Journal of Venomous Animals and Toxins including Tropical Diseases

Published by SciELO

Online ISSN: 1678-9199

Articles


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).
A bradykinin-potentiating peptide (BPP-10c) from Bothrops jararaca induces changes in seminiferous tubules
  • Article
  • Full-text available

November 2013

·

258 Reads

Joyce M Gilio

·

·

·

[...]

·

Carlos Alberto-Silva
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.
Download
Share

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 of wild-caught Indian cobra Naja naja (Linnaeus, 1758)

April 2014

·

284 Reads

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
Clinical aspects of envenomation caused by Tityus obscurus (Gervais, 1843) in two distinct regions of Pará state, Brazilian Amazon basin: A prospective case series

February 2014

·

322 Reads

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.

Figure 1: Adult female of Rophalurus amazonicus.
Figure 2: Scorpion sting site on the middle of the thumb 56 hours after the accident.
Figure 3: 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.
Envenomation caused by Rhopalurus amazonicus Lourenço, 1986 (Scorpiones, Buthidae) in Pará State, Brazil

December 2014

·

3,919 Reads

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.
A historical approach to scorpion studies with special reference to the 20 and 21st centuries

March 2014

·

512 Reads

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.

Characterization ofLeiurus abdullahbayrami (Scorpiones: Buthidae) venom: Peptide profile, cytotoxicity and antimicrobial activity

November 2014

·

579 Reads

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.

Hemolytic activity of venom from crown-of-thorns starfish Acanthaster planci spines

September 2013

·

709 Reads

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.
Potentiation of a novel palladium (II) complex lethality with bee venom on the human T-cell acute lymphoblastic leukemia cell line (MOLT-4)

October 2013

·

131 Reads

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).
Resistance of cervical adenocarcinoma cells (HeLa) to venom from the scorpion Centruroides limpidus limpidus

September 2013

·

273 Reads

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.

Use of antivenoms for the treatment of envenomation by Elapidae snakes in Guinea, Sub-Saharan Africa

March 2013

·

538 Reads

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.

Outbreaks of Ebola virus disease in Africa: The beginnings of a tragic saga

October 2014

·

1,630 Reads

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.
Public health aspects of snakebite care in West Africa: Perspectives from Nigeria

October 2013

·

1,146 Reads

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.
Vasoconstrictor effect of Africanized honeybee (Apis mellifera L.) venom on rat aorta

September 2013

·

78 Reads

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).
Lesions caused by Africanized honeybee stings in three cattle in Brazil

August 2013

·

1,353 Reads

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 surface antigen seroconversion in HIV-infected individual after pegylated interferon-alpha treatment: a case report

December 2013

·

57 Reads

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 
Seroprevalence of flaviviruses antibodies in water buffaloes (Bubalus bubalis) in Brazilian Amazon

March 2014

·

59 Reads

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.
Current challenges for confronting the public health problem of snakebite envenoming in Central America

March 2014

·

1,245 Reads

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).
Anaphylactic shock following the bite of a wild Kayan slow loris (Nycticebus kayan): Implications for slow loris conservation

October 2014

·

1,739 Reads

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.

Figure 1: 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.
Table 1 Antiparasitic activity and cytotoxicity of SaFr1, the viability was determined by the colorimetric assay of MTT at 570 nm
Figure 2: 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.
Figure 3: 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.
Antileishmanial and antitrypanosomal activity of the cutaneous secretion of Siphonops annulatus

November 2014

·

111 Reads

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.

Table 2 Total distribution of Helicobacter pylori in various types of milk and dairy products 
Table 3 Distribution of putative virulence factors in Helicobacter pylori strains isolated from various types of milk and dairy products 
Virulence factors and antibiotic resistance of Helicobacter pylori isolated from raw milk and unpasteurized dairy products in Iran

December 2014

·

204 Reads

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.

Table 1 Estimated percentage of animals with diagnosis for Leishmania spp. by location and species 
Table 2 Blood cultures and IFAT according to PCR for Leishmania spp 
Evaluation of canine and feline leishmaniasis by the association of blood culture, immunofluorescent antibody test and polymerase chain reaction

February 2014

·

94 Reads

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.

Figure 1: 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).
Figure 2: 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).
Table 2 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
Antimycobacterial and cytotoxicity activity of microcystins

December 2015

·

183 Reads

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.

In vitro screening and evaluation of antivenom phytochemicals from Azima tetracantha Lam. leaves against Bungarus caeruleus and Vipera russelli

April 2014

·

1,280 Reads

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.

Characteristics of hemolytic activity induced by the aqueous extract of the Mexican fire coral Millepora complanata

November 2014

·

185 Reads

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.

Purification and characterization of a hyaluronidase from venom of the spider Vitalius dubius (Araneae, Theraphosidae)

February 2014

·

1,377 Reads

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.

Top-cited authors