Butantan Institute Research Articles

Using crystallography tools to improve vaccine formulations.

This article summarizes developments attained in oral vaccine formulations based on the encapsulation of antigen proteins inside porous silica matrices. These vaccine vehicles show great efficacy in protecting the proteins from the harsh acidic stomach medium, allowing the Peyer's patches in the small intestine to be reached and consequently enhancing immunity. Focusing on the pioneering research conducted at the Butantan Institute in Brazil, the optimization of the antigen encapsulation yield is reported, as well as their distribution inside the meso- and macroporous network of the porous silica. As the development of vaccines requires proper inclusion of antigens in the antibody cells, X-ray crystallography is one of the most commonly used techniques to unveil the structure of antibody-combining sites with protein antigens. Thus structural characterization and modelling of pure antigen structures, showing different dimensions, as well as their complexes, such as silica with encapsulated hepatitis B virus-like particles and diphtheria anatoxin, were performed using small-angle X-ray scattering, X-ray absorption spectroscopy, X-ray phase contrast tomography, and neutron and X-ray imaging. By combining crystallography with dynamic light scattering and transmission electron microscopy, a clearer picture of the proposed vaccine complexes is shown. Additionally, the stability of the immunogenic complex at different pH values and temperatures was checked and the efficacy of the proposed oral immunogenic complex was demonstrated. The latter was obtained by comparing the antibodies in mice with variable high and low antibody responses.

Lonomia obliqua Envenoming and Innovative Research.

As a tribute to Butantan Institute in its 120th anniversary, this review describes some of the scientific research efforts carried out in the study of Lonomia envenoming in Brazil, a country where accidents with caterpillars reach over 42,000 individuals per year (especially in South and Southeast Brazil). Thus, the promising data regarding the studies with Lonomia’s toxins contributed to the creation of new research centers specialized in toxinology based at Butantan Institute, as well as to the production of the antilonomic serum (ALS), actions which are in line with the Butantan Institute mission “to research, develop, manufacture, and provide products and services for the health of the population”. In addition, the study of the components of the Lonomia obliqua bristle extract led to the discovery of new molecules with peculiar properties, opening a field of knowledge that could lead to the development and innovation of new drugs aimed at cell regeneration and inflammatory diseases.

Zebrafish Beyond the Bench: The 'Plataforma Zebrafish Open Doors' Programme.

The Butantan Institute is a pioneering Brazilian health sciences institution, which also houses a large science park with museums that contribute to ongoing science education for schools and the wider community. In recent years, as part of Butantan Institute's Plataforma Zebrafish™, zebrafish embryos have been used for the dissemination of scientific knowledge during on-site events and as part of outreach campaigns to non-scientific audiences, mostly children. The aim of this work is mainly to demystify the activities of the scientific researcher, highlight the role of science in the furthering of knowledge, and increase public interest and confidence in science. In this article, the Institute's 'Plataforma Zebrafish Open Doors' programme is described, which offered guided tours of the laboratory facilities. The tours gave visitors the opportunity to observe zebrafish research and embryo development, and to use the knowledge gained from this experience as a framework for understanding fundamental ethical issues. During the 2-day event, around 800 visitors (most of them school-age children) attended. Together with the guided tours, our experience of outreach offered meaningful opportunities to bring children and members of the public closer to science and 'real-life' scientists, hopefully inspiring and encouraging the next generation of scientists. It also gave the scientists an opportunity to engage more closely with wider society. We believe that these activities also substantially contribute to the wider dissemination of relevant experimental results that have been obtained with public funding and that impact society in general.

Clinical implications of ontogenetic differences in the coagulotoxic activity of Bothrops jararacussu venoms

Is snake venom activity influenced by size? This is a long-standing question that can have important consequences for the treatment of snake envenomation. Ontogenetic shifts in venom composition are a well-documented characteristic of numerous snake species. Although snake venoms can cause a range of pathophysiological disturbances, establishing the coagulotoxic profiles related to such shifts is a justified approach because coagulotoxicity can be deadly, and its neutralisation is a challenge for current antivenom therapy. Thus, we aimed to assess the coagulotoxicity patterns on plasma and fibrinogen produced by B othrops jararacussu venoms from individuals of different sizes and sex, and the neutralisation potential of SAB (anti bothropic serum produced by Butantan Institute). The use of a metalloproteinase inhibitor (Prinomastat) and a serine proteinase inhibitor (AEBSF) enabled us to determine the toxin class responsible for the observed coagulopathy: activity on plasma was found to be metalloprotease driven, while the activity on fibrinogen is serine protease driven. To further explore differences in venom activity, the activation of Factor X and prothrombin as a function of snake size was also evaluated. All the venoms exhibited a potent procoagulant effect upon plasma and were less potent in their pseudo-procoagulant clotting effect upon fibrinogen. On human plasma, the venoms from smaller snakes produced more rapid clotting than the larger ones. In contrast, the venom activity on fibrinogen had no relation with size or sex. The difference in procoagulant potency was correlated with the bigger snakes being proportionally better neutralized by antivenom due to the lower levels of procoagulant toxins, than the smaller. Thus, while the antivenom ultimately neutralized the venoms, proportionally more would be needed for an equal mass of venom from a small snake than a large one. Similarly, the neutralisation by SAB of the pseudo-procoagulant clotting effects was also correlated with relative potency, with the smaller and bigger snakes being neutralized proportional to potency, but with no correlation to size. Thromboelastography (TEG) tests on human and toad plasma revealed that small snakes’ venoms acted quicker than large snakes’ venom on both plasmas, with the action upon amphibian plasma consistent with smaller snakes taking a larger proportion of anuran prey than adults. Altogether, the ontogenetic differences regarding coagulotoxic potency and corresponding impact upon relative antivenom neutralisation of snakes with different sizes were shown, underscoring the medical importance of investigating ontogenetic changes in order to provide data crucial for evidence-based design of clinical management strategies.

How the Mainstream Media Help to Spread Disinformation about Covid-19

How the Mainstream Media Help to Spread Disinformation about Covid-19

Maintenance of venomous snakes in captivity for venom production at Butantan Institute from 1908 to the present: a scoping history.

Maintenance of snakes at Butantan Institute started in the last century, intending to produce a different antivenom serum to reduce death caused by snakebites. Through a successful campaign coordinated by Vital Brazil, farmers sent venomous snakes to Butantan Institute by the railway lines with no cost. From 1908 to 1962, the snakes were kept in an outdoor serpentarium, where venom extraction was performed every 15 days. During this period, the snake average survival was 15 days. In 1963, the snakes were transferred to an adapted building, currently called Laboratory of Herpetology (LH), to be maintained in an intensive system. Although the periodicity of venom extraction remained the same, animal average survival increased to two months. With the severe serum crisis in 1983, the Ministry of Health financed remodeling for the three public antivenom producers, and with this support, the LH could be improved. Air conditioning and exhausting systems were installed in the rooms, besides the settlement of critical hygienic-sanitary managements to increase the welfare of snakes. In the early 1990s, snake survival was ten months. Over the years to the present day, several improvements have been made in the intensive serpentarium, as the establishment of two quarantines, feeding with thawed rodents, an interval of two months between venom extraction routines, and monitoring of snake health through laboratory tests. With these new protocols, average snake survival increased significantly, being eight years for the genus Bothrops, ten years for genus Crotalus and Lachesis, and four years for the genus Micrurus. Aiming the production of venoms of good quality, respect for good management practices is essential for the maintenance of snakes in captivity. New techniques and efficient management must always be sought to improve animal welfare, the quality of the venom produced, and the safety of those working directly with the venomous snakes.

Projeto S: A Stepped-Wedge Randomized Trial to Assess CoronaVac Effectiveness in Serrana, Brazil

Background: A stepped-wedge trial is an approach for assessing vaccine effectiveness in the real world. By the end of the study, all participants could receive the intervention, eliminating the ethical dilemma of placebo, especially during a pandemic. Methods: We evaluated the effectiveness of CoronaVac in Serrana, Brazil, amid an uncontrolled community Covid-19 epidemic using a stepped-wedge randomized trial. The city was separated into 25 subareas, divided into four groups, and randomized to receive CoronaVac in a two-dose scheme with a four-week interval. Intervention was initiated in each group with a one-week interval. The primary endpoint was the incidence of symptomatic cases in fully immunized individuals. The secondary endpoints were Covid-19-related hospitalizations and deaths and incidence according to immunization coverage. Findings: The study occurred during epidemiological weeks 6 to 19 in 2021. Up to 27,406 participants received the first dose of the study vaccine, corresponding to 81·3% of the adults and 60·9% of the urban population. Among fully immunized individuals, the vaccine effectiveness was 80·5 (95% CI, 75·1 to 84·7) for preventing symptomatic Covid-19 cases, 95% (95% CI, 86·9 to 98·1) and 94·9% (95% CI, 76·4 to 98·9) for preventing Covid-19-related hospitalizations and deaths, respectively. There was a significant indirect protective effect in unvaccinated people when 52% of the adult population was fully vaccinated. The Gamma variant was dominant during the study. Interpretation: CoronaVac effectively prevented symptomatic Covid-19 cases and protected against severe disease and death during Gamma variant circulation. Unvaccinated individuals benefited from high vaccine coverage levels. Trial Registration: This study was registered with (ClinicalTrials.gov Identifier, NCT04747821) Funding: Fundacao Butantan and Sao Paulo Research Foundation (FAPESP). Declaration of Interest: MCB, BMC, GJV, NNF, PMMG, RH, BALF, and RTC received research funding from Butantan Institute during the conduct of this study. RP, MTRPC, APB, JPS, and ROP were employees of Butantan Institute during the conduct of this study. HAB, EGP, GGP, SCSV and DC are employees of Butantan Institute. All other authors declare no competing interests Ethical Approval: The study was reviewed and approved by the Ethics Committee of the Clinical Hospital, Ribeirao Preto Medical School, University of Sao Paulo (CAAE 42390621.1.0000.5440).

Modelling non-proportional hazard for survival data with different systematic components

We propose a new extended regression model based on the logarithm of the generalized odd log-logistic Weibull distribution with four systematic components for the analysis of survival data. This regression model can be very useful and could give more realistic fits than other special regression models. We obtain the maximum likelihood estimates of the model parameters for censored data and address influence diagnostics and residual analysis. We prove empirically the importance of the proposed regression by means of a real data set (survival times of the captive snakes) from a study carried out at the Herpetology Laboratory of the Butantan Institute in Sao Paulo, Brazil.

Preparedness against pandemic influenza: Production of an oil-in-water emulsion adjuvant in Brazil.

Increasing pandemic influenza vaccine manufacturing capacity is considered strategic by WHO. Adjuvant use is key in this strategy in order to spare the vaccine doses and by increasing immune protection. We describe here the production and stability studies of a squalene based oil-in-water emulsion, adjuvant IB160, and the immune response of the H7N9 vaccine combined with IB160. To qualify the production of IB160 we produced 10 consistency lots of IB160 and the average results were: pH 6.4±0.05; squalene 48.8±.0.03 mg/ml; osmolality 47.6±6.9 mmol/kg; Z-average 157±2 nm, with polydispersity index (PDI) of 0.085±0.024 and endotoxin levels <0.5 EU/mL. The emulsion particle size was stable for at least six months at 25°C and 24 months at 4–8°C. Two doses of H7N9 vaccine formulated at 7.5 μg/dose or 15 μg/dose with adjuvant IB160 showed a significant increase of hemagglutination inhibition (HAI) titers in sera of immunized BALB/c mice when compared to control sera from animals immunized with the H7N9 antigens without adjuvant. Thus the antigen-sparing capacity of IB160 can potentially increase the production of the H7N9 pandemic vaccine and represents an important achievement for preparedness against pandemic influenza and a successful North (IDRI) to South (Butantan Institute) technology transfer for the production of the adjuvant emulsion IB160.

Safety and immunogenicity of the tetravalent, live-attenuated dengue vaccine Butantan-DV in adults in Brazil: a two-step, double-blind, randomised placebo-controlled phase 2 trial

The Butantan Institute has manufactured a lyophilised tetravalent live-attenuated dengue vaccine Butantan-DV, which is analogous to the US National Institutes of Health (NIH) TV003 admixture. We aimed to assess the safety and immunogenicity of Butantan-DV. We did a two-step, double-blind, randomised placebo-controlled phase 2 trial at two clinical sites in São Paulo, Brazil. We recruited healthy volunteers aged 18-59 years; pregnant women, individuals with a history of neurological, heart, lung, liver or kidney disease, diabetes, cancer, or autoimmune diseases, and individuals with HIV or hepatitis C were excluded. Step A was designed as a small bridge-study between Butantan-DV and TV003 in DENV-naive participants. In step A, we planned to randomly assign 50 dengue virus (DENV)-naive individuals to receive two doses of Butantan-DV, TV003, or placebo, given 6 months apart. In step B, we planned to randomly assign 250 participants (DENV-naive and DENV-exposed) to receive one dose of Butantan-DV or placebo. Participants were randomly assigned, by computer-generated block randomisation (block sizes of five); participants in step A were randomly assigned (2:2:1) to receive Butantan-DV, TV003, or placebo and participants in step B were randomly assigned (4:1) to receive Butantan-DV or placebo. Participants and study staff were unaware of treatment allocation. The primary safety outcome was the frequency of solicited and unsolicited local and systemic adverse reactions within 21 days of the first vaccination, analysed by intention to treat. The primary immunogenicity outcome was seroconversion rates of the DENV-1-4 serotypes measured 91 days after the first vaccination, analysed in the per-protocol population, which included all participants in step A, and all participants included in step B who completed all study visits with serology sample collection. This trial is registered with ClinicalTrials.gov, NCT01696422. Between Nov 5, 2013, and Sept 21, 2015, 300 individuals were enrolled and randomly assigned: 155 (52%) DENV-naive participants and 145 (48%) DENV-exposed participants. Of the 155 DENV-naive participants, 97 (63%) received Butantan-DV, 17 (11%) received TV003, and 41 (27%) received placebo. Of the 145 DENV-exposed participants, 113 (78%) received Butantan-DV, three (2%) received TV003, and 29 (20%) received placebo. Butantan-DV and TV003 were both immunogenic, well-tolerated, and no serious adverse reactions were observed. In step A, rash was the most frequent adverse event (16 [845] of 19 participants in the Butantan-DV group and 13 [76%] of 17 participants in the TV003 group). Viraemia was similar between the Butantan-DV and TV003 groups. Of the 85 DENV-naive participants in the Butantan-DV group who attended all visits for sample collection for seroconversion analysis and thus were included in the per-protocol analysis population, 74 (87%) achieved seroconversion to DENV-1, 78 (92%) to DENV-2, 65 (76%) to DENV-3, and 76 (89%) to DENV-4. Of the 101 DENV-exposed participants in the Butantan-DV group who attended all visits for sample collection for seroconversion analysis, 82 (81%) achieved seroconversion to DENV-1, 79 (78%) to DENV-2, 83 (82%) to DENV-3, and 78 (77%) to DENV-4. Butantan-DV and TV003 were safe and induced robust, balanced neutralising antibody responses against the four DENV serotypes. Efficacy evaluation of the Butantan-DV vaccine is ongoing. Intramural Research Program US NIH National Institute of Allergy and Infectious Diseases, Brazilian National Bank for Economic and Social Development, Fundação de Amparo à Pesquisa do Estado de São Paulo, and Fundação Butantan.

Geographic variation of individual venom profile of Crotalus durissus snakes.

Background: South American rattlesnakes are represented in Brazil by a single species, Crotalus durissus, which has public health importance due to the severity of its envenomation and to its wide geographical distribution. The species is subdivided into several subspecies, but the current classification is controversial. In Brazil, the venoms of C. d. terrificus and C. d. collilineatus are used for hyperimmunization of horses for antivenom production, even though the distinction of these two subspecies are mostly by their geographical distribution. In this context, we described a comparative compositional and functional characterization of individual C. d. collilineatus and C. d. terrificus venoms from three Brazilian states.Methods: We compared the compositional patterns of C. d. terrificus and C. d. collilineatus individual venoms by 1-DE and RP-HPLC. For functional analyzes, the enzymatic activities of PLA2, LAAO, and coagulant activity were evaluated. Finally, the immunorecognition of venom toxins by the crotalic antivenom produced at Butantan Institute was evaluated using Western blotting.Results: The protein profile of individual venoms from C. d. collilineatus and C. d. terrificus showed a comparable overall composition, despite some intraspecific variation, especially regarding crotamine and LAAO. Interestingly, HPLC analysis showed a geographic pattern concerning PLA2. In addition, a remarkable intraspecific variation was also observed in PLA2, LAAO and coagulant activities. The immunorecognition pattern of individual venoms from C. d. collilineatus and C. d. terrificus by crotalic antivenom produced at Butantan Institute was similar.Conclusions: The results highlighted the individual variability among the venoms of C. durissus ssp. specimens. Importantly, our data point to a geographical variation of C. durissus ssp. venom profile, regardless of the subspecies, as evidenced by PLA2 isoforms complexity, which may explain the increase in venom neurotoxicity from Northeastern through Southern Brazil reported for the species.

Crotamine in Crotalus durissus: distribution according to subspecies and geographic origin, in captivity or nature.

Background: Crotalus durissus is considered one of the most important species of venomous snakes in Brazil, due to the high mortality of its snakebites. The venom of Crotalus durissus contains four main toxins: crotoxin, convulxin, gyroxin and crotamine. Venoms can vary in their crotamine content, being crotamine-negative or -positive. This heterogeneity is of great importance for producing antivenom, due to their different mechanisms of action. The possibility that antivenom produced by Butantan Institute might have a different immunorecognition capacity between crotamine-negative and crotamine-positive C. durissus venoms instigated us to investigate the differences between these two venom groups. Methods:The presence of crotamine was analyzed by SDS-PAGE, western blotting and ELISA, whereas comparison between the two types of venoms was carried out through HPLC, mass spectrometry analysis as well as assessment of antivenom lethality and efficacy. Results:The results showed a variation in the presence of crotamine among the subspecies and the geographic origin of snakes from nature, but not in captive snakes. Regarding differences between crotamine-positive and -negative venoms, some exclusive proteins are found in each pool and the crotamine-negative pool presented more phospholipase A2 than crotamine-positive pool. This variation could affect the time to death, but the lethal and effective dose were not affected.Conclusion:These differences between venom pools indicate the importance of using both, crotamine-positive and crotamine-negative venoms, to produce the antivenom.

Experimental antivenom against serine proteases from the Bothrops jararaca venom obtained in mice, and its comparison with the antibothropic serum from the Butantan Institute

In Brazil, snakes from the Bothrops genus are responsible for thousands of accidents, and their venoms are mainly made up of proteolytic enzymes. Although the antibothropic serum produced by the Butantan Institute is remarkable in saving lives, studies show that some symptoms observed in cases of envenoming are not efficiently neutralized. Moreover, our group has shown that the commercial antivenom does not fully neutralize in vitro some serine proteases present in the Bothrops jararaca venom. Therefore, this study focuses on a new method in the production of specific immunoglobulins capable of neutralizing the activities of these enzymes in vitro. For this, a pool of serine proteases that was not inhibited by the commercial antivenom, made up of four enzymes (KN-BJ2, BjSP, HS112 and BPA) from the B. jararaca venom was obtained through two chromatographic steps (DEAE-HPLC and C8-RP-HPLC). The identities of these proteases were confirmed by SDS-PAGE, followed by tryptic digestion and mass spectrometry analysis. This pool was inoculated into BALB/c and C57BL/6 mice, using SBA-15 as adjuvant, and the produced IgGs were purified by affinity chromatography. The sera were characterized by ELISA, avidity and proteolytic neutralization assays. Both animal models responded to the immunization, producing higher IgGs titers when compared to the commercial antivenom. The experimental serum from BALB/c mice presented a better hydrolysis inhibition of the selective fluorescent substrate for serine proteases (~80%) when compared to C57BL/6 (~25%) and the commercial antivenom (<1%) at the dose of 500:1 (weight of antivenom:weight of venom). These results show that a different immunization method using isolated serine proteases improves the toxins neutralizing efficacy and could lead to a better end product to be used as a supplemental medicine to the currently used immunotherapy.

Frederico Carlos Hoehne e o Horto Oswaldo Cruz

O Instituto Butantan (SP) teve ao longo de sua existência algumas atividades voltadas ao estudo da botânica. O Horto Oswaldo Cruz, criado no local, surgiu em meio às discussões sobre a saúde pública e tinha como objetivo o estudo e o cultivo de plantas tóxicas e medicinais. A iniciativa foi chefiada por Frederico Carlos Hoehne, botânico que se notabilizou pelos estudos na área e pela atuação em instituições governamentais paulistas. Neste artigo, analisamos a atuação de Hoehne quando foi responsável pelo horto entre os anos de 1917 e 1924. O estudo aborda as atividades realizadas no local, procurando entender o cotidiano das práticas científicas a partir dos cultivos e da criação de uma rede de interlocutores. A análise dos relatórios anuais do Instituto Butantan e das publicações de autoria de Hoehne explicita como os objetivos traçados foram traduzidos em ações cotidianas, colocando em destaque os interesses ligados à saúde e aos diversos usos que poderiam ser feitos das espécies cultivadas. Os resultados indicam que as orientações governamentais, ainda que importantes, não foram unicamente consideradas. Os interlocutores do horto botânico, as preferências de Hoehne e os limites impostos pelas condições materiais também influenciaram e ajudaram a delimitar o que foi realizado.

Cutaneous mycobiota of boid snakes kept in captivity

ABSTRACT Boids are large, constrictor snakes that feed mostly on mammals, reptiles, and birds. These animals are commonly raised as pets, and their improper handling can favor the emergence of fungal infections, which can lead to dermatological diseases that are undiagnosed in nature. Here, we isolate and identify the filamentous fungi that compose the mycobiota of the scales of boid snakes kept in captivity at the Biological Museum of the Butantan Institute. Thirty individuals of four species were evaluated: four Eunectes murinus, twelve Boa constrictor constrictor, seven Corallus hortulanus, and seven Epicrates crassus. Microbiological samples were collected by rubbing small square carpets on the snake scales. We isolated five genera of fungi: Penicillium sp. (30%), Aspergillus sp. (25%), Mucor sp. (25%), Acremonium sp. (10%), and Scopulariopsis sp. (10%). Approximately half of the snakes evaluated had filamentous fungi on the scales, but only 12% of the individuals were colonized by more than one fungal genus. We found no dermatophytes in the evaluated species. Our results provide an overview of the fungal mycobiota of the population of boids kept in the Biological Museum, allowing the identification of possible pathogens.

Identification and purification of Phospholipase A2 Inhibitors (PLIs) from snake blood maintained in the Laboratory of Herpetology of Butantan Institute

Identification and purification of Phospholipase A2 Inhibitors (PLIs) from snake blood maintained in the Laboratory of Herpetology of Butantan Institute

Purification and Biochemical Characterization of TsMS 3 and TsMS 4: Neuropeptide-Degrading Metallopeptidases in the Tityus serrulatus Venom.

Although omics studies have indicated presence of proteases on the Tityus serrulatus venom (TsV), little is known about the function of these molecules. The TsV contains metalloproteases that cleave a series of human neuropeptides, including the dynorphin A (1-13) and the members of neuropeptide Y family. Aiming to isolate the proteases responsible for this activity, the metalloserrulase 3 and 4 (TsMS 3 and TsMS 4) were purified after two chromatographic steps and identified by mass spectrometry analysis. The biochemical parameters (pH, temperature and cation effects) were determined for both proteases, and the catalytic parameters (Km, kcat, cleavage sites) of TsMS 4 over fluorescent substrate were obtained. The metalloserrulases have a high preference for cleaving neuropeptides but presented different primary specificities. For example, the Leu-enkephalin released from dynorphin A (1-13) hydrolysis was exclusively performed by TsMS 3. Neutralization assays using Butantan Institute antivenoms show that both metalloserrulases were well blocked. Although TsMS 3 and TsMS 4 were previously described through cDNA library studies using the venom gland, this is the first time that both these toxins were purified. Thus, this study represents a step further in understanding the mechanism of scorpion venom metalloproteases, which may act as possible neuropeptidases in the envenomation process.

Bites by Tomodon dorsatus (serpentes, dipsadidae): Clinical and epidemiological study of 86 cases

A total of 86 proven cases of Tomodon dorsatus bites admitted to Hospital Vital Brazil (HVB) of Butantan Institute, in São Paulo, Brazil, between 1945 and 2018, were retrospectively analyzed. The cases included were those in which the snake was brought to HVB and was correctly identified. Of the 86 cases of snake bites, it was possible to describe the sex of the snake in 52 cases; 31 (59.6%) snakes were male. Only 52 snakes out of 86 could be studied because of their preservation status. The length of snakes (snout–vent length) ranged from 180 to 770 mm. Of the 86 snakes, 72 could be distinguished as adults (n = 63, 87.5%) or juveniles (n = 9, 12.5%). Most bites occurred in the spring and summer seasons (n = 57, 66.3%) and during warmer periods of the day (n = 61, 72.6%), between 9 a.m. and 3 p.m.The mean (±standard deviation) age of the victims was 26.9 ± 17.2 years, and 60 (69.8%) were men. Approximately 90% of the patients sought medical care within 6 h after the bite. Both upper (n = 45, 52.3%) and lower (n = 37, 43.0%) limbs were the most frequently bitten, particularly the feet and hands (n = 54, 62.8%). The local clinical manifestations were pain (n = 55, 64.0%), transitory bleeding (n = 23, 26.7%), erythema (n = 22, 25.6%), edema (n = 14, 16.3%), paresthesia (n = 9, 10.5%), and ecchymosis (n = 3, 3.5%). Only 10 (11.6%) patients reported non-specific systemic symptoms characterized by transient dizziness or mild headache, and 21 (24.4%) patients showed no evidence of envenomation. A 20 min whole blood clotting test was performed in 31 (36.0%) patients on admission and all of them had coagulable blood. Supportive treatment was offered to 38 (44.2%) patients, namely, antiseptic (n = 20, 23.3%), antihistamines (n = 12, 14.0%), and analgesics (n = 9, 10.5%). Four (4.7%) patients were inappropriately treated with Bothrops antivenom before their admission to HVB. No sequelae or relevant complications were observed in patients, and the prognosis was benign. Therefore, although T. dorsatus bites can cause mild local symptomatology, it is important that health professionals know how to make the correct diagnosis to avoid unnecessary use of antivenom.

Kn-Ba: a novel serine protease isolated from Bitis arietans snake venom with fibrinogenolytic and kinin-releasing activities

BackgroundBitis arietans is a venomous snake found in sub-Saharan Africa and in parts of Morocco and Saudi Arabia. The envenomation is characterized by local and systemic reactions including pain, blistering, edema and tissue damage, besides hemostatic and cardiovascular disturbances, which can cause death or permanent disabilities in its victims. However, the action mechanisms that provoke these effects remain poorly understood, especially the activities of purified venom components. Therefore, in order to elucidate the molecular mechanisms that make the Bitis arietans venom so potent and harmful to human beings, this study reports the isolation and biochemical characterization of a snake venom serine protease (SVSP).MethodsSolubilized venom was fractionated by molecular exclusion chromatography and the proteolytic activity was determined using fluorescent substrates. The peaks that showed serine protease activity were determined by blocking the proteolytic activity with site-directed inhibitors. In sequence, the fraction of interest was submitted to another cycle of molecular exclusion chromatography. The purified serine protease was identified by mass spectrometry and characterized biochemically and immunochemically.ResultsA serine protease of 33 kDa with fibrinogen-degrading and kinin-releasing activities was isolated, described, and designated herein as Kn-Ba. The experimental Butantan Institute antivenom produced against Bitis arietans venom inhibited the Kn-Ba activity.ConclusionsThe in vitro activities of Kn-Ba can be correlated with the capacity of the venom to provoke bleeding and clotting disorders as well as hypotension, which are common symptoms presented by envenomed victims. Obtaining satisfactory Kn-Ba inhibition through the experimental antivenom is important, given the WHO’s recommendation of immunotherapy in cases of human accidents with venomous snakes.

PARASITOLOGICAL DIAGNOSIS OF ISLAND SNAKES KEPT IN CAPTIVITY AT THE BUTANTAN INSTITUTE

The aim of this study was to identify the endoparasites and to verify the efficacy of their prophylactic control of Bothrops insularis and B. alcatraz kept in captivity. Snakes were treated ex-situ with ivermectin and praziquantel, and parasitological examinations were performed on fresh feces. We evaluated 43 individuals of B. insularis (26 wild-caught adults and 17 young born in captivity) and 9 of B. alcatraz (4 wild-caught adults and 5 young born in captivity) kept in the Laboratory of Ecology and Evolution, Butantan Institute, Sao Paulo, Brazil. Using direct examination and the Willis Mollay technique, we observed the presence of the cestode Ophiotaenia sp. (eggs and proglottids) in 33.3% of B. alcatraz individuals . However, cestodes were found only in wild-born adult females. Eggs from a mouse parasite ( Syphacia obvelata ) were found in 27.9% of B. insularis individuals. We conclude that ivermectin and praziquantel were effective to treat nematodes and cestodes. Wild-caught snakes need to be treated against cestodes because, in captivity, this parasite causes anorexia, anemia, and weight loss. The presence of S. obvelata (a non-pathogenic mouse parasite) was due to feeding the snakes with laboratory mice.