Platelet participation in the pathogenesis of dermonecrosis induced by Loxosceles gaucho venom.

Loxosceles gaucho spider venom induces in vitro platelet activation and marked thrombocytopenia in rabbits. Herein, we investigated the involvement of platelets in the development of the dermonecrosis induced by L. gaucho venom, using thrombocytopenic rabbits as a model. L. gaucho venom evoked a drop in platelet and neutrophil counts 4 h after venom injection. Ecchymotic areas at the site of venom inoculation were noticed as soon as 4 h in thrombocytopenic animals but not in animals with initial normal platelet counts. After 5 days, areas of scars in thrombocytopenic animals were also larger, evidencing the marked development of lesions in the condition of thrombocytopenia. Histologically, local hemorrhage, collagen fiber disorganization, and edema were more severe in thrombocytopenic animals. Leukocyte infiltration, predominantly due to polymorphonuclears, was observed in the presence or not of thrombocytopenia. Thrombus formation was demonstrated by immunohistochemistry at the microvasculature, and it occurred even under marked thrombocytopenia. Taken together, platelets have an important role in minimizing not only the hemorrhagic phenomena but also the inflammatory and wound-healing processes, suggesting that cutaneous loxoscelism may be aggravated under thrombocytopenic conditions.

Biochemical and biological analysis of Philodryas baroni (Baron's green racer; Dipsadidae) venom: relevance to the findings of human risk assessment.

Philodryas baroni--an attractively colored snake--has become readily available through the exotic pet trade. Most people consider this species harmless; however, it has already caused human envenomation. As little is known about the venom from this South American opisthoglyphous "colubrid" snake, herein, we studied its protein composition by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), as well as its effects on the hemostatic system. Both reducing and nonreducing SDS-PAGE analysis demonstrated that the venom exhibits greatest complexity in the range of 50-80 kDa. The venom displayed proteolytic activity toward azocollagen, with a specific activity of 75.5 U mg⁻¹, and rapidly hydrolyzed the Aα-chain of fibrinogen, exhibiting lower activity toward the Bß- and γ-chains. The venom from P. baroni showed no platelet proaggregating activity per se, but it inhibited collagen- and thrombin-induced platelet aggregation. Prominent hemorrhage developed in mouse skin after intradermal injection of the crude venom, and its minimum hemorrhagic dose was 13.9 µg. When injected intramuscularly into the gastrocnemius of mice, the venom induced local effects such as hemorrhage, myonecrosis, edema, and leucocyte infiltration. Due to its venom toxicity shown herein, P. baroni should be considered dangerous to humans and any medically significant bite should be promptly reviewed by a qualified health professional.

Efficacy of Argentine propolis formulation for topical treatment of canine otitis externa / Eficácia da formulação da propólis Argentina para o tratamento tópico de otite externa canina

The therapeutic effects of Argentine propolis ear drop formulation on canine otitis externa were evaluated. Forty-eight dogs with symptoms of otitis externa were randomly assigned to double-blinded, controlled clinical trial to evaluate the efficacy of topical formulation with propolis versus a topical placebo in the treatment of otitis externa. The propolis preparation and placebo were administrated into both external ear canals, twice daily for 14 days. Throughout the study, clinical examination and microbiological analysis of dogs ear exudates were made. The most frequent microorganisms isolated in culture media were: Malassezia pachydermatis (54.2 percent), Staphylococcus aureus (43.8 percent), coagulase-negative Staphylococcus (25.0 percent), Pseudomonas aeruginosa (20.8 percent), Candida albicans (18.8 percent), Proteus mirabilis (16.7 percent), Streptococcus spp. (16.7 percent), Enteroccocus faecalis (12.5 percent), Escherichia coli (12.5 percent), Staphylococcus intermedius (6.3 percent), Klebsiella spp. (4.2 percent), andCandida glabrata (2.1 percent). Whereas the control group did not recover from the infectious ear disease, the propolis preparation exhibited antimicrobial activity against most of the microorganisms isolated from samples of the treated group. In addition, no propolis-adverse effects were observed. This allowed propolis-treated patients to show a significant improvement of the clinical parameters. Thus, this new Argentine propolis ear drop formulation may be used for topical treatment of otitis externa in dogs.

Os efeitos terapêuticos da formulação em gotas óticas de própolis procedentes da Argentina foram avaliados no tratamento da otite externa canina. Quarenta e oito cães com sintomas de otite externa foram distribuídos aleatoriamente em ensaio clínico duplo-cego controlado para avaliar a eficácia da formulação tópica com a própolis contra um placebo tópico no tratamento da otite externa. A preparação de própolis e placebo foi administrada em ambos os canais da orelha externa, duas vezes por dia, durante 14 dias. Ao longo do estudo, os cães foram submetidos a exame físico e à análise microbiológica de exsudatos auriculares. Os mais frequentes microrganismos isolados em meios de cultura foram: Malassezia pachydermatis (54,2 por cento), Staphylococcus aureus (43,8 por cento), Staphylococcus coagulase-negativo (25,0 por cento), Pseudomonas aeruginosa (20,8 por cento), Candida albicans (18,8 por cento), Proteus mirabilis (16,7 por cento), Streptococcus spp.(16,7 por cento), Enterococcus faecalis (12,5 por cento), Escherichia coli (12,5 por cento), Staphylococcus intermedius (6,3 por cento), Klebsiella spp.(4,2 por cento) e Candida glabrata (2,1 por cento). A preparação de própolis apresentou atividade antimicrobiana contra a maioria dos microrganismos isolados de amostras do grupo de tratamento, sendo que os do grupo-controle não se recuperaram da doença infecciosa auricular, e não foram observados efeitos adversos à própolis. Isso permitiu aos pacientes tratados com própolis melhora significativa dos parâmetros clínicos. Essa nova formulação da própolis argentina para o ouvido apresenta potencial utilidade no tratamento tópico da otite externa em cães.

Purification and characterization of patagonfibrase, a metalloproteinase showing alpha-fibrinogenolytic and hemorrhagic activities, from Philodryas patagoniensis snake venom.

Venoms of Colubridae snakes are a rich source of novel compounds, which may have applications in medicine and biochemistry. In the present study, we describe the purification and characterization of a metalloproteinase (patagonfibrase), the first protein to be isolated from Philodryas patagoniensis (Colubridae) snake venom. Patagonfibrase is a single-chain protein, showing a molecular mass of 53,224 Da and an acidic isoelectric point (5.8). It hydrolyzed selectively the Aalpha-chain of fibrinogen and when incubated with fibrinogen or plasma, the thrombin clotting time was prolonged. Prominent hemorrhage developed in mouse skin after intradermal injection of patagonfibrase. When administered into mouse gastrocnemius muscle, it induced local hemorrhage and necrosis, and systemic bleeding in lungs. Patagonfibrase showed proteolytic activity toward azocasein, which was enhanced by Ca(2+) and inhibited by Zn(2+), cysteine, dithiothreitol and Na(2)EDTA. Patagonfibrase impaired platelet aggregation induced by collagen and ADP. Thus, patagonfibrase may play a key role in the pathogenesis of disturbances that occur in P. patagoniensis envenomation, and may be used as a biological tool to explore many facets of hemostasis.

Duvernoy's gland secretion of Philodryas patagoniensis from the northeast of Argentina: its effects on blood coagulation.

Duvernoy's gland secretion of Philodryas patagoniensis exhibits high hemorrhagic activity, containing enzymes that are able to degrade the vascular wall. In this work we aim to determine if the secretion can also affect the hemostatic system by causing changes in blood coagulation. Procoagulant and coagulant activities were evaluated on plasma and fibrinogen, respectively. The delay in the thrombin clotting time of fibrinogen previously incubated with the secretion was also determined. Specific hydrolysis of fibrinogen and fibrin incubated with the secretion at different time intervals was shown by electrophoresis on polyacrylamide gel. To determine the structural characteristics of the enzymes degrading fibrinogen and fibrin, secretion were incubated in the presence of 45 mM Na(2)EDTA, 40 mM Benzamidine, and/or 2 mM PMSF before the incubation with fibrinogen or fibrin, respectively. The effect in vivo was investigated in adult male rats injected with different dose of secretion, aliquots of blood were withdrawn at different time intervals, and the fibrinogen concentration was determined. Duvernoy's gland secretion of P. patagoniensis did not clot plasma or fibrinogen. It exhibited a potent fibrinogenolytic activity degrading the Aalpha-chain faster than the Bbeta-chain, whereas gamma-chain was resistant. This latter corresponded with a strong delay in the thrombin clotting time of fibrinogen (4 mg/ml) pre-incubated with the secretion, being 9.53 microg the amount of protein from Duvernoy's gland secretion that increased the thrombin clotting time from 20 to 60 s. In vivo, the loss of rat plasma fibrinogen was proportional to the amount of secretion injected. The secretion also hydrolyzed fibrin degrading the alpha-monomer. Inhibition studies with Na(2)EDTA, Benzamidine, and/or PMSF showed that metalloproteinases and serinoproteinases are the main enzymes responsible for the hydrolyzing activity on fibrinogen and fibrin. All these results demonstrate that Duvernoy's gland secretion of P. patagoniensis possesses enzymes able to hydrolyze plasma components playing a relevant role in the blood coagulation. These hydrolyzing activities and those acting on the wall of blood vessels let the secretion exhibit a high hemorrhagic activity, which may result in permanent sequelae or even cause the death of the victims bitten by this colubrid snake.

Muscle and skin necrotizing and edema-forming activities of Duvernoy's gland secretion of the xenodontine colubrid snake Philodryas patagoniensis from the north-east of Argentina.

Philodryas patagoniensis is a colubrid snake spread by all South America, but very little is known about the composition and biological activities of its Duvernoy's gland secretion. In order to characterize it, we studied edematogenic, dermonecrotic and myonecrotic activities. For edematogenic activity, solutions containing different amounts of secretion were injected s.c. in the right foot pad of mice, both feet were subsequently cut off and weighed individually. For myonecrotic activity, mice were injected i.m. with solutions containing 40 microg of secretion, and at various time intervals mice were bled to determine serum creatine kinase activity and gastrocnemius muscles were removed for microscopic examination (Hematoxylin-Eosin stain). For dermonecrotic activity, solutions containing different amounts of secretion were injected into the shaved dorsal skin of mice; the necrotic lesion was measured on the inner surface of the skin and trimmed for microscopic examination (Hematoxylin-Eosin stain). Phospholipase A(2) activity was evaluated using a kinetic method. Results showed that P. patagoniensis Duvernoy's gland secretion exhibits a high edematogenic activity and moderate myonecrotic and dermonecrotic activities, while lacking phospholipase A(2) effect. Regarding edema, a 30% increase in the weight was produced by injecting 0.26 microg of Duvernoy's gland secretion. Microscopically, myonecrosis reached its highest intensity 12 h after injection, which was also demonstrated by serum creatine kinase levels. Dermonecrosis was proportional to the amount of secretion injected, with a minimum necrotizing dose of 15.7 microg. Myonecrotic, edematogenic and dermonecrotic activities were inhibited when the secretion was pre-incubated with 1 mM Na(2)EDTA. This suggests that the enzymes responsible for those activities are mostly metalloproeinases. All the studies carried out up to now demonstrate the potential toxicity of P. patagoniensis Duvernoy's gland secretion (which inhabits the north-east region of Argentina) and that the local lesions caused by this colubrid snake are very similar to those found in bothropic accidents. This latter suggests a more careful evaluation of the victims when considering the medical treatment to be adopted.

Edematogenic and myotoxic activities of the Duvernoy's gland secretion of Philodryas olfersii from the north-east region of Argentina

Philodryas olfersii is found in South America, from Amazonas to Patagonia. It is important to characterize the venom of P. olfersii, who inhabits the North-East region of Argentina, since snake venoms are known to exhibit considerable variability in composition and biological activities. In this work, mice weighing 18-20 g (n = 4 for each experimental group) were used. For the edematogenic activity mice were injected s.c. in the right foot pad with 50 microl of solutions containing different amounts of venom, whereas the left foot pad was injected with 50 microl of PBS. Two hours after injection mice were killed by cervical dislocation and both feet were cut off and weighed individually. For the myotoxic activity mice were injected i.m. with 100 microl of solutions containing 40 microg of venom. Blood samples were extracted after 1, 3, 6, 8, 10, 12, 14, 16 and 24 h of venom injection to determinate serum CPK activity and mice were sacrificed at the same time intervals to obtain the inoculated gastrocnemius muscle. They were fixed with Bouin solution and stained with Hematoxylin-Eosin. Results showed that P. olfersii venom exhibits a high edematogenic activity (MED = 0.31 microg) and a moderate myotoxic activity. Myonecrosis reached its highest level after 12 h of venom injection as shown by plasmatic CPK levels (5,401 +/- 330 IU/l) and microscopic assay. It demonstrates the potential toxicity of the venom of P. olfersii, who inhabits the North-East region of Argentina. It also reinforces the original warning concerning the potential danger of bites by colubrids.

Edematogenic and myotoxic activities of the Duvernoys gland secretion of Philodryas olfersii from the north-east region of Argentina

Philodryas olfersii is found in South America, from Amazonas to Patagonia. It is important to characterize the venom of P. olfersii, who inhabits the North-East region of Argentina, since snake venoms are known to exhibit considerable variability in composition and biological activities. In this work, mice weighing 18-20 g (n = 4 for each experimental group) were used. For the edematogenic activity mice were injected s.c. in the right foot pad with 50 microl of solutions containing different amounts of venom, whereas the left foot pad was injected with 50 microl of PBS. Two hours after injection mice were killed by cervical dislocation and both feet were cut off and weighed individually. For the myotoxic activity mice were injected i.m. with 100 microl of solutions containing 40 microg of venom. Blood samples were extracted after 1, 3, 6, 8, 10, 12, 14, 16 and 24 h of venom injection to determinate serum CPK activity and mice were sacrificed at the same time intervals to obtain the inoculated gastrocnemius muscle. They were fixed with Bouin solution and stained with Hematoxylin-Eosin. Results showed that P. olfersii venom exhibits a high edematogenic activity (MED = 0.31 microg) and a moderate myotoxic activity. Myonecrosis reached its highest level after 12 h of venom injection as shown by plasmatic CPK levels (5,401 +/- 330 IU/l) and microscopic assay. It demonstrates the potential toxicity of the venom of P. olfersii, who inhabits the North-East region of Argentina. It also reinforces the original warning concerning the potential danger of bites by colubrids. (AU)

Hemorrhagic activity of the Duvernoy's gland secretion of the xenodontine colubrid Philodryas patagoniensis from the north-east region of Argentina.

Colubrid snakes belonging to Philodryas genus, widespread all over South America, bring about lesions (swelling, ecchymosis, transient bleeding from the bite site punctures), that are similar to those produced by Bothrops species (yarará). In the present work we began the characterization of Philodryas patagoniensis venom. We examined if this venom produces hemorrhagic lesions as those observed in victims bitten by Philodryas olfersii. Hemorrhagic, proteolytic and fibrinogenolytic activities were evaluated, and histological observations in samples of gastrocnemius muscle were carried out. Inhibition studies were carried out in metal chelator (ethylenediaminetetraacetic acid) presence. Our results show a small Minimum Hemorrhagic Dose (MHD=0.035 microg) and a high proteolytic activity (143 U/mg), and prove the capacity of this venom to degrade fibrinogen in vitro rendering it unclottable by thrombin, supporting the presence of proteases, principally metalloproteases, in P. patagoniensis venom that are able to alterate the vascular wall and degrade fibrinogen, being both activities responsible of a high hemorrhagic activity.