Butantan Institute Research Articles

Butantan Institute: strategies to obtain scorpions for the production of anti-scorpion serum

Dear Editor,Continuing previous communications on the importance of Butantan Institute to Brazilian science, we would like to report through this letter its initiative on scorpion antivenom production. In 1918, the first director of Butantan Institute, Vital Brazil, exposed at the Second Conference of the South American Society of Hygiene, Microbiology and Pathology the following: “In 1905, the Butantan Institute has begun the study of scorpion venoms. The main problem was the acquisition of material. In fact, where could we get those arachnids in so large quantities? Several times we sent representatives to the state of Minas Gerais with the intention of looking for this material in places where they are common, and the results were every time so meagre that we could not begin the immunization of animals in order to fulfill our intent. At the beginning of 1907, we accidentally met a hunter of those animals, and he could offer us thousands of them. This man was working on empty plots around the city and observed that he could find some scorpions inside termitaries. After this observation, he had the idea of becoming a ‘scorpion hunter’. These animals are cannibals and for this reason, we can rarely find more than two in the same shelter. The venom glands are situated in the last tail segment. To prepare the antigen in large quantities we must cut off the segment from live or recently dead animals using a scissor” (1, 2).In 1939, the biologist Wolfgang Bucherl was hired by the Butantan Institute. Shortly after, he became the head of the Laboratory of Medical Zoology and responsible for the supply of scorpion venom. Since 1952, he was involved in the maintenance of scorpions in captivity and, for the first time, he employed the electric method of venom extraction. This represented a progress, since animals were not sacrificed anymore, enabling the extraction of venom from the same scorpion several times. The technique also improved the antigen quality (3). During the following years, the Institute asked suppliers numerous times to send live scorpions. Bucherl was grateful to many institutions of Sao Paulo and Minas Gerais states for animal collection. However, the main problem was that during transportation, around 30% of the collected scorpions died. Additionally, many of them were intoxicated by pesticides.From 1951 to 1953, Butantan Institute received 1,400 live scorpions, an insufficient amount to ensure the antivenom production. To solve such problem, Felisberto Miguel Arcanjo (from Santa Barbara de Nova Era, Minas Gerais state) was hired and paid per delivered scorpion; thus, the Institute would have a permanent supply of live animals. The specimens used to be transported for free by the railway company in wooden boxes containing humid pieces of smashed sugar cane.By the end of 1967, Bucherl had retired. Sylvia Lucas, the new responsible for the

Destruction of the collection of reptiles and arthropods at Butantan Institute: a view from the United Kingdom

Destruction of the collection of reptiles and arthropods at Butantan Institute: a view from the United Kingdom

General biochemical and immunological characterization of the venom from the scorpion Tityus trivittatus of Argentina

Tityus trivittatus is the Argentinean scorpion reported to cause the majority of human fatalities in the country, however no systematic studies have been conducted with the venom of this species. This communication describes a general biochemical and immunological characterization of the venom obtained from T. trivittatus scorpions collected in the city of Buenos Aires and various provinces of Argentina: Catamarca, Cordoba, Entre Rios, La Rioja, Santa Fe and Santiago del Estero. These are places where human accidents were reported to occur due to this scorpion. For comparative purposes two types of samples were assayed: whole soluble venom obtained by electrical stimulation and supernatant from homogenized venomous glands. Two strains of mice (NIH and CF-1) were used for LD 50 determinations by two distinct routes of administration (intravenously and intraperitoneally). Important variations were found that goes from 0.5 to 12 mg/kg mouse body weight. Samples of soluble venom were always more potent than Telson homogenates. More complex pattern was observed in homogenates compared to soluble venom, as expected. This was supported by gel electrophoretic analysis and high performance liquid chromatographic (HPLC) separations. Additionally, the HPLC profile was enriched in proteins resolved at similar elution times as other known toxins from scorpion venoms studied. Immune enzymatic assays were also conducted comparatively, using four different anti-venoms commercially available for treatment of scorpion stings (Argentinean antidote from INPB, two anti-venoms from Butantan Institute of Brazil and Alacramyn from the Mexican Bioclon Institute). Cross-reactivities were observed and are reported among the various venoms and anti-venoms used. Lung, heart, liver and pancreas pathological modifications were observed on tissues of intoxicated mice. It seems that there are important variations on the venom compositions of the various samples studied and reported here, depending on the geographical area where the scorpions were captured. The results reported here are important for the clinical outcome of human accidents.

Cloning of serine protease cDNAs from Crotalus durissus terrificus venom gland and expression of a functional Gyroxin homologue in COS-7 cells

Gyroxin is one of main serine proteases of Crotalus durissus terrificus venom, representing about 2% of the protein content in the crude venom. It is a 33 kDa glycoprotein with 3.8% by weight of sugar moiety. This toxin induces hemotoxicity in mice and a neurological condition called barrel rotation syndrome. In the present work, we report the molecular cloning of five new nucleotide sequences from a cDNA library of the venom glands of a single specimen of C. d. terrificus. These sequences have been analyzed in silico with respect to their cDNA organization and similarity with other snake venom serine proteases (SVSPs). We also describe a rapid and efficient method for screening vectors for mammalian cell expression, based on the fact that SVSPs are difficult-to-express toxins due to the presence of several disulfide bonds and glycosylation in their structures. Thus, one of the Gyroxin cDNAs was subcloned into pSectag2 HygroA and pED vectors and used to transfect COS-7 cells. Expression of the functional recombinant Gyroxin isoform was achieved with this cell line with esterase activity in the conditioned culture medium, as revealed by immunoblot of secreted protein and standard anti-crotalic serum from Butantan Institute.

Determinação sérica de colesterol e triglicérides em cavalos soroprodutores alimentados com concentrados com teores de 15% ou 22% de proteína bruta

A produção de soro-hiperimune em eqüinos ainda é fundamental para obtenção do soro utilizado no tratamento de algumas enfermidades como o tétano e a difteria. Para verificar o possível benefício da utilização de concentrados hiperprotéicos na dieta dos cavalos soroprodutores da Fazenda São Joaquim do Instituto Butantan-SP, foram analisados os teores séricos de colesterol e triglicérides de 32 animais, pertencentes ao plantel de produção de soro antidiftérico. Estes foram distribuídos, ao acaso, em dois grupos de 16 eqüinos. Os animais do grupo A receberam concentrado com 15% de proteína bruta (PB) e os animais do grupo B receberam concentrado com 22% de PB. As amostras de soro foram obtidas em dois ciclos de produção nos momentos anteriores à inoculação do antígeno, antes das sangrias de produção e nos dias 15, 30 e 45 de descanso. As concentrações de triglicérides foram maiores no grupo B no dia 58 do primeiro ciclo (p<0,001) e, no segundo ciclo, foram maiores no grupo A nos dias 95 e 137 (p<0,04) e no grupo B no dia 109 (p<0,04). Não houve diferença entre os grupos para as concentrações séricas de colesterol. Este trabalho concluiu que embora as concentrações séricas de triglicérides e colesterol sejam sensíveis ao processo de produção de plasma-hiperimune antidifitérico, os resultados encontrados não foram suficientes para afirmar se há benefício ou não na utilização de concentrado hiperprotéico (22% PB) em cavalos produtores de soro-hiperimune anti-difteria em dois ciclos de produção

Interspecific variation in venom composition and toxicity of Brazilian snakes from Bothrops genus

The genus Bothrops spp. is responsible for 90% of envenomation by snakes in Brazil, and the standard treatment for snakebites is the antivenom therapy. The anti-bothropic serum produced by Butantan Institute is prepared by the hyperimmunization of horses with a pool of venoms from Bothrops alternatus, Bothrops jararaca, Bothrops jararacussu, Bothrops moojeni and Bothrops neuwiedi. In this study, the biochemical and biological characteristics of the venoms from nineteen snakes of the genus Bothrops, responsible for human accidents in Brazil, were analysed. Venoms, particularly from Crotalidae and Viperidae snakes, are rich sources of serine proteases and metalloproteases and the ability of the Brazilian anti-bothropic serum to neutralize the proteolytic activity of these venoms were also tested. The results obtained here show the existence of a large range of variation in the composition and activities in Bothrops spp. toxins and demonstrate that the anti-bothropic serum is not able to fully neutralize the toxic activities of all analysed venoms. These suggest that for the preparation of a fully effective therapeutic anti-bothropic serum, other venoms should be included in the immunization mixture.

Epidemiologic and clinical survey of victims of centipede stings admitted to Hospital Vital Brazil (São Paulo, Brazil)

We retrospectively analyzed 98 proven cases of centipede stings admitted to Hospital Vital Brazil, Butantan Institute, São Paulo, Brazil, between 1990 and 2007. Most stings occurred at the metropolitan area of São Paulo city ( n = 94, 95.9%), in the domiciles of patients ( n = 67, 68.4%), and during the warm-rainy season ( n = 60, 61.2%). The mean age of the victims was 32.0 ± 18.8-years-old. Cryptops and Otostigmus genera were responsible for most cases. Around 86% of the patients sought medical care within 6 h after the sting. Both lower (56.1 %) and upper limbs (41.8 %) were most frequently bitten, especially the feet and hands (89.8%). The most frequent local clinical manifestations were pain (94.9%), erythema (44.9%) and edema (21.4%), and the latter was mainly observed in patients bitten by Otostigmus spp. Supportive treatment was used in only 28.6% of the patients, namely administration of local anesthesia (9.2%) and systemic analgesia (13.3%). No sequels or complications were observed in patients, and the prognostic was benign.

Comparação entre a epidemiologia do acidente e a clínica do envenenamento por serpentes do gênero Bothrops, em adultos idosos e não idosos

This study had the aim of ascertaining epidemiological and clinical differences in envenoming caused by Bothrops spp between elderly adults (>60 years) and non-elderly adults (20 to 59 years). The data were obtained from 1,930 medical records of patients attended at the Butantan Institute between 1981 and 1992. The greater the patients age was, the higher the frequency of bites on the hands rather than on the feet was (p < 0.05). A slightly higher percentage of the elderly patients (17%, versus 11% of the non-elderly group) were attended >12 hours after the bite (p < 0.05). Necrosis was more common among the elderly patients (p < 0.05) and renal failure was more common among patients aged 50 years or over (p < 0.05), in relation to younger patients. It was concluded that elderly individuals are more often bitten on the hands and less often on the feet, and that they develop local necrosis and renal failure more frequently than do younger individuals.

Diet of the rattlesnake Crotalus durissus in southeastern Brazil (Serpentes, Viperidae)

Gut contents of 633 live rattlesnakes from southeastern Brazil received at the Butantan Institute, SP, Brazil between 1993 and 1995 were studied. The snakes were weighed, measured and sexed. Two hundred and fifty‐nine rattlesnakes had stomach and/or intestinal contents. Prey size was estimated by comparison of prey items with specimens from museum collections. Rodents and small marsupials were the main prey eaten by the rattlesnakes, and only 1% of the items were found in the stomach, whereas 41% of the individuals in the sample had feces in the intestine. There was low correlation between size of snake and prey size. No seasonal difference in frequency was found between fed and not fed males, but the occurrence of fed females was significantly lower during summer than winter months (28.9% and 51.8%, respectively). Fed newborn rattlesnakes had the lowest frequency, and also fed on rodents. O conteúdo do trato digestivo de 633 cascavéis do sudeste brasileiro recebidas pelo Instituto Butantan, SP, Brasil entre 1993–1995 foram estudados. As serpentes foram pesadas, medidas e tiveram seu sexo determinado. Duzentas e cinqüenta e nove serpentes apresentaram conteúdo estomacal e/ou intestinal. O tamanho das presas foi estimado comparando‐os com espécimes de museu. Roedores e pequenos marsupiais foram as principais presas ingeridas pelas cascavéis, e somente 1% dos itens foram encontrados no estômago, entretanto 41% das cascavéis tinham fezes no trato intestinal. Houve pequena correlação entre o tamanho da serpente e da presa ingerida. Não foi observada diferença sazonal na freqüência de machos com e sem conteúdo, mas a ocorrência de fêmeas sem conteúdo foi significativamente menor nos meses de verão do que nos meses de inverno (28,9% e 51,8%, respectivamente). Cascavéis recém‐nascidas também se alimentam de roedores e têm a mais baixa taxa de animais com conteúdo.

Microbiological monitoring of clean rooms in development of vaccines

The aim of the present work was to evaluate an environmental monitoring program for clean rooms, or classified environments, involved in the filling and quality control of biological products produced by Butantan Institute, Sao Paulo, Brazil. This monitoring established the quantification, characterization and seasonality of the microorganisms in air and operators and, moreover, determined the alert and action limits. The total detectable microbial number showed some contrasts in installed air purification systems and in the operational impact on adopted procedures. The typical microbial population consisted of Staphylococcus sp, Micrococcus sp, Bacillus sp and Penicillium sp. The highest microorganism concentration occurred during summer and springtime. The established internal alert and action limits supported the operational procedures. Therefore, the environmental monitoring program is recommended for other laboratories involved in the production of vaccines, hyperimmune sera and biopharmaceuticals.

Effect of a single tetanus-diphtheria vaccine dose on the immunity of elderly people in São Paulo, Brazil

Epidemiological data regarding tetanus and diphtheria immunity in elderly people in Brazil are scarce. During the First National Immunization Campaign for the Elderly in Brazil in April 1999, 98 individuals (median age: 84 years) received one tetanus-dyphtheria (Td) vaccine dose (Butantan Institute, lot number 9808079/G). Inclusion criteria were elderly individuals without a history of severe immunosuppressive disease, acute infectious disease or use of immunomodulators. Blood samples were collected immediately before the vaccine and 30 days later. Serum was separated and stored at -20 degrees C until analysis. Tetanus and diphtheria antibodies were measured by the double-antigen ELISA test. Tetanus and diphtheria antibody concentrations lower than 0.01 IU/mL were considered to indicate the absence of protection, between 0.01 and 0.09 IU/mL were considered to indicate basic immunity, and values of 0.1 IU/mL or higher were considered to indicate full protection. Before vaccination, 18% of the individuals were susceptible to diphtheria and 94% were susceptible to tetanus. After one Td dose, 78% became fully immune to diphtheria, 13% attained basic immunity, and 9% were still susceptible to the disease. In contrast, 79% remained susceptible to tetanus, 4% had basic immunity and 17% were fully immune. Although one Td dose increases immunity to diphtheria in many elderly people who live in Brazil, a complete vaccination series appears to be necessary for the prevention of tetanus.

Scientists and Their Pursuit of Excellence: Crossroads and Gateways (or Who is Who in the Festschrift)

Scientists and Their Pursuit of Excellence: Crossroads and Gateways (or Who is Who in the Festschrift)

Redescription of Amblyomma fuscum Neumann, 1907 (Acari: Ixodidae), a rare South America tick confirmed in Brazil

The species Amblyomma fuscum Neumann, 1907 is a rare tick found on the Neotropical Region, but it has not been recorded as a valid taxon in some lists proposed by current taxonomists. After a comparison between the Brazilian material of A. fuscum deposited in the Acari Collection of the Butantan Institute, São Paulo, Brazil, and the male type deposited in Leiden Museum of Natural History, The Netherlands, we confirm the taxonomic validity of A. fuscum and redescribe the adult specimens based on light and scanning electron microscope studies.

Redescrição de Prorachias bristowei Mello-Leitão, 1924 (Araneae, Mygalomorphae, Nemesiidae)

Prorachias Mello-Leitão, 1924 é conhecido apenas pela espécie-tipo P. bristowei Mello-Leitão, 1924, descrita com base em uma fêmea do estado do Rio de Janeiro, Brasil. A descrição é sumária e incompleta, sendo omitidos caracteres importantes como o formato da espermateca. O exemplar-tipo, que deveria estar depositado no Museu Nacional do Rio de Janeiro, não foi localizado e está provavelmente perdido. O estudo de material coletado pelo Projeto "Biodiversidade de Arachnida e Myriapoda do Estado de São Paulo" e de material depositado na coleção do Instituto Butantan permitiu complementar a diagnose do gênero, redescrever a fêmea e descrever pela primeira vez o macho desta espécie. Além disto, dados sobre biologia são apresentados e a distribuição geográfica da espécie é ampliada para os estados de Goiás, Minas Gerais e São Paulo.

222 Analysis of The Immunogenicity of A New Porcine Lung Surfactant Using The Rabbit Model

Background: The introduction of foreign proteins from animal lung-based surfactants into the airways of premature infants may provide an antigenic stimulus with a possible immunological response to these proteins. Recently the Butantan Institute (Brazil) produced a porcine pulmonary surfactant preparation obtained by organic extraction coupled with adsorption on a cellulose derivative, composed mainly by phospholipids with two hydrophobic polypeptides, SP-B and SP-C. This new surfactant contains 76% of phosphatidylcholine, 6–8% of phosphatidylethanolamine, 6% of phosphatidylinositol + phosphatidylserine, and 4–6% of sphingomyelin; 30–35% of the phosphatidylcholine was dipalmitoylphosphatidylcholine. The total content of protein is 5.6% of the surfactant preparation. The objective of this study was to analyze in rabbits the immunogenicity of the Butantan Institute surfactant preparation compared with commercial available lung surfactant preparations.

223 Effect Of Storage Time in the In Vivo Function of a New Exogenous Pulmonary Surfactant

Background: The function of exogenous pulmonary surfactant depends of an adequate stability of its components, a highly complex mixture of lipids (mainly phospholipids) and two hydrophobic polypeptides, SP-B and SP-C. This composition could become instable with long time surfactant storage. The Butantan Institute (Brazil) produced a new porcine pulmonary surfactant preparation composed mainly of 76% of phosphatidylcholine (30–35% of the phosphatidylcholine is saturated dipalmitoyl phosphatidylcholine), 6–8% of phosphatidyllethanolamine, 6% of phosphatidylinositol + phosphatidylserine, and 4–6% of sphingomyelin, with 5.6% of two hydrophobic polypeptides, SP-B and SP-C. The objective of this study was to evaluate the Butantan exogenous surfactant function one year after its production, using the premature rabbit model.

O Laboratório de Artrópodes do Instituto Butantan e os aracnídeos peçonhentos

O presente relato é um histórico sobre a origem do Laboratório de Artrópodes do Instituto Butantan, desde a época de sua fundação até a aposentadoria de Wolfgang Bücherl, um dos maiores aracnológos do Brasil, em meados de 1967. Baseia-se nos relatórios anuais de Vital Brazil, diretor do instituto durante o período de 1900 a 1919 e de 1924 a 1927 nos trabalhos publicados nas Memórias do Instituto Butantan e também no meu convívio, a partir de 1961, como assistente de Bücherl, com pesquisadores, pessoal técnico e colaboradores do instituto.

Prognostic factors for local necrosis in Bothrops jararaca (Brazilian pit viper) bites

The prognostic factors related to envenoming are not very well known. This study aims to identify prognostic factors for necrosis in envenoming by Bothrops jararaca. We analysed 779 medical records of patients bitten by B. jararaca and treated at the Hospital Vital Brazil, Butantan Institute, São Paulo, Brazil, between 1982 and 1990: 111 cases with necrosis were compared with the remaining cases. The length of the snake, the bite site, the month of the accident, pain, oedema, ecchymosis, blisters, systemic bleeding, shock, and the use of tourniquet were statistically associated with the presence of necrosis ( P < 0·05) in the univariate analysis. The size of the snake, the bite site (leg and finger), the sex of the patient, the month of the accident, systemic bleeding, and the use of tourniquet were independent prognostic factors within the variables tested in the multivariate analysis. The size of the snake was the most important independent prognostic factor related to the presence of necrosis.

Development and evaluation of the neutralizing capacity of horse antivenom against the Brazilian spider Loxosceles intermedia

Spider bites due to Loxosceles intermedia are currently a major public health problem in South Brazil. About 3000 cases are reported annually. Specific treatment for spider bites is provided by the polyvalent anti-arachnidic antiserum produced by Butantan Institute, São Paulo, Brazil by immunizing horses with mixtures of venoms from Tityus serrulatus and T. bahiensis scorpions, as well as Phoneutria nigriventer and L. gaucho spiders. Due to the large amounts of the anti-arachnidic antivenom required and since L. intermedia venom has some biochemical and pharmacological variations, we have produced a specific anti- L. intermedia antivenom. This study shows that horses immunized with crude L. intermedia venom produced IgG antibodies that neutralized the dermonecrotic and lethal activities of the venom. The neutralizing potency of the anti-loxoscelic antivenom was compared with that of the anti-arachnidic antivenom. Our results indicate that both antivenoms were effective in terms of neutralization. However, the anti-loxoscelic antivenom was more efficient than the anti-arachnidic.

Influenza eqüina: avaliação da resposta imune humoral, através das reações de inibição da hemaglutinação e de hemólise radial simples, em soro de animais vacinados com vacina comercial e experimental

Foram avaliadas, através de testes de inibição da hemaglutinação (Hl) e da hemólise radial simples (HRS),as respostas de anticorpos contra influenza em 4 lotes de eqüinos adultos. O grupo do lote 01 recebeu a imunizaçãocom 2 doses de vacina contra influenza, preparada experimentalmente no Instituto Butantan. Noslotes 02 e 03, regularmente imunizados contra a influenza, foram administradas doses de reforço anual coma vacina comercial e com a vacina experimental, respectivamente. O lote 04 foi o grupo controle da avaliação.Os resultados dos testes demonstraram que as médias de títulos de HRS e IH do lote 01 apresentaram diferenças ao nível de p &lt; 0,001, com significante aumento das médias de títulos detectados nos soros após a 2- imunização. Não foram observadas diferenças significantes entre as médias de títulos de anticorpos em soros obtidos antes e após a dose de reforço anual dos eqüinos dos lotes 02 e 03, atribuindo-se a persistênciade nível de anticorpos protetores mantida após 1 ano da imunização regular com vacina comercial. A conversão sorológica dos eqüinos do lote 01, à persistência dos títulos de anticorpos nos eqüinos dos lotes 02 e 03 e, ainda, os baixos títulos de anticorpos verificados nos eqüinos não vacinados do lote 04 comprovam o resultado da resposta sorológica das duas vacinas, a experimental e a comercial, avaliadas neste trabalho.