Unveiling the secrets of snakes: Analysis of environmental, socioeconomic, and spatial factors associated with snakebite risk in Paraná, Southern Brazil.

The state of Paraná is home to three out of the five medically significant snake genera in Brazil and lacks of snakebite epidemiology studies. This study aimed to ascertain the spatial, environmental, and socioeconomic factors associated with snakebite risk by analyzing notification data of cases in the state of Paraná. Notification and socioeconomic data were gathered from the online platforms of the National System of Notifiable Diseases (SINAN) and the Brazilian Institute of Geography and Statistics (IBGE). Land cover and land use maps were obtained from the Mapbiomas platform in raster format and subsequently converted into vectors using QGis software. The proportions of land use and land cover in square kilometers (km2) were then calculated. All acquired data were tabulated using Microsoft Excel 365 software. For spatial analysis, GeoDa software version 1.20 was utilized to calculate the Global and Local Moran indices, assessing spatial correlations. Between 2007 and 2021, 12,877 notifications were recorded, with an average incidence of 8.22/100,000 inhabitants in the state, 8166 (63.41%) caused by Bothrops, 1534 (11.91%) caused by Crotalus, 56 (0.43%) caused by Micrurus. 1703 (13.22%) caused by non-venomous snake species, and the remaining cases did not have the identified causative species. The incidents caused by Bothrops and Crotalus showed different distribution patterns. Spatial analysis revealed that key factors contributing to snakebite risk included the presence of native forests, mangroves, apicuns, and monospecific planted forests. The population group at the highest risk comprised rural residents and workers. Furthermore, the absence of basic sanitation and proper garbage collection and disposal exhibited positive correlations with snakebites. Conversely, intensive farming practices with substantial mechanization and pastures demonstrated negative spatial correlations. This study has enabled the identification of the primary factors associated with snakebite risk, facilitating more targeted efforts to prevent snakebite accidents among vulnerable populations.

Snakebites in Northeastern Brazil: accessing clinical-epidemiological profile as a strategy to deal with Neglected Tropical Diseases.

BACKGROUND: Brazil ranks first in the number of snakebites in South America. A detailed analysis of these cases is required to improve the public health planning. In this study, we retrospectively examined the clinical and epidemiological profiles of snakebites in Maranhão between January 2009 and December 2019. METHODS: Data were obtained from the compulsory notification forms provided by the Health Department of Maranhão. RESULTS: A total of 17,658 cases were recorded during the study period. Most of the bites were from snakes belonging to the genus Bothrops. Medical care was mostly within three hours after the bite. Most cases were classified as mild and most victims recovered; however, 139 deaths were recorded. Most bites occurred among people aged 20-39 years, mainly among rural workers. The most frequent local clinical manifestations were pain, edema, and ecchymosis. The most common systemic clinical manifestations include neuroparalysis, vagal syndrome, and myolysis. Most snakebites occurred between January and March. The municipalities with the highest number of notifications were Buriticupu (936 cases), Arame (705 cases), and Grajaú (627 cases). CONCLUSIONS: The clinical profile of snakebites in Maranhão is similar to that observed in other states of Northeast Brazil. However, we found that some systemic manifestations are not compatible with the etiology of snakebites, which leads us to believe that the problem could be the lack of knowledge of the health professionals at the site of envenomation, who may not be ready for attendance, and an important lack of health centers with snake antivenom to treat snakebites.

First molecular assessment of two digenean parasites of the lancehead snake Bothrops moojeni Hoge, 1966 (Serpentes, Viperidae) in Brazil.

Two digenean species, Infidum infidum Faria, 1910 (Dicrocoeliidae) and Travtrema stenocotyle Cohn, 1902 (Plagiorchiidae), were collected in the large pit viper Bothrops moojeni Hoge, 1966 from Reserva Particular do Patrimônio Natural Cisalpina, municipality of Brasilândia, Mato Grosso do Sul State, Brazil. In this study, we provide the first molecular characterisation using the 28S rDNA and phylogenetic position data of these two common digeneans from B. moojeni. The molecular framework revealed topologies with strongly supported clades using maximum likelihood and Bayesian inference methods, positioned I. infidum among Plagiorchiidae and not among Dicrocoeliidae as expected and T. stenocotyle (Plagiorchiidae) surprisingly grouped as a sister group to Allassogonoporidae, Microphallidae, Pleurogenidae, and Prosthogonimidae, not related to plagiorchids. Our molecular phylogenetic data showed that these species may not correspond to their assigned families and encourage future studies on the systematic of these understudied groups.

Snakebite envenoming in French Guiana: Assessment of the preclinical efficacy against the venom of Bothrops atrox of two polyspecific antivenoms.

Snakebite envenoming is a relevant public health problem in French Guiana, and Bothrops atrox is responsible for the vast majority of envenomings in this overseas French territory. The preclinical efficacy of freeze-dried antivenoms manufactured in Costa Rica (Polival-ICP®) and Mexico (Antivipmyn Tri®) was assessed against the lethal, hemorrhagic, in vitro coagulant, and myotoxic effects of Bothrops atrox venom from French Guiana. Antivenoms differ in protein concentration and in the type of active principle (IgG and F (ab')2, respectively). Polival-ICP® showed significantly higher neutralizing activity against lethal, hemorrhagic and in vitro coagulant activities of the venom. Antivenoms neutralized myotoxic effect to a similar extent. In the case of lethal activity, Antivipmyn Tri® did not neutralize the effect at the highest antivenom level tested (1 mg venom/mL antivenom).

Clinical assessment and pathophysiology of Bothrops venom-related acute kidney injury: a scoping review

Bothrops are one of the most common medically important snakes found in Latin America. Its venom is predominantly hemotoxic and proteolytic, which means that local lesion (edema and redness) and hemorrhagic symptoms are recurrent in envenoming by this snake. Although hemorrhage is usually the major cause of death, snakebite-related acute kidney injury is another potentially fatal clinical complication that may lead to chronic kidney disease. The present review highlights the main studies on Bothrops venom-related acute kidney injury, including observational, cross-sectional, case-control and cohort human studies available up to December 2019. The following descriptors were used according to Medical Subject Headings (MeSH): on Medline/Pubmed and Google Scholar "acute kidney injury" or "kidney disease" and "Bothrops"; on Lilacs and SciELO "kidney disease" or "acute kidney injury" and "Bothrops". Newcastle-Ottawa quality assessment scale was used to appraise the quality of the cross-sectional and cohort studies included. The selection of more severe patients who looked for health care units and tertiary centers is a risk of bias. Due to the methodological heterogeneity of the studies, a critical analysis of the results was performed based on the hypothesis that the design of the included studies influences the incidence of acute kidney injury. Fifteen human studies (total participants 4624) were included according to stablished criteria. The coagulation abnormalities (hemorrhagic symptoms, abnormal fibrinogen and activated partial thromboplastin time) were associated with acute kidney injury in the most recent studies reported. The findings observed in this review provide up-to-date evidence about the acute kidney injury pathogenesis following Bothrops syndrome. Studies pointed out that coagulation abnormalities comprise the major pathway for acute kidney injury development. This review may improve patient management by primary healthcare providers, allowing earlier diagnosis and treatment of Bothrops venom-related acute kidney injury.(AU)

Expression of an scFv antibody fragment in Nicotiana benthamiana and in vitro assessment of its neutralizing potential against the snake venom metalloproteinase BaP1 from Bothrops asper.

Human accidents with venomous snakes represent an overwhelming public health problem, mainly in rural populations of underdeveloped countries. Their high incidence and the severity of the accidents result in 81,000 to 138,000 deaths per year. The treatment is based on the administration of purified antibodies, produced by hyper immunization of animals to generate immunoglobulins (Igs), and then obtained by fractionating hyper immune plasma. The use of recombinant antibodies is an alternative to conventional treatment of snakebite envenoming, particularly the Fv fragment, named the single-chain variable fragment (scFv). We have produced recombinant single chain variable fragment scFv against the venom of the pit viper Bothrops asper at high levels expressed transiently and stably in transgenic plants and in vitro cultures that is reactive to BaP1 (a metalloproteinase from B. asper venom). The yield from stably transformed plants was significantly (p > 0.05) higher than the results in from transient expression. In addition, scFvBaP1 yields from systems derived from stable transformation were: transgenic callus 62 µg/g (±2); biomass from cell suspension cultures 83 µg/g (±0.2); culture medium from suspensions 71.75 mg/L (±6.18). The activity of scFvBaP1 was confirmed by binding and neutralization of the fibrin degradation induced by BnP1 toxins from B. neuwiedi and by Atroxlysin Ia from B. atrox venoms. In the present work, we demonstrated the potential use of plant cells to produce scFvBaP1 to be used in the future as a biotechnological alternative to horse immunization protocols to produce anti-venoms to be used in human therapy against snakebites.

Snakebite accidents in Rio Grande do Norte state, Brazil: Epidemiology, health management and influence of the environmental scenario.

OBJECTIVES: Brazil is home to large variety of snake species, of which about 17% are venomous. A large proportion of reported snakebite accidents in the country take place in northeast Brazil. We aimed to analyse the epidemiology of snakebites as a public health concern in Rio Grande do Norte state. METHODS: A retrospective epidemiologic analysis was performed using data from the Brazilian Case Registry Database pertaining to the period 2007-2016, and considering environmental climate characteristics as an influential factor. RESULTS: A number of 3909 cases were reported in total. Among those cases, 58% involved venomous species and more than 80% of these were caused by Bothrops species. An association between environmental characteristics and the incidence of reported cases was found. The Borborema Potiguar region was the most affected area. Apart from that, deficiencies in managing the victims were described. CONCLUSION: This study highlights snakebite accidents as a public health concern in Rio Grande do Norte state. Snake bites are most likely being influenced by climate change and human activities. Continuous training of involved medical personnel could help optimise patient care and avoid under reporting in the accident reporting system.

OBJECTIFS: Le Brésil abrite de très nombreuses espèces de serpents, dont environ 17% sont venimeuses. Une grande partie des accidents rapportés de morsures de serpent dans le pays ont lieu dans le nord-est du pays. Notre objectif était d'analyser l'épidémiologie des morsures de serpents en tant que problème de santé publique dans l'Etat du Rio Grande do Norte. MÉTHODES: Une analyse épidémiologique rétrospective a été réalisée à l'aide des données de la Base de Données du Registre Brésilien des cas couvrant la période 2007-2016 et prenant en compte les caractéristiques climatiques de l'environnement comme facteur d'influence. RÉSULTATS: 3.909 cas au total ont été rapportés. 58% concernaient des espèces venimeuses et plus de 80% d'entre elles étaient causées par l'espèce Bothrops. Une association entre les caractéristiques environnementales et l'incidence des cas rapportés a été mise en évidence. La région de Borborema Potiguar était la plus touchée. Outre cela, les déficiences dans la prise en charges des victimes ont été décrites. CONCLUSION: Cette étude souligne que les accidents causés par des morsures de serpent constituent un problème de santé publique dans l'Etat de Rio Grande do Norte. Les morsures de serpents sont très probablement influencées par les changements climatiques et les activités humaines. La formation continue du personnel médical impliqué pourrait aider à optimiser les soins prodigués aux patients et à éviter une sous-notification dans le système de notification des accidents.

Assessment of the anti-snakebite properties of extracts of Aniba fragrans Ducke (Lauraceae) used in folk medicine as complementary treatment in cases of envenomation by Bothrops atrox.

ETHNOPHARMACOLOGICAL RELEVANCE: Extracts of leaves and bark of Aniba fragrans are used as tea (decoction) to treat snakebites in communities in the Brazilian Amazon. The aqueous extract of the leaves of A. fragrans has been proven to be effective against Bothrops venom, but only when pre-incubated with the venom. This study sought to assess the potential of different types of extract of this species to inhibit the biological activities of Bothrops atrox venom (BaV) when used the same way as in folk medicine. The main classes of secondary metabolites and the concentrations of phenolics in the extracts were also determined. MATERIALS AND METHODS: Four types of extract of A. fragrans were prepared: aqueous extract of the leaf (AEL), aqueous extract of the bark (AEB), hydroalcoholic leaf extract (HLE) and extract of the residue from hydrodistillation of the leaf (ERHL). The phytochemical profiles of the aqueous extracts were determined using thin layer chromatography (TLC), and the concentrations of phenolics were measured by colorimetric assays. To investigate the potential of the extracts to inhibit the biological activities of BaV, in vitro tests for antiphospholipase and antifibrinolytic activities were performed. In vivo tests for antihemorrhagic and antidefibrinating activities were also carried out, as well as antimicrobial tests for activity against the main bacteria found in the oral cavity of snakes. Interaction between the extracts and the proteins in BaV was assessed by electrophoresis (SDS-PAGE) and Western blot (WB). The cytotoxicity of the extracts was assessed in a strain of MRC-5 human fibroblasts. RESULTS: Terpenoids, flavonoids and condensed and hydrolysable tannins were detected in all the extracts. Metabolites such as coumarins, fatty acids and alkaloids were present in some extracts but not in others, indicating different phytochemical profiles. Phenolics content varied between extracts, and there were more tannins in AEB and HLE. In the in vitro tests, the extracts inhibited the phospholipase and fibrinolytic activities of BaV in the two ratios of venom to extract used. HLE exhibited effective antimicrobial action as it inhibited growth of 11 of the 15 bacteria investigated, including Morganella morganii, the main bacteria described in the oral cavity of snakes. The extracts failed to inhibit the defibrinating activity of BaV, and only the Bothrops antivenom had a significant effect (96.1%) on this activity. BaV-induced hemorrhage was completely inhibited by AEL and AEB when the pre-incubation (venom:extract) protocol was used. When administered orally, as in folk medicine, both AEB and AEL produced significant inhibition of hemorrhagic activity (maximum inhibition 46.5% and 39.2%, respectively). SDS-PAGE and WB of the extracts pre-incubated with BaV showed that the main proteins in the venom had been precipitated by the extracts. None of the four extracts showed cytotoxic effects in the tests carried out with a human fibroblast cell line. CONCLUSION: In addition to being effective in reducing hemorrhage when administered orally, the extracts displayed a high antimicrobial potential against microorganisms involved in secondary infections at the site of the snakebite. Once the extracts have been tested in accordance with the appropriate regulations, this species could potentially be used to produce a phytomedicine for complementary treatment of the secondary infections due to bacteria that aggravate the local signs and symptoms after snakebite envenomation.

MICROCOMPUTED TOMOGRAPHIC, MORPHOMETRIC, AND HISTOPATHOLOGIC ASSESSMENT OF CONGENITAL BONE MALFORMATIONS IN TWO NEOTROPICAL VIPERIDS.

Congenital malformations have been reported in all classes of vertebrates and may be a determinant of life span and survival. In reptiles, the incidence of congenital malformations can be associated with genetic and environmental causes, including pollution. The characterization of pathological processes involved in the development of congenital malformations of bone in snakes is rare in the literature, but is of great relevance in the field of reptile conservation and environmental health. We describe congenital bone lesions in 50 newborn jararaca (Bothrops jararaca) and 26 South American rattlesnakes (Crotalus durissus terrificus) born from wild-caught pregnant females in Southeastern Brazil. Lesions were evaluated by morphometric quantitative analysis, x-ray microtomography, and histopathologic descriptive analysis. Morphometric analysis showed that jararaca presented more severe axial lesions (kyphosis, scoliosis, and kyphoscoliosis) than rattlesnakes. Female rattlesnakes presented more severe axial lesions than did males. In rattlesnakes, spinal deformities were more frequently diagnosed in the caudal segment of the body. We present x-ray microtomographic assessments and images of malformed snakes (n=9) and characterized novel malformations, such as the agenesis of frontal, parietal, and supraoccipital bones in a jararaca specimen. Histopathologic findings included vertebral body fusion, myositis, coagulation necrosis, and disorganization of periaxial muscle fibers. The new methods and results presented in this study will be useful and informative for future research in pathology, teratology, embryology, and ecotoxicology in snakes.

A new l-amino acid oxidase from Bothrops jararacussu snake venom: Isolation, partial characterization, and assessment of pro-apoptotic and antiprotozoal activities.

A new l-amino acid oxidase (LAAO) from Bothrops jararacussu venom (BjussuLAAO-II) was isolated by using a three-step chromatographic procedure based on molecular exclusion, hydrophobicity, and affinity. BjussuLAAO-II is an acidic enzyme with pI=3.9 and molecular mass=60.36kDa that represents 0.3% of the venom proteins and exhibits high enzymatic activity (4884.53U/mg/mim). We determined part of the primary sequence of BjussuLAAO-II by identifying 96 amino acids, from which 34 compose the N-terminal of the enzyme (ADDRNPLEECFRETDYEEFLEIARNGLSDTDNPK). Multiple alignment of the partial BjussuLAAO-II sequence with LAAOs deposited in the NCBI database revealed high similarity (95-97%) with other LAAOs isolated from Bothrops snake venoms. BjussuLAAO-II exerted a strong antiprotozoal effect against Leishmania amazonensis (IC50=4.56µg/mL) and Trypanosoma cruzi (IC50=4.85µg/mL). This toxin also induced cytotoxicity (IC50=1.80µg/mL) and apoptosis in MCF7 cells (a human breast adenocarcinoma cell line) by activating the intrinsic and extrinsic apoptosis pathways, but were not cytotoxic towards MCF10A cells (a non-tumorigenic human breast epithelial cell line). The results reported herein add important knowledge to the field of Toxinology, especially for the development of new therapeutic agents.

Inhibition of local effects induced by Bothrops erythromelas snake venom: Assessment of the effectiveness of Brazilian polyvalent bothropic antivenom and aqueous leaf extract of Jatropha gossypiifolia.

Bothrops erythromelas is a snake of medical importance responsible for most of the venomous incidents in Northeastern Brazil. However, this species is not included in the pool of venoms that are used in the Brazilian polyvalent bothropic antivenom (BAv) production. Furthermore, it is well known that antivenom therapy has limited efficacy against venom-induced local effects, making the search for complementary alternatives to treat snakebites an important task. Jatropha gossypiifolia is a medicinal plant widely indicated in folk medicine as an antidote for snakebites, whose effectiveness against Bothrops jararaca venom (BjV) has been previously demonstrated in mice. In this context, this study assessed the effectiveness of the aqueous extract (AE) of this plant and of the BAv against local effects induced by B. erythromelas venom (BeV). Inhibition of BeV-induced edematogenic and hemorrhagic local effects was assayed in mice in pre-treatment (treatment prior to BeV injection) and post-treatment (treatment post-envenomation) protocols. Inhibition of proteolytic, phospholipase A2 (PLA2) and hyaluronidase enzymatic activities of BeV were evaluated in vitro. BAv cross-reactivity and estimation of antibody titers against BeV and BjV were assessed by Ouchterlony double diffusion test. The results show that in pre-treatment protocol AE and BAv presented very similar effects (about 70% of inhibition for edematogenic and 40% for hemorrhagic activities). However, BAv poorly inhibited edema and hemorrhage in post-envenomation protocol, whilst, in contrast, AE was significantly active even when used after BeV injection. AE was able to inhibit all the tested enzymatic activities of BeV, while BAv was active only against hyaluronidase activity, which could justify the low effectiveness of BAv against BeV-induced local effects in vivo. Ouchterlony's test showed positive cross-reactivity against BeV, but the antibody titers were slightly higher against BjV. Together, these data indicate that despite the presence of immunological cross-reactivity, Brazilian polyvalent bothropic antivenom presented low inhibitory potential against biological and enzymatic effects of BeV, illustrating the need for new strategies in the production of antivenom with broad neutralizing potential in the treatment of Bothrops spp. envenomation throughout the country. Together, the results highlight the antiophidic potential of J. gossypiifolia, suggesting that it can be considered a potential adjuvant in the treatment of bothropic envenomation local effects.

Plants used to treat snakebites in Santarém, western Pará, Brazil: an assessment of their effectiveness in inhibiting hemorrhagic activity induced by Bothrops jararaca venom.

ETHNOPHARMACOLOGICAL RELEVANCE: The poor distribution and limited availability of antivenoms in Brazil have led to greater use of plants to treat snakebites. Very often such plants are the only alternative available to riverside communities. MATERIALS AND METHODS: Direct questionnaire-based interviews were conducted with members of the Cucurunã, São Pedro and Alter do Chão communities in Santarém, Pará, Brazil. For each of the 12 most frequently mentioned species aqueous extracts were prepared and the phytochemical profiles determined by thin layer chromatography. The concentrations of phenolic compounds (tannins and flavonoids) in the aqueous extracts were determined by colorimetric assays. To assess inhibition of the hemorrhagic activity of Bothrops jararaca venom, solutions containing the venom mixed with aqueous extracts in the ratios 1:12 and 1:48 were tested (w/w). SDS-PAGE and Western blot were used to assess the action of the extracts on Bothrops jararaca venom. RESULTS: In all, 24 plants belonging to 19 families were mentioned in the survey as being used to treat snakebites. Leaves (84%), seeds (60.9%) and inner bark (53%) were cited as the most frequently used parts in folk remedies, which were usually prepared in the form of a decoction (62.5%), tincture (45%) or maceration (22.5%). Hemorrhage induced by Bothrops jararaca venom was completely inhibited by aqueous extracts of Bellucia dichotoma, Connarus favosus, Plathymenia reticulata and Philodendron megalophyllum, which had a high phenolic content and contained condensed and hydrolyzable tannins. The results of SDS-PAGE showed that some venom protein bands were not visible when the venom was preincubated with the extracts that had completely inhibited hemorrhagic activity of the venom. Western blot showed that the extracts did not have any enzymatic action on the proteins in the venom as it failed to detect low-molecular-weight bands, which are indicative of possible enzymatic cleavage. CONCLUSIONS: Traditional use of plants to treat snakebites is a common practice in the western region of Pará, Brazil. Our findings show that some plant extracts were able to inhibit snake venom-induced hemorrhage in vitro. In vivo studies are being carried out to validate the traditional use of these species to treat snakebites.

Toxicity of Bothrops sp snake venoms from Ecuador and preclinical assessment of the neutralizing efficacy of a polyspecific antivenom from Costa Rica.

The toxicological profile of the venoms of the snakes Bothrops asper and Bothrops atrox from Ecuador was investigated, together with the venom of a population of B. asper formerly classified as 'Bothrops xanthogrammus'. The three venoms exerted lethal, hemorrhagic, myotoxic, coagulant and defibrinogenating effects, in agreement with the characteristic toxicological profile of Bothrops sp venoms. A polyspecific antivenom (bothropic-crotalic-lachesic) manufactured in Costa Rica was assessed for its preclinical efficacy against the toxic activities of these Ecuadorian venoms. Antivenom was effective in the neutralization of the five activities tested in the three venoms. These observations are in agreement with previous reports on the extensive cross-reactivity and paraspecific neutralization of antivenoms manufactured in Latin America against the venoms of Bothrops sp snakes.

Preliminary assessment of Hedychium coronarium essential oil on fibrinogenolytic and coagulant activity induced by Bothrops and Lachesis snake venoms

The search for new inhibitors of snake venom toxins is essential to complement or even replace traditional antivenom therapy, especially in relation to compounds that neutralize the local effects of envenomations. Besides their possible use as alternative to traditional antivenom therapy, some plant species possess bioactive secondary metabolites including essential oils, which can be extracted from weeds that are considered substantial problems for agriculture, such as Hedychium coronarium. The essential oils of leaves and rhizomes from H. coronarium were extracted by hydrodistillation, and their potential inhibitory effects on the coagulant and fibrinogenolytic activities induced by the venoms of Lachesis muta, Bothrops atrox and Bothrops moojeni were analyzed. Citrated human plasma was used to evaluate the clotting time whereas changes in fibrinogen molecules were visualized by electrophoresis in polyacrylamide gel. The experimental design used for testing coagulation inhibition was randomized in a 3 × 2 factorial arrangement (concentration × essential oils), with three replications. The essential oils were compared since they were extracted from different organs of the same botanical species, H. coronarium. The results suggest that the oils interact with venom proteases and plasma constituents, since all oils evaluated, when previously incubated with venoms, were able to inhibit the clotting effect, with less inhibition when oils and plasma were preincubated prior to the addition of venoms. Thus, after extensive characterization of their pharmacological and toxicological effects, the essential oils can be used as an alternative to complement serum therapy, especially considering that these plant metabolites generally do not require specific formulations and may be used topically immediately after extraction.

Preliminary assessment of Hedychium coronarium essential oil on fibrinogenolytic and coagulant activity induced by Bothrops and Lachesis snake venoms

Background The search for new inhibitors of snake venom toxins is essential to complement or even replace traditional antivenom therapy, especially in relation to compounds that neutralize the local effects of envenomations. Besides their possible use as alternative to traditional antivenom therapy, some plant species possess bioactive secondary metabolites including essential oils, which can be extracted from weeds that are considered substantial problems for agriculture, such as Hedychium coronarium.Methods The essential oils of leaves and rhizomes from H. coronarium were extracted by hydrodistillation, and their potential inhibitory effects on the coagulant and fibrinogenolytic activities induced by the venoms of Lachesis muta,Bothrops atrox and Bothrops moojeniwere analyzed. Citrated human plasma was used to evaluate the clotting time whereas changes in fibrinogen molecules were visualized by electrophoresis in polyacrylamide gel. The experimental design used for testing coagulation inhibition was randomized in a 3 × 2 factorial arrangement (concentration × essential oils), with three replications. The essential oils were compared since they were extracted from different organs of the same botanical species, H. coronarium.Results The results suggest that the oils interact with venom proteases and plasma constituents, since all oils evaluated, when previously incubated with venoms, were able to inhibit the clotting effect, with less inhibition when oils and plasma were preincubated prior to the addition of venoms.Conclusions Thus, after extensive characterization of their pharmacological and toxicological effects, the essential oils can be used as an alternative to complement serum therapy, especially considering that these plant metabolites generally do not require specific formulations and may be used topically immediately after extraction.(AU)

Preclinical assessment of the neutralizing capacity of antivenoms produced in six Latin American countries against medically-relevant Bothrops snake venoms.

Species of the genus Bothrops induce the vast majority of snakebite envenomings in Latin America. A preclinical study was performed in the context of a regional network of public laboratories involved in the production, quality control and development of antivenoms in Latin America. The ability of seven polyspecific antivenoms, produced in Argentina, Brazil, Peru, Bolivia, Colombia and Costa Rica, to neutralize lethal, hemorrhagic, coagulant, defibrinogenating and myotoxic activities of the venoms of Bothrops neuwiedi (diporus) (Argentina), Bothrops jararaca (Brazil), B. neuwiedi (mattogrossensis) (Bolivia), Bothrops atrox (Peru and Colombia) and Bothrops asper (Costa Rica) was assessed using standard laboratory tests. Despite differences in the venom mixtures used in the immunization of animals for the production of these antivenoms, a pattern of extensive cross-neutralization was observed between these antivenoms and all the venoms tested, with quantitative differences in the values of effective doses. This study reveals the capacity of these antivenoms to neutralize, in preclinical tests, homologous and heterologous Bothrops venoms in Central and South America, and also highlight quantitative differences in the values of Median Effective Doses (ED50s) between the various antivenoms.

Bothrops lanceolatus bites: guidelines for severity assessment and emergent management.

Approximately 20-30 declared snakebite cases occurin Martinique each year. Bothrops lanceolatus, a member of the Crotalidae family, is considered to be the only involved snake. B. lanceolatus, commonly named "Fer-de-Lance", is endemic and only found on this Caribbean island. Envenomation local features include the presence of fang marks, swelling, pain, bleeding from punctures, and ecchymosis. Severe envenomation is associated with multiple systemic thromboses appearing within 48 h of the bite and resulting in cerebral, myocardial or pulmonary infarctions. Diagnosis requires first of all identification of the snake. Coagulation tests are helpful to identify thrombocytopenia or disseminated intravascular coagulation. A clinical score based on 4 grades is helpful to assess envonimation severity. A specific monovalent equine anti-venom (Bothrofav(®), Sanofi-Pasteur, France) to neutralize B. lanceolatus venom is available. Its early administration within 6h from the biting in case of progressive local injures, general signs or coagulation disturbances is effective to prevent severe thrombosis and coagulopathy. Its tolerance is considered to be good. Despite an increasing incidence of bites, no deaths have been recently attributed to B. lanceolatus in Martinique, probably due to the currently recommended strategy of early antivenom administration when required.

Contribution of mast cells to the oedema induced by Bothrops moojeni snake venom and a pharmacological assessment of the inflammatory mediators involved.

The ability of Bothrops moojeni venom (BmV) to induce oedema in mice, the involvement of principal inflammatory mediators and mast cells (MCs) were investigated. The intraplantar injection of BmV (0.3-6 microg/paw) caused a dose- and time-dependent oedema with a peak between 30 and 60 min after venom injection (0.3-1 microg/paw), disappearing within 24h. Either MCs granule inhibition or depletion by cromoglycate or C48/80, respectively, markedly reduced BmV-induced oedema. MCs depletion by imatinib also reduced oedema. Intraperitoneal BmV injection (2.5-10 microg/site) induced MCs degranulation and release of PGD(2). Treatment with promethazine, cimetidine or thioperamide, histamine H1, H2 and H3/H4 receptor antagonists, respectively, markedly reduced the initial phase of oedema. Combined treatment with these antagonists further reduced, but not abrogated oedema. Indomethacin or eterocoxib (cyclooxygenase inhibitors) reduced oedema until 180 min, whereas zileuton (lipoxygenase inhibitor) affected this event until 60 min. Dexamethazone caused a long lasting reduction of oedema. However, L-NAME and aminoguanidine (NO synthase inhibitors) significantly increased BmV-induced oedema. In conclusion, BmV induces oedema, mediated by MCs degranulation, histamine by H1, H2, H3/H4 receptors, prostaglandins and leukotrienes, and down-regulated by NO. Partial neutralization of oedema was observed even when polyspecific bothropic antivenom was injected immediately after venom.

Assessment of the impact of solvent/detergent treatment on the quality and potency of a whole IgG equine antivenom.

We have evaluated for the first time the impact of a solvent/detergent (S/D) treatment on the quality and in vivo neutralization potency of horse-derived whole IgG antivenom used in the treatment of viperid snake bite envenoming in Central America. The S/D treatment by 1% tri (n-butyl) phosphate (TnBP) - 1% Triton X-45 at 22-25 degrees C was applied either on starting plasma or on purified immunoglobulins. The S/D agents were removed from both fractions by extractions with oil. S/D-treated plasma was subjected to caprylic acid precipitation to purify the immunoglobulins. Products were formulated, sterile-filtered, and filled into 10-mL vials, stored at 5+/-3 degrees C, and subjected to routine quality controls, SDS-PAGE, determination of anti-Bothrops asper venom antibody titre by ELISA, in vivo B. asper venom-neutralization potency tests, and safety test, comparatively with an antivenom manufactured by caprylic acid fractionation without S/D treatment. Results indicate that these conditions of S/D treatment on purified immunoglobulin yielded an antivenom of high turbidity that induced weight loss in animals. In contrast, antivenom fractionated from the S/D-treated plasma had physico-chemical and biological characteristics indistinguishable from those of the non-S/D-treated antivenom. S/D treatment of horse plasma may be considered to increase the viral safety of antivenoms.