Journal of Venomous Animals and Toxins including Tropical Diseases

Published by BioMed Central
Online ISSN: 1678-9199
Morphological analysis of the seminiferous epithelium of male adult mice treated with BPP-10c and captopril. Photomicrographs of the seminiferous tubules of male adult mice treated with vehicle (A), captopril (B) and BPP-10c (C, D, E, F) using Mallory’s trichrome stain. The seminiferous tubule morphological data obtained from the animals treated with BPP-10c indicated the presence of atypical cells in the lumen (arrow), disruption of the epithelium (*), and a loss of elongated spermatids (#). No alteration was observed in the seminiferous epithelium from mice treated with captopril or vehicle.
Morphometric analysis of the seminiferous epithelium of male adult mice treated with BPP-10c or captopril. (A) Morphometric aspects of the seminiferous tubules of control and treated animals (epithelium height, tubule diameter and lumen diameter). (B) Total support capacity of Sertoli cell increased during stages I, V, VII/VIII and XII of the seminiferous epithelium cycle when animals were treated with BPP-10c. Data are presented as mean ± SEM, and the criteria for statistical significance were set at p < 0.05.
Effects of BPP-10c and captopril on the distribution of claudin-1 in the seminiferous epithelium of adult mouse testis. (A) Immunoblot analysis of mouse testis lysate using an antibody to claudin-1. (B, C, D) Immunohistochemical staining of mouse testis transverse cross-sections treated with vehicle. (E) Non-specific staining was detected only in the basal and adluminal compartments of seminiferous epithelium of control sections – negative control. (F and G) Immunostaining of claudin-1 following treatment with captopril or (H and I) BPP-10c demonstrated no difference in the distribution of claudin-1 when compared with control. Hematoxylin was used for counterstaining. Scale bar: 50 μm.
Maintenance of the blood-testis barrier (BTB) in male adult mice treated with vehicle, captopril or BPP-10c. Photomicrographs of 0.5-μm thick – plastic-embedded transverse sections of (A) vehicle, (B) captopril, (C) BPP-10c and (D) LPS – of seminiferous tubules following fixation with hyperosmotic fixative and staining with hematoxylin and eosin. The adluminal meiotic cells are protected from fixative-induced condensation, maintaining normal morphology and cellular contacts in A, B and C (arrow). The BTB is not maintained in the seminiferous tubules of mice treated with LPS (D, see arrow).
The testis-specific isoform of angiotensin-converting enzyme (tACE) is exclusively expressed in germ cells during spermatogenesis. Although the exact role of tACE in male fertility is unknown, it clearly plays a critical function in spermatogenesis. The dipeptidase domain of tACE is identical to the C-terminal catalytic domain of somatic ACE (sACE). Bradykinin potentiating peptides (BPPs) from snake venoms are the first natural sACE inhibitors described and their structure--activity relationship studies were the basis for the development of antihypertensive drugs such as captopril. In recent years, it has been showed that a number of BPPs -- including BPP-10c -- are able to distinguish between the N- and C-active sites of sACE, what is not applicable to captopril. Considering the similarity between tACE and sACE (and since BPPs are able to distinguish between the two active sites of sACE), the effects of the BPP-10c and captopril on the structure and function of the seminiferous epithelium were characterized in the present study. BPP-10c and captopril were administered in male Swiss mice by intraperitoneal injection (4.7 mumol/kg for 15 days) and histological sections of testes were analyzed. Classification of seminiferous tubules and stage analysis were carried out for quantitative evaluation of germ cells of the seminiferous epithelium. The blood-testis barrier (BTB) permeability and distribution of claudin-1 in the seminiferous epithelium were analyzed by hypertonic fixative method and immunohistochemical analyses of testes, respectively. The morphology of seminiferous tubules from animals treated with BPP-10c showed an intense disruption of the epithelium, presence of atypical multinucleated cells in the lumen and degenerated germ cells in the adluminal compartment. BPP-10c led to an increase in the number of round spermatids and total support capacity of Sertoli cell in stages I, V, VII/VIII of the seminiferous epithelium cycle, without affecting BTB permeability and the distribution of claudin-1 in the seminiferous epithelium. Interestingly, no morphological or morphometric alterations were observed in animals treated with captopril. The major finding of the present study was that BPP-10c, and not captopril, modifies spermatogenesis by causing hyperplasia of round spermatids in stages I, V, and VII/VIII of the spermatogenic cycle.
Morphology and staining characteristic of (a) large lymphocytes (Lly), small lymphocytes (Sly), (b) erythrocytes (Er), normocytes (Mo), (c) eosinophils, (d) basophils and (e) heterophils (Ne) of the circulating blood of 
Naja naja.
Hematology and plasma biochemistry parameters are useful in the assessment and management of snake physiological status. Although reference ranges are readily available for many snake species, they are lacking for most venomous ophidians. We determined hematology and plasma biochemistry reference ranges for the wild-caught Indian cobra, Naja naja. Blood samples, taken from the ventral tail vein, were assessed for erythrocyte count, total leukocyte count, hemoglobin concentration, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration, considering the sex of snakes. Results revealed the erythrocyte numbers (male, 390000 +/- 12503.33/mm3 and female, 347500 +/- 7505.55/mm3), shapes and the centrally located oval nuclei. Leukocytes were round, circular or disk-shaped, and the mean size was larger in male than female snakes. The maximum number of leukocytes was found to be 11700 +/- 100/mm3 in male and 12100 +/- 200/mm3 in female snakes, and mean values of differential leukocyte count differed statistically between male and female snakes. The total leukocyte levels were found to be higher in female snakes, but the levels of hemoglobin, hematocrit, and MCV values were higher in male snakes. However, the MCH and MCHC values remained higher in female snakes throughout the study period. Mean protein and cholesterol contents differed significantly between male (45.32 +/- 1.76 and 3.76 +/- 0.06 mg/mL) and female (12.47 +/- 0.82 and 4.72 +/- 0.2 mg/mL) snakes. In conclusion, monitoring snake hematological and biochemical parameters can serve as a means to evaluate the physiological and health status of N. naja populations, which may be a useful indicator of their environmental status.
Study areas in Pará state, northern Brazil.
Female specimen of Tityus obscurus (Gervais, 1843) with length of 10 cm from the eastern region of Pará state, Brazil. It had been provided by a patient who was showing systemic manifestations upon hospital admission.
findings of patients envenomed by Tityus obscurus (Gervais, 1843) in two study areas of Pará state, Brazil
Scorpion envenomations are a major public health problem in Brazil, whose most dangerous cases are attributable to the genus Tityus. This study was designed to compare the clinical and demographic features of envenomations by Tityus obscurus in two areas of the state of Para located in the Amazon basin.Were compared demographic findings, local and systemic signs and symptoms of human envenomations caused by T. obscurus that occurred in western and eastern areas of the state. Forty-eight patients with confirmed envenomation by T. obscurus were evaluated from January 2008 to July 2011. Most of them came from the eastern region, where male and female patients were present in similar numbers, while males predominated in the west. Median age groups were also similar in both areas. Most scorpion stings took place during the day and occurred significantly more frequently on the upper limbs. The time between the sting and admission to the health center was less than three hours in both areas. Most eastern patients had local manifestations while in the west, systemic manifestations predominated. Local symptoms were similar in both areas, but systemic signs and symptoms were more common in the west. Symptoms frequently observed at the sting site were local and radiating pain, paresthesia, edema, erythema, sweating, piloerection and burning. The systemic manifestations were significantly higher in patients from the west. Futhermore, neurological symptoms such as general paresthesia, ataxia, dysarthria, myoclonus, dysmetria, and electric shock-like sensations throughout the body were reported only by patients from the west. The present study shows that two regions of Para state differ in the clinical manifestations and severity of confirmed envenomation by T. obscurus which suggests a toxicity variation resulting from the diversity of T. obscurus venom in different areas of the Brazilian Amazon basin, and that T. serrulatus antivenom can be successfully used against T. obscurus.
Adult female of Rophalurus amazonicus.
Scorpion sting site on the middle of the thumb 56 hours after the accident.
Map of the Pará state, in the northern Brazil. Inset emphasizes the hydrographic basin of the Tapajós river. The black star indicates Tapari, where the envenomation provoked by R. amazonicus occurred. Distance is shown on a relative scale bar. The map was created using the free software QGis 2.2.
Scorpions, mainly those belonging to the genus Tityus cause many deaths and injuries in Brazil, with tens of thousands of envenomations notified every year. However, injuries involving other scorpion species are scarcely registered. Among the sixteen species of the genus Rhopalurus, Thorell, 1876, described up to date, nine are found in this country, with only a confirmed case of human envenomation provoked by R. agamemnon Koch, 1839. The present case reports, for the first time, a case of scorpion sting in a human victim involving Rhopalurus amazonicus, endemic species of the west region of the Pará state, Amazon, Brazil. The symptoms of envenomation were local pain and paresthesia. This study contributes to develop the knowledge on venomous scorpions, particularly those that may cause envenomations in this region.
Very old photo showing Louis Fage (seated) and Max Vachon (standing behind him), examining scorpions in the National Museum of Natural History, Paris. Photo published in the newspaper Le Figaro Litteraire on July 19, 1952.
One of the photos taken during the IV International Congress of Arachnology which took place in Paris in 1968. M. Vachon (1) is next to P. Bonnet (2).
Official photo of the VII International Congress of Arachnology which took place in Exeter (UK) in 1977. A number of scorpiologists were present: (1) M. Vachon, (2) B. Lamoral, (3) J. L. Cloudsley-Thompson, (4) N. F. Hadley, (5) H. W. C. Couzijn, (6) W. R. Lourenço, (7) M. R. Warburg.
Official photo of the 1st International Congress on Envenomations and Their Treatments which took place at the Pasteur Institute, Paris, in 1995. (1) C. Diniz, (2) M. Goyffon, (3) W. R. Lourenço.
This work provides historical context about scorpion studies from the end of the 19th century to the present day. The content is mainly addressed to non-zoologists, working in research fields that embrace scorpion biology, notably to those working with venoms and toxins. The historical aspects described include academic professional scholars who worked on scorpion classification and general distribution patterns; and to a lesser extent, on studies of ecology and natural history. The aim is not to provide an exhaustive description of all scholars who in one way or another became involved with scorpions, but rather of those who greatly contributed during a given period to the research of these organisms. No critical analysis of the work of previous researchers is undertaken, but some comments are proposed to bring clarification on 'who's who'. Since a global consensus in relation to classification and/or distribution patterns has not been reached among modern experts, these different approaches are also presented without judgment. Consequently, distinct approaches remain open for discussion.
Scorpion venoms are rich bioactive peptide libraries that offer promising molecules that may lead to the discovery and development of new drugs. Leiurus abdullahbayrami produces one of the most potent venoms among Turkish scorpions that provokes severe symptoms in envenomated victims. In the present study, the peptide profile of the venom was investigated by electrophoretic methods, size-exclusion and reversed-phase chromatography and mass spectroscopy. Cytotoxic and antimicrobial effects were evaluated on a breast cancer cell line (MCF-7) and various bacterial and fungal species. Proteins make up approximately half of the dry weight of L. abdullahbayrami crude venom. Microfluidic capillary electrophoresis indicated the presence of 6 to 7 kDa peptides and proved to be a highly practical peptidomics tool with better resolution when compared to conventional polyacrylamide gel electrophoresis. Mass spectroscopy analysis helped us to identify 45 unique peptide masses between 1 to 7 kDa with a bimodal mass distribution peaking between molecular weights of 1 to 2 kDa (29%) and 3 to 4 kDa (31%). L. abdullahbayrami crude venom had a proliferative effect on MCF-7 cells, which may be explained by the high concentration of polyamines as well as potassium and calcium ions in the arachnid venoms. Antimicrobial effect was stronger on gram-negative bacteria. This work represents the first peptidomic characterization of L. abdullahbayrami venom. Considering the molecular weight-function relationship of previously identified venom peptides, future bioactivity studies may lead to the discovery of novel potassium and chloride ion channel inhibitors as well as new antimicrobial peptides from L. abdullahbayrami venom.
The crown-of-thorns starfish Acanthaster planci is a venomous species from Taiwan whose venom provokes strong hemolytic activity. To understand the hemolytic properties of A. planci venom, samples were collected from A. planci spines in the Penghu Islands, dialyzed with distilled water, and lyophilized into A. planci spine venom (ASV) powder. Both crude venom and ASV cause 50% hemolysis at a concentration of 20 mug/mL. The highest hemolytic activity of ASV was measured at pH 7.0-7.4; ASV-dependent hemolysis was sharply reduced when the pH was lower than 3 or greater than 8. There was almost no hemolytic activity when the Cu2+ concentration was increased to 10 mM. Furthermore, incubation at 100[degree sign]C for 30 to 60 minutes sharply decreased the hemolytic activity of ASV. After treatment with the protease alpha-chymotrypsin, the glycoside hydrolase cellulase, and the membrane component cholesterin, the hemolytic activity of ASV was significantly inhibited. The results of this study provide fundamental information about A. planci spine venom. The hemolytic activity was affected by pH, temperature, metal ions, EDTA, cholesterin, proteases, and glycoside hydrolases. ASV hemolysis was inhibited by Cu2+, cholesterin, alpha-chymotrypsin, and cellulose, factors that might prevent the hemolytic activity of venom and provide the medical treatment for sting.
Growth inhibition and cell death were determined via MTT assay. (A) Dose- and time-dependent effect of BV and (B) dose effect of BV/Pd (II) complex on viability of MOLT-4 cells. Results are presented as mean ± S.E.M. n = 3; ** p > 0.01 and *** p > 0.001 significantly different from the control.
Effect of BV and BV/Pd (II) complex on the morphology of MOLT-4 cells. Photomicrographs from inverted microscope. Condensed nuclei obviously indicate apoptosis. (A) Controls, (B) cell treated with BV, (C) cells treated with BV/Pd (II) complex.
Characterization of BV and BV/Pd (II) complex-induced apoptosis in MOLT-4 cells by flow cytometry. Cells were cultured (A) without any component (control), (B) with BV and (C) with BV/Pd (II) complex for 24 hours. Note that 32.30% of the cells exposed to Cc50 value of these two components simultaneously [1 μg/mL BV/0.85 μM Pd (II) complex] enter early apoptosis stage.
Results of caspase-3 enzyme activity assay. Cells treated (A) with BV and (B) with BV/Pd (II) complex. The optical density was measured at 405 nm. The OD values were not altered by increasing BV, while the OD values rose following a dose increase from 1/2Cc50 BV/Pd (II) complex [0.5 μg/mL BV/0.425 μM Pd (II) complex] to Cc50 BV/Pd (II) complex [1 μg/mL BV/0.85 μM Pd (II) complex] compared to the control. Data represent mean ± SEM of three different experiments. n = 3; *** p > 0.001 significantly different from the control.
Although honeybee venom (BV) has been reported to induce apoptosis in different types of cancerous cells, its synergistic effects with customary anti-cancer drugs remain largely unknown. In the present study, we evaluated the cytotoxic effect of BV alone (as a natural product) and the synergistic cytological effects of this component in combination with [Pd (bpy) (Pi-Pydtc)]NO3 -- a novel palladium complex on human T-cell lymphoblastic leukemia cells. To investigate the cytotoxic effect of the BV alone and in combination with palladium complex on MOLT-4 cells MTT assay was performed. In order to determine the apoptotic effects of BV separately and in combination with Pd (II) complex on these cells and its ability to induce apoptosis, morphological examination, flowcytometric analysis and caspase-3 colorimetric assay were done. We found that BV induced morphological changes, namely nuclear shrinkage, and inhibited MOLT-4 cell proliferation; both effects were dose- and time-dependent. Flow cytometry by Annexin-V antibody demonstrated that BV induced apoptosis in MOLT-4 cells. Furthermore, BV induced apoptosis independently of caspase-3 in these cells. In addition, we proved a clear synergistic effect of BV on [Pd (bpy) (Pi-Pydtc)]NO3. The apoptotic pathway activated by BV in combination with Pd complex was caspase-3-dependent. These observations provide an explanation for the anti-proliferative properties of BV, and suggest that this agent may be useful for treating lymphoblastic leukemia alone or in combination with chemotherapy drugs pending further investigations on animal models as preclinical tests.
Venom bioactivity at different days post-collection differed statistically (p < 0.05) in four-week-old Balb/c mice among treatments despite a lower bioactivity tendency found in venom 8 days post-collection.
Morphology of HeLa cells 24 hours after incubation with MFS: only one dose is shown since no differences were observed among different doses. (A) Venom dose of 400 μg/100 μL and (B) untreated control cells.
MTS metabolism in Hela cells after treatment with different doses of venom (50-400 μg/100 μL of medium), positive control (CFF, cyclophosphamide 400 μg/100 μL) and negative control (CTRL–). Optical density (OD) was read at 490 nm, the data were plotted according to the mean and SD. Treatment means differed significantly with respect to the positive control but not for the negative control (p < 0.05).
MTS metabolism by macrophages and viability percentage showed significant difference between treatment means at p < 0.05.
Images showing Hela cells after treatment with scorpion venom. (A) Cells treated with scorpion venom up to 400 μg/100 μL, and (B) positive controls showing apoptotic cells treated with cyclophosphamide (400 μg/100 μL).
The venom of Centruroides limpidus limpidus (Cll) is a mixture of pharmacologically active principles. The most important of these are toxic proteins that interact both selectively and specifically with different cellular targets such as ion channels. Recently, anticancer properties of the venom from other scorpion species have been described. Studies in vitro have shown that scorpion venom induces cell death, inhibits proliferation and triggers the apoptotic pathway in different cancer cell lines. Herein, after treating human cervical adenocarcinoma (HeLa) cells with Cll crude venom, their cytotoxic activity and apoptosis induction were assessed. Cll crude venom induced cell death in normal macrophages in a dose-dependent manner. However, through viability assays, HeLa cells showed high survival rates after exposure to Cll venom. Also, Cll venom did not induce apoptosis after performing ethidium bromide/acridine orange assays, nor was there any evidence of chromatin condensation or DNA fragmentation. Crude Cll venom exposure was not detrimental to HeLa cell cultures. This may be partially attributable to the absence of specific HeLa cell membrane targets for molecules present in the venom of Centruroides limpidus limpidus. Although these results might discourage additional studies exploring the potential of Cll venom to treat human papilloma cervical cancer, further research is required to explore positive effects of crude Cll venom on other cancer cell lines.
In Guinea Elapids are responsible for 20% of envenomations. The associated case fatality rate (CFR) ranged 15-27%, irrespective of treatment. We studied 77 neurotoxic envenomations divided in 3 groups: a set of patients that received only traditional or symptomatic treatments, and two other groups that received either 2 or 4 initial vials of Antivipmyn® Africa renewed as necessary. CFR was 27.3%, 15.4% and 17.6%, respectively. Although antivenom treatment was likely to reduce CFR, it didn't seem to have an obvious clinical benefit for the patients, suggesting a low treatment efficacy. Mean delay to treatment or clinical stages were not significantly different between the patients who recovered and the patients who died, or between groups. Interpretation of these results is complicated by the lack of systematic studies under comparable conditions. Of particular importance is the absence of assisted ventilation, available to patients in all the other clinical studies of neurotoxic envenomation. The apparent lack of clinical benefit may have several causes. The hypothesis of a limited therapeutic window, i.e. an insufficient formation of antigen-antibody complexes once toxins are bound to their targets and/or distributed beyond the reach of antivenom, should be explored.
The tremendous outbreak of Ebola virus disease occurring in West Africa since the end of 2013 surprises by its remoteness from previous epidemics and dramatic extent. This review aims to describe the 27 manifestations of Ebola virus that arose after its discovery in 1976. It provides an update on research on the ecology of Ebola viruses, modes of contamination and human transmission of the disease that are mainly linked to close contact with an infected animal or a patient suffering from the disease. The recommendations to contain the epidemic and challenges to achieve it are reminded.
Carpet viper, African spitting cobra and puff adder.
Hub-and-spoke model for snakebite care. Full meaning of acronyms: ADR: Adverse Drug Reactions; ASV: Anti-Snake Serum AntiVenom; CHC: Comprehensive Health Centre; CHEW: Community Health Extension Worker; MD: Medical Doctors; PCV: Packed Cell Volume; QA-QC: Quality Assurance Quality Control; TOT: Training of Trainers; 20WBCT: 20 minute Whole Blood Clotting Test.
Snakebite envenoming is a major public health problem among rural communities of the Nigerian savanna. The saw-scaled or carpet viper (Echis ocellatus) and, to a lesser extent, the African cobras (Naja spp.) and puff adders (Bitis arietans) have proved to be the most important cause of mortality and morbidity. The main clinical features of E. ocellatus envenoming are systemic hemorrhage, incoagulable blood, shock, local swelling, bleeding and, occasionally, necrosis. Bites may be complicated by amputation, blindness, disability, disfigurement, mutilation, tissue destruction and psychological consequences. Antivenom remains the hallmark and mainstay of envenoming management while studies in Nigeria confirm its protection of over 80% against mortality from carpet-viper bites. However, the availability, distribution and utilization of antivenom remain challenging although two new antivenoms (monospecific EchiTab G and trispecific EchiTab ICP-Plus) derived from Nigerian snake venoms have proven very effective and safe in clinical trials. A hub-and-spoke strategy is suggested for broadening antivenom access to endemic rural areas together with instituting quality assurance, standardization and manpower training. With the advent of antivenomics, national health authorities must be aided in selecting and purchasing antivenoms appropriate to their national needs while manufacturers should be helped in practical ways to improve the safety, efficacy and potential coverage against snake venoms and pricing of their products.
Fractionation of AmV. (A) Chromatography of whole protein extract from honeybee venom using a molecular exclusion column packed with Superdex®75. The chromatographic run was carried out at a flow rate of 0.6 mL/h and monitored at 280 nm. (B) Reverse phase HPLC run yielded five main fractions designated from VI-1 to VI-5. Fraction VI-3 was confirmed as PLA2 by specific phospholipase A2 assay and fraction VI-4 as melittin after MALDI-TOFF analysis. (C) SDS-PAGE analysis of the purified proteins.
Characterization of the vasoconstrictor effect of AmV. (A) Vasoconstrictor effect of AmV (0.1-50 μg/mL) (●, n = 5) and the fractions: melittin (■, n = 5); PLA2 (Ο, n = 5); and the complex PLA2 + melittin (□, n = 5). (B) Effect of endothelium removal on AmV vasoconstrictor effect. The concentration response curve of AmV (0.1-50 μg/mL) on basal tone in endothelium-containing aorta preparations (●, n = 5), and in endothelium-denuded aorta preparations (Ο, n = 4). Vasoconstrictor effects are expressed as a percentage of the contractile response to K+ (60 mM). Data are expressed as mean ± SEM and analyzed by ANOVA followed by the Holm-Sidak post hoc test.
Study on the probable action mode of AmV. Vasoconstrictor effects of AmV: (A) Ca2+-free medium (O, n = 5); (B) pretreatment with phentolamine (5 μM; □, n = 5); (C) pretreatment with verapamil (10 μM; Δ, n = 5). Vasoconstrictor effects are expressed as a percentage of the contractile response to K+ (60 mM). Data are expressed as mean ± SEM and analyzed by ANOVA followed by the Holm-Sidak posttest.
Study on the probable action mode of AmV. Vasoconstrictor effects of AmV: (A) pretreatment with losartan (100 μM; O, n = 5); (B) pretreatment with U-73122 (10 μM; □, n = 5). Vasoconstrictor effects are expressed as a percentage of the contractile response to K+ (60 mM). Data are expressed as mean ± SEM and analyzed by ANOVA followed by the Holm-Sidak posttest.
Apis mellifera stings are a problem for public health worldwide, particularly in Latin America due to the aggressiveness of its Africanized honeybees. Massive poisoning by A. mellifera venom (AmV) affects mainly the cardiovascular system, and several works have described its actions on heart muscle. Nevertheless, no work on the pharmacological action mechanisms of the AmV in isolated aorta has been reported. Thus, the present work aimed to investigate the actions of AmV and its main fractions, phospholipase A2 (PLA2) and melittin, on isolated aorta rings and a probable action mechanism. AmV and the complex PLA2 + melittin (0.1-50 mug/mL) caused contraction in endothelium-containing aorta rings, but neither isolated PLA2 nor melittin were able to reproduce the effect. Endothelium removal did not change the maximum vasoconstrictor effect elicited by AmV. Ca2+-free medium, as well as treatment with phentolamine (5 muM), verapamil (10 muM), losartan (100 muM), and U-73122 (10 muM, a phospholipase C inhibitor), eliminated the AmV-induced contractile effects. In conclusion, AmV caused contractile effect in aorta rings probably through the involvement of voltage-operated calcium channels, AT1 and alpha-adrenergic receptors via the downstream activation of phospholipase C. The protein complex, PLA2 + melittin, was also able to induce vasoconstriction, whereas the isolated proteins were not.
Crossbred cow 17 days after the bee swarm attack. Observe the necrosis and detachment of the skin on the dewlap and lower portion of the chest.
Crossbred cow 17 days after the attack swarm of bees. Observe the necrosis and detachment of skin on the hind limbs.
Crossbred cow 45 days after the bee swarm attack. Observe the healed lesions on the dewlap and lower portion of the chest.
Cicatricial retraction of the ear, popularly known in Brazil as “cartridge-shaped ears” (calf).
We report three cases of stings by Africanized bees in cattle in the state of Rio de Janeiro, Brazil. Erythema, subcutaneous edema, necrosis accompanied by skin detachment, and subsequent skin regeneration were observed, especially on the head and dewlap. Histopathological examinations performed 45 days later revealed complete skin reepithelialization with moderate dermal fibrosis. The clinical picture and differential diagnosis are discussed in the present manuscript, with a focus on photosensitization, which causes cutaneous lesions on the head (sequela) with cicatricial curving of the ears and can be very similar to what is observed in cattle attacked by swarms of bees. The distinction between photosensitization and bee sting lesions can be made with a focus on history and clinical and pathological aspects.
Evolution of biochemical and serological tests before and after treatment.
Hepatitis B virus (HBV) infects from 6 to 14% of HIV-infected individuals. Concurrent HIV/HBV infection occurs due to the overlapping routes of transmission, particularly sexual and parenteral. HIV-infected patients that have acute hepatitis B have six times greater risk of developing chronic hepatitis B, with higher viral replication, rapid progression to end-stage liver disease and shorter survival. The coinfection is also associated with poor response to hepatitis B treatment with interferon-alpha and increased liver toxicity to the antiretroviral therapy. Herein, we describe the case of a 35-year-old man who engages in sex with men and presented with newly diagnosed HIV-1, serological markers for acute hepatitis B and progression to chronic hepatitis B infection (HBsAg+ > 6 months, high alanine aminotransferase levels and moderate hepatitis as indicated by liver biopsy). Lacking indication of antiretroviral treatment (CD4 768 cells/mm3), he was treated with pegylated-interferon alpha2b (1.5 mg/kg/week) by subcutaneous injection for 48 weeks. Twelve weeks after treatment, the patient presented HBeAg seroconversion to anti-HBe. At the end of 48 weeks, he presented HBsAg seroconversion to anti-HBs. One year after treatment, the patient maintained sustained virological response (undetectable HBV-DNA). The initiation of antiretroviral therapy with nucleosides and nucleotides is recommended earlier for coinfected individuals. However, this report emphasizes that pegylated interferon remains an important therapeutic strategy to be considered for selected patients, in whom the initiation of HAART may be delayed.
of hemagglutination-inhibition antibodies against antigens of six flaviviruses in serum samples of water buffaloes from different regions of Pará state, Brazilian Amazon 
The state of Para encompasses 26% of Brazilian Amazon where an enormous diversity of arboviruses has been found. This study aimed to assess the prevalence and distribution of hemagglutination-inhibition antibodies against antigens of six Flavivirus (yellow fever virus, Ilheus virus, Saint Louis encephalitis virus, Cacipacore virus, Bussuquara virus and Rocio virus) in water buffaloes in Para state, Brazil. The prevalence of antibodies in these farm animals is important to determine the circulating arboviruses. All investigated arboviruses were detected in the species studied and our results indicate that water buffaloes are susceptible to Flavivirus infection. Furthermore, there is solid evidence of active circulation of these viruses in the Brazilian Amazon. Water buffaloes showed higher prevalence of heterotypic antibody reactions and we hypothesized that they can serve as sentinels to detect the movement of such arboviruses in the Brazilian Amazon.
Estimated absolute number of snakebite cases in the various Central American countries. These numbers are based on hospital data. Incidences are presented in Table 1.
Bothrops asper (family Viperidae) is the most important snake from the medical standpoint in Central America. It is distributed in the humid tropical lowlands and provokes the majority of cases, and the most serious ones. It is also responsible for the majority of fatal cases in the region. This snake adapts very well to altered environments, such as agricultural fields and pastures. Photo by Mahmood Sasa and reprinted from “Confronting the neglected problem of snake bite envenoming: the need for a global partnership” by Gutiérrez et al., PLOS Medicine, 2006, 3(6), e150 [7]. Creative Commons Attribution License (CCAL).
Incidence of snakebite in Costa Rica per district per 100,000 population (1990-2007). Prepared by Erik Hansson and reprinted from “Using geographical information systems to identify populations in need of improved accessibility to antivenom treatment for snakebite envenoming in Costa Rica” by Hansson et al., PLOS Neglected Tropical Diseases, 2013, 7 (1), e2009 [37]. Creative Commons Attribution License (CCAL).
Distribution of hospitals, clinics and ambulance stations in Costa Rica, and estimated time to reach hospitals or clinics in various regions of the country. As shown in Figure 3, regions where transportation to health facilities takes longer correspond to regions of high snakebite incidence. Prepared by Erik Hansson and reprinted from Hansson et al.[37].
Cover of a publication used in education campaigns to promote the prevention of snakebites in indigenous communities of the Cabécar ethnic group in Costa Rica. This material was prepared in the Spanish and Cabécar languages by a cooperative project coordinated by Dr. Laura Monturiol (Instituto Clodomiro Picado) and developed by Instituto Clodomiro Picado (Universidad de Costa Rica), Caja Costarricense del Seguro Social, the Ministry of Education, and the Cabécar people. It was widely distributed in the Cabécar communities and used in local schools as part of a prevention campaign [42]. Serpientes de Costa Rica y prevención de mordeduras: Guía de información, 2009, Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica. Reprinted approved by the General Director of the Institute.
Snakebite envenoming is a serious public health problem in Central America, where approximately 5,500 cases occur every year. Panama has the highest incidence and El Salvador the lowest. The majority, and most severe, cases are inflicted by the pit viper Bothrops asper (family Viperidae), locally known as 'terciopelo', 'barba amarilla' or 'equis'. About 1% of the bites are caused by coral snakes of the genus Micrurus (family Elapidae). Despite significant and successful efforts in Central America regarding snakebite envenomings in the areas of research, antivenom manufacture and quality control, training of health professionals in the diagnosis and clinical management of bites, and prevention of snakebites, much remains to be done in order to further reduce the impact of this medical condition. This essay presents seven challenges for improving the confrontation of snakebite envenoming in Central America. Overcoming these challenges demands a coordinated partnership of highly diverse stakeholders though inter-sectorial and inter-programmatic interventions.
This subadult slow loris bit the victim’s finger intensely resulting in a severe wound. (Panel A) Subadult Nycticebus kayan before the victim handled it – already a large drop of saliva can be seen protruding from the animal’s mouth. (Panel B) The bite site 12 days after the bite. (Photos by G. Madani).
After the bite, the patient showed extreme swelling, worsening over time. (Panel A) 33 minutes after the bite; (Panel B) 54 minutes after the bite; (Panel C) one hour and 39 minutes after the bite; and (Panel D) one week after the bite (photos by G. Madani).
Background Asian slow lorises (Nycticebus spp.) are one of few known venomous mammals, yet until now only one published case report has documented the impact of their venomous bite on humans. We describe the reaction of a patient to the bite of a subadult Nycticebus kayan, which occurred in the Mulu District of Sarawak in 2012. Findings Within minutes of the bite, the patient experienced paraesthesia in the right side of the jaw, ear and right foot. By 40 minutes, swelling of the face was pronounced. The patient was admitted to Mulu National Park Health Clinic/Klinik Kesihatan Taman Mulu Tarikh, at which time he was experiencing: swollen mouth, chest pain, mild abdominal pain, nausea, numbness of the lips and mouth, shortness of breath, weakness, agitation and the sensation of pressure in the ears due to swelling. The blood pressure was 110/76, the heart ratio was 116 and oxygen saturation was 96%. The patient was treated intramuscularly with adrenaline (0.5 mL), followed by intravenous injection of hydrocortisone (400 mg) and then intravenous fluid therapy of normal saline (500 mg). By 8 h10 the next day, the patient’s condition had significantly improved with no nausea, and with blood pressure and pulse rate stable. Conclusions A handful of anecdotes further support the real danger that slow loris bites pose to humans. As the illegal pet trade is a major factor in the decline of these threatened species, we hope that by reporting on the danger of handling these animals it may help to reduce their desirability as a pet.
Chromatographic profile of hexane and ethyl acetate unified fractions obtained by RP-UPLC-PDA in a C18 column (ACE C18, 5 μm, 100 Å, 250 mm × 4.6 mm) at a constant flow rate of 1 mL/min−1 using water as solvent A and MeOH as solvent B. The chromatogram was obtained at wavelengths of 214 and 254 nm.
Antiparasitic activity and cytotoxicity of SaFr1, the viability was determined by the colorimetric assay of MTT at 570 nm
Evaluation of ultrastructural damage by transmission electron microscopy (TEM). Promastigotes of L. (L.) infantum were incubated with SaFr1 at different times: (A) control (untreated); (B) one-hour incubation; (C) two-hour incubation; (D) and (E) four-hour incubation and (F) 16-hour incubation. m: mitochondrial damage; v: vacuole; pm: plasma membrane integrity. Bar represents (A) 0.5 μm; (B) 1 μm; (C) 0.1 μm; (D) 0.5 μm; (E) 100 nm; (F) 100 nm.
Evaluation of the plasma membrane permeability of L. (L.) infantum incubated with SaFr1 by SYTOX Green®. The fluorescence intensity was determined using a fluorimetric microplate reader with excitation and emission wavelengths of 485 and 520 nm, respectively. Triton X-100 was used as the positive control of the assay.
Among the tropical parasitic diseases, those caused by protozoans are considered a challenge to public health, being represented by leishmaniasis and Chagas disease. In view of the low effectiveness and toxicity of the current therapy, animal venoms such as amphibian secretions have been used as a promising source of new drug prototypes. The present work aimed to achieve bioguided fractionation of metabolites present in a cutaneous secretion of the caecilian Siphonops annulatus (Amphibia: Gymnophiona: Siphonopidae) with antileishmanial and antitrypanosomal activity. Through liquid-liquid partition and chromatographic techniques, the secretion was fractionated using bioguided assays. The 50% inhibitory concentration (IC50) of the main fraction (SaFr1) was studied against Leishmania (L.) infantum promastigotes and intracellular amastigotes, trypomastigotes of Trypanosoma cruzi and mammalian cells; viability was detected by the colorimetric MTT assay. By using a spectrofluorimetric assay with the probe SYTOX® Green and transmission electron microscopy (TEM), we also investigated the potential damage caused by SaFr1 in the plasma membrane and mitochondria of Leishmania. The bioguided assay enabled isolation of a highly purified fraction (SaFr1) with an IC50 of 0.065 μg/mL against promastigotes and 2.75 μg/mL against trypomastigotes. Due to its high toxicity to peritoneal macrophages, SaFr1 showed no selectivity towards the intracellular forms of Leishmania. Ultrastructural studies with Leishmania demonstrated severe mitochondrial damage and the formation of large cytoplasmic vacuoles, leading to the parasite's death within a few hours. Nevertheless, it caused no alteration in the plasma membrane permeability as detected by the fluorescent probe and TEM. The present study demonstrated for the first time the antiparasitic activity of the skin secretion of the caecilian S. annulatus against Leishmania and T. cruzi, confirming that skin secretions of these amphibians, similarly to those of anurans and salamanders, are also potential tools for the development of new drug candidates against neglected diseases.
Total distribution of Helicobacter pylori in various types of milk and dairy products 
Distribution of putative virulence factors in Helicobacter pylori strains isolated from various types of milk and dairy products 
Despite the high importance of Helicobacter pylori, the origin and transmission of this bacterium has not been clearly determined. According to controversial theories and results of previous studies, animal source foods - especially milk - play an important role in the transmission of H. pylori to humans. The aim of the present study was to determine the distribution of vacA, cagA, iceA and oipA virulence factors in H. pylori strains isolated from milk and dairy products and study their antimicrobial resistance properties. A total of 520 raw milk and 400 traditional dairy product samples were cultured and tested. Those that were H. pylori-positive were analyzed for the presence of vacA, cagA, iceA and oipA virulence factors. Antimicrobial susceptibility testing was performed by the disk diffusion method. One hundred and three out of 520 milk samples (19.8%) and 77 out of 400 dairy products samples (19.2%) were contaminated with H. pylori. The most frequently contaminated samples were ovine milk (35%) and traditional cheese (30%). Total prevalence of vacA, cagA, iceA and oipA factors were 75%, 76.6%, 41.6% and 25%, respectively. H. pylori strains of milk and dairy products harbored high levels of resistance to ampicillin (84.4%), tetracycline (76.6%), erythromycin (70.5%) and metronidazole (70%). High presence of antibiotic-resistant strains of H. pylori suggest that milk and dairy samples may be the sources of bacteria that can cause severe infection. Our findings should raise awareness about antibiotic resistance in H. pylori strains in Iran.
Estimated percentage of animals with diagnosis for Leishmania spp. by location and species 
Blood cultures and IFAT according to PCR for Leishmania spp 
This study aimed to evaluate the occurrence of Leishmania spp. in dogs and cats from Botucatu, Sao Paulo state, and Campo Grande, Mato Grosso do Sul state, Brazil, by the association of three diagnostic tests: blood culture in liver infusion tryptose medium, immunofluorescent antibody test and polymerase chain reaction. Fifty blood samples of dogs and cats from the Center for Zoonosis Control in Campo Grande, an area endemic for canine visceral leishmaniasis, were collected randomly, as well as canine and feline blood samples from the Municipal Kennel and Animal Protection Association in Botucatu, currently considered a transmission-free, non-endemic area. Of the 50 dog blood cultures from Botucatu, three (6%) were positive and of the 50 cats, two (4%) were positive. In Campo Grande, 29 dog blood cultures (58%) were positive and all (100%) cats negative by this test. Polymerase chain reaction detected Leishmania spp. in 100% of dog and cat samples from Botucatu but found all the cats from Campo Grande to be negative. On the other hand, 36 dogs from Campo Grande were positive (72%) by the same technique. Immunofluorescent antibody test in Botucatu found 100% of dogs and cats non-reactive, while in Campo Grande, it detected positivity in 32 dogs (64%) and 15 cats (30%). The results show the importance of not only continuous epidemiological surveillance in areas not endemic for leishmaniasis, but also research for accurate diagnosis of this zoonosis.
General structure of microcystins (LR) with leucine (L) in the amino acid position 2 and arginine (R) in the amino acid position 4 and the structural differences in the position 1 of MC-LR (left) and [D-Leu 1 ] MC-LR (right).
Number of HTC viable cell by trypan blue exclusion test 24 hours after exposure to different concentrations of Microcystis aeruginosa strain 9501 (578.15 to 93.36 μM) which produces [D-Leu 1 ] MC-LR. Data are expressed as mean + standard error. *Indicates significant difference from the control (p < 0.05).
Minimum inhibitory concentration (MIC) of the microcystin variant [D-Leu 1 ] MC-LR from extracts of M. aeruginosa RST 9501 and MC-LR (commercially available) against M. tuberculosis strains
The present work aimed to evaluate the antimycobacterial activity and cytotoxicity of Microcystis aeruginosa toxins, the MC-LR variant and purified extract of [D-Leu(1)] microcystin-LR. 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. 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-Leu(1)] 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. These preliminary results suggest that [D-Leu(1)] MC-LR is a promising candidate for the development of a new antimycobacterial agent.
Snakebites are considered a neglected tropical disease that affects thousands of people worldwide. Although antivenom is the only treatment available, it is associated with several side effects. As an alternative, plants have been extensively studied in order to obtain an alternative treatment. In folk medicine, Azima tetracantha Lam. is usually used to treat snakebites. The present study aims to provide a scientific explanation for the use of this plant against snakebite. The extracts of shade dried leaves of A. tetracantha were tested for in vitro inhibitory activity on toxic venom enzymes like phosphomonoesterase, phosphodiesterase, acetylcholinesterase, hyaluronidase etc. from Bungarus caeruleus and Vipera russelli venoms. The ethylacetate extract rendered a significant inhibitory effect on the phosphomonoesterase, phosphodiesterase, phospholipase A2 andacetylcholinesterase enzymes. The present study suggests that ethylacetate extract of A. tetracantha leaves possesses compounds that inhibit the activity of toxic enzymes from Bungarus caeruleus and Vipera russelli venom. Further pharmacological in vivo studies would provide evidence that this substance may be lead to a potential treatment against these venoms.
Millepora complanata is a plate-like fire coral common throughout the Caribbean. Contact with this species usually provokes burning pain, erythema and urticariform lesions. Our previous study suggested that the aqueous extract of M. complanata contains non-protein hemolysins that are soluble in water and ethanol. In general, the local damage induced by cnidarian venoms has been associated with hemolysins. The characterization of the effects of these components is important for the understanding of the defense mechanisms of fire corals. In addition, this information could lead to better care for victims of envenomation accidents. An ethanolic extract from the lyophilized aqueous extract was prepared and its hemolytic activity was compared with the hemolysis induced by the denatured aqueous extract. Based on the finding that ethanol failed to induce nematocyst discharge, ethanolic extracts were prepared from artificially bleached and normal M. complanata fragments and their hemolytic activity was tested in order to obtain information about the source of the heat-stable hemolysins. Rodent erythrocytes were more susceptible to the aqueous extract than chicken and human erythrocytes. Hemolytic activity started at ten minutes of incubation and was relatively stable within the range of 28-50°C. When the aqueous extract was preincubated at temperatures over 60°C, hemolytic activity was significantly reduced. The denatured extract induced a slow hemolytic activity (HU50 = 1,050.00 ± 45.85 μg/mL), detectable four hours after incubation, which was similar to that induced by the ethanolic extract prepared from the aqueous extract (HU50 = 1,167.00 ± 54.95 μg/mL). No significant differences were observed between hemolysis induced by ethanolic extracts from bleached and normal fragments, although both activities were more potent than hemolysis induced by the denatured extract. The results showed that the aqueous extract of M. complanata possesses one or more powerful heat-labile hemolytic proteins that are slightly more resistant to temperature than jellyfish venoms. This extract also contains slow thermostable hemolysins highly soluble in ethanol that are probably derived from the body tissues of the hydrozoan.
Venom hyaluronidase (Hyase) contributes to the diffusion of venom from the inoculation site. In this work, we purified and characterized Hyase from the venom of Vitalius dubius (Araneae, Theraphosidae), a large theraphosid found in southeastern Brazil. Venom obtained by electrical stimulation of adult male and female V. dubius was initially fractionated by gel filtration on a Superdex® 75 column. Active fractions were pooled and applied to a heparin-sepharose affinity column. The proteins were eluted with a linear NaCl gradient. Active fractions were pooled and assessed for purity by SDS-PAGE and RP-HPLC. The physicochemical tests included optimum pH, heat stability, presence of isoforms, neutralization by flavonoids and assessment of commercial antivenoms. Hyase was purified and presented a specific activity of 148 turbidity-reducing units (TRU)/mg (venom: 36 TRU/mg; purification factor of ~4). Hyase displayed a molecular mass of 43 kDa by SDS-PAGE. Zymography in hyaluronic-acid-containing gels indicated an absence of enzyme isoforms. The optimum pH was 4-5, with highest activity at 37°C. Hyase was stable up to 60°C; but its activity was lost at higher temperatures and maintained after several freeze-thaw cycles. The NaCl concentration (up to 1 M) did not influence activity. Hyase had greater action towards hyaluronic acid compared to chondroitin sulfate, and was completely neutralized by polyvalent antiarachnid sera, but not by caterpillar, scorpion or snakes antivenoms. The neutralization by arachnid but not scorpion antivenom indicates that this enzyme shares antigenic epitopes with similar enzymes in other spider venoms. The biochemical properties of this Hyase are comparable to others described.
Clinical score 2 and corresponding histopathological analyses in a hind paw from a C57BL/6 mouse infected with enterotoxin C producer S. aureus strain. (a’) normal mouse; (b’) mouse with arthritis. (c’, d’) Histopathological micrographics are shown with 10x magnification. JC: joint cavity; C: cartilage; B: bone; BE: bone erosion; CE: cartilage erosion; P: pannus formation and IC: inflammatory cells.
Experimental treatments in septic arthritis
Schematic outline of the parameters that are most frequently analyzed in the murine SA experimental model.
Septic arthritis is an inflammatory disease of the joints that is started by an infection whose most common agent is Staphylococcus aureus. In this review we discuss some of the most arthritogenic bacterial factors and the contribution of innate and specific immune mechanisms to joint destruction. Special emphasis is given to the induction of experimental arthritis by S. aureus in mice. The improvement of therapy by association of antibiotics with down-modulation of immunity is also included.
Conus vexillum venom (1.25 mg/kg) collected from Hurgada and Sharm El-Shaikh induced intracellular oxidative stress in EAC cells assessed by the level of (A) lipid peroxidation, (B) protein carbonyl content and (C) level of nitric oxide.*Significant difference between control (EAC control group) and treated group using Student’s unpaired t-test (p < 0.05). #Represents a significant difference between Hurgada and Sharm El-Shaikh groups using Student’s unpaired t-test (p < 0.05). £Significant difference between treated groups using one-way ANOVA (p < 0.05).
Changes in the antioxidant measurements GSH (panel A), CAT (panel B), Cu/Zn SOD (panel C) and TAC (panel D) of EAC cells after administration of Conus vexillum venom (1.25 mg/kg) collected from Hurgada and Sharm El-Shaikh.
It is estimated that venoms of marine cone snails (genus Conus) contain more than 100,000 different small peptides with a wide range of pharmacological and biological actions. Some of these peptides were developed into potential therapeutic agents and as molecular tools to understand biological functions of nervous and cardiovascular systems. In this study we examined the cytotoxic and anticancer properties of the marine vermivorous cone snail Conus vexillum (collected from Hurgada and Sharm El-Shaikh, Red Sea, Egypt) and suggest the possible mechanisms involved. The in vitro cytotoxic effects of Conus venom were assessed against Ehrlich's ascites carcinoma (EAC) cells. Conus venom treatment resulted in concentration-dependent cytotoxicity as indicated by a lactate dehydrogenase leakage assay. Apoptotic effects were measured in vivo by measuring levels of reactive oxygen species and oxidative defense agents in albino mice injected with EAC cells. Conus venom (1.25 mg/kg) induced a significant increase (p < 0.05) in several oxidative stress biomarkers (lipid peroxidation, protein carbonyl content and reactive nitrogen intermediates) of EAC cells after 3, 6, 9 and 12 hours of venom injection. Conus venom significantly reduced (p < 0.05) the activities of oxidative defense enzymes (catalase and superoxide dismutase) as well as the total antioxidant capacity of EAC cells, as evidenced by lowered levels of reduced glutathione. These results demonstrate the cytotoxic potential of C. vexillum venom by inducing oxidative stress mediated mechanisms in tumor cells and suggest that the venom contains novel molecules with potential anticancer activity.
Stingrays are a group of rays -- cartilaginous fish related to sharks -- that have whiplike tails with barbed, usually venomous spines and are found around the world, especially the marine species. Despite recent reports of accidents involving these fish, they are not aggressive, reacting only when stepped on or improperly handled. Injuries by stingrays are seldom mentioned by historians, although they have always been present in riverine communities of inland waters and in South American coasts. Indeed, envenomations by stingrays are quite common in freshwater and marine fishing communities. Although having high morbidity, such injuries are neglected because they have low lethality and usually occur in remote areas, which favor the use of folk remedies. In the present review article, historical aspects of injuries caused by stingrays in Brazil and their distribution on the coast of Sao Paulo state and riverine communities of the North, Midwest and Southeast regions were studied. In addition, other aspects were analyzed such as clinical features, therapeutic methods, preventive measures and trends in occurrence of these accidents in the country, particularly in areas in which freshwater stingrays had not been previously registered, being introduced after breaching of natural barriers.
Primers and condition of amplification used in the study 
Frequency of genes cagA and dupA and genotypes of vacA in H. pylori strains isolated from 105 adults and 100 children with gastric chronic and normal gastric mucosa 
Only a few Helicobacter pylori-infected individuals develop severe gastric diseases and virulence factors of H. pylori appear to be involved in such clinical outcomes. Duodenal ulcer promoting gene A (dupA) is a novel virulence factor of Helicobacter pylori that is associated with duodenal ulcer development and reduced risk for gastric carcinoma in some populations. The aims of the present study were to determine the presence of dupA gene and evaluate the association among dupA and other virulence factors including cagA and vacA in Brazilian patients. Gastric biopsies were obtained from 205 dyspeptic patients (100 children and 105 adults). DNA was extracted and analyzed for the presence of H. pylori and its virulence factors using the polymerase chain reaction method. Patients with gastritis tested positive for H. pylori more frequently. The dupA gene was detected in 41.5% of them (85/205); cagA gene was found in 98 isolates (47.8%) and vacA genotype s1/m1 in 50.2%, s1/m2 in 8.3%, s2/m2 in 36.6%, s2/m1 in 0.5% and s1/s2/m1/m2 in 4.4%. We also verified a significant association between cagA and dupA genes [p = 0.0003, relative risk (RR) 1.73 and confidence interval [CI] = 1.3-2.3]. The genotypes s1/m1 were also associated with dupA gene (p = 0.0001, RR: 1.72 and CI: 1.3-2.2). The same associations were found when analyzing pediatric and adult groups of patients individually. Ours results suggest that dupA is highly frequent in Brazilian patients and is associated with cagA gene and vacA s1/m1 genotype, and it may be considered an important virulence factor in the development of gastric diseases in adults or children.
Kinetic of cytokines release in sera following Aam or Aah injection. (A) IL-1β, (B) IL-6, (C) IL-10 *p < 0.05, **p < 0.01, ***p < 0.001, NS: not significant, compared to control.
Effect of Androctonus amoreuxi and Androctonus australis hector venoms on myeloperoxydase activity in the presence or absence of atropine (At) and propranolol (Pr). *p < 0.05, **p < 0.01, **p < 0.001.
Effects of Androctonus amoreuxi and Androctonus australis hector venoms on pulmonary parenchyma. (A1 and B1) Magnification of 400×, (A’1 and B’1) magnification of 100×, in the presence of atropine (A2, B2) or propranolol (A3, B3 and B’3), (C) control injected with saline. A: alveolus, E: edema, H: hemorrhage, IC: inflammatory cells, Hematoxylin-Eosin.
Previous works had shown that scorpion venom induced neurotransmitter elevation and an inflammatory response associated with various anatomo-pathological modifications. The most dangerous scorpions species in Algeria responsible for these effects are Androctonus australis hector (Aah) and Androctonus amoreuxi (Aam). Comparison of the physiopathological effects induced by the two venoms showed differences in the kinetic of cytokine release and in lung injury.The lung edema was only observed in response to Aah venom and it was correlated with cell infiltration. In order to better understand the involved mechanism in inflammatory response, we used two antagonists, atropine (non-selective muscarinic antagonist) and propranolol (β adrenergic antagonist), which lead to a decrease of cell infiltration but has no effect on edema forming. These results suggest another pathway in the development of lung injury following envenomation with Aam or Aah venom.
Numerous spider toxins are of interest as tools for neurophysiological research or as lead molecules for the development of pharmaceuticals and insecticides. Direct detection and identification of the interacting proteins of a spider toxin are helpful for its action-mechanism analysis and practical application. The present study employed a combinative strategy for the analysis of interacting proteins of huwentoxin-IV (HWTX-IV), a peptidic neurotoxin from the venom of the spider Selenocosmia huwena. HWTX-IV was first lightly labeled with biotin under the optimized mild experimental conditions and the toxin labeled with a single biotin group (monobiotinylated HWTX-IV) was demonstrated by electrophysiological experiments to retain its original bioactivity and was used in combination with far-western blotting to detect its interacting proteins. Comparative experiments indicated that some membrane proteins from rat neuromuscular junction preparations bind to monobiotinylated HWTX-IV after being transferred onto a PVDF membrane from the SDS-gel. With capillary high performance liquid chromatography-tandem mass spectrometry, several membrane proteins with which HWTX-IV potentially interacted were identified from the preparations and then bioinformatically analyzed. This work has provided not only a new insight into the action mechanism of HWTX-IV but also a reference technology for the relevant researches.
Background: Toxic cyanobacterial blooms are recurrent in Patos Lagoon, in southern Brazil. Among cyanotoxins, [D-Leu(1)] 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-Leu(1)] 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-Leu(1)] microcystin-LR was isolated and purified from batch cultures of Microcystis aeruginosa strain 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-Leu(1)] 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-Leu(1)] microcystin-LR. The phylogenetic analysis of the 16S rRNA gene from DMXS strain classified the organism as Pseudomonas aeruginosa. DMXS strain incubated with [D-Leu(1)] microcystin-LR lowered the amount of toxin from 1 μg.L(-1) to < 0.05 μg.L(-1). Besides, an increase in the bacterial count-from 71 × 10(5) bacteria.mL(-1) to 117 × 10(5) 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-Leu(1)] microcystin-LR, the most frequent microcystin variant in Brazilian freshwaters.
Only seven types of mammals are known to be venomous, including slow lorises (Nycticebus spp.). Despite the evolutionary significance of this unique adaptation amongst Nycticebus, the structure and function of slow loris venom is only just beginning to be understood. Here we review what is known about the chemical structure of slow loris venom. Research on a handful of captive samples from three of eight slow loris species reveals that the protein within slow loris venom resembles the disulphide-bridged heterodimeric structure of Fel-d1, more commonly known as cat allergen. In a comparison of N. pygmaeus and N. coucang, 212 and 68 compounds were found, respectively. Venom is activated by combining the oil from the brachial arm gland with saliva, and can cause death in small mammals and anaphylactic shock and death in humans. We examine four hypotheses for the function of slow loris venom. The least evidence is found for the hypothesis that loris venom evolved to kill prey. Although the venom's primary function in nature seems to be as a defense against parasites and conspecifics, it may also serve to thwart olfactory-orientated predators. Combined with numerous other serpentine features of slow lorises, including extra vertebra in the spine leading to snake-like movement, serpentine aggressive vocalisations, a long dark dorsal stripe and the venom itself, we propose that venom may have evolved to mimic cobras (Naja sp.). During the Miocene when both slow lorises and cobras migrated throughout Southeast Asia, the evolution of venom may have been an adaptive strategy against predators used by slow lorises as a form of Mullerian mimicry with spectacled cobras.
Body weight gain (in grams) of dams treated with subcutaneous injection of 1.46% (w/v) NaCl (control group) or 2.5 mg/kg Tityus bahiensis scorpion venom on the 5th (GD5) and/or 10th (GD10) gestational day. Values represent the mean ± SEM.
Photograph of skeletal alterations. (A) and (B) Deformity of the interparietal bone of the skull from GD10 group. (C) Longer fetuses from GD5 group (snout and head). (D) Detail of larger and malformed paw.
Scorpion envenoming is a public health problem in Brazil, where Tityus serrulatus and T. bahiensis are considered the most dangerous scorpions. They are well adapted to urbanized environments, and there is an increasing probability of human exposure to these venoms, including during pregnancy. Not much is known about the effects of prenatal exposure to the venom, and no information is available to aid in the rational treatment of victims stung during pregnancy. Thus, this study aimed to investigate whether venom from the scorpion T. bahiensis administered once to pregnant female rats at a dose that causes a moderate envenomation may lead to deleterious effects on the reproductive performance of the dams and on the development of their offspring. This is the first work demonstrating that T. bahiensis venom, when administered experimentally to rats, alters maternal reproductive performance and the morphological development of fetuses. The venom was given to dams on the 5th (GD5) or on the 10th (GD10) gestational day. After laparotomy, on GD21, fetuses and placentas were counted, weighed and externally analyzed. The corpora lutea were counted. The sex and vitality of fetuses were evaluated, and each litter was then randomly divided for visceral or skeletal analyses. Data were analyzed by ANOVA followed by the Tukey-Kramer test and Fisher's exact test. The significance level for all tests was set at p < 0.05. GD5 group presented an increased number of pre-implantation losses. Weight gains in fetuses and placentas were observed in the GD5 and GD10 groups. Weights of the heart and lungs were elevated in GD5 and GD10 and liver weight in GD10. Moderate envenomation by T. bahiensis scorpion venom alters maternal reproductive performance and fetal development. However, these are preliminary results whose causes should be investigated more carefully in future studies.
Polycystic ovarian syndrome (PCOS) is a low-grade inflammatory disease characterized by hyperandrogenemia, hirsutism, chronic anovulation and vascular disorder. Interleukin-6 (IL-6), cyclooxygenase-2 (COX-2) and vascular endothelial growth factor (VEGF) are triggered by inflammatory stimuli and lead to angiogenesis and pathogenesis of the ovary. Honeybee venom (HBV) contains an array of biologically active components possessing various pharmaceutical properties. This study was designed to assess the possibility of HBV application as an anti-inflammatory therapeutic agent to suppress levels of the main inflammatory mediators IL-6, COX-2 and VEGF.To induce PCOS, 1 mg of estradiol valerate (EV) per 100 g of body weight was subcutaneously (SC) injected into eight-week-old rats. After 60 days, 0.5 mg/kg of HBV was administered SC for 14 consecutive days, and the results of PCOS treatment were investigated. Rats were then anesthetized with chloroform, and the ovaries were surgically removed. Serum IL-6 was detected by the ELISA kit. Immunoexpression of COX-2 and VEGF were examined in three groups: EV-induced PCOS, HBV-treated PCOS and control animals. Thickness of theca layer, number and diameter of cysts and levels of IL-6 significantly decreased in HBV group relative to PCOS group. The immunohistochemical analysis showed an increase in COX-2 and VEGF expression in PCOS group whereas HBV-treated rats presented weak and irregular immunostaining. Our results suggest that the beneficial effect of HBV may be mediated through its inhibitory effect on serum IL-6 level and ovarian COX-2 and VEGF expression.
Monthly distribution of bee sting cases in Campina Grande, Paraíba state, between 2007 and 2012 (n = 459)
Descriptive analyses of bee sting cases in Campina Grande, Paraíba state, between 2007 and 2012, according to epidemiological variables (n = 459)
Studied area of bee sting cases in Paraíba state, Brazil.
The present study aims to investigate the clinical-epidemiological characteristics of bee sting cases recorded between 2007 and 2012 in the city of Campina Grande, Paraiba state, Brazil. Data were collected from the database of the Injury Notification Information System of the Brazilian Ministry of Health. A total of 459 bee sting cases were retrospectively analyzed. The average annual incidence was 19 cases per 100,000 inhabitants. Cases were distributed in all months of the year, with higher prevalence in September and February. Most victims were men aged between 20 and 29 years. The highest incidence of cases was recorded in urban areas. Victims were stung mainly on the head and torso and received medical assistance predominantly 1 to 3 hours after being stung. The most frequent clinical manifestations were pain, edema and itching. Most cases were classified as mild, and three deaths were reported. The high incidence of envenomations provoked by bees in Campina Grande suggests that it may be an important risk area for accidents. Since several medical records lacked information, clinical-epidemiological profile of bee sting cases in the studied region could not be accurately determined.The current study provides relevant data for the development of strategies to promote control and prevention of bee stings in this area. Further training for health professionals seems to be necessary to improve their skills in recording clinical-epidemiological information as well as in treating bee sting victims.
Biomolecules from Cerastes cerastes venom have been purified and characterized. Two phospholipases isolated from Cerastes cerastes venom share 51% of homology. CC2-PLA2 exhibits antiplatelet activity that blocks coagulation. CCSV-MPase, a non-hemorrhagic Zn2+-metalloproteinase, significantly reduced the plasmatic fibrinogen level and hydrolyzes only its Bβ chain. Serine proteinases such as RP34, afaâcytin and CC3-SPase hydrolyze the fibrinogen and are respectively α, αβ and αβ fibrinogenases. In deficient human plasma, afaâcytin replaces the missing factors VIII and IX, and activates purified human factor X into factor Xa. It releases serotonin from platelets and directly aggregates human (but not rabbit) blood platelets. RP34 proteinase also had no effect on both human and rabbit blood platelet aggregation. CC3-SPase revealed a pro-coagulant activity. However, the insolubility of the obtained clot indicates that CC3-SPase does not activate factor XIII. In addition, CC3-SPase clotting activity was carried out with human plasmas from volunteer patients deficient in clotting factors. Results showed that CC3-SPase shortens clotting time of plasma deficient in factors II and VII but with weaker clotting than normal plasma. The clotting time of plasma deficient in factor II is similar to that obtained with normal plasma; suggesting that CC3-SPase is able to replace both factors IIa and VII in the coagulation cascade and thus could be involved in the blood clotting process via an extrinsic pathway. These results imply that CC3-SPase and afaâcytin could repair hemostatic abnormalities and may replace some factors missing in pathological deficiency. Afaâcytin also exhibits α fibrinase property similar to a plasmin-like proteinase. Despite its thrombin-like characteristics, afaâcytin is not inhibited by plasmatic thrombin inhibitors. The procoagulant properties of afaâcytin might have potential clinical applications.
Total number of sandflies, by species and gender, captured with automatic light traps in areas of Atlantic Forest and cerrado, between September 2001 and January 2005, in Rubião Júnior, Botucatu municipality, São Paulo state, Brazil 
The study of the distribution and ecology of sandfly species is essential for epidemiological surveillance and estimation of the transmission risk of Leishmania spp. infection. In the present study, sandflies were captured in native fragmented forest areas in Rubião Júnior district, Botucatu municipality, São Paulo state, Brazil, between September 2001 and January 2005. A minimum of two automatic light traps were installed per night from 6 pm to 8 am, in different months, resulting in approximately 900 collecting hours. During this period, 216 sandfly specimens of sixteen species were captured. Pintomyia monticola and Brumptomyia guimaraesi were the most abundant with 56 specimens (25.93%) captured per species, followed by Pintomyia fischeri 28 (12.96%) and Psathyromyia pascalei 18 (8.33%). Other captured species were Lutzomyia amarali, Sciopemyia sordellii, Psathyromyia aragaoi, Nyssomyia whitmani, Migonemyia migonei, Pintomyia bianchigalatiae, Pintomyia misionensis, Brumptomyia carvalheiroi, Brumptomyia cardosoi, Brumptomyia cunhai, Brumptomyia nitzulescui, Brumptomyia brumpti and Brumptomyia spp. represented by 58 (26.85%) specimens. Although less frequently found, the presence of Pintomyia fischeri, Nyssomyia whitmani and Migonemyia migonei, known vectors of Leishmania braziliensis, indicates risk of American cutaneous leishmaniasis occurrence. Moreover, the absence of Lutzomyia longipalpis-the main vector of Leishmania infantum chagasi, which is the agent of American visceral leishmaniasis-suggests that there is no risk of introduction and establishment of this disease in the studied area.
Prevalence of the antibodies anti-Toxoplasma gondii, anti-Leptospira spp. and anti-Brucella canis, by sex, in serum samples from dogs housed in a private shelter in the municipality of Umuarama, Paraná, 2011 
Leptospirosis, toxoplasmosis and brucellosis are diseases with worldwide distribution. Among stray dogs, these zoonoses are facilitated by direct contact with other animal species, by the habit of scavenging garbage and hunting in search of food, drinking standing water, smelling other animals' urine, licking female genitalia and the sexual act itself. The objective of this study was to detect antibodies anti-Toxoplasma gondii, anti-Leptospira spp., anti-Brucella canis and anti-Brucella abortus in stray dogs housed in shelters at Umuarama city, Parana, Brazil. In order to detect toxoplasmosis, indirect immunofluorescence assay (IFA) was performed, agglutination microscopic (MAT) test for leptospirosis and agar gel immunodiffusion (AGID) and buffered acidified antigen (BAA) tests for brucellosis. Of the 175 serum samples analyzed, 70.85% were considered positive for toxoplasmosis by IFA, 20% by MAT for leptospirosis and 2.85% by AGID for Brucella canis. The serological results of this study showed that stray dogs housed at the private shelter are potential carriers of these three different zoonoses and contribute to the spread and maintenance of these etiologic agents in the urban area of Umuarama (PR), Brazil.
Entry of 
Bothrops jararaca 
Crotalus durissus 
(Serpentes, Viperidae) per year at Butantan Institute, São Paulo, SP, Brazil.
Crotalus durissus are found from Mexico to northern Argentina in a highly disjunct distribution. According to some studies, this species is prone to occupy areas disturbed by human activities and floods comprise a plausible method of dispersal as inferred for some North American rattlesnakes. Based on the literature, it seems plausible that Crotalus durissus expanded their natural distribution in Brazil due to floods, but only in a few municipalities in Rio de Janeiro State. Data entries of Butantan Institute, in Sao Paulo, Brazil, from 1998 to 2012 show a declining tendency of snakes brought by donors. In addition, research shows no evidence of Crotalus durissus being an expanding species in the Brazilian territory.
Being distant from Brazil's great natural diversity, from its long tradition in the study of snakebites and from the fact that it is one of the few countries which has a national information system for monitoring incidents involving venomous animals, non-Brazilian researchers face risks when estimating the incidence of these accidents in the country. The present work offers a critical review of the main estimates undertaken since 1954. It is interesting to note contradictions between textual and graphic information within the same article, variations over time in the work of a same researcher and differences among distinct authors, and that all these issues remain unmentioned or undiscussed. Comparison among such estimates and the data available at the Brazilian Information System on Diseases of Compulsory Declaration (Sistema de Informacao de Agravos de Notificacao -- SINAN) creates an opportunity to identify the degree of imprecision present in those articles, and draws attention to the need for the production of studies at both the regional and national levels, based on concrete data collected at national, state and municipal levels, which has been available on the internet since 2001.
With the aim of studying Leptospira spp. infection in sheep herds, blood samples and respective kidney and liver fragments were collected from 100 animals from twenty different properties during slaughter at a meat company in the Sorocaba region, São Paulo state, southeast Brazil. The microscopic agglutination test (MAT) was performed with 29 strains of Leptospira spp. To identify the agent in the liver and kidney, 100 samples of each tissue were submitted to culture in Fletcher medium and analyzed by the polymerase chain reaction (PCR) for Leptospira spp. MAT detected 23 samples serologically positive for one or more Leptospira spp. serovars and significantly more for Autumnalis. Eight (4%) samples were positive in culture (four kidneys and four livers), corresponding to five animals with positive serology (one animal simultaneously positive for both kidney and liver) and two negatives. PCR detected Leptospira spp. in 14 samples (seven kidneys and seven livers) corresponding to 12 positive animals (two animals simultaneously positive for kidney and liver), of which ten were serologically positive and two negative. PCR was faster, more practical and more sensitive than culture for detecting leptospires. The results reinforce the importance of sheep in the epidemiological context of leptospirosis.
Difference in the prevalence of hepatitis B viral infection among first-time blood donors between 2001 and 2010, Campo Grande, Brazil 
The aim of the present study was to estimate hepatitis B virus seroprevalence among first-time blood donors in the city of Campo Grande, Mato Grosso do Sul State, in the central-western region of Brazil. A retrospective analysis of first-time voluntary blood donor records, from January 2010 to December 2010, was conducted at the Hematology Center of Mato Grosso do Sul. The prevalence of the HBsAg and anti-HBc serological markers and their respective 95% confidence intervals were calculated. Chi-square analysis was performed between the seroprevalence previously found in 2001 and the one determined by the current study. Results were considered statistically significant if p < 0.05. Among 8,840 subjects, 269 (3.04%, 95% CI: 2.7-3.4) were positive for HBV markers. The prevalence rate of HBsAg was 0.19% (95% CI: 0.1-0.3) and anti-HBc alone was 2.85% (95% CI: 2.5-3.2). There was no statistically significant difference regarding gender. However, an important association was observed between HBV infection and older age (p < 0.01). The seroprevalence of HBV infection in first-time blood donors diminished from 2001 to 2010 (p < 0.01). Such decrease suggests an improvement in the recruitment of safe donors, the positive impact of vaccination programs and the decreasing of HBV infection prevalence in the general population.
Map of the state of São Paulo showing the collection sites and events related to yellow fever in 2000 and 2008. Black dots represent the collection sites by number. The larger polygons represent the main river basins of the state. The smaller polygons not filled in indicate municipalities with entomological collections or autochthonous human cases of yellow fever in 2000 (H0) and 2008 (H8). When filled in gray, the area indicates deaths of monkeys in 2008. The dashed line represents the region where the epizootic outbreaks have been confirmed by Adolfo Lutz Institute/State Health Secretariat of São Paulo, Brazil.
Number of collected and engorged females by collection site and municipality
Percentages of blood-source types for most abundant species of the total processed, by species. H: human (exclusive), NH: non-human primate (exclusive), P: primate (human + non-human), B: bird, C: cow, Ho: horse, R: rat and NR: not reagent. Aedes serratus (G): group including Ae. serratus and Ae. serratus/aenigmaticus.
Number of samples by combinations and numbers of types of blood source
Number of samples by type of blood source, collection point and river basin
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. 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. 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/capricornii was 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. 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.
immune response in cats and dogs vaccinated against rabies in Dracena and Presidente Prudente, southeastern Brazil, using the RFFIT test* 
Background Brazil holds annual nationwide public campaigns to vaccinate dogs and cats against rabies. The presence of rabies antibodies in these animals, which are among the main transmitters of rabies to humans, is a good indicator that they are immunized and protected. Methods In the present study we analyzed 834 serum samples from dogs and cats from the Southeast of Brazil (Presidente Prudente and Dracena cities), 12 months after the 2009 vaccination campaign. We used the technique known as rapid fluorescent focus inhibition test (RFFIT) and considered reactant those sera with values higher 0.5 IU/mL. Results and discussion Reactant sample results in Presidente Prudente were 153 (51.0%) for dogs and 59 (32.6%) for cats, and in Dracena 110 (52.1%) for dogs and 71 (50.0%) for cats. We discussed vaccine coverage of animals involved in this experiment, and observed low titers < 0.5 IU/mL, especially in cats from Presidente Prudente. Conclusion According to the results presented in our experiment, we suggest that titers below 0.5 IU/mL are worrisome and that, for multiple reasons, animals should be immunized against rabies in the period between public vaccination campaigns. Hence, the desired vaccine coverage was not accomplished, especially among cats from Presidente Prudente.
Retention times and molecular masses of venom components
Map of Cuba showing the enlarged area (Guantanamo - Gtmo) were samples were collected.
HPLC separation of soluble venom from different geographical areas: A 2-mg protein portion from each venom was separated into a C18 reverse-phase column eluted with a linear gradient from solution A (0.12% TFA in water) to 60% solution B (0.10% TFA in acetonitrile), run for 60 minutes: (A) venom of scorpions collected in Moa and La Poa; (B) venom collected in Limonar and El Chote; (C) venom collected in Farallones.
SDS-PAGE gel electrophoresis for detection of hyaluronidase activity. Venom from male scorpions (1, 3, 5, 7 and 9) and female scorpions (2, 4, 6, 8 and 19) containing 50 μg of protein each were run in gels containing hyaluronic acid, as described in Materials and Methods. The white band at approximately 46 kDa indicates the presence of hyaluronidase activity. Positive control was a venom sample from the spider Brachypelma vagans.
The venom of the Cuban scorpion Rhopalurus junceus is poorly study from the point of view of their components at molecular level and the functions associated. The purpose of this article was to conduct a proteomic analysis of venom components from scorpions collected in different geographical areas of the country. Venom from the blue scorpion, as it is called, was collected separately from specimens of five distinct Cuban towns (Moa, La Poa, Limonar, El Chote and Farallones) of the Nipe-Sagua-Baracoa mountain massif and fractionated by high performance liquid chromatography (HPLC); the molecular masses of each fraction were ascertained by mass spectrometry analysis. At least 153 different molecular mass components were identified among the five samples analyzed. Molecular masses varied from 466 to 19755 Da. Scorpion HPLC profiles differed among these different geographical locations and the predominant molecular masses of their components. The most evident differences are in the relative concentration of the venom components. The most abundant components presented molecular weights around 4 kDa, known to be K+-channel specific peptides, and 7 kDa, known to be Na+-channel specific peptides, but with small molecular weight differences. Approximately 30 peptides found in venom samples from the different geographical areas are identical, supporting the idea that they all probably belong to the same species, with some interpopulational variations. Differences were also found in the presence of phospholipase, found in venoms from the Poa area (molecular weights on the order of 14 to 19 kDa). The only ubiquitous enzyme identified in the venoms from all five localities studied (hyaluronidase) presented the same 45 kD molecular mass, identified by gel electrophoresis analysis. The venom of these scorpions from different geographical areas seem to be similar, and are rich in peptides that have of the same molecular masses of the peptides purified from other scorpions that affect ion-channel functions.
Prothrombin time test for different concentrations of crude venom of Ec
Prothrombin time test on mouse plasma by using fractions obtained from gel chromatography
Results of PT, PTT and FT tests before and after the injection of F 1 B subfraction
Gel chromatography (Sephadex G-75) of 50 mg of 
Echis carinatus 
crude venom.
Ion exchange chromatography of F
The venom of the family Viperidae, including the saw-scaled viper, is rich in serine proteinases and metalloproteinases, which affect the nervous system, complementary system, blood coagulation, platelet aggregation and blood pressure. One of the most prominent effects of the snake venom of Echis carinatus (Ec) is its coagulation activity, used for killing prey. Subfractions F1A and F1B were isolated from Ec crude venom by a combination of gel chromatography (Sephadex G-75) and ion exchange chromatography on a DEAE-Sepharose (DE-52). These subfractions were then intravenously (IV) injected into NIH male mice. Blood samples were taken before and after the administration of these subfractions. Times for prothrombin, partial thromboplastin and fibrinogen were recorded. Comparison of the prothrombin time before and after F1A and F1B administrations showed that time for blood coagulation after injection is shorter than that of normal blood coagulation and also reduced coagulation time after Ec crude venom injection. This difference in coagulation time shows the intense coagulation activity of these subfractions that significantly increase the coagulation cascade rate and Causes to quick blood coagulation. The LD50 of the Ec crude venom was also determined to be 11.1 μg/mouse. Different crude venom doses were prepared with physiological serum and injected into four mice. Comparison of the prothrombin times after injection of subfractions F1A and F1B showed that the rate of mouse blood coagulation increases considerably. Comparing the partial thromboplastin times after injecting these subfractions with this normal test time showed that the activity rate of intrinsic blood coagulation system rose sharply in mice. Finally, by comparing the fibrinogen time after subfraction injections and normal test time, we can infer intense activation of coagulation cascade and fibrin production.
Medical imaging tests 
Erucism is a skin reaction to envenomation from certain poisonous caterpillar bristles. In Brazil, most reports of erucism provoked by Lonomia caterpillars are from the southern region. Most manifestations of erucism are local and include burning pain, itching, local hyperthermia and, rarely, blisters (benign symptoms with spontaneous regression in a few hours). General symptoms such as nausea and vomiting, headache, fever, myalgia, abdominal pain and conjunctivitis may also occur. Uncommon symptoms include arthritis, coagulation disorders (manifested as bruising and bleeding), intracerebral hemorrhage and acute renal failure, which comprise serious complications. The present study reports the case of 60-year-old patient from Rio de Janeiro state, Brazil, who came into contact with a caterpillar and developed, a few days later, chronic renal disease.
Photography of the 
Echinometra lucunter 
spine tip: (A) intact spine; (B) regeneration process.
Enzymatic characterization of the spine extract. Kinetic data of velocity over concentration of substrate, after the incubation of spine aqueous extract (12 μg) with (A) Z-R-MCA and (B) Abz-GIVRAK(Dnp)-OH. (C) Enzyme titration using E-64 inhibitor, performed over Abz-GIVRAK(Dnp)-OH substrate. (D) Determination of pH for optimum activity of spine aqueous extract, over Abz-GIVRAK(Dnp)-OH substrate. (E) HPLC profile, in λ = 365 nm, of the products of complete hydrolysis of the Abz-GIVRAK(Dnp)-OH substrate by spine aqueous extract. (F) Mass spectrometry analysis of the products of Abz-GIVRAK(Dnp)-OH hydrolysis by spine aqueous extract.
Proteic characterization of the spine extract. (A) SDS-PAGE (10%) of aqueous extract of spine. M = molecular mass standard, SE = spine extract. Arrows indicate the molecular masses. (B) Western blotting of 5, 10 and 20 μg aqueous extract of spine, incubated with anti-cathepsin B antibody. Left lane, molecular mass standard. (C) Chromatogram obtained by gel filtration of spine aqueous extract. Dashed lines indicate the enzymatic activity.
Immunohistochemical test for anti-cathepsin B antibody was performed in transversal spine sections. (A) Spine section stained with toluidin-fuchsin. (B) Spine section incubated with anti-cathepsin B. (C and D) zoomed images, corresponding to A and B, respectively. It is possible to observe the positive (brownish staining) along the decalcified matrix (B), in the same location where cells were observed in the sections stained by toluidine-fuchsin (A).
Echinometra lucunter is a common American sea urchin responsible for the majority of the marine accidents in Brazil. Although not lethal, these accidents are reported to be extremely painful. Recently, our group described the presence of toxins in its spines that contribute to the pathological reactions. Additionally, we have observed that the E. lucunter spines can regenerate when broken. In the present work we evaluated the enzymatic activities of sea urchin spine extracts in order to identify an enzyme that could contribute not only to the toxicity, but also participate in the spine growth and regeneration. The spine aqueous extract was tested for peptidase activity, with synthetic substrates, in the presence and absence of inhibitors and activators. For proper enzyme classification, the FRET-substrate cleavage pattern, pH-dependency activity and Western-blot analyses were performed. The spine extract was able to cleave Z-R-MCA and Abz-GIVRAK(Dnp)-OH following pre-incubation with DTT, and was inhibited by E-64. Furthermore, the double-peaked pH curve (5 and 7) and the cleavage site proportion (4:6, R[downwards arrow]A:A[downwards arrow]K) indicate the presence of both mono and dicarboxypeptidase activities. Moreover, in Western-blot analysis, the spine extract was positive for anti-cathepsin B antibody. E. lucunter spines extracts presented a cysteine peptidase activity that was identified as cathepsin B/X that would participate in the remodeling and growth processes of the spine, as well as in the inflammatory response to the accident.
Journal of Venomous Animals and Toxins including Tropical Diseases Center for the Study of Venoms and Venomous Animals (CEVAP) Sao Paulo State University (UNESP), Fazenda Experimental Lageado, Caixa Postal 577, Rua Jose Barbosa de Barros, 1780, Botucatu, SP, 18610-307
Top-cited authors
Jean-Philippe Chippaux
  • Institute of Research for Development
Benedito Barraviera
  • São Paulo State University
Rui Seabra Ferreira Jr
  • São Paulo State University
Helio Langoni
  • São Paulo State University
Elghzizal Yassir
  • Faculty of science and techology, fès