New immunization protocol to produce crotalic antivenom combining Crotalus durissus terrificus venom and its PLA2.

Antivenoms are usually obtained by animal immunization with successive inoculations of increasing sublethal amounts of venom, which may impair the animal health. The high lethality of venom requires prolonged immunization plans with small amounts of venom. Thus, we propose an alternative plan that includes a pre-immunization of the animal with phospholipase A2, the main crotoxin component, which is responsible for the whole venom lethality. For comparison, three different immunization schemes were designed: high dose protocol (HDP; 0.5-27 mg of venom), low dose protocol (LDP; 0.1-7 mg of venom) and Mix protocol (MP; preimmunization 0.1-1.2 mg of crotalic PLA2, and then 4.5-8 mg of venom). Antibody titers were determined by ELISA, in blood plasma obtained from the marginal vein of the ear. The neutralizing ability of the different sera obtained by all protocols (HDS, LDS and MS) was tested against the most important pharmacological activities of whole venom: PLA2 activity, myotoxicity, thrombin like activity and lethality. MS showed the best neutralizing efficacy and at the same time, it was obtained by an immunization protocol that takes account of animal health care, since it requires low quantities of venoms in comparison to traditional protocols.

Local and systemic effects of BtaMP-1, a new weakly hemorrhagic Snake Venom Metalloproteinase purified from Bothriopsis taeniata Snake Venom.

A new weak hemorrhagic metalloproteinase named BtaMP-1 was purified from Bothriopsis taeniata snake venom by molecular exclusion followed by anion exchange chromatographies. This protein showed a molecular mass of 25,968.16 Da and is composed of 218 amino acid residues. The multiple alignments of its partial amino acid sequence showed high structural identity with other P-I class SVMP. BtaMP-1 showed caseinolytic activity that was enhanced by Ca2+ ion, completely inhibited by chelating and reducing agents and can be classified as an α-fibrinogenolytic enzyme. Locally, BtaMP-1 induces hemorrhage and edema, but not myotoxicity. These findings were confirmed by histological analysis of mouse gastrocnemius muscle. "In vitro" studies suggest that BtaMP-1 induce cytotoxicity in myoblast C2C12 but not in the myotubes cell line. BtaMP-1 induced systemic alterations in mice with one MHD and two hours exposure; histological analysis of lungs showed hemorrhagic areas, congestion, and increase the thickness of alveolar septum. Also, this protein induced mild effects on kidney and disruption of coagulation by depletion of fibrinogen plasma levels. This work provides insights into the importance of BtaMP-1 biological effects in envenomation by Bothropsis taeniata snake venom and providing further evidence to understand the role of P-I class SVMP in ophidian envenomation.

The effects of Cissampelos pareira extract on envenomation induced by Bothropsdiporus snake venom.

ETHNOPHARMACOLOGICAL RELEVANCE: Ophidian accidents are a serious public health problem in Argentina; the Bothrops species is responsible for 97% of these accidents, and in particular, B. diporus is responsible for 80% of them. In the northeast of the country (Corrientes Provinces), Cissampelos pareira L. (Menispermaceae) is commonly used against the venom of B. diporus; its use is described in almost all ethnobotanical literature from countries where the plant grows. AIM OF THE STUDY: In this study, the in vitro and in vivo antivenom activities of C. pareira extracts were evaluated against B. diporus venom, with a particular focus on the local effects associated with envenoming. The seasonal influence on the chemical composition of the active extracts was also studied, in order determine the associated range of variability and its influence on the antivenom activity. MATERIALS AND METHODS: This research was conducted using aerial parts (leaves, flowers, tender stems) and roots of Cissampelos pareira collected from two different phytogeographic regions of Corrientes (Argentina); Paso de la Patria and Lomas de Vallejos. In addition, to perform a seasonal analysis and to evaluate the metabolic stability, material was collected at three different growth stages. In vivo and in vitro anti-snake venom activities were tested, and a bio-guided chromatographic separation was performed in order to determine the active chemicals involved. The fractions obtained were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and the chemical profile of the most active constituent was analyzed by ultra high-performance liquid chromatography coupled to quadrupole/high-resolution mass spectrometry (Q-Orbitrap). (UHPLC-MS). RESULTS: The alcoholic extract was found to be the most active The bio-guided fractionation allowed selection one fraction to be analyzed by UHPLC-MS in order to identify the components responsible for the activities found; this identified five possible flavonoids. CONCLUSIONS: Our studies of the activity of C. pareira against the venom of B. diporus have confirmed that this species possesses inhibitory effects in both in vitro and in vivo models. Moreover, the present data demonstrate that certain flavonoids may mitigate some of the venom-induced local tissue damage.

Systemic pathological alterations caused by Philodryas patagoniensis colubrid snake venom in rats.

Very little is known about the systemic effects caused by Philodryas patagoniensis colubrid snake venom. In this work, this venom was tested for its ability to induce histopathological changes in rats after its intramuscular, subcutaneous or intravenous administration, by light microscopic examination of some organs (cerebellum, cerebrum, lung, liver, kidney and heart). Four rats were used for each dose of 0.23, 0.45 and 0.90 mg of venom in 0.3 ml of phosphate-buffered saline solution (pH 7.4). Aliquots of blood were withdrawn at different time intervals for enzymatic determination of alanine aminotransferase, aspartate aminotransferase and creatine kinase levels. After 2h the animals were killed by an overdose of anesthetic, and samples of kidney, heart, liver, lung, cerebrum and cerebellum were taken to microscopic examination (hematoxylin and eosin stain). Histologically, no abnormality was observed in heart tissue, in none of the administration routes of the venom used. However, histological observations showed multifocal hemorrhage in cerebellum, cerebrum and lung sections, severe peritubular capillary congestion in kidney sections and hydropic degeneration in liver sections, when venom was administrated intravenously. The subcutaneous route showed similar results to the previous one, with the exception of cerebellar hemorrhage. Intramuscularly, neither cerebral nor cerebellar hemorrhage was observed. Plasma alanine aminotransferase and aspartate aminotransferase increased levels were demonstrated, mainly when venom was administered intravenously or subcutaneously. Our results suggest that P. patagoniensis venom induces moderate histopathological changes in vital organs of rats. These changes are initiated at early stages of the envenomation and may be associated with a behavioral or functional abnormality of those organs during envenoming.

Equine laminitis: bites by Bothrops spp cause hoof lamellar pathology in the contralateral as well as in the bitten limb.

The envenoming caused by Bothrops snakebite includes local symptoms, such as pronounced edema, hemorrhage, intense pain, vesicles, blisters and myonecrosis. The principal systemic symptom consists in the alteration of blood clotting, due to fibrinogen consumption and platelet abnormalities. The horses involved in this study had this symptomatology and one of them exhibited symptoms consistent with laminitis in the bitten and in the contralateral limbs. Laminitis lesions were characterized by separation of the hoof lamellar basement membrane (BM) from basal cells of the epidermis. These results demonstrated that Bothrops snake venom can induce acute laminitis. We conclude that components of the venom, probably metalloproteinases, cause severe lesions in the hoof early in the envenoming process. Antivenom therapy must be initiated as soon as possible in order to prevent complications, not only to save the life of an envenomed horse, but also to avoid the dysfunctional sequels of laminitis.

First report of parasitism by Hexametra boddaertii (Nematoda: Ascaridae) in Oxyrhopus guibei (Serpentes: Colubridae).

The current study summarizes the postmortem examination of a specimen of Oxyrhopus guibei (Serpentes, Colubridae) collected in Iguazu National Park (Argentina), and found deceased a week following arrival to the serpentarium of the National Institute of Tropical Medicine (Argentina). Although the snake appeared to be in good health, a necropsy performed following its death identified the presence of a large number of roundworms in the coelomic cavity, with indications of peritonitis and serosal adherence. Additional observations from the necropsy revealed small calcifications in the mesothelium of the coelomic cavity; solid and expressive content in the gallbladder; massive gastrointestinal obstruction due to nematodes; and lung edema and congestion. Histopathological analyses of lung sections also showed proliferative heterophilic and histiocytic pneumonia. Parasites isolated from both the intestine and coelomic cavity were identified as Hexametra boddaertii by a combination of light and scanning electron microscopic examination. Results from this necropsy identify O. guibei as a new host for H. boddaertii, and is the first report of a natural infection by Hexametra in Argentina. Since Hexametra parasites may contribute to several pathological conditions in humans, and with the recent availability of O. guibei specimens through the illegal pet trade, it is necessary to consider the possibility of zoonotic helminth transmission of Hexametra from snake to human.

P9a(Cdt-PLA2) from Crotalus durissus terrificus as good immunogen to be employed in the production of crotalic anti-PLA2 IgG.

Four proteins with phospholipase A2 (PLA2) activity, designated P9a(Cdt-PLA2), P9b(Cdt-PLA2), P10a(Cdt-PLA2) and P10b(Cdt-PLA2) were purified from the venom of Crotalus durissus terrificus by two chromatographic steps: a gel filtration and reversed phase HPLC. The profile obtained clearly shows that three of them have a similar abundance. The molecular mass, 14193.8340Da for P9a(Cdt-PLA2), 14134.9102Da for P9b(Cdt-PLA2), 14242.6289Da for P10a(Cdt-PLA2) and 14183.8730Da for P10b(Cdt-PLA2), were initially evaluated by SDS-PAGE and confirmed by ESI-Q-TOF spectrometry, and all of them displayed a monomeric conformation. Also, partial amino acid sequence of each protein was obtained and their alignments with other crotalic PLA2 revealed a high degree of identity among them. Additionally, we studied some pharmacological activities like neurotoxicity, myotoxicity and lethality, which prompted us to pick two of them, P9a(Cdt-PLA2) and P10a(Cdt-PLA2) that resulted to be less toxic that the others, and further characterize them to be used as immunogen. We next injected these last proteins in mice to produce antitoxins against them and ELISA and dot blots reveled that both toxins do not show immunogenic differences, unlike those other pharmacologic activities tested. Furthermore, the antibodies produced cross-reacted with all the isoforms purified demonstrating the feasibility of using only one of them and ensuring the cross-reaction of all. The results obtained show that P9a(Cdt-PLA2) isoform has the lowest toxicity and also a good purification performance; thus this protein may be a promising candidate to be employed in the production of crotalic antitoxins.

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.

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. "Gyroxin syndrome" 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.

Effect of monospecific antibodies against baltergin in myotoxicity induced by Bothrops alternatus venom from northeast of Argentina. Role of metalloproteinases in muscle damage.

Myotoxicity, one of the most relevant local manifestations in envenomation by Bothrops genus, may result from a direct action of myotoxins or be due to an indirect vascular degeneration and ischemia. Baltergin, a snake venom metalloproteinase (SVMP), isolated from Bothrops alternatus venom has been used to obtain monospecific IgG, in order to determine the relative role of toxin in myotoxicity induced by whole venom. Bothrops diporus venom, another medical relevant genus of the northeastern region of Argentina, was also studied. Anti-baltergin IgG was able to neutralize completely the hemorrhagic activity of B. alternatus venom at an antibodies:venom ratio of 30:1 (w:w). However, mice injected with B. diporus venom showed a small spot remaining even at the highest ratio of IgG:venom assayed (50:1; w:w). Specific antibodies were efficient to neutralize the myotoxicity of B. alternatus venom at ratio 30:1 (w:w) but did not neutralize the same effects in B. diporus venom. Anti-baltergin polyclonal antibodies were useful tools for revealing the central role of SVMPs in the development of myotoxicity of B. alternatus venom, as well as, helping to suggest indirectly presence of potent myotoxic phospholipases A2 (PLA2s) in B. diporus venom.

Purification and characterization of a cysteine-rich secretory protein from Philodryas patagoniensis snake venom.

Cysteine-rich secretory proteins (CRiSPs) are widespread in reptile venoms, but most have functions that remain unknown. In the present study we describe the purification and characterization of a CRiSP (patagonin) from the venom of the rear-fanged snake Philodryas patagoniensis, and demonstrate its biological activity. Patagonin is a single-chain protein, exhibiting a molecular mass of 24,858.6 Da, whose NH(2)-terminal and MS/MS-derived sequences are nearly identical to other snake venom CRiSPs. The purified protein hydrolyzed neither azocasein nor fibrinogen, and it could induce no edema, hemorrhage or inhibition of platelet adhesion and aggregation. In addition, patagonin did not inhibit contractions of rat aortic smooth muscle induced by high K(+). However, it caused muscular damage to murine gastrocnemius muscle, an action that has not been previously described for any snake venom CRiSPs. Thus, patagonin will be important for studies of the structure-function and evolutionary relationships of this family of proteins that are widely distributed among snake venoms.

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.

Edema Y mionecrosis inducidos por veneno de Bothrops jararaca de Argentina en ratones / Edema and myonecrosis induced by Bothrops jararaca venom of Argentina in rats

Se estudiaron las actividades edematizante y mionecrótica inducidas por veneno de Bothrops jararaca de Argentina. Para la actividad edematizante se inocularon ratones con distintas dosis de veneno diluído en 0.05 ml de solución fisiológica. La dosis de 0.86 mug/20g ratón provocó un edema del 30 por ciento respecto al miembro opuesto en 1 hora. La evaluación de los efectos mionecróticos se realizó en ratones inoculados en el músculo gastroenemio con 70 mug de veneno em 0.1 ml de solución fisiológica, los que se sacrificaron en distinos tiempos de exprosición. Del área de inoculación se obtuvo material para procesamiento histopatológico. Los animales sacrificados a los 30 y 60 minutos revelaron edema y mionecrosis. Aquellos que se sacrificaron a las tres horas después de la inoculación, edemas de edema y hemorragia, presentaron necrosis e infiltrado inflamatorio.

Efectos del veneno de Bothrops alternatus de Argentina sobre músculo y distintos organos de ratones / Effect of Bothrops alternatus poison of Argentina on muscle and different organs in mices

Se inucularon ratones de 18 1 20 g de peso, en el músculo gastrocnemius con 50 mug de veneno de Bothrops alternatus, en 0,1 ml de solución fisiológica. Se conformaron grupos de 5 animales los que fueron sacrificados a las 3,6 y 12 hs posteriores a la inoculación. Se extrajo el músculo inoculado y muestras de diferentes órganos, los que fueron fijados en Bouin y procesados para histopatología. El veneno provocó una reacción inflamatoria local intensa con gran edema, congestión y hemorragia. La observación microscópica del tejido muscular, mostró minecrosis de tipo coagulativa y miolítica. A las 3 y 6 hs de exposición, en tejido hepático se observó tumefacción celular y degeneración hidrópica en reas de venas centrales. En el tejido renal se presentó congestión cortical, tumefacción hidrópica de túbulos contorneados proximales y distales. En cambio no se hallaron anormalidades en corazón, pulmón y cerebro.

Hemorragia inducida por venenos de serpientes de Argentina / Hemorrhage induced by snake venoms in Argentina

Se inocularon ratones de 18 a 20 g de peso por vía intradérmica con 0.1 ml de diluciones seriadas de veneno en solución salina amortiguada a pH 7.2. Se conformaron grupos de 4 animales los que se sacrificaron a las 2 horas de la inoculación. Se quitó la piel y se midió el área hemorrágica. Se utilizaron los venenos de Bothrops alternatus, Bothrops jararaca, Bothrops jararacussu y Bothrop neuwiedii. Todos ellos presentaron actividad hemorrágica. Crotalus durissus terrifcus no manifestó actividad hemorrágica.

Estudios de toxicidad del senecio grisebachii en ratones e identificación de componentes volatiles potencialmente toxicos / Toxicity studies of senecio grisebachii in mice and potentially toxic volatile components identification

El Senecio grisebachii es una planta de amplia distribución en el nordeste argentino que causa intoxicaciones en animales. En nuestro trabajo se alimentaron ratones con flores y hojas secas (20 por ciento de la ración) El vegetal causó intoxicación que se manisfestó por cambios en el comportamiento, perdida de peso y la histopatología revelò megalocitos y degeneración hidrópìca en zona centro lobulillar y periportal con mayor tiempo de consumo del vegetal. De distintas partes del vegetal se aislaron compuestos volátiles aún no detectados en esta planta, analizados por cromatografía gaseosa e identificados por espectrometría de masas. Los componentes identificados fueron: indol, b-mirceno, o-cimeno, ylangeno, cariofileno y a-cariofileno, que podrían sumarse a los tóxicos ya conocidos, los alcaloides pirrolizidinicos.

Lesines locales y sistemicas inducidas por veneno de Bothrops alternatus (víbora de la cruz) de Argentina / Local and sistemic lesions induced by Bothrops alternatus venom (víbora de la cruz) of Argentine

Se inocularon ratas de 220+= 20 g de peso, en grupos de 5 animales, con 800 ug de veneno de Bothrops alternatus de Argentina desecado y homogeneizado, diluído en 0,1 ml de solución salina, pore via intramuscular. Para la obtención de sangre y su posterior sacrificio, se anestesiaron a las 3, 9 y 24 horas, tomándose muestras del músculo inoculado, hígado y riñón. Se realizaron determinaciones enzimáticas y los tejidos se procesaron para histopatología. A las 3 horas, se observaron necrosis de fibras musculares confirmadas por métodos histoquímicos, hemorragia e infiltrado inflamatorio, los que se intensificaron a las 9 y 24 horas. Paralelamente se observó un incremento plasmático de lñas actividades enzimáticas de creatin fosfoquinasa (CPK), aspartato aminotransferasa (AST) y la alanina aminotransferasa (ALT); siendo máximo el aumento de CPK entre las 3 y 9 horas. La respuesta inflamatoria estudiada en ratón, mostró una reacción rápida cuya recuperación fue lenta. La necrosis de fibras musculares fue acompañada por peroxidación de lípidos y precipitación de calcio en las células. Las lesiones de te4jido hepático no fueron relevantes y en riñón se detectaron alteraciones en zona yuxtamedular y en el intersticio cortical

Intoxicación por veneno de crotalus durissus terrificus (cascabel) en ratas / Crotalus durissus terrificus (cascabel) nenom poisoning in rats

Se inocularon ratas de 180 a 220 g de peso con 120 ug/100 g de neneno desecado de Crotalus durissus terrrificus de Argentina, en 0,1 ml de soloución salina, por via i.m en grupos de 5 animales. Para la obtención de sangre y posterior sacrificio fueron anestesiados a las 3, 9 y 24 horas, tomándose muestras del músculo inoculado, del contralateral, de hígado y riñón. Se realizaron determinaciones enzimáticas y los tejidos se procesaron para histopatología. Se observó daño del tejido muscular esquelético a partir de las 3 horas, con necrosis miolítica y coagulativa. Paralelamente se pbservó un aumento plasmático de CK, AST yALT, siendo el aumento de CK máximo a las 9 horas posteriores a la inoculación. El tejido hepático se vió afectado, con congestión y dilastación de los vasos sanguíneos grandes. Los sinusoides periféricos a tales vasos presentaron hemosiderina a las 24 horas; las pequeñas vénulas contenian trombos. Los incrementos de AST y alt fueron máximos a las 24 horas. En tejido renal se observó congentión cortical a partir de 3 horas. A las 24 horas la congestión se extendió a las zonas yuxtamedulares con escasa cantidad de cilindros hialinos, Todos los animales manifestaron alteraciones neurológicasd con ptosis palpebral y midriasis bilateral, dificultad respiratoria, insensibilidad y parálisis progresiva. Se demostró que Crotalus durissus terrrificus de Argentina no solo posee actividad neurotóxica, sino también miolítica, hepatotóxica y nefrotóxica