Scielo RSS <![CDATA[Journal of Venomous Animals and Toxins including Tropical Diseases]]> vol. 21 num. lang. es <![CDATA[SciELO Logo]]> <![CDATA[Post-exposure treatment of Ebola virus using passive immunotherapy: proposal for a new strategy]]> Background Better treatments are urgently needed for the management of Ebola virus epidemics in Equatorial Africa. Methods We conducted a systematic review of the literature on the use of passive immunotherapy for the treatment or prevention of Ebola virus disease. We placed findings from this review into the context of passive immunotherapy currently used for venom-induced disease, and recent improvements in manufacturing of polyvalent antivenom products. Results Passive immunotherapy appears to be one of the most promising specific treatments for Ebola. However, its potential has been incompletely evaluated, considering the overall experience and recent improvement of immunotherapy. Development and use of heterologous serum derivatives could protect people exposed to Ebola viruses with reasonable cost and logistics. Conclusion Hyperimmune equine IgG fragments and purified polyclonal whole IgG deserve further consideration as treatment for exposure to the Ebola virus. <![CDATA[What do we know about some of the most conspicuous scorpion species of the genus <italic>Tityus</italic>? A historical approach]]> <title>Abstract</title><p>In the present study, comments are proposed on historical aspects of the most conspicuous scorpion species of the genus <italic>Tityus</italic> found in Brazil. Both <italic>Tityus bahiensis</italic> (Perty) and <italic>Tityus serrulatus</italic> Lutz &amp; Mello are better known for their infamous reputation of noxious species. However, the original discovery and description of both species are associated with interesting historical episodes. A short comment is also provided on <italic>Tityus costatus</italic> (Karsch), the species possibly involved in the first record of a scorpion incident in Brazil.</p> <![CDATA[The unfulfilled promises of scorpion insectotoxins]]> <title>Abstract</title><p>Since the description and biochemical characterization of the first insect-specific neurotoxins from scorpion venoms, almost all contributions have highlighted their potential application as leads for the development of potent bioinsecticides. Their practical use, however, has been hindered by different factors, some of which are intrinsically related to the toxins and other external determinants. Recent developments in the understanding of the action mechanisms of the scorpion insectotoxins and their bioactive surfaces, coupled with the exploration of novel bioinsecticide delivery systems have renewed the expectations that the scorpion insectotoxins could find their way into commercial applications in agriculture, as part of integrated pest control strategies. Herein, we review the current arsenal of available scorpion neurotoxins with a degree of specificity for insects, the progress made with alternative delivery methods, and the drawbacks that still preclude their practical use.</p> <![CDATA[The History of Venomous Spider Identification, Venom Extraction Methods and Antivenom Production: a Long Journey at the Butantan Institute, São Paulo, Brazil]]> <title>Abstract</title><p>The article provides a historical report on venomous spider identification, venom obtainment methods and serum production at the Butantan Institute, São Paulo, Brazil. It is based on literature and personnal experience during the last 50 years. This result is the discovery that the real species causing potential severe human accidents were the spiders of the genus <italic>Loxosceles</italic> and <italic>Phoneutria</italic>.</p> <![CDATA[A checklist of the scorpions of Ecuador (Arachnida: Scorpiones), with notes on the distribution and medical significance of some species]]> Ecuador harbors one of the most diverse Neotropical scorpion faunas, hereby updated to 47 species contained within eight genera and five families, which inhabits the “Costa” (n= 17), “Sierra” (n= 34), “Oriente” (n= 16) and “Insular” (n= 2) biogeographical regions, corresponding to the western coastal, Andean, Amazonian, and the Galápagos archipelago regions, respectively. The genusTityus Koch, in the family Buthidae, responsible for severe/fatal accidents elsewhere in northern South America and the Amazonia, is represented in Ecuador by 16 species, including T. asthenes, which has caused fatalities in Colombia and Panama, and now in the Ecuadorian provinces of Morona Santiago and Sucumbíos. Underestimation of the medical significance of scorpion envenoming in Ecuador arises from the fact thatCentruroides margaritatus (Gervais) (family Buthidae) andTeuthraustes atramentarius Simon (family Chactidae), whose venoms show low toxicity towards vertebrates, frequently envenom humans in the highly populated Guayas and Pichincha provinces. This work also updates the local scorpion faunal endemicity (74.5 %) and its geographical distribution, and reviews available medical/biochemical information on each species in the light of the increasing problem of scorpionism in the country. A proposal is hereby put forward to classify the Ecuadorian scorpions based on their potential medical importance. <![CDATA[Arachnids of medical importance in Brazil: main active compounds present in scorpion and spider venoms and tick saliva]]> Arachnida is the largest class among the arthropods, constituting over 60,000 described species (spiders, mites, ticks, scorpions, palpigrades, pseudoscorpions, solpugids and harvestmen). Many accidents are caused by arachnids, especially spiders and scorpions, while some diseases can be transmitted by mites and ticks. These animals are widely dispersed in urban centers due to the large availability of shelter and food, increasing the incidence of accidents. Several protein and non-protein compounds present in the venom and saliva of these animals are responsible for symptoms observed in envenoming, exhibiting neurotoxic, dermonecrotic and hemorrhagic activities. The phylogenomic analysis from the complementary DNA of single-copy nuclear protein-coding genes shows that these animals share some common protein families known as neurotoxins, defensins, hyaluronidase, antimicrobial peptides, phospholipases and proteinases. This indicates that the venoms from these animals may present components with functional and structural similarities. Therefore, we described in this review the main components present in spider and scorpion venom as well as in tick saliva, since they have similar components. These three arachnids are responsible for many accidents of medical relevance in Brazil. Additionally, this study shows potential biotechnological applications of some components with important biological activities, which may motivate the conducting of further research studies on their action mechanisms. <![CDATA[Neuroactive compounds obtained from arthropod venoms as new therapeutic platforms for the treatment of neurological disorders]]> The impact of neurological disorders in society is growing with alarming estimations for an incidence increase in the next decades. These disorders are generally chronic and can affect individuals early during productive life, imposing real limitations on the performance of their social roles. Patients can have their independence, autonomy, freedom, self-image, and self-confidence affected. In spite of their availability, drugs for the treatment of these disorders are commonly associated with side effects, which can vary in frequency and severity. Currently, no effective cure is known. Nowadays, the biopharmaceutical research community widely recognizes arthropod venoms as a rich source of bioactive compounds, providing a plethora of possibilities for the discovery of new neuroactive compounds, opening up novel and attractive opportunities in this field. Several identified molecules with a neuropharmacological profile can act in the central nervous system on different neuronal targets, rendering them useful tools for the study of neurological disorders. In this context, this review aims to describe the current main compounds extracted from arthropod venoms for the treatment of five major existing neurological disorders: stroke, Alzheimer’s disease, epilepsy, Parkinson’s disease, and pathological anxiety. <![CDATA[Seroepidemiological analysis of toxoplasmosis in college students]]> Background Toxoplasmosis is a zoonosis caused by an obligate intracellular parasite, Toxoplasma gondii, which affects warm-blooded animals including humans. Its prevalence rates usually vary in different regions of the planet. Methods In this study, an analysis of the seroprevalence of toxoplasmosis among Brazilian students was proposed by means of IgG specific antibodies detection. The presence of anti-Toxoplasma gondiiantibodies by indirect fluorescent antibody test (IFAT) was also evaluated in order to compare it with enzyme-linked immunosorbent assay (ELISA) and to assess the use of 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) and o-phenylenediamine dihydrochloride chromogens. Results The IFAT method showed a seroprevalence of 22.3%. These results were similar to those obtained by ELISA (24.1%). The seroprevalence was directly estimated from the IgG avidity, which showed that in a sample of 112 students, three of them had acute infection, an incidence of 1.6% in the studied population. Conclusion In this study, the use of different chromogenic substrates in immunoenzymatic ELISA assays did not display different sensitivity in the detection of T. gondii-reagent serum. The extrapolation of results to this population must be carefully considered, since the investigation was conducted on a reduced sample. However, it allows us to emphasize the importance of careful and well prepared studies to identify risk factors for toxoplasmosis, to adopt preventive measures and to offer guidance to at-risk populations about the disease. <![CDATA[The red seaweed <em>Plocamium brasiliense</em> shows anti-snake venom toxic effects]]> Background Snakebite is considered a neglected tropical disease by the World Health Organization. In Brazil, about 70% of the envenomation cases are caused by Bothrops snakes. Its venom may provoke hemorrhage, pain, necrosis, hemolysis, renal or cardiac failure and even death in victims. Since commercial antivenom does not efficiently neutralize the local toxic effects of venoms, natural products have been tested in order to provide alternative or complementary treatment to serum therapy. Therefore, the present study aimed to evaluate the ability of the seaweed Plocamium brasiliense and its active derivatives to neutralize hemorrhagic, edematogenic, hemolytic, coagulant and proteolytic activities of B. jararaca venom. Methods Specimens of P. brasiliense were collected in Rio de Janeiro state, Brazil, dried and submitted to oil extraction using four solvents of increasing polarities, n-hexane (HEX), dichloromethane (DCM), ethyl acetate (ETA) and hydroalcoholic solution (HYD). The solvents were evaporated, yielding HEX, DCM, ETA and HYD extracts. Further, all extracts were dissolved in dimethylsulfoxide. In addition, two monoterpenes (8-bromo-3,4,7-trichloro-3,7-dimethyl-1E, 5E-octadiene and 1,8-dibromo-3,4,7-trichloro-3,7-dimethyl-1E, 5E-octadiene) and a cholesterol fraction were isolated from the extract of P. brasiliense prepared in hexane. Algal samples were incubated for 30 minutes with B. jararaca venom, and then tested for lethality; hemorrhagic, edematogenic, hemolytic, coagulant and proteolytic effects. Results Most of the algal extracts inhibited the toxic effects with different potencies. The DCM extract was the most effective, since it inhibited all types of toxic activity. On the other hand, the HYD extract failed to inhibit any effect. Moreover, the isolated products inhibited proteolysis and protected mice from hemorrhage in 30% of the cases, whereas 8-bromo-3,4,7-trichloro-3,7-dimethyl-1E, 5E-octadiene inhibited 100% and 20% of the hemorrhagic and proteolytic activities, respectively. None of the algal products were toxic to mice. Conclusion Seaweeds may be a promising source of inhibitors against toxic effects caused by B. jararacaenvenomation, which may contribute to antivenom treatment. <![CDATA[Biodegradation of [D-Leu<sup>1</sup>] microcystin-LR by a bacterium isolated from sediment of Patos Lagoon estuary, Brazil]]> Background Toxic cyanobacterial blooms are recurrent in Patos Lagoon, in southern Brazil. Among cyanotoxins, [D-Leu1] microcystin-LR is the predominant variant whose natural cycle involves water and sediment compartments. This study aimed to identify and isolate from sediment a bacterial strain capable of growing on [D-Leu1] microcystin-LR. Sediment and water samples were collected at two distinct aquatic spots: close to the Oceanographic Museum (P1), in Rio Grande City, and on São Lourenço Beach (P2), in São Lourenço do Sul City, southern Brazil. Methods [D-Leu1] microcystin-LR was isolated and purified from batch cultures of Microcystis aeruginosastrain RST9501. Samples of water and sediment from Rio Grande and São Lourenço do Sul were collected. Bacteria from the samples were allowed to grow in flasks containing solely [D-Leu1] microcystin-LR. This strain named DMSX was isolated on agar MSM with 8 g L−1 glucose and further purified on a cyanotoxin basis growth. Microcystin concentration was obtained by using the ELISA immunoassay for microcystins whereas bacterial count was performed by epifluorescence microscopy. The genus Pseudomonas was identified by DNA techniques. Results Although several bacterial strains were isolated from the samples, only one, DMXS, was capable of growing on [D-Leu1] microcystin-LR. The phylogenetic analysis of the 16S rRNA gene from DMXS strain classified the organism as Pseudomonas aeruginosa. DMXS strain incubated with [D-Leu1] microcystin-LR lowered the amount of toxin from 1 μg.L−1 to &lt; 0.05 μg.L−1. Besides, an increase in the bacterial count–from 71 × 105 bacteria.mL−1 to 117 × 105 bacteria.mL−1–was observed along the incubation. Conclusions The use of bacteria isolated from sediment for technological applications to remove toxic compounds is viable. Studies have shown that sediment plays an important role as a source of bacteria capable of degrading cyanobacterial toxins. This is the first Brazilian report on a bacterium–of the genus Pseudomonas–that can degrade [D-Leu1] microcystin-LR, the most frequent microcystin variant in Brazilian freshwaters. <![CDATA[<em>Paederus</em> beetles: the agent of human dermatitis]]> Background Rove beetles of the genus Paederus cause dermatitis when they come in contact with human skin. This condition is prevalent in some tropical and subtropical regions, such as in northern Pakistan, where it was recorded for the first time by US troops. Despite much research from other countries on this subject, few studies, mostly clinical, have been performed in a Pakistani context. A survey was carried out in villages, towns and cities of Punjab province, Pakistan, to explore the rove beetle population dynamics and to develop a model to elucidate the symptoms, preventive measures and treatment strategies for this dermatitis. Methods The prospective observational and patient surveys were performed bimonthly over a period of two years, in different districts of Punjab province. Collection was carried out in fields, gardens and houses during every visit with the aid of a pitfall trap, light trap, flight intercept trap, Berlese funnel trap and sweep netting. These traps were installed for four days during every visit. Interviews of ten individuals of different ages and sexes from each site were recorded during each visit. Results Out of 980 individuals, 26.4% were found to suffer from Paederus dermatitis. Lesions were most commonly found on the neck followed by the face. In July-August during the rainy season, this skin irritation was most prevalent and the population of these beetles peaked (36.2%). During May-June, the beetle population was lowest (7.85%) due to soil dryness. About 70% of such irritation cases were from individuals living in farming villages or in farmhouses. Their houses typically (80%) had broken doors and screen-less windows while 97% of the residents were unaware of how they may have come into contact with these beetles. In most cases (91% from villages/small towns and 24% from cities and adjoining areas) the local residents were unaware of modern treatment strategies. Conclusions Paederus dermatitis is extremely frequent in villages with poor housing facilities and could be avoided via community awareness. <![CDATA[Antimycobacterial and cytotoxicity activity of microcystins]]> Background The present work aimed to evaluate the antimycobacterial activity and cytotoxicity of Microcystis aeruginosa toxins, the MC-LR variant and purified extract of [D-Leu1] microcystin-LR. Methods The antimicrobial activity of M. aeruginosa extract and microcystin was evaluated by resazurin microtiter assay against Mycobacterium tuberculosis, M. terrae, M. chelonae and M. kansasii. The cytotoxicity assay was performed by trypan blue exclusion against the HTC cell line. Results Antimicrobial activity was observed in the hexanic extract of M. aeruginosa (RST 9501 strain) against M. tuberculosis, including sensitive and resistant strains with minimal inhibitory concentrations (MIC) between 1.93 μM and 0.06 μM. The high activity of M. aeruginosa hexanic extract could be attributed to the major presence of the toxins MC-LR and [D-Leu1] MC-LR that showed activity at MIC between 53 and 0.42 μM against tested mycobacterial strains. Even at the highest concentration tested, no toxicity of M. aeruginosa extracts was identified against HTC cells. Conclusions These preliminary results suggest that [D-Leu1] MC-LR is a promising candidate for the development of a new antimycobacterial agent. <![CDATA[Feeding habits of mosquitoes (Diptera: Culicidae) in an area of sylvatic transmission of yellow fever in the state of São Paulo, Brazil]]> AbstractBackground The reintroduction of sylvatic yellow fever in the state of São Paulo after about six decades was confirmed in the Northwestern region in 2000, where in 2008 there also occurred an important epizootic. The purpose of this study was to investigate the feeding habits of culicids potentially involved in the sylvatic transmission of the virus in this region.Methods Specimens were collected in 24 forested localities at ground level with hand nets and mouth aspirators. Collections were made quarterly between October 2006 and July 2008 during daylight hours. Blood-meal identification was carried out in mosquitoes of the tribes Aedini, Mansoniini and Sabethini. The biotin/avidin sandwich ELISA was employed to determine six source types: bird, bovine, equine, rat, human and monkey.Results A total of 24,879 females of the three tribes were obtained, 245 (0.98%) of which were engorged. The presence of three different blood sources per engorged female was the predominant situation, and included 35.10% of the total of samples processed. Samples with two or four different sources were represented by 25.31% and 25.71%, of the specimens, respectively, while just 9.39% had only one type and 1.22%, five different sources. Aedes scapularis, Ae. serratus(Group),Psorophora albigenu and Ps. ferox were the most abundant species and accounted for about 95% of the engorged specimens. Of the principal vector species,Haemagogus janthinomys/capricorniiwas found with bird, bovine and primate blood. These sources were predominant and alternated top ranking as the most frequent source according to the mosquito species and collection site. In general, primate blood was the most prevalent source.Conclusions The human population of the region visits this ecotone frequently, which indicates the need for the periodical assessment of vaccination coverage against yellow fever. The frequency of non-human primate blood source in mosquito species that show minor vector importance in yellow fever virus transmission deserves attention. The eclectic feeding habits and some aspects of the interactions between potential vectors and reservoirs of yellow fever may be associated with the habitat fragmentation characteristic of the region. We recommend that further studies on the capacity and vector competence be performed on secondary vectors in extra-Amazonian region. <![CDATA[DNA damage in BALB/c mice infected with <em>Lacazia loboi</em> and its relation to nutritional status]]> AbstractBackground Jorge Lobo’s disease, also known as lacaziosis, is a cutaneous-subcutaneous mycosis with chronic evolution. It is caused by the fungus Lacazia loboi. Herein we report a study that relates the genotoxicity caused by L. loboi in isogenic mice with nutritional status, through a normal or restricted diet.Methods DNA damage was assessed in the peripheral blood by the comet assay (tail intensity).Results The results for leukocytes showed increases in the mean tail intensity in mice under dietary restriction, in infected mice under dietary restriction and in infected mice ingesting a normal diet.Conclusion These results indicate that dietary restriction and L. loboi infection may increase DNA damage levels in mice, as detected by the comet assay. <![CDATA[First report of brown widow spider sightings in Peninsular Malaysia and notes on its global distribution]]> <title>Abstract</title><sec><title>Background:</title><p>The brown widow spider (<italic>Latrodectus geometricus</italic> Koch, 1841) has colonised many parts of the world from its continent of origin, Africa. By at least 1841, the species had successfully established populations in South America and has more recently expanded its range to the southern states of North America. This highly adaptable spider has been far more successful in finding its niche around the world than its famous cousins, the black widow, <italic>Latrodectus mactans,</italic> found in the south-eastern states of North America, and the red-back, <italic>Latrodectus hasselti</italic>, found mostly in Australia, New Zealand and Japan.</p></sec><sec><title>Methods:</title><p>We performed an extensive web search of brown widow sightings and mapped the location of each sighting using ArcGIS. Specimens reputedly of the species <italic>L. geometricus</italic> were collected at three localities in Peninsular Malaysia. The spiders were identified and documented based on an examination of morphological characteristics and DNA barcoding.</p></sec><sec><title>Results:</title><p>The spiders found in Peninsular Malaysia were confirmed to be <italic>Latrodectus geometricus</italic> based on their morphological characteristics and DNA barcodes. We recorded 354 sightings of the brown widow in 58 countries, including Peninsular Malaysia.</p></sec><sec><title>Conclusion:</title><p>Reports from the Americas and the Far East suggest a global-wide invasion of the brown widow spider. Herein we report the arrival of the brown widow spider in Peninsular Malaysia and provide notes on the identification of the species and its recently expanded range.</p></sec> <![CDATA[Epidemiology of envenomations by terrestrial venomous animals in Brazil based on case reporting: from obvious facts to contingencies]]> <title>Abstract</title><sec><title>Background:</title><p>Envenomation remains a neglected public health issue in most tropical countries. A better understanding of the epidemiology of bites and stings by venomous animals should facilitate their prevention and management. This study aimed to explore the benefits that could be derived from the compulsory notification of cases as it is now routinely practiced in Brazil.</p></sec><sec><title>Methods:</title><p>The Brazilian Notifiable Diseases Information System (SINAN) was consulted online for the 2001-2012 period on all envenomations by venomous terrestrial animals. We studied the incidence, severity, number of deaths, gender, season of accident and time between the accident and hospital consultation.</p></sec><sec><title>Results:</title><p>In total, 1,192,667 accidents and 2,664 deaths from terrestrial venomous animals (snakes, scorpions, spiders, bees and caterpillars) were reported in Brazil during these 12 years, the circumstances of which are detailed in this study. Most envenomations and deaths were caused by snakebites and scorpion stings. However, incidence and mortality showed high regional variations. During this period, the steady and parallel increase of the cases from all the species resulted from several factors including the human population increase, gradual improvement of data collection system and, probably, environmental and socioeconomic factors affecting in a different way the incidence of envenomation by each zoological group and by region.</p></sec><sec><title>Conclusion:</title><p>Mandatory reporting of cases appears to be a useful tool to improve the management of envenomations. However, local studies should be continued to account for the variability of accident circumstances and refine measures necessary for their management.</p></sec> <![CDATA[Epidemiological characterization and prognostic factors in patients with confirmed cerebral cryptococcosis in central Taiwan]]> <title>Abstract</title><sec><title>Background</title><p>Cryptococcal meningitis is a deadly fungal infection. This study aimed to characterize the epidemiology of cerebral cryptococcosis and to define its prognostic factors.</p></sec><sec><title>Methods</title><p>This cross-sectional study collected clinical information from cryptococcal meningitis patients with confirmed cerebral cryptococcosis from 2006 to 2012 at the Changhua Christian Healthcare System to access prognostic factors.</p></sec><sec><title>Results</title><p>Fifty-nine adult cryptococcal meningitis patients were studied. The incidence at Changhua Christian Healthcare System was approximately 170 episodes per 100,000 patients within the studied period. Forty-one of 59 cryptococcal meningitis patients developed complications. Overall, 12 of 59 patients died, for a three-month mortality rate of 20.3 %. Prognostic factors positively associated with the three-month mortality included age (&gt;55 years), patient delay, prolonged delay by the doctor in administering antifungal agent therapy, duration of intensive care unit stay, chronic lung disease, cryptococcemia, headache, altered mental status, positive blood cultures, and high cerebrospinal fluid opening pressure (&gt;250 mm H<sub>2</sub>O).</p></sec><sec><title>Conclusions</title><p>We strongly recommend early administration of an antifungal agent to each suspected cryptococcal meningitis patient to decrease both the delay by doctors in administering therapy and the mortality risk. Aggressive and supportive care for severe cryptococcal meningitis patients is critical to decrease overall mortality from this infection.</p></sec> <![CDATA[New insights into the structural characteristics of irradiated crotamine]]> <title>Abstract</title><sec><title>Background:</title><p>Since ionizing radiation has the potential to alter the molecular structure and affect the biologica properties of biomolecules, it has been successfully employed to attenuate animal toxins. The present study aimed to characterize the structural modifications on irradiated crotamine, a toxin from <italic>Crotalus durissus terrificus</italic> venom, using circular dichroism (CD), fluorescence, Fourier transformed infrared spectroscopy (FTIR), atomic force microscopy (AFM) and differential scanning calorimetry (DSC).</p></sec><sec><title>Methods:</title><p>A combination of size exclusion and ion-exchange chromatography was used to purify the peptide using crude venom. The pure toxin was then submitted to 2 kGy gamma irradiation doses from a cobalt-60 source. Native and irradiated crotamine were analyzed using a fluorescence spectrophotometer. Wavelength was fixed at 295 nm and fluorescence emission scans were collected from 300 to 400 nm. CD and FTIR techniques were used to identify the secondary structure of both samples. DSC analyses were performed at a starting temperature of 20 °C up to a final temperature of 90 °C. AFM provided a 3D profile of the surfaces of both crotamine forms adsorbed on mica.</p></sec><sec><title>Results:</title><p>Fluorescence spectroscopy showed that the quantum yield of the irradiated form decreased. CD spectra of native and irradiated crotamine solutions showed differences between the samples in wavelength, indicating that irradiation induced a transition of a small portion of the random coil regions towards an a-helical conformation. FTIR and CD showed that the native and irradiated crotamine spectra were different with regard to secondary structure. The thermodynamic analysis showed that irradiation caused changes in the calorimetric profile and CD showed that temperature-induced changes also occur in the secondary structure. Finally, AFM showed the possible formation of insoluble aggregates.</p></sec><sec><title>Conclusions:</title><p>Our results indicate that irradiation leads to progressive changes in the structure of the toxin, which could explain a decrease in myotoxic activity.</p></sec> <![CDATA[Antibacterial potential of a basic phospholipase A<sub>2</sub>(VRV-PL-VIIIa) from Daboia russelii pulchella (Russell's viper) venom]]> <title>Abstract</title><sec><title>Background:</title><p>Microbial/bacterial resistance against antibiotics poses a serious threat to public health. Furthermore, the side effects of these antibiotics have stimulated tremendous interest in developing new molecules from diverse organisms as therapeutic agents. This study evaluates the antibacterial potential of a basic protein, <italic>Vipera russellii</italic> venom phospholipase A<sub>2</sub> fraction VIIIa (VRV-PL-VIIIa), from Daboia <italic>russelii pulchella</italic> venom against gram-positive and gram-negative bacteria.</p></sec><sec><title>Methods:</title><p>The antibacterial potential of VRV-PL-VIIIa in the presence and absence of an inhibitor (p-bromophenacyl bromide) was tested against gram-positive and gram-negative bacteria and the minimum inhibitory concentration was determined by microdilution tests.</p></sec><sec><title>Results:</title><p>VRV-PL-VIIIa demonstrated potent antibacterial activities against all the human pathogenic strains tested. It more effectively inhibited such gram-positive bacteria as <italic>Staphylococcus aureus</italic> and <italic>Bacillus subtilis</italic>, when compared to the gram-negative bacteria <italic>Escherichia coli, Vibrio cholerae, Klebsiella pneumoniae</italic> and <italic>Salmonella paratyphi</italic>. It inhibited bacterial growth at minimum inhibitory concentration values ranging from 11.1 to 19.2 μg/mL. The anti-bacterial potential of VRV-PL-VIIIa was comparable to the standards gentamycin, chlorophenicol and streptomycin. The PLA<sub>2</sub>'s hemolytic and antibacterial activities were strongly correlated. Furthermore, even in the presence of p-bromophenacyl bromide, intense antibacterial activity was observed, suggesting a dissociation or partial overlapping of the bactericidal/antimicrobial domains.</p></sec><sec><title>Conclusion:</title><p>VRV-PL-VIIIa demonstrated potent antibacterial activities against all the human pathogenic strains tested. The study shows that despite a strong correlation between enzymatic and antimicrobial activities of VRV-PL-VIIIa, it may possess additional properties that mimic the bactericidal/membrane permeability-increasing protein. This study encourages further in-depth studies on the molecular mechanisms of antibacterial properties of VRV-PL-VIIIa, which would thereby facilitate development of this protein into a possible therapeutic lead molecule for treating bacterial infections.</p></sec> <![CDATA[Pharmacological characterization of venoms from three theraphosid spiders: Poecilotheria regalis, Ceratogyrus darlingi and Brachypelma epicureanum]]> <title>Abstract</title><sec><title>Background</title><p>Tarantulas (Theraphosidae) represent an important source of novel biologically active compounds that target a variety of ion channels and cell receptors in both insects and mammals. In this study, we evaluate and compare the pharmacological activity of venoms from three taxonomically different theraphosid spiders bred in captivity: Poecilotheria regalis, an aggressive arboreal tarantula from southeastern India; Ceratogyrus darlingi, an aggressive tarantula from southern Africa; and Brachypelma epicureanum, a docile tarantula from the Yucatan dry forest of Mexico. Prior to this study, no research had been conducted with regard to the composition and pharmacological activity of these venoms.</p></sec><sec><title>Methods</title><p>The pharmacological characterization of the venoms was described for the first time by the assessment of their toxicity in crickets (LD<sub>50</sub>) along with their nociceptive (by using the formalin test), hyaluronidase, phospholipase A<sub>2</sub>, edematogenic and caseinolytic activity.</p></sec><sec><title>Results</title><p>P. regalis and B. epicureanum venoms induced a similar lethal effect on crickets (LD<sub>50</sub> = 5.23 ± 3.1 and 14.4 ± 5.0 μg protein/g 48 h post-injection, respectively), whereas C. darlingi venom (119.4 ± 29.5 μg protein/g 48 h post-injection) was significantly less lethal than the other two venoms. All three venoms induced similar edematogenic activity on rats but did not induce nociceptive behavior. The assessment of enzymatic activity indicated that P. regalis venom induces significantly higher hyaluronidase activity (27.6 ± 0.9 TRU/mg) than both C. darlingi (99.7 ± 1.9 TRU/mg) and B. epicureanum (99.6 ± 1.6 TRU/mg); these latter venoms did not display phospholipase A<sub>2</sub>or caseinolytic activity.</p></sec><sec><title>Conclusions</title><p>This study demonstrates that these theraphosid spiders of different habitats produce venoms with different activities. P. regalis venom displays a high level of hyaluronidase activity, which may be associated with its potentially medically significant bite.</p></sec> <![CDATA[A comparison between the recombinant expression and chemical synthesis of a short cysteine-rich insecticidal spider peptide]]> <title>Abstract</title><sec><title>Background:</title><p>The choice between heterologous expression versus chemical synthesis for synthesizing short cysteine-rich insecticidal peptides from arthropods may impact the obtainment of yields and well-folded bioactive molecules for scientific research. Therefore, two recombinant expression systems were compared to that of chemical synthesis for producing Ba1, a cysteine-rich spider neurotoxin.</p></sec><sec><title>Methods:</title><p>The transcription of the insecticidal neurotoxin Ba1 was obtained from a cDNA library of venom glands of the spider <italic>Brachypelma albiceps.</italic>It was cloned into the pCR®2.1-TOPO® cloning vector and then introduced in two different expression vectors, pQE40 and pET28a<sup>+</sup>. Each vector was transfected into E. <italic>coli</italic> M15 and BL21 cells, respectively, and expressed under induction with isopropyl thiogalactoside (IPTG). The chemical synthesis of Ba1 was performed in an Applied Biosystems 433A peptide synthesizer.</p></sec><sec><title>Results:</title><p>Both expression systems pQE40 and pET28a<sup>+</sup> expressed the His-tagged recombinant protein products, HisrDFHRBa1 and HisrBa1, respectively, as inclusion bodies. The recombinant proteins HisrDFHRBa1 and HisrBa1 presented respective molecular masses of 28,289 and 8274.6 Da, and were not biologically active. These results suggested that both HisrDFHRBa1 and HisrBa1 were oxidized after cell extraction, and that their insecticidal activities were affected by their N-terminal pro-peptides and different disulfide bridge arrangements. The respective protein expression yields for HisrDFHRBa1 and HisrBa1 were 100 μg/L and 900 μg/L of culture medium. HisrBa1 was reduced and folded under <italic>in vitro</italic>conditions. The <italic>in vitro</italic> folding of HisrBa1 produced several isoforms, one of which, after removing its N-terminal pro-peptide by enzymatic cleavage, presented elevated insecticidal activities compared to the native Ba1. Furthermore, the His-tagged protein HisrDFHRBa1 underwent enzymatic cleavage to obtain recombinant Ba1 (rBa1). As expected, the molecular mass of rBa1 was 4406.4 Da. On the other hand, Ba1 was chemically synthesized (sBa1) with a yield of 11 mg per 0.1 mmol of amino acid assembly.</p></sec><sec><title>Conclusions:</title><p>The two recombinant insecticidal peptides and the one synthesized chemically were as active as the native Ba1; however, toxin yields differed drastically.</p></sec> <![CDATA[Molecular detection and antimicrobial resistance of Pseudomonas aeruginosa from houseflies (<italic>Musca domestica</italic>) in Iran]]> <title>Abstract</title><sec><title>Background</title><p>Pseudomonas aeruginosa is a common bacterium that can cause disease in humans and other animals. This study was conducted to screen for molecular detection and antimicrobial-resistant P. aeruginosa in Musca domestica in different locations in the Iranian provinces of Shahrekord and Isfahan.</p></sec><sec><title>Methods</title><p>Musca domestica were captured by both manual and sticky trap methods, during the daytime, from household kitchens, cattle farms, animal hospitals, human hospitals, slaughterhouses and chicken farms at random locations in Shahrekord and Isfahan provinces of Iran, and subsequently transported to the laboratory for detection of P. aeruginosa. In the laboratory, flies were identified and killed by refrigeration in a cold chamber at −20 °C, then placed in 5 mL peptone water and left at room temperature for five hours before being processed. Pseudomonas isolates were preliminarily identified to genus level based on colony morphology and gram staining, and their identity was further confirmed by polymerase chain reaction.</p></sec><sec><title>Results</title><p>Overall blaTEM gene was recovered from 8.8 % (53/600) of the P. aeruginosa isolated from houseflies collected from the two provinces. A slightly higher prevalence (10.7 %; 32/300) was recorded in Shahrekord province than Isfahan province (7.0 %; 21/300). The locations did not differ statistically (p &lt; 0.05) in bacterial prevalence in flies. Seasonal prevalence showed a significantly lower infection frequency during autumn.</p></sec><sec><title>Conclusions</title><p>Houseflies are important in the epidemiology of P. aeruginosa infections.</p></sec> <![CDATA[Tissue apoptosis in mice infected with <italic>Leptospira interrogans</italic> serovar Icterohaemorrhagiae]]> <title>Abstract</title><sec><title>Background:</title><p>This investigation aimed to evaluate the occurrence of some apoptotic features induced by <italic>Leptospira interrogans</italic> serovar Icterohaemorrhagiae infection in young BALB/c mice during 2, 4, 7, 10, 14 and 21 days post-infection (dpi).</p></sec><sec><title>Methods:</title><p>The animals were euthanized and lung, liver and kidneys were harvested to histopathology analysis and immunohistochemistry to caspase-3 antigen detection was performed.</p></sec><sec><title>Results:</title><p>Chromatin condensation in kidney and liver tissues, but not in lung tissue, was observed. Caspase-3 reactive cells, mainly characterized as renal epithelial cells, were detected in the days 14 and 21 at high levels when compared to days 2,4 and 7 (p = 0.025; p &lt;0.05). Lung sections revealed caspase-3 labeled alveolar cells in 10 and 14 days post-infection was higher than observed at 7 days (p = 0.0497; p &lt; 0.05). Liver sections demonstrated reactive cells at a highest level at 14 and 21 days post-infection when comparison to 2,4, 7 and 10 days (p = 0.0069; p&lt;0.05).</p></sec><sec><title>Conclusions:</title><p>Our results suggest that infection of L <italic>interrogans</italic> induce in kidney, liver and lung an activation of apoptosis mediated by caspase-3 dependent pathway in later phases of infectious process.</p></sec> <![CDATA[Biological characterization of compounds from <em>Rhinella schneideri</em> poison that act on the complement system]]> Background The skin secretions of toads of the family Bufonidae contain biogenic amines, alkaloids, steroids (bufotoxins), bufodienolides (bufogenin), peptides and proteins. The poison of Rhinella schneideri, formerly classified as Bufo paracnemis, presents components that act on different biological systems, including the complement system. The aim of this study was to isolate and examine the activity ofRhinella schneideri poison (RsP) components on the complement system.Methods The components active on the complement system were purified in three chromatographic steps, using a combination of cation-exchange, anion-exchange and gel filtration chromatography. The resulting fractions were analyzed by SDS-PAGE and screened for their activity in the hemolytic assay of the classical/lectin complement pathways. Fractions active on the complement system were also assessed for their ability to generate C3 fragments evaluated by two dimensional immunoelectrophoresis assay, C3a and C5a by neutrophil chemotaxis assay and SC5b-9 complex by ELISA assay.Results The fractionation protocol was able to isolate the component S5 from theRsP, as demonstrated by SDS-PAGE and the RP-FPLC profile. S5 is a protein of about 6000 Da, while S2 presents components of higher molecular mass (40,000 to 50,000 Da). Fractions S2 and S5 attenuated the hemolytic activity of the classical/lectin pathways after preincubation with normal human serum. Both components stimulated complement-dependent neutrophil chemotaxis and the production of C3 fragments, as shown by two-dimensional immunoelectrophoresis. S2 showed a higher capacity to generate the SC5b- 9 complex than the other fractions. This action was observed after the exposure of normal human serum to the fractions.Conclusions This is the first study to examine the activity of RsP components on the complement system. Fractions S2 and S5 reduced the complement hemolytic activity, stimulated complement-dependent neutrophil chemotaxis and stimulated the production of C3 fragments, indicating that they were able to activate the complement cascade. Furthermore, fraction S2 was also able to generate the SC5b-9 complex. These components may be useful tools for studying dysfunction of the complement cascade. <![CDATA[Bordonein-L, a new L-amino acid oxidase from <em>Crotalus durissus terrificus</em> snake venom: isolation, preliminary characterization and enzyme stability]]> BackgroundCrotalus durissus terrificus venom (CdtV) is one of the most studied snake venoms in Brazil. Despite presenting several well known proteins, its L-amino acid oxidase (LAAO) has not been studied previously. This study aimed to isolate, characterize and evaluate the enzyme stability of bordonein-L, an LAAO from CdtV.Methods The enzyme was isolated through cation exchange, gel filtration and affinity chromatography, followed by a reversed-phase fast protein liquid chromatography to confirm its purity. Subsequently, its N-terminal amino acid sequence was determined by Edman degradation. The enzyme activity and stability were evaluated by a microplate colorimetric assay and the molecular mass was estimated by SDS-PAGE using periodic acid-Schiff staining and determined by mass spectrometry.Results The first 39 N-terminal amino acid residues exhibited high identity with other snake venom L-amino acid oxidases. Bordonein-L is a homodimer glycoprotein of approximately 101 kDa evaluated by gel filtration. Its monomer presents around 53 kDa estimated by SDS-PAGE and 58,702 Da determined by MALDI-TOF mass spectrometry. The enzyme exhibited maximum activity at pH 7.0 and lost about 50 % of its activity after five days of storage at 4 °C. Bordonein-L’s activity was higher than the control when stored in 2.8 % mannitol or 8.5 % sucrose.Conclusions This research is pioneering in its isolation, characterization and enzyme stability evaluation of an LAAO from CdtV, denominated bordonein-L. These results are important because they increase the knowledge about stabilization of LAAOs, aiming to increase their shelf life. Since the maintenance of enzymatic activity after long periods of storage is essential to enable their biotechnological use as well as their functional studies. <![CDATA[<em>Bothrops</em> snake venoms and their isolated toxins, an L-amino acid oxidase and a serine protease, modulate human complement system pathways]]> Background Activation of the complement system plays an important role in the regulation of immune and inflammatory reactions, and contributes to inflammatory responses triggered by envenomation provoked byBothrops snakes. The present study aimed to assess whether Bothrops jararacussuand Bothrops pirajai crude venoms and their isolated toxins, namely serine protease (BjussuSP-I) and L-amino acid oxidase (BpirLAAO-I), modulate human complement system pathways.Methods Lyophilized venom and toxin samples solubilized in phosphate buffered saline were diluted in appropriate buffers to evaluate their hemolytic activity on the alternative and classical pathways of the complement system. Venom- and toxin-treated normal human serum was added to the erythrocyte suspension, and the kinetic of hemolysis was measured spectrophotometrically at 700 nm. The kinetic 96-well microassay format was used for this purpose. We determined the t ½values (time required to lyse 50 % of target erythrocytes), which were employed to calculate the percentage of inhibition of the hemolytic activity promoted by each sample concentration. To confirm complement system activation, complement-dependent human neutrophil migration was examined using the Boyden chamber model.Results At the highest concentration tested (120 μg/mL), B. jararacussu and B. pirajai crude venoms inhibited the hemolytic activity of the classical pathway (65.3 % and 72.4 %, respectively) more strongly than they suppressed the hemolytic activity of the alternative pathway (14.2 and 13.6 %, respectively). BjussuSP-I (20 μg/mL) did not affect the hemolytic activity of the classical pathway, but slightly decreased the hemolytic activity of the alternative pathway (13.4 %). BpirLAAO-I (50 μg/mL) inhibited 24.3 and 12.4 % of the hemolytic activity of the classical and alternative pathways, respectively. Normal human serum treated with B. jararacussu and B. pirajai crude venoms induced human neutrophil migration at a level similar to that induced by zymosan-activated normal human serum.Conclusion Together, the results of the kinetics of hemolysis and the neutrophil chemotaxis assay suggest that pre-activation of the complement system byB. jararacussu and B. pirajai crude venoms consumes complement components and generates the chemotactic factors C3a and C5a. The kinetic microassay described herein is useful to assess the effect of venoms and toxins on the hemolytic activity of the complement system. <![CDATA[Purification procedure for the isolation of a P-I metalloprotease and an acidic phospholipase A <sub>2</sub> from<em>Bothrops atrox</em> snake venom]]> Background Snake venoms are complex mixtures of inorganic and organic components, mainly proteins and peptides. Standardization of methods for isolating bioactive molecules from snake venoms is extremely difficult due to the complex and highly variable composition of venoms, which can be influenced by factors such as age and geographic location of the specimen. Therefore, this study aimed to standardize a simple purification methodology for obtaining a P-I class metalloprotease (MP) and an acidic phospholipase A2 (PLA 2 ) from Bothrops atroxvenom, and biochemically characterize these molecules to enable future functional studies.Methods To obtain the toxins of interest, a method has been standardized using consecutive isolation steps. The purity level of the molecules was confirmed by RP-HPLC and SDS-PAGE. The enzymes were characterized by determining their molecular masses, isoelectric points, specific functional activity and partial amino acid sequencing.Results The metalloprotease presented molecular mass of 22.9 kDa and pI 7.4, with hemorrhagic and fibrin(ogen)olytic activities, and its partial amino acid sequence revealed high similarity with other P-I class metalloproteases. These results suggest that the isolated metalloprotease is Batroxase, a P-I metalloprotease previously described by our research group. The phospholipase A 2 showed molecular mass of 13.7 kDa and pI 6.5, with high phospholipase activity and similarity to other acidic PLA2 s from snake venoms. These data suggest that the acidic PLA2 is a novel enzyme from B. atrox venom, being denominated BatroxPLA 2 .Conclusions The present study successfully standardized a simple methodology to isolate the metalloprotease Batroxase and the acidic PLA 2 BatroxPLA2 from the venom of B. atrox, consisting mainly of classical chromatographic processes. These two enzymes will be used in future studies to evaluate their effects on the complement system and the inflammatory process, in addition to the thrombolytic potential of the metalloprotease. <![CDATA[Angiotensin-converting enzyme inhibitors of <em>Bothrops jararaca</em> snake venom affect the structure of mice seminiferous epithelium]]> Background Considering the similarity between the testis-specific isoform of angiotensin-converting enzyme and the C-terminal catalytic domain of somatic ACE as well as the structural and functional variability of its natural inhibitors, known as bradykinin-potentiating peptides (BPPs), the effects of different synthetic peptides, BPP-10c (&lt;ENWPHQIPP), BPP-11e (&lt;EARPPHPPIPP), BPP-AP (&lt;EARPPHPPIPPAP) and captopril were evaluated in the seminiferous epithelium of male mice.Methods The adult animals received either one of the synthetic peptides or captopril (120 nmol/dose per testis) via injection into the testicular parenchyma. After seven days, the mice were sacrificed, and the testes were collected for histopathological evaluation.Results BPP-10c and BPP-AP showed an intense disruption of the epithelium, presence of atypical multinucleated cells in the lumen and high degree of seminiferous tubule degeneration, especially in BPP-AP-treated animals. In addition, both synthetic peptides led to a significant reduction in the number of spermatocytes and round spermatids in stages I, V and VII/VIII of the seminiferous cycle, thickness of the seminiferous epithelium and diameter of the seminiferous tubule lumen. Interestingly, no morphological or morphometric alterations were observed in animals treated with captopril or BPP-11e.Conclusions The major finding of the present study was that the demonstrated effects of BPP-10c and BPP-AP on the seminiferous epithelium are dependent on their primary structure and cannot be extrapolated to other BPPs. <![CDATA[Snakes and snakebite envenoming in Northern Tanzania: a neglected tropical health problem]]> Background Snakebites cause considerable human and livestock injuries as well as deaths worldwide, and particularly have a high impact in sub-Saharan Africa. Generating a basic platform of information on the characteristics of snakes and snakebites in various countries is relevant for designing and implementing public health interventions.Methods This study was performed to identify types of snakes and some of the characteristics of snakebite cases in two communities, an agricultural and a pastoralist, in Arusha region, northern Tanzania. A total of 30 field visits were carried out in areas considered by local inhabitants to be potential microhabitats for snakes. Direct observation of snake types based on morphological features and a structured questionnaire were employed for data collection.Results A total of 25 live and 14 dead snakes were encountered. Among the dead ones, the following species were identified: two black-necked spitting cobras (Naja nigricollis); five puff adders (Bitis arietans), one common egg-eater (Dasypeltis scabra); two rufous-beaked snakes (Ramphiophis rostratus); two brown house snakes (Lamprophis fuliginosus); one Kenyan sand boa (Eryx colubrinus), and one black mamba (Dendroaspis polylepis). The frequency of snake encounters was significantly higher (χ2 = 4.6; p= 0.03) in the pastoral than in the agricultural area; there were more snakebite cases in the former, but the differences were not statistically significant (p= 0.7). A total of 242 snakebite victims attended at the Meserani Clinic, located in the study area, between the years 2007 to 2012. Of all cases, 146 (61.6 %) and 96 (38.4 %) were male and female patients, respectively. As for age distribution, 59.1 % of snakebite victims were from the economically active age groups between 15 and 55 years.Conclusion Snakebites are a threat to rural communities and public health in general. The burden of snakebites in Tanzania presents an epidemiologically similar picture to other tropical countries. Livestock keeping and agriculture are the major economic activities associated with snakebites. Community-based public education is required to create awareness on venomous snakes and predisposing factors to snakebites. These tasks demand integration of diverse stakeholders to achieve a common goal of reducing the impact of human suffering from these envenomings in Tanzania. <![CDATA[Epidemiological characteristics of human rabies in Henan province in China from 2005 to 2013]]> Background Rabies is very common in People’s Republic of China. Henan province, in the central portion of China, is one of the most densely populated provinces in the entire country. With the new rabies epidemic trend noted in northern and western China, it is necessary to investigate the characteristics of human rabies in this area and control the disease.Methods We chose patients in hospital isolation in 18 municipalities in Henan province as the investigation subjects. Data were collected through systematic reporting from these hospitals, whereas a questionnaire was applied to the relatives of patients.Results A total of 1022 rabies cases were reported. The incidence of human rabies in Henan has increased rapidly since 2005, having peaked in 2007, and maintained a high level in the remaining years. The cases were found mainly in rural areas in the south and east of the province. Rabies was often noted in summer and with the highest number in August. Most cases were noted in males and often in farmers. The patients aged between 40 and 60 years accounted for 36.8 % of all the cases. The wound treatment rate (12.2 %) and vaccination rate (2.6 %) of rabies cases after exposure were relatively low, while the rabies immunoglobulin utilization rate was only 2.8 %.Conclusions Rabies epidemic cases at the county level overall were increasing in Henan province during the period of 2005–2013; the epidemic has spread quickly. The data in this study imply that the disease could be better managed by more integrated surveillance across human health and veterinary sectors, improved education and better government policies. <![CDATA[Report of 15 injuries caused by lionfish (<em>pterois volitans</em>) in aquarists in Brazil: a critical assessment of the severity of envenomations]]> AbstractLionfish are venomous fish that belong to the Scorpaenidae family. Individuals of this family and those of the Synanceiidae family comprise most of the existing venomous fish in the world. Lionfish are originally found in the Indo-Pacific, but they have received special attention in the last years for their dissemination in the Atlantic Ocean, with the emergence of large populations in the USA, Caribbean and South America. Because of its beauty, this fish has always been present in private and commercial aquariums around the world. Herein, we describe 15 envenomations in aquarists registered in a period of eighteen years (1997–2014). The stings caused excruciating pain and marked inflammation, with local erythema, edema, heat, paleness and cyanosis. In one case, it was possible to observe vesicles and blisters. There were no skin necroses or marked systemic manifestations. We discuss the possible coming of the fish to South America and the circumstances and clinical impact of the envenomations. <![CDATA[<em>Mycobacterium marinum</em> infection: a case report]]> AbstractThe infection by Mycobacterium marinum in humans is relatively uncommon. When it occurs, it mainly affects the skin, usually with a chronic, indolent and benign evolution. The diagnosis requires a high index of suspicion, and a significant delay may be observed between the first symptoms to the final diagnosis. This present case reports a M. marinum infection in an immunocompetent patient that had a chronic undiagnosed injury on the dominant hand for at least five years. The patient had several medical consultations, without proper suspicion, hampering adequate diagnostic investigation. Histopathology detected tuberculoid granulomas, but showed no acid-fast bacilli. The culture in appropriate medium and the polymerase chain reaction-restriction enzyme analysis (PRA)-hsp65 confirmed the diagnosis. Treatment with clarithromycin (1 g/day) for three months was effective. Although uncommon, this infection is a contact zoonosis. Therefore, it is important for clinicians to be aware of this diagnosis and properly guide preventable measures to professionals that are in risk group. <![CDATA[First serine protease inhibitor isolated from <em>Rhinella schneideri</em> poison]]> Background Toad secretions are a source of molecules with potential biotechnological application on a wide spectrum of diseases. Toads from theRhinella family have two kinds of poisonous glands, namely granular and mucous glands. Rhinella schneideritoads produce granular secretions that comprise a great number of molecules, including serine proteases inhibitors. Serine proteases, such as trypsin, chymotrypsin and elastase, are enzymes that have a serine amino acid into its catalytic site and can be found in a large number of vertebrate species and pathogenic microorganisms. Therefore, the present work aims to purify a serine protease inhibitor from Rhinella schneiderigranular secretions.Findings This study presents the protocol used to purify a serine protease inhibitor from the Rhinella schneideri poison. The granular secretion was submitted to dialysis in order to separate the low molecular weight compounds, which were submitted to a reversed phase-fast protein liquid chromatography fractionation step in a C2C18 column. The major fractions were tested over trypsin, chymotrypsin and elastase through colorimetric assay. The inhibition tests were performed with the enzyme in absence (positive control) and presence of fractions, denatured enzyme (negative control) and the respective chromogenic substrate. Rs20 was the compound with the major inhibitory activity over chymotrypsin, inducing a delay in the formation of the chromogenic enzymatic product. The structure characterization of Rs20 was performed by high resolution electronspray ionization-mass spectrometry (HRESI-MS) and gas chromatography coupled with mass spectrometry (GC-MS). HRESI showed an intense signal suggesting the presence of bufadienolide with less than 10 ppm error. In addition, it was observed a low intense signal at m/z399 that could be lithocholic acid, a biosynthetic precursor of bufadienolide. Finally, GC-MS analysis applying NIST library identification reinforced this hypothesis.Conclusions The current study have isolated and partially characterized the function and structure of the first bufadienolide with inhibitory action over chymotrypsin.