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)

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.

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.

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.

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

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.

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

Se estudiaron las actividades hemorrágica, edematizante, proteolítica, hemolítica indirecta (in vitro) y por microscopía óptica la mionecrosis y regeneración de fibras musculares inducidas por veneno de Bothrops moojeni de Argentina. El veneno demostró poseer las actividades mensionadas. La actividad mionecrótica fue intensa a partir de los 60 minutos de exposición incrementando su intensidad a las 3 horas con predominio de lesiones de fibras musculares de tipo miolótico, respecto a necrosis coagulativa. En los cortes histológicos, la hemorragia fue mínima al igual que el edema, mientras que a las 3 horas se observó infiltrado inflamatorio polimorfonuclear con predominio de neutrófilos y a partir de las 48 horas se sumaron los macrófagos. Al tercer día se observaron fibroblastos, los que se incrementaron paulatinamente; a los 7 días se observó regeneración de algunas fibras musculares que presentaban estriaciones, a partir de la curta semana, éstas alternaban con áreas calcificadas. La recuperación de las lesiones fue parcial, con fibras musculares de menor diámetro que las normales, alterando con áreas de calcificación y otras de fibrosis. Las lesiones causadas por el veneno de Bothrops moojeni son intensas y por la recuperación parcial de las mismas, puede generar pérdida importante de tejidos, con secuelas funcionales.