for SpCTx: a lethal factor from scorpionfish venom ( Scorpaena plumieri )

Lethal factors are multifunctional oligomeric proteins found in the venomous apparatus of Scorpaeniformes fish. These toxins elicit not only an array of biological responses in vitro but also cardiovascular disorders and strong hemolytic, nociceptive and edematogenic activities in vivo. This work describes the cloning and molecular identification of two toxin subunits, denominated Sp-CTx-α and Sp-CTx-ß, from scorpionfish venom ( Scorpaena plumieri ). Methods: The primary structures were deduced after cDNA amplification by PCR with primers from conserved sequences described in Scorpaeniformes toxins. Following DNA sequencing and bioinformatic analysis, the tridimensional structures of both subunits were modeled. Results: The translated sequences (702 amino acids, each subunit) show homology with other lethal factors, while alignment between Sp-CTx-α and Sp-CTx-ß shows 54% identity. The subunits lack N-terminal signal sequences and display masses of approximately 80 kDa each. Both Sp-CTx subunits display a B30.2/SPRY domain at the C-terminal region with typically conserved motifs as described in these toxins. Secondary structure prediction identified six α-helices 18 residues long in both α and ß subunits, some of them amphiphilic with their N-terminal flanked by many basic residues, creating a cationic site associated with the cytolytic activity of these toxins. Antimicrobial potential sites were identified in Sp-CTx and share some features with other peptides presenting variable and broad-spectrum activity. A phylogenetic tree built to represent these toxins supports the proximity between scorpionfish, lionfish and stonefish. Conclusion: The study identified a putative toxin protein whose primary structure is similar to other fish toxins and with potential for production of antivenom against scorpionfish envenomation in Brazil. As a prelude to structure-function studies, we propose that the toxin is structurally related to pore-forming marine toxins.(AU)

Development of Equine IgG Antivenoms against Major Snake Groups in Mozambique.

BACKGROUND: Snake envenoming is a significant public health problem in underdeveloped and developing countries. In sub-Saharan Africa, it is estimated that 90,000-400,000 envenomations occur each year, resulting in 3,500-32,000 deaths. Envenomings are caused by snakes from the Viperidae (Bitis spp. and Echis spp.) and Elapidae (Naja spp. and Dendroaspis spp.) families. The African continent has been suffering from a severe antivenom crisis and current antivenom production is only sufficient to treat 25% of snakebite cases. Our aim is to develop high-quality antivenoms against the main snake species found in Mozambique. METHODS: Adult horses primed with the indicated venoms were divided into 5 groups (B. arietans; B. nasicornis + B. rhinoceros; N. melanoleuca; N. mossambica; N. annulifera + D. polylepis + D. angusticeps) and reimmunized two times for antivenom production. Blood was collected, and plasma was separated and subjected to antibody purification using caprylic acid. Plasmas and antivenoms were subject to titration, affinity determination, cross-recognition assays and in vivo venom lethality neutralization. A commercial anti-Crotalic antivenom was used for comparison. RESULTS: The purified antivenoms exhibited high titers against B. arietans, B. nasicornis and B. rhinoceros (5.18 x 106, 3.60 x 106 and 3.50 x 106 U-E/mL, respectively) and N. melanoleuca, N. mossambica and N. annulifera (7.41 x 106, 3.07 x 106 and 2.60 x 106 U-E/mL, respectively), but lower titers against the D. angusticeps and D. polylepis (1.87 x 106 and 1.67 x 106 U-E/mL). All the groups, except anti-N. melanoleuca, showed significant differences from the anti-Crotalic antivenom (7.55 x 106 U-E/mL). The affinity index of all the groups was high, ranging from 31% to 45%. Cross-recognition assays showed the recognition of proteins with similar molecular weight in the venoms and may indicate the possibility of paraspecific neutralization. The three monospecific antivenoms were able to provide in vivo protection. CONCLUSION: Our results indicate that the anti-Bitis and anti-Naja antivenoms developed would be useful for treating snakebite envenomations in Mozambique, although their effectiveness should to be increased. We propose instead the development of monospecific antivenoms, which would serve as the basis for two polyvalent antivenoms, the anti-Bitis and anti-Elapidae. Polyvalent antivenoms represent an increase in treatment quality, as they have a wider range of application and are easier to distribute and administer to snake envenoming victims.

An investigation of snakebite antivenom usage in Taiwan.

BACKGROUND/PURPOSE: Four types of antivenom are used to treat snakebites by the six species of venomous snakes native to Taiwan. Research into antivenom use in Taiwan and its outcomes, as well as the utility of current Taiwan Poison Control Center guidelines for antivenom use, has been limited. We aimed to provide increased understanding by investigating the treatment and outcomes of patients treated for snakebite in Taiwan. METHODS: On the basis of data collected from the 2009 Taiwan National Health Insurance database, patients with snakebites were identified and categorized into two sets of groups according to types of antivenom administered. The relationships between antivenom types, dosage and the variables of antibiotic use, surgical intervention, acute respiratory failure acute, renal failure, antivenom-related allergic reaction, mortality, need for hospital admission, and length of hospitalization were analyzed by multivariate logistic regression and the Kruskal-Wallis test. RESULTS: The majority of patients were successfully treated by administration of 1 vial of antivenom and discharged without complications. However, patients treated for neurotoxic-type venom snakebite required administration of larger doses of antivenom and > 30% required surgical intervention, particularly those treated for Chinese cobra snakebite. Approximately 10% of patients were administered two types of antivenom. CONCLUSION: The results partially support Taiwan Poison Control Center guidelines for treating the hemorrhagic-type venom snakebite. However, deficit in the guidelines for treatment of neurotoxic-type venom snakebite is obvious and new guidelines for treatment of neurotoxic-type venom snakebite and diagnosis should be developed.

Successful snakebite treatment in three juvenile African wild dogs (Lycaon pictus) with polyvalent antivenom: a Namibian case report.

This article reports the first documented treatment of venomous snakebite with a polyvalent snake antivenom from the South African Institute for Medical Research in endangered African wild dogs (Lycaon pictus). Three juvenile male animals (6.5 months of age) showed clinical signs after being bitten by an unidentified venomous snake. The signs included loss of appetite, disorientation, impaired locomotion, excessive facial swelling, profuse salivation, reduced respiratory effort and an apparent depressed mental state. Intravenous treatment with isotonic Ringer lactate solution, hetastarch 6% and dexamethazone, subcutaneous administration of procaine benzylpenicillin and benzathine benzylpenicillin, and ultimately intravenous administration of the polyvalent snake antivenom resulted in the complete recovery of all three wild dogs.

Evaluation of the effects of various chemicals on discharge of and pain caused by jellyfish nematocysts.

Jellyfish tentacles in contact with human skin can produce pain swelling and redness. The pain is due to discharge of jellyfish nematocysts and associated toxins and discharge can be caused by a variety of mechanical and chemical stimuli. A series of tests were carried out with chemicals traditionally used to treat jellyfish stings e.g. acetic acid ammonia meat tenderizer baking soda and urea to determine if these chemicals stimulated or inhibited nematocyst discharge and if they brought relief to testers who were exposed to jellyfish tentacles. Chrysaora quinquecirrha (sea nettle) Chiropsalmus quadrumanus (sea wasp) and Physalia physalis (Portuguese man-of-war) were used in the study. It was found that many of the chemicals traditionally used to treat jellyfish stings stimulated nematocyst discharge and did not relieve the pain. However there was immediate relief when a common anesthetic lidocaine was sprayed on the skin of testers in contact with jellyfish tentacles. Initial exposure of tentacle suspensions to lidocaine prevented the nematocyst discharge by subsequent exposure to acetic acid ethanol ammonia or bromelain. Thus lidocaine in addition to acting as an anesthetic on skin in contact with jellyfish tentacles inhibited nematocyst discharge possibly by blocking sodium and/or calcium channels of the nematocytes.

Características biológicas e inmunológicas del veneno de Bothrops cotiara (Serpentes: Viperidae) / Biological and immunological characteristics of the poison of Bothrops cotiara (Serpentes: Viperidae)

Bothrops cotiara is a venomous snake sporadically found in the province of Misiones in Argentina, South of Brazil and Paraguay. Data on the clinics of the envenomation produced by its bite and on its venom are scarce. There is no information on the neutralizing capacity of the antivenoms available. In this study, the lethal potency, hemorrhagic, necrotizing, coagulant and thrombin-like, defibrinogenating, indirect hemolytic and fibrinolytic activities of the venom of B. cotiara specimens from the province of Misiones were determined. The toxic activities were within the range of those described for the other Bothrops species from Argentina, and the electrophoretic and chromatographic studies showed similarities with those described for the other bothropic venoms. The immunochemical reactivity of six South American anti Viper antivenoms (ELISA) have a strong reactivity with all the antivenoms studied. The neutralizing capacity of three of these therapeutic antivenoms against the lethal potency and hemorrhagic, necrotizing, coagulant, thrombin-like and hemolytic activities showed a very close neutralizing capacity. Our data strongly suggest that the antivenoms for therapeutic use available in this area of South America are useful to neutralize the toxic and enzymatic activities of the venom of this uncommon specie of Bothrops.

Bothrops cotiara es una serpiente que se encuentra en la provincia de Misiones (Argentina), el Sur de Brasil y Paraguay. La información sobre las características clínicas de los accidentes por esta serpiente es muy escasa y existen pocos datos sobre su veneno y la capacidad neutralizante de las actividades tóxicas del mismo por antivenenos terapéuticos. En este trabajo se estudiaron características bioquímicas, actividades tóxicas y la reactividad inmunoquímica del veneno de B. cotiara. Seis antivenenos anti Viperinos Sudamericanos fueron estudiados frente a este veneno por el método ELISA y se probó la capacidad neutralizante de tres de estos frente a las actividades hemorrágica, necrotizante, procoagulante, trombina-símil, hemolítica indirecta y la potencia letal de veneno de ejemplares de B. cotiara de la provincia de Misiones. Los patrones cromatográficos y electroforéticos mostraron características similares a los de otros venenos de Bothrops. Las actividades tóxicas estuvieron dentro de los ámbitos descritos para los venenos botrópicos. Los seis antivenenos mostraron gran reactividad inmunoquímica por ELISA y las potencias neutralizantes de los tres estudiados fueron muy próximas para las actividades letal, hemorrágica, necrotizante, hemolítica indirecta, coagulante y trombina-símil. Los resultados de los estudios de neutralización indicarían que ante la mordedura de esta poco común especie de Bothrops, pueden usarse los diferentes tipos de antivenenos botrópicos o botrópico-crotálicos para uso terapéutico disponibles en esa región.

[Biological and immunological characteristics of the poison of Bothrops cotiara (Serpentes: Viperidae)]. / Características biológicas e inmunológicas del veneno de Bothrops cotiara (Serpentes: Viperidae).

Bothrops cotiara is a venomous snake sporadically found in the province of Misiones in Argentina, South of Brazil and Paraguay. Data on the clinics of the envenomation produced by its bite and on its venom are scarce. There is no information on the neutralizing capacity of the antivenoms available. In this study, the lethal potency, hemorrhagic, necrotizing, coagulant and thrombin-like, defibrinogenating, indirect hemolytic and fibrinolytic activities of the venom of B. cotiara specimens from the province of Misiones were determined. The toxic activities were within the range of those described for the other Bothrops species from Argentina, and the electrophoretic and chromatographic studies showed similarities with those described for the other bothropic venoms. The immunochemical reactivity of six South American anti Viper antivenoms (ELISA) have a strong reactivity with all the antivenoms studied. The neutralizing capacity of three of these therapeutic antivenoms against the lethal potency and hemorrhagic, necrotizing, coagulant, thrombin-like and hemolytic activities showed a very close neutralizing capacity. Our data strongly suggest that the antivenoms for therapeutic use available in this area of South America are useful to neutralize the toxic and enzymatic activities of the venom of this uncommon specie of Bothrops.

Enrichment of the antibodies against the C-terminus of Taiwan cobra cobrotoxin using dimeric glutaraldehyde-modified toxin as an immunogen.

The repertoire of antibodies producing by immunizing rabbits with cobrotoxin and dimeric glutaraldehyde-modified cobrotoxin (dGA-cobrotoxin) was analyzed by studying the immunoreactivity of the two antibody preparations toward cobrotoxin, GA-cobrotoxin and recombinant cobrotoxin. The results of enzyme-linked immunoassay revealed that the two antibody preparations exhibited a higher reactivity against their cognate antigen. Moreover, different behavior was observed for the reactivity of the two antibody preparations against GA-cobrotoxin and recombinant cobrotoxin. Notably, distortion of disulfide linkages at the C-terminus resulted in a reduced decrease in the antigenic activity of recombinant cobrotoxin toward anti-cobrotoxin antibodies compared to anti-dGA-cobrotoxin antibodies. Affinity purification of the antibodies against the C-terminus of cobrotoxin revealed that its amount represented 77% and 35.5% of the total anti-dGA-cobrotoxin antibodies and the total anti-cobrotoxin antibodies, respectively. These findings suggest that the antibody preparation elicited by dGA-cobrotoxin enriches the content of antibodies recognizes the C-terminal region of native cobrotoxin.

[Acute management of patients bitten by poisonous snakes]. / Acute opvang van patiënten gebeten door een gifslang.

The management of poisonous snake bites includes first aid and clinical medical treatment. First aid consists of reassurement of the patient, immobilisation of the bitten limb and rapid transport to the nearest hospital to monitor the vital functions. In no case suction, incision or tight bandages should be applied. The degree of envenomation can be classified in three categories depending on the spread of the symptoms: no symptoms, only local non-progressive symptoms, and systemic or local rapidly progressive symptoms (severe envenomation). Antivenin therapy is indicated in severe envenomation. Because of the risk of anaphylactic reactions or serum sickness, antivenin should be given with great caution. Antibiotic therapy and tetanus prophylaxis are advised in all cases. Immediate consultation with the National Intoxication Centre of the Rijksinstitut voor Volksgezondheid en Milieu (RIVM) is always warranted; telephone: 030-2748888. In a national protocol, which is available at the RIVM, the Harbour Hospital Rotterdam, the Academic Medical Centre and Artis Zoo Amsterdam, the correct management of snake bites is described. An overview of all antivenins available in the Netherlands is also given in this protocol.

Evidence showing a different repertoire of antibodies against unfolded cobrotoxin in anticobrotoxin and anti-reduced and S-carboxymethylated cobrotoxin antisera.

Antibodies which specifically reacted with reduced and S-carboxy-methylated(RCM) cobrotoxin were purified from anticobrotoxin antisera and anti-RCM-cobrotoxin antisera, respectively. Results using a competitive immunoassay revealed that the antibodies from anti-RCM-cobrotoxin antisera had a greater affinity for RCM-cobrotoxin than for cobrotoxin. Whereas the reactivity of the antibodies from the anticobrotoxin antisera toward cobrotoxin and RCM-cobrotoxin had a reversed order of binding. In contrast to observations made with S. aureus V8 protease-digest hydrolysates, the antigenic structures of RCM-cobrotoxin recognized by antibodies from anti-RCM-cobrotoxin antisera were notably affected following hydrolysis of RCM-cobrotoxin with chymotrypsin. Moreover, the chymotryptic hydrolysates showed a comparable reactivity as RCM-cobrotoxin toward the antibodies purified from anticobrotoxin antibodies, but a decrease in antigenicity with the V8 protease hydrolysates. These results reveal that the repertoire of antibodies against the unfolded cobrotoxin are not the same in anticobrotoxin and anti-RCM-cobrotoxin antisera. Moreover, it suggests that the repertoire of antibodies from the different sources against the same antigen can be differentiated by measurement of their reactivities with the proteolytic hydrolysates of the antigen.