Pulmonary mechanic and lung histology injury induced by Crotalus durissus terrificus snake venom

In the present work we investigated the effects of Crotalus durissus terrificus venom (CdtV) on the pulmonary mechanic events [static and dynamic elastance, resistive (Delta P1) and viscoelastic pressures (Delta P2)] and histology after intramuscular injection of saline solution (control) or venom (0.6 mu g/g). The static and dynamic elastance values were increased significantly after 3 It of venom inoculation, but were reduced at control values in the other periods studied. The Delta P1 values that correspond to the resistive properties of lung tissue presented a significant increase after 6 h of CdtV injection, reducing to basal levels 12 h after the venom injection. In Delta P2 analysis, correspondent to viscoelastic components, an increase occurred 12 h after the venom injection, returning to control values at 24 h. CdtV also caused an increase of leukocytes recruitment (3-24 h) to the airways wall as well as to the lung parenchyma. In conclusion, C. durissus terrificus rattlesnake venom leads to lung injury which is reverted, after 24 h of inoculation. (c) 2008 Elsevier Ltd. All rights reserved.

Loxosceles venom-induced cytokine activation, hemolysis, and acute kidney injury

Herein, we describe a confirmed case of Loxosceles spider bite that illustrates the critical complications seen in loxoscelism, including skin necrosis, rhabdomyolysis, hemolysis, coagulopathy, acute kidney failure, and electrolyte disorders. Upon initial assessment, laboratory studies revealed the following: the white blood cell count was 29 400 WBCs/mm(3), hemoglobin was 9.2g/dL, and the platelet count was 218000cells/mm(3). Coagulation studies revealed the following: international normalized ratio, 1.83; activated partial-thromboplastin time, 62s; D-dimer, 600 ng/mL (normal range < 500 ng/mL); free protein S, 37% (normal range = 64-114%); protein C, negative; and antithrombin III, negative. Various serum levels were abnormal: urea, 110mg/dL; creatinine, 3.1 mg/dL; indirect bilirubin, 3.8 mg/dL; creatine kinase, 1631 U/L, lactate dehydrogenase, 6591 U/L; potassium 6.2mmol/L. Urine tests were positive for hemoglobin and bilirubin. In addition, concentrations of interleukin-6 and tumor necrosis factor-alpha were notably elevated in the serum. In conclusion, physicians must be alert to the possibility of loxoscelism when a patient presents with the clinical and laboratory findings described above, especially if the patient resides in an endemic area. Advances in our understanding of multiple pathways and mediators that orchestrate the response to Loxosceles venom might reveal new possibilities for the management of loxoscelism. (C) 2007 Elsevier Ltd. All rights reserved.

Bites by the colubrid snake Philodryas patagoniensis: A clinical and epidemiological study of 297 cases

We retrospectively analyzed 297 proven cases of Philodryas patagoniensis bites admitted to Hospital Vital Brazil (HVB), Butantan Institute, Sao Paulo, Brazil, between 1959 and 2008. Only cases in which the causative animal was brought and identified were included. Part of the snakes brought by the patients was still preserved in the collection maintained by the Laboratory of Herpetology. Of the 297 cases, in 199 it was possible to describe the gender of the snake, and seventy three (61.3%) of them were female. The length of snakes (snout-vent length) ranged from 160 to 1080 mm. In 117 snakes their state of preservation enabled the dissection and examination of their stomach contents. The stomach was empty in 106 snakes (89.1%). Most bites occurred in the seasons of spring and summer (n = 196, 66.0%) and during warmer periods of the day. The mean age of the victims was 24.1 +/- 15.1 years old and 206 (69.4%) patients were men. Around 92% of the patients sought medical care within 6 h after the bite. Both lower (n = 188, 63.3%) and upper limbs (n = 102, 34.3%) were most frequently bitten, especially the feet and hands (n = 205, 69.0%). The local clinical manifestations were pain (n = 151, 50.8%), transitory bleeding (n = 106, 35.7%), erythema (n = 47, 15.8%) and edema (n = 39, 13.1%). Ecchymosis was not observed. Only 7 (2.4%) patients reported systemic symptoms characterized by mild dizziness and 88 patients (29.6%) showed no evidence of envenoming. The whole blood clotting time was performed in 76 (25.6%) patients on admission and all of them had coagulable blood. Supportive treatment was offered to only 13.4% of patients, namely administration of antihistamines (n = 19, 6.4%) and analgesics (n = 12, 4.1%). Eight patients (2.7%) were mistreated with Bothrops antivenom before their admission to HVB. No sequels or relevant complications were observed in patients, and the prognostic was benign. Therefore, although P. patagoniensis accidents can cause mild local symptomatology, it is very important that health professionals know how to make the correct diagnosis to avoid unnecessary use of antivenom. (C) 2010 Elsevier Ltd. All rights reserved.

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

We retrospectively analyzed 98 proven cases of centipede stings admitted to Hospital Vital Brazil, Butantan Institute, Sao Paulo, Brazil, between 1990 and 2007. Most stings occurred at the metropolitan area of Sao Paulo city (n = 94, 95.9%), in the domiciles of patients (n = 67, 68.4%), and during the warm-rainy season (n = 60, 61.2%). The mean age of the victims was 32.0 +/- 18.8-years-old. Cryptops and Otostigmus genera were responsible for most cases. Around 86% of the patients sought medical care within 6 h after the sting. Both lower (56.1%) and upper limbs (41.8%) were most frequently bitten, especially the feet and hands (89.8%). The most frequent local clinical manifestations were pain (94.9%), erythema (44.9%) and edema (21.4%), and the latter was mainly observed in patients bitten by Otostigmus spp. Supportive treatment was used in only 28.6% of the patients, namely administration of local anesthesia (9.2%) and systemic analgesia (13.3%). No sequels or complications were observed in patients, and the prognostic was benign. (c) 2008 Elsevier Ltd. All rights reserved.

Antibothropic action of Casearia sylvestris Sw. (flacourtiaceae) extracts

Casearia sylvestris Sw., popularly known in Brazil as `guacatonga`, has been used as antitumor, antiseptic, antiulcer, local anaesthetic and healer in folk medicine. Snakebite envenomation by Bothrops jararacussu (Bjssu) constitutes a relevant public health hazard capable of inducing serious local damage in victims. This study examined the pharmacological action of apolar and polar C sylvestris leaf extracts in reverting the neuromuscular blockade and myonecrosis, which is induced by Bjssu venom and its major toxin bothropstoxin-I on the mouse phrenic nerve-diaphragm preparations. The polar methanol extract (ME) was by far the most efficacious. ME not only prevented myonecrosis and abolished the blockade, but also increased ACh release. Such facilitation in neuromuscular transmission was observed with ME alone, but was accentuated in preparations incubated with ME plus venom or toxin. This established synergy opens an interesting point of investigation because the venom or toxin in contact with ME changes from a blocking to a facilitating effect. It is suggested that rutin, known to have potent antioxidant properties, and one of the components present in the ME, could have a role in the observed effects. Since commercial rutin did not reproduce the ME effects, it is likely that a rutin-containing phytocomplex is neutralizing the bothropic envenoming effects. Copyright (C) 2008 John Wiley & Sons, Ltd.

Species and regional variations in the effectiveness of antivenom against the in Vitro neurotoxicity of death adder (Acanthophis) venoms

Although viperlike in appearance and habit, death adders belong to the Elapidae family of snakes. Systemic envenomation represents a serious medical problem with antivenom, which is raised against Acanthophis antarcticus venom, representing the primary treatment. This study focused on the major Acanthophis variants from Australia and islands in the Indo-Pacific region. Venoms were profiled using liquid chromatography-mass spectrometry, and analyzed for in vitro neurotoxicity (0.3-10 mug/ml), as well as the effectiveness of antivenom. (1-5 units/ml; 10 min prior to the addition of 10 mug/ml venom). The following death adder venoms were examined: A. antarcticus (from separate populations in New South Wales, Queensland, South Australia, and Western Australia), A. hawkei, A. praelongus, A. pyrrhus, A. rugosus, A. wellsi, and venom from an unnamed species from the Indonesian island of Seram. All venoms abolished indirect twitches of the chick isolated biventer cervicis nerve-muscle preparation in a dose-dependent manner. In addition, all venoms blocked responses to exogenous acetylcholine (1 m-M) and carbachol (20 muM), but not KCl (40 mM), suggesting postsynaptic neurotoxicity. Death adder antivenom (1 unit/ml) prevented the neurotoxic effects of A. pyrrhus, A. praelongus, and A. hawkei venoms, although it was markedly less effective against venoms from A. antarcticus (NSW, SA, WA), A. rugosus, A. wellsi, and A. sp. Scram. However, at 5 units/ml, antivenom was effective against all venoms tested. Death adder venoms, including those from A. antarcticus geographic variants, differed not only in their venom composition but also in their neurotoxic activity and susceptibility to antivenom. For the first time toxicological aspects of A. hawkei, A. wellsi, A. rugosus, and A. sp. Seram venoms were studied. (C) 2001 Academic Press.

Cross-reactivity of Sydney funnel-web spider antivenom: neutralization of the in vitro toxicity of other Australian funnel-web (Atrax and Hadronyche) spider venoms

Australian funnel-web spiders are recognized as one of the most venomous spiders to humans world-wide. Funnel-web spider antivenom (FWS AV) reverses clinical effects of envenomation from the bite of Atrax robustus and a small number of related Hadronyche species. This study assessed the in vitro efficacy of FWS AV in neutralization of the effects of funnel-web spider venoms, collected from various locations along the eastern seaboard of Australia, in an isolated chick biventer cervicis nerve-muscle preparation. Venoms were separated by SDS-PAGE electrophoresis to compare protein composition and transblotted for Western blotting and incubation with FWS AV. SDS-PAGE of venoms revealed similar low and high molecular weight protein bands. Western blotting with FWS AV showed similar antivenom binding with protein bands in all the venoms tested. Male funnel-web spider venoms (7/7) and female venoms (5110) produced muscle contracture and fasciculation when applied to the nerve-muscle preparation. Venom effects were reversed by subsequent application of FWS AV or prevented by pretreatment of the preparation with antivenom. FWS AV appears to reverse the in vitro toxicity of a number of funnel-web spider venoms from the eastern seaboard of Australia. FWS AV should be effective in the treatment of envenomation from most, if not all, species of Australian funnel-web spiders. (C) 2001 Elsevier Science Ltd. All rights reserved.

Synthesis and characterization of delta-atracotoxin-ar1a, the lethal neurotoxin from venom of the Sydney funnel-web spider (Atrax robustus)

delta-Atracotoxin-Ar1a (delta-ACTX-Ar1a) is the major polypeptide neurotoxin isolated from the venom of the male Sydney funnel-web spider, Atrax robustus. This neurotoxin targets both insect and mammalian voltage-gated sodium channels, where it competes with scorpion alpha-toxins for neurotoxin receptor site-3 to slow sodium-channel inactivation. Progress in characterizing the structure and mechanism of action of this toxin has been hampered by the limited supply of pure toxin from natural sources. In this paper, we describe the first successful chemical synthesis and oxidative refolding of the four-disulfide bond containing delta-ACTX-Ar1a. This synthesis involved solid-phase Boc chemistry using double coupling, followed by oxidative folding of purified peptide using a buffer of 2 M GdnHCl and glutathione/glutathiol in a 1:1 mixture of 2-propanol (pH 8.5). Successful oxidation and refolding was confirmed using both chemical and pharmacological characterization. Ion spray mass spectrometry was employed to confirm the molecular weight. H-1 NMR analysis showed identical chemical shifts for native and synthetic toxins, indicating that the synthetic toxin adopts the native fold. Pharmacological studies employing whole-cell patch clamp recordings from rat dorsal root ganglion neurons confirmed that synthetic delta-ACTX-Ar1a produced a slowing of the sodium current inactivation and hyperpolarizing shifts in the voltage-dependence of activation and inactivation similar to native toxin. Under current clamp conditions, we show for the first time that delta-ACTX-Ar1a produces spontaneous repetitive plateau potentials underlying the clinical symptoms seen during envenomation. This successful oxidative refolding of synthetic delta-ACTX-Ar1a paves the way for future structure-activity studies to determine the toxin pharmacophore.

Comparison of the in vitro neuromuscular activity of venom from three Australian snakes (Hoplocephalus stephensi, Austrelaps superbus and Notechis scutatus): Efficacy of tiger snake antivenom

1. Tiger snake antivenom, raised against Notechis scutatus venom, is indicated not only for the treatment of envenomation by this snake, but also that of the copperhead (Austrelaps superbus ) and Stephen's banded snake (Hoplocephalus stephensi ). The present study compared the neuromuscular pharmacology of venom from these snakes and the in vitro efficacy of tiger snake antivenom. 2. In chick biventer cervicis muscle and mouse phrenic nerve diaphragm preparations, all venoms (3-10 mug/mL) produced inhibition of indirect twitches. In the biventer muscle, venoms (10 mug/mL) inhibited responses to acetylcholine (1 mmol/L) and carbachol (20 mumol/L), but not KCl (40 mmol/L). The prior (10 min) administration of 1 unit/mL antivenom markedly attenuated the neurotoxic effects of A. superbus and N. scutatus venoms (10 mug/mL), but was less effective against H. stephensi venom (10 mug/mL); 5 units/mL antivenom attenuated the neurotoxic activity of all venoms. 3. Administration of 5 units/mL antivenom at t(90) partially reversed, over a period of 3 h, the inhibition of twitches produced by N. scutatus (10 mug/mL; 41% recovery), A. superbus (10 mug/mL; 25% recovery) and H. stephensi (10 mug/mL; 50% recovery) venoms. All venoms (10-100 mug/mL) also displayed signs of in vitro myotoxicity. 4. The results of the present study indicate that all three venoms contain neurotoxic activity that is effectively attenuated by tiger snake antivenom.

Neostigmine in the treatment of snake accidents caused by Micrurus frontalis: report of two cases

Antivenom in order to be effective in the treatment of coral snake accidents must be injected very soon after the bite owing to the rapid rate of absorption of the venom neurotoxins. As this is not always possible, other forms of treatment besides serotherapy must be employed to avoid asphyxia and death. Neostigmine and artificial respiration are used for this purpose. Neostigmine restores neuromuscular transmission if the venom-induced blockade results from a reversible interaction of its neurotoxins with the end-plate receptors. This is the mechanism of the neuromuscular blockade produced by the venom of M. frontalis snakes from centereastern and southern Brazil, and Argentine. Neostigmine is able, therefore, to antagonize the blockade, and has been shown to be very effective in the treatment of the experimental envenomation of dogs and monkeys. In the present communication, two cases of M. frontalis accidents treated with antivenom and neostigmine are reported. In both, neostigmine was successful in producing regression of the paralysis, confirming the effectiveness shown in the treatment of the poisoning induced in animals by M. frontalis venom.

A clinico-epidemiological study of bites by spiders of the genus Phoneutria

From January, 1984 to December, 1996, 422 patients (ages 9 m-99 y, median 29 y) were admitted after being bitten by spiders which were brought and identified as Phoneutria spp. Most of the bites occurred at March and April months (29.2%), in the houses (54.5%), during the day (76.5%), and in the limbs (feet 40.9%, hands 34.3%). Upon hospital admission, most patients presented only local complaints, mainly pain (92.1%) and edema (33.1%) and were classified as presenting mild (89.8%), moderate (8.5%) and severe (0.5%) envenomation. Few patients (1.2%) did not present signs of envenomation. Severe accidents were only confirmed in two children (9 m, 3 y). Both developed acute pulmonary edema, and the older died 9 h after the accident. Patients more than 70 year-old had a significantly greater (p<0.05) frequency of moderate envenomations compared to the 10-70-year-old individuals. Proceedings to relief local pain were frequently performed (local anesthesia alone 32.0%, local anesthesia plus analgesics 20.6% and oral analgesics alone 25.1%). Only 2.3% of the patients (two cases classified as severe and eight as moderate, eight of them in children) were treated with i.v. antiarachnid antivenom. No antivenom early reaction was observed. In conclusion, accidents involving the genus Phoneutria are common in the region of Campinas, with the highest risk groups being children under 10 years of age and adults over 70 years of age. Cases of serious envenomation are rare (0.5%).

Immunohistochemical changes in kidney glomerular and tubular proteins caused by rattlesnake (Crotalus vegrandis) venom

Renal damage is an important cause of death in patients who have survived the early effects of severe crotalid envenomation. Extracellular matrix of renal tissue is altered by Crotalus toxin activities. The aim of this study was to describe how cytoskeletal proteins and basal membrane components undergo substantial alterations under the action of Crotalus vegrandis crude venom and its hemorrhagic fraction (Uracoina-1) in mice. To detect the proteins in question, the immunoperoxidase method with monoclonal and polyclonal antibodies was used. Cell types within renal lesions were characterized by phenotypic identification, by means of immunohistologic analysis of marker proteins using different primary antibodies against mesangial cells, endothelial cells, cytoskeletal proteins (intermediate filament), extracellular matrix and basal membranes. Samples for morphological study by standard procedures (biotin-streptavidin-peroxidase technique) using light microscopy were processed. Positive and negative controls for each antigen tested in the staining assay were included. After crude venom and hemorrhagic fraction inoculation of mice, the disappearance of cytoskeletal vimentin and desmin and collagen proteins in the kidney was observed. In extracellular matrix and basal membranes, collagen type IV from envenomed animals tends to disappear from 24 h to 120 h after venom injection.

A low-cost method to test cytotoxic effects of Crotalus vegrandis (Serpentes: Viperidae) venom on kidney cell cultures

The pathogenesis of the renal lesion upon envenomation by snakebite has been related to myolysis, hemolysis, hypotension and/or direct venom nephrotoxicity caused by the venom. Both primary and continuous cell culture systems provide an in vitro alternative for quantitative evaluation of the toxicity of snake venoms. Crude Crotalus vegrandis venom was fractionated by molecular exclusion chromatography. The toxicity of C. vegrandis crude venom, hemorrhagic, and neurotoxic fractions were evaluated on mouse primary renal cells and a continuous cell line of Vero cells maintained in vitro. Cells were isolated from murine renal cortex and were grown in 96 well plates with Dulbecco's Modified Essential Medium (DMEM) and challenged with crude and venom fractions. The murine renal cortex cells exhibited epithelial morphology and the majority showed smooth muscle actin determined by immune-staining. The cytotoxicity was evaluated by the tetrazolium colorimetric method. Cell viability was less for crude venom, followed by the hemorrhagic and neurotoxic fractions with a CT50 of 4.93, 18.41 and 50.22 µg/mL, respectively. The Vero cell cultures seemed to be more sensitive with a CT50 of 2.9 and 1.4 µg/mL for crude venom and the hemorrhagic peak, respectively. The results of this study show the potential of using cell culture system to evaluate venom toxicity.

Bites by coral snakes (Micrurus spp.) in Campinas, State of São Paulo, Southeastern Brazil

Coral snakes (Micrurus spp.) are the main representatives of the Elapidae in South America. However, bites by these snakes are uncommon. We retrospectively reviewed the data from 11 individuals bitten by coral snakes over a 20-year period; four were confirmed (snake brought for identification) and seven were highly suspected (neuromuscular manifestations) cases of elapid envenoming. The cases were classified as dry-bite (n = 1, caused by M. lemniscatus; did not receive antivenom), mild (n = 2, local manifestations with no acute myasthenic syndrome; M. frontalis and Micrurus spp.), moderate (n = 5, mild myasthenia) or severe (n = 3, important myasthenia; one of them caused by M. frontalis). The main clinical features upon admission were paresthesia (local, n = 9; generalized, n = 2), local pain (n = 8), palpebral ptosis (n = 8), weakness (n = 4) and inability to stand up (n = 3). No patient developed respiratory failure. Antivenom was used in ten cases, with mild early reactions occurring in three. An anticholinesterase drug was administered in the three severe cases, with a good response in two. No deaths were observed. Despite the high toxicity of coral snake venoms, the prognosis following envenoming is good. In serious bites by M. frontalis or M. lemniscatus, the venom of which acts postsynaptically, anticholinesterases may be useful as an ancillary measure if antivenom is unavailable, if there is a delay in obtaining a sufficient amount, or in those patients given the highest recommended doses of antivenom without improvement of the paralysis or with delayed recovery.

Sea urchin granuloma

Injuries caused by venomous and poisonous aquatic animals may provoke important morbidity in humans. The phylum Echinoderma include more than 6000 species of starfish, sea urchins, sand dollars, and sea cucumbers some of which have been found responsible for injuries to humans. Initial injuries by sea urchins are associated with trauma and envenomation, but later effects can be observed. Sea urchin granuloma is a chronic granulomatous skin disease caused by frequent and successive penetration of sea urchin spines which have not been removed from wounds. The authors report a typical case of sea urchin granuloma in a fisherman and its therapeutic implications.