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.

Systemic alterations induced by a Bothrops alternatus hemorrhagic metalloproteinase (baltergin) in mice.

Systemic alterations induced by a Bothrops alternatus hemorrhagin, named baltergin, a 55kDa fibrinogenolytic metalloproteinase isolated from venom of north-eastern Argentina specimens, were studied in mice. It caused macroscopic hemorrhagic spots in lungs which was injected intravenously with a minimum pulmonary hemorrhagic dose of 10microg. Histological observations of lungs showed mainly hemorrhagic areas, evidenced by the presence of erythrocytes in the alveolar spaces, congestion and increase of thickness of alveolar septum due to polymorphonuclear infiltrate and mononuclear cells. Neither macroscopic hemorrhage in other organs nor histological alterations in heart and cerebrum/cerebellum were observed at doses assayed. However, kidney and liver were mildly affected. Kidney examination revealed congestion, subcapsular hemorrhage with local capsule detachment, inflammatory infiltrate and degeneration of tubular cells. Congestion of blood vessels and hydropic degeneration of hepatocytes were observed in liver. Besides, baltergin was able to further hydrolyze type IV collagen. Although the enzyme showed to be less lethal than whole venom, it induced severe pulmonary bleeding and affected kinder and liver in minor grade. In conclusion, baltergin is able to alter the integrity of capillary vessels and simultaneously, to interfere on the hemostatic system. Thus, this metalloproteinase contribute markedly to systemic alterations characteristic of B. alternatus envenomations.

IgG antibodies against phospholipase A2 from Crotalus durissus terrificus: cross-reaction with venoms from Bothrops species from Argentina

We examined the ability of IgG anti-crotalic PLA2 to cross-react with Bothrops spp. venoms, from snakes found in the northeast of Argentina. Immunoblotting and ELISA tests showed that IgG anti-crotalic PLA2 recognize antigens of bothropic venoms. Indirect hemolytic activity tests showed that the quantity of antibodies that neutralized 50% of Crotalus durissus terrificus venom (ED50: 2.1 mg IgG anti-crotalic PLA2/100 µg of venom) were also able to neutralize venom from other snakes in the following proportion: 34% of B. alternatus, 18% of B. diporus and 12% of B. jararacussu. Likewise, direct PLA2 activity neutralization tests showed a similar cross-neutralization pattern including 56% of B. alternatus, 29% of B. diporus and 30% of B. jararacussu. In addition, in a myotoxic activity neutralization test, measured by plasma activity of creatine kinase, 35% of B. alternatus venom and 26% of B. diporus venom were neutralized, while no neutralization was detected with B. jararacussu venom. This study presents original data concerning cross-reactions between bothropic venoms from Argentina and IgG anti-crotalic PLA2. Our results suggest that anti-crotalic PLA2 antibodies should not be used to neutralize PLA2 activity of B. alternatus, B. diporus and especially B. jararacussu venoms; nor to enrich commercial antivenoms against these Bothrops species.(AU)

IgG antibodies against phospholipase A2 from Crotalus durissus terrificus: cross-reaction with venoms from Bothrops species from Argentina

We examined the ability of IgG anti-crotalic PLA2 to cross-react with Bothrops spp. venoms, from snakes found in the northeast of Argentina. Immunoblotting and ELISA tests showed that IgG anti-crotalic PLA2 recognize antigens of bothropic venoms. Indirect hemolytic activity tests showed that the quantity of antibodies that neutralized 50% of Crotalus durissus terrificus venom (ED50: 2.1 mg IgG anti-crotalic PLA2/100 µg of venom) were also able to neutralize venom from other snakes in the following proportion: 34% of B. alternatus, 18% of B. diporus and 12% of B. jararacussu. Likewise, direct PLA2 activity neutralization tests showed a similar cross-neutralization pattern including 56% of B. alternatus, 29% of B. diporus and 30% of B. jararacussu. In addition, in a myotoxic activity neutralization test, measured by plasma activity of creatine kinase, 35% of B. alternatus venom and 26% of B. diporus venom were neutralized, while no neutralization was detected with B. jararacussu venom. This study presents original data concerning cross-reactions between bothropic venoms from Argentina and IgG anti-crotalic PLA2. Our results suggest that anti-crotalic PLA2 antibodies should not be used to neutralize PLA2 activity of B. alternatus, B. diporus and especially B. jararacussu venoms; nor to enrich commercial antivenoms against these Bothrops species.

Isolation and biological characterization of a basic phospholipase A2 from Bothrops jararacussu snake venom

A phospholipase A2 has been isolated from Bothrops jararacussu venom from snakes that inhabit the northeast region of Argentina. The present study describes in vivo and in vitro biological activities of phospholipase A2 from B. jararacussu as well as isolation details of its. Venom was obtained by milking of adult snakes which were housing in wood reptile cages of varying dimensions in heated (20-30ºC) rooms. Snakes received a weekly diet of mice and water was available ad libitum for drinking and soaking. The enzyme was purified by gel filtration on a Sephadex G-75 column followed by ion exchange chromatography on a SP-Sephadex C25 column. The major peak belonging to proteins was retained in the cation exchanger and then eluted using a concentration gradient of KCl that exhibited phospholipase activity. This basic PLA2 consists of a single polypeptide chain with a molecular mass of 15.6 kDa. It had a high indirect hemolytic activity and produced a significant paw edema reaction in mice. The enzyme showed a low lethality (LD50 148.6 mg) when was administered i.p. but exhibited elevated myotoxic effects in vivo by increasing plasma CK activity of injected mice, corroborated results by the histological observations of samples of gastrocnemius muscle. Myonecrosis is the result of intense destruction of muscular fibers that involves local infiltration of inflammatory cells and leads to the highest peak of CK level just after 1 hour mice injection. Moreover, the isolated enzyme showed anticoagulant activity, evaluated on sheep platelet-poor plasma which recalcification time was prolonged after incubation with the isolated phospholipase A2. These findings showed that this phospholipase, isolated by only two simple chromatographic steps, possesses high edematogenic and myotoxic activities. However, despite the low lethal activity, this enzyme would contribute markedly to the pathophysiology of the bothropic envenomation.(AU)

Isolation and biological characterization of a basic phospholipase A2 from Bothrops jararacussu snake venom

A phospholipase A2 has been isolated from Bothrops jararacussu venom from snakes that inhabit the northeast region of Argentina. The present study describes in vivo and in vitro biological activities of phospholipase A2 from B. jararacussu as well as isolation details of its. Venom was obtained by milking of adult snakes which were housing in wood reptile cages of varying dimensions in heated (20-30ºC) rooms. Snakes received a weekly diet of mice and water was available ad libitum for drinking and soaking. The enzyme was purified by gel filtration on a Sephadex G-75 column followed by ion exchange chromatography on a SP-Sephadex C25 column. The major peak belonging to proteins was retained in the cation exchanger and then eluted using a concentration gradient of KCl that exhibited phospholipase activity. This basic PLA2 consists of a single polypeptide chain with a molecular mass of 15.6 kDa. It had a high indirect hemolytic activity and produced a significant paw edema reaction in mice. The enzyme showed a low lethality (LD50 148.6 mg) when was administered i.p. but exhibited elevated myotoxic effects in vivo by increasing plasma CK activity of injected mice, corroborated results by the histological observations of samples of gastrocnemius muscle. Myonecrosis is the result of intense destruction of muscular fibers that involves local infiltration of inflammatory cells and leads to the highest peak of CK level just after 1 hour mice injection. Moreover, the isolated enzyme showed anticoagulant activity, evaluated on sheep platelet-poor plasma which recalcification time was prolonged after incubation with the isolated phospholipase A2. These findings showed that this phospholipase, isolated by only two simple chromatographic steps, possesses high edematogenic and myotoxic activities. However, despite the low lethal activity, this enzyme would contribute markedly to the pathophysiology of the bothropic envenomation.

Proteolytic, edematogenic and myotoxic activities of a hemorrhagic metalloproteinase isolated from Bothrops alternatus venom.

A hemorrhagic metalloproteinase has been isolated from Bothrops alternatus venom from specimens that inhabit the north-east region of Argentina. The present study aimed at evaluating the proteolytic, hemorrhagic, edematogenic and myotoxic activities of the purified metalloproteinase, in order to consider its participation on the phatophysiology of the intoxication by Bothrops alternatus venom. The hemorrhagic metalloproteinase was isolated by a combination of DEAE-Cellulose chromatography and gel filtration on Sephadex G-75. The enzyme showed a molecular mass around 55k Da, it exhibited a hemorrhagic activity with a minimal hemorrhagic dose of 1.9 microg, almost two fold minor than the whole venom (3.6 microg). The enzyme showed a weak proteolytic activity on casein (18.72 U/mg enzyme), similar to the one exhibited by the whole venom (20 U/mg venom). Besides, the ability to degrade casein could be detected by SDS-PAGE; beta-casein was the fraction that showed the higher degradation, followed by alphas(1)-casein and kappa-casein degradation. The hemorrhagic metalloproteinase rapidly hydrolysed the A alpha-chain of fibrinogen, followed by B beta-chain degradation and leaving the gamma-chain unaffected. Proteolytic activities were inhibited by EDTA whereas they were not inhibited by benzamidine and PMSF. The metalloproteinase showed several polypeptides chains after autocatalytic processing, including a chain of 28k Da, it could be the processed disintegrin-like and cysteine-rich domains. The isolated enzyme exhibited myotoxic activity with high CK levels at 6h, due to local ischemia resulting of its hemorrhagic activity, and a significant edema-inducing effect (MED=1.3 microg), corroborated both results by the histological observations of samples of gastrocnemius muscle. These findings showed that this hemorrhagic metalloproteinase, possesses high edematogenic and myotoxic activities and, in despite of exhibiting a weak proteolytic activity, it is able to degrade fibrinogen. So, this enzyme would contribute markedly to the phatophysiology of the bothropic envenomation.

Mice plasma fibrinogen consumption by thrombin-like enzyme present in rattlesnake venom from the north-east region of Argentina

Due to variability of venom components from the same species of snakes that inhabit different regions, particular properties of the venom of Crotalus durissus terrificus that inhabits the North-East of Argentina were studied. Gyroxin, a thrombin-like enzyme, was isolated from this venom by gel filtration and affinity chromatography, it was found to be homogeneous according to SDS-PAGE, with a molecular weight of 33 kDa. [quot ]Gyroxin syndrome[quot ] in mice was tested and it showed changes in the animal behavior, confirming that the isolated thrombin-like enzyme is gyroxin. Effects of this enzyme and the crude venom on mice plasmatic fibrinogen levels were determined. The mice plasma fibrinogen decreased rapidly until incoagulability during the first hour after thrombin-like enzyme injection, then reaching its normal level 10 hours after injection; whereas crude venom resulted in a 60% decrease of the mice plasma fibrinogen, reaching its normal level after the same period of time. After 1 hour of gyroxin inoculation, intravascular coagulation was observed in histological cuttings of lung, cardiac muscle and liver. The isolated enzyme showed strong hydrolyzing activity on fibrinogen and fibrin in vitro, whereas the crude venom exhibited weak hydrolyzing activity on both substrates. It is probable that this very low activity is due to the low percentage of the enzyme in the crude venom. Decreasing of plasmatic fibrinogen levels may be due to either the coagulant or hydrolyzing actions of the enzyme.

Teniendo en cuenta la variabilidadde los componentes del veneno de serpientes de una misma especie que habitan regiones diferentes, se decidióestudiar las propiedades particulares del veneno de Crotalus durissus terrificus que habita el nordeste de Argentina, Giroxina, una enzima con actividad trombínica, fue aislada del veneno por cromatografía de filtración por gel y de afinidad; se comprobó su homogeneidad y se determinó su peso molecular, 33 kDa, por SDSPAGE. Se ensayó el síndrome giroxina en ratones, los que mostraron cambios en el comportamiento, confirmandoque la enzima tipo trombina aislada es giroxina. Se evaluó la acción de esta enzima sobre los niveles de fibrinógeno plasmático en ratones, comparándola con la del veneno crudo. Se comprobó que la enzima provoca una disminución de los niveles plasmáticos de fibrinógeno hasta la incoagulabilidad, durante la primer hora de inoculación, mientras que el veneno entero produjo una reducción del nivel plasmático en un 60%; sin embargo, en ambos casos, se evidenció una rápida reposición de fibrinógeno plasmático, alcanzando sus valores normales en un plazo de 10 horas. Se observó coagulación intravascular con la administración de giroxina una hora después de la inoculación, evidenciados en estudios histológicos de tejido pulmonar, cardíaco y hepático. En ensayos realizados in vitro, la enzima aislada mostró capacidad de degradar fibrinógeno como así también coágulos de fibrina, mientras que el veneno exhibió débil actividad hidrolítica sobre ambos sustratos. Es probable que esta baja actividad sea debida a la baja concentración de la enzima en el veneno. La disminución de los niveles de fibrinógeno plasmático observado en ratones se debería a la acción coagulante de la enzima, sin embargo no se descarta que también contribuya a este proceso una acción hidrolítica sobrefibrinógeno y fibrina por parte de la enzima.

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.

Mice plasma fibrinogen consumption by thrombin-like enzyme present in rattlesnake venom from the north-east region of Argentina

Due to variability of venom components from the same species of snakes that inhabit different regions, particular properties of the venom of Crotalus durissus terrificus that inhabits the North-East of Argentina were studied. Gyroxin, a thrombin-like enzyme, was isolated from this venom by gel filtration and affinity chromatography, it was found to be homogeneous according to SDS-PAGE, with a molecular weight of 33 kDa. [quot ]Gyroxin syndrome[quot ] in mice was tested and it showed changes in the animal behavior, confirming that the isolated thrombin-like enzyme is gyroxin. Effects of this enzyme and the crude venom on mice plasmatic fibrinogen levels were determined. The mice plasma fibrinogen decreased rapidly until incoagulability during the first hour after thrombin-like enzyme injection, then reaching its normal level 10 hours after injection; whereas crude venom resulted in a 60% decrease of the mice plasma fibrinogen, reaching its normal level after the same period of time. After 1 hour of gyroxin inoculation, intravascular coagulation was observed in histological cuttings of lung, cardiac muscle and liver. The isolated enzyme showed strong hydrolyzing activity on fibrinogen and fibrin in vitro, whereas the crude venom exhibited weak hydrolyzing activity on both substrates. It is probable that this very low activity is due to the low percentage of the enzyme in the crude venom. Decreasing of plasmatic fibrinogen levels may be due to either the coagulant or hydrolyzing actions of the enzyme.(AU)

Teniendo en cuenta la variabilidadde los componentes del veneno de serpientes de una misma especie que habitan regiones diferentes, se decidióestudiar las propiedades particulares del veneno de Crotalus durissus terrificus que habita el nordeste de Argentina, Giroxina, una enzima con actividad trombínica, fue aislada del veneno por cromatografía de filtración por gel y de afinidad; se comprobó su homogeneidad y se determinó su peso molecular, 33 kDa, por SDSPAGE. Se ensayó el síndrome giroxina en ratones, los que mostraron cambios en el comportamiento, confirmandoque la enzima tipo trombina aislada es giroxina. Se evaluó la acción de esta enzima sobre los niveles de fibrinógeno plasmático en ratones, comparándola con la del veneno crudo. Se comprobó que la enzima provoca una disminución de los niveles plasmáticos de fibrinógeno hasta la incoagulabilidad, durante la primer hora de inoculación, mientras que el veneno entero produjo una reducción del nivel plasmático en un 60%; sin embargo, en ambos casos, se evidenció una rápida reposición de fibrinógeno plasmático, alcanzando sus valores normales en un plazo de 10 horas. Se observó coagulación intravascular con la administración de giroxina una hora después de la inoculación, evidenciados en estudios histológicos de tejido pulmonar, cardíaco y hepático. En ensayos realizados in vitro, la enzima aislada mostró capacidad de degradar fibrinógeno como así también coágulos de fibrina, mientras que el veneno exhibió débil actividad hidrolítica sobre ambos sustratos. Es probable que esta baja actividad sea debida a la baja concentración de la enzima en el veneno. La disminución de los niveles de fibrinógeno plasmático observado en ratones se debería a la acción coagulante de la enzima, sin embargo no se descarta que también contribuya a este proceso una acción hidrolítica sobrefibrinógeno y fibrina por parte de la enzima. (AU)

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.

Muscular regeneration after myonecrosis induced by Bothrops jararacussu snake venom from Argentina.

Venom from Bothrops snake produces severe local symptoms on the envenomed victim, such as hemorrhage, edema and myonecrosis. The latter is perhaps the most important of all, since antivenom therapy is not effective for it, even when antivenom is injected only a few minutes after the accident. In this work, mice weighing 18-20 g (n = 5) were inoculated with 70 micrograms Bothrops jararacussu venom in 0.1 ml PBS in the gastrocnemius muscle. Mice were sacrificed using ether after 1, 12 hours, 3, 5, 7 days and 2, 3, 5, 6 weeks after the injection of the venom to obtain gastrocnemius muscles. They were fixed with Bouin's solution and stained using Hematoxylin--Eosin and Mason's trichromic stain was applied to visualize collagen fibers. Results showed that inflammatory reaction was evident after a few minutes of the venom injection, which was not evident after 6 weeks. Muscular fiber necrosis reached its highest level on the seventh day. Even thought regeneration of muscular fibers was important, they never reached the size of the control. We conclude that Bothrops jararacussu venom causes severe necrosis on muscle fibers with partial recovery, showing low hemorrhage and abundance of granulation tissue. This points that not all fibers were regenerated, which can be seen as a functional sequel for injured muscle.

Muscular regeneration after myonecrosis induced by Bothrops jararacussu snake venom from Argentina.

Venom from Bothrops snake produces severe local symptoms on the envenomed victim, such as hemorrhage, edema and myonecrosis. The latter is perhaps the most important of all, since antivenom therapy is not effective for it, even when antivenom is injected only a few minutes after the accident. In this work, mice weighing 18-20 g (n = 5) were inoculated with 70 micrograms Bothrops jararacussu venom in 0.1 ml PBS in the gastrocnemius muscle. Mice were sacrificed using ether after 1, 12 hours, 3, 5, 7 days and 2, 3, 5, 6 weeks after the injection of the venom to obtain gastrocnemius muscles. They were fixed with Bouins solution and stained using Hematoxylin--Eosin and Masons trichromic stain was applied to visualize collagen fibers. Results showed that inflammatory reaction was evident after a few minutes of the venom injection, which was not evident after 6 weeks. Muscular fiber necrosis reached its highest level on the seventh day. Even thought regeneration of muscular fibers was important, they never reached the size of the control. We conclude that Bothrops jararacussu venom causes severe necrosis on muscle fibers with partial recovery, showing low hemorrhage and abundance of granulation tissue. This points that not all fibers were regenerated, which can be seen as a functional sequel for injured muscle.

Acidente por mordida de cascavel (Crotalus durissus terrificus) em cäes na Argentina / American rattlesnake (Crotalus durissus terrificus) bite accidents in dogs in Argentina

Descreve-se a sintomatologIa e o tratamento de dois cäes picados por cascavel da America do Sul (Crotalus durissus terrificus) na Argentina. Sinais neurológicos foram evidentes poucos minutos após o acidente, incluindo anestesia local, ataxia do membro afetado e fascia neurotóxica. As enzimas aminotransferase da alanina, aminotransferase do aspartato, quinase da creatinina, desidrogenase de lactato e o cálcio foram determinados para analisar dano muscular. Falha rEnal näo foi observada, mas algumas alteraçöes foram detectadas na urina, incluindo densidade baixa, sedimento com grumos granulares e células pequenas redondas. Biópsias de músculo foram obtidas de ambas as pernas para histopatología, revelando edema e fibras necróticas isoladas. Ambos os cäes receberam tratamento quatro horas após o acidente, pela via intravenosa. A antitoxina foi administrada diluída em 250ml de soluçäo Ringer lactato na dose capaz de neutralizar 8mg de toxina. Dexametasona foi aplicada antes de administrar a antitoxina. A evoluçäo clínica foi boa e ambos os pacientes estavam com boa saúde no segundo dia após a acidente

American rattlesnake (Crotalus durissus terrificus) bite accidents in dogs in Argentina

The symptomatology and treatment of two dogs bitten by Crotalus durissus terrificus are described. Neurological signs were present few minutes after the accident with local anesthesia and ataxia of the affected limb and neurotoxic fascia. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine kinase (CK), lactate dehydrogenase (LDH) and calcium were evaluated in an attempt to investigate muscle damage. Renal failure was not observed but some alterations were detected in urine. Urine density was low and the urinary sediment contained granular clumps and small round cells. Muscle samples were obtained from both legs for histopathological study, showing edema and isolated necrotic fibers. Both dogs received treatment within four hours after the accident by intravenous route. The antivenom was administered diluted in 250ml of Ringer solution in a dose enough to inactivate more than 8mg of venom. Dexamethasone was applied previously to the antivenom. Clinical evolution was good and both patients were in good health condition on the second day after the accident.

Descreve-se a sintomatología e o tratamento de dois cães picados por cascavel da America do Sul (Crotalus durissus terrificus) na Argentina. Sinais neurológicos foram evidentes poucos minutos após o acidente, incluindo anestesia local, ataxia do membro afetado e fascia neurotóxica. As enzimas aminotransferase da alanina, aminotransferase do aspartato, quinase da creatinina, desidrogenase de lactato e o cálcio foram determinados para analisar dano muscular. Falha renal não foi observada, mas algunas alterações foram detectadas na urina, incluindo densidade baixa, sedimento com grumos granulares e células pequenas redondas. Biópsias de músculo foram obtidas de ambas as pernas para histopatología, revelando edema e fibras necróticas isoladas. Ambos os cães receberam tratamento quatro horas após o acidente, pela via intravenosa. A antitoxina foi administrada diluída em 250ml de solução Ringer lactato na dose capaz de neutralizar 8mg de toxina. Dexametasona foi aplicada antes de administrar a antitoxina. A evolução clínica foi boa e ambos os pacientes estavam com boa saúde no segundo dia após o acidente.

[Hemorrhagic, edema-forming, proteolytic and myonecrotic activities of viper venoms of Bothrops alternatus (Vibora de la Cruz)]. / Actividades hemorrágica, edematizante, proteolítica y mionecrótica de venenos de viboreznos de Bothrops alternatus (Víbora de la Cruz).

A study of hemorrhagic, edema-forming, proteolytic and myonecrotic activities induced by newborn Bothrops alternatus snake venoms from Argentina was performed. The analyzed venom had these activities with markedly differences when compared with the venom of adult specimens. Hemorrhagic activity was four times higher, while edema forming activity was ten times or more. But proteolytic activity was similar in both groups. Hystological analysis showed intense hemorrhage and muscular fiber myolisis after 60 minutes of venom injection. Myonecrosis and inflammatory exudate were higher in the following hours. Hemorrhage decreased after 24 hours and muscular fiber regeneration started after the first week with granulation tissue formation. On the fourth week regenerating muscular fiber areas and granulation tissue were still observed. As a conclusion, we can assume that the injury induced in the snake accident by newborn Bothrops alternatus snakes would be more intense than those induced by the adult ones.

[Toxic and enzymatic activities of the Bothrops moojeni venom from Argentina]. / Actividades tóxicas y enzimáticas del veneno de Bothrops moojeni de Argentina.

Toxic and enzymatic activity of Bothrops moojeni venom from Argentine. Hemorrhagic, edema forming, proteolytic and indirect hemolytic activities, myonecrosis and muscle regeneration using optic microscopy induced by Bothrops moojeni snake venom from Argentina were studied. The described activities were present in this venom. Myonecrosis was intense 60 minutes after venom injection, increasing its activity during the first 3 hours. Myolytic lesions rise above coagulative ones. Hemorrhage was minimum the same as the edema. After 3 hours polymorphonuclear inflammatory infiltrate was observed, with neutrophils. Macrophages were visible after 48 hours. After 3 days fibroblasts were present. At the 7 day muscle fiber regeneration were present, showing striations alternating with calcified areas. Bothrops moojeni venom injuries are intense and the recuperation of them can generate important tissue lose, with functional consequences.

Actividades hemorrágica, edematizante, proteolítica y mionecrótica de venenos de viboreznos de Bothrops alternatus (víbora de la Cruz) / Hemorrhagic, edema-forming, proteolytic and myonecrotic activities of viper venoms of Bothrops alternatus (Víbora de la Cruz)

Se realizó el estudio de las actividades hemorrágica, edematizante, proteolítica y mionecrótica inducida por veneno de viboreznos de Bothrops alternatus de Argentina. Los venenos estudiados poseen las actividades mensionadas, mostrando marcadas diferencias respecto al veneno de ejemplares adultos. Cuadriplica la capacidad hemorrágica, supera en más de diez veces la edematizante. La actividad proteolítica es la que menor diferencia posee. El análisis histológico mostró intensa hemorragia y miólisis de fibras musculares a los 60 minutos de exposición al veneno. La mionecrosis y el exudado inflamatório fueron más altos en las siguientes horas. La hemorragia disminuyó de 24 horas y la regeneración de las fibras musculares se inició en la primer semana con formación de tejido de granulación. A la cuarta semana se observaban áreas de regeneración de fibras musculares y aún se observada tejido de granulación. Se concluye que la injuria producida en los accidentes por viboreznos de Cothrops alternatus es más intensas que la inducida por ejemplares adultos de la misma especie. (AU)