Clinical, hematological and coagulative changes consequent to antibody level increase in horses destined to antivenom production

El objetivo del trabajo consistió en estudiar los efectos clínicos, hemáticos y coagulativos, así como la producción de anticuerpos en tres equinos inmunizados para la producción de suero antiofídico monovalente contra Bothrops alternatus y evaluar los resultados obtenidos correlacionando la respuesta inmune humoral con el estado de salud de los animales. Los resultados indicaron que la inmunización de los equinos con el protocolo utilizado, indujo alteraciones clínicas menores y cambios en ciertos parámetros del hemograma y pruebas coagulativas. Se registraron variaciones de la capacidad neutralizante sobre distintos componentes del veneno, en especial las toxinas que afectan la coagulación de la sangre. Asimismo, se constató una correlación directa entre las tasas séricas de leucocitos y la producción de inmunoglobulinas. Los resultados indicaron que el período de descanso de un trimestre resulta extenso y causa una franca disminución del título de anticuerpos, perdiendo el suero capacidad neutralizante de la toxicidad del veneno; no obstante, al reanudar las inoculaciones los equinos presentaron una rápida reposición de los anticuerpos anti-toxinas de B. alternatus, tolerando altas dosis de veneno en el segundo período de inmunización.The aim of this work consisted to study the clinical and hematological effects that included blood coagulation and antibody production of a group of three horses immunized for the production of monovalent serum against Bothrops alternatus venom, in order to correlate the results with the humoral immune response of the animals. The results showed that immunization of horses with the essayed protocol induce slight clinic alterations and discrete changes in some parameters of the hemogram. Results also showed variations in the neutralizing capacity of the serum on different venom components, particularly toxins that affect blood coagulation. Also, it was verified a direct relationship between the rate of white blood cells and serum immunoglobulin production. Furthermore, they also indicate that the three-month rest period may be considered as too long as it causes a marked decrease in antibody titles, losing the serum its neutralizing capacity to venom compounds. Nevertheless, after re-administration of venom horses provided a rapid reposition of anti-toxins against B. alternatus venom, tolerating high venom doses during this second period of immunization.Fil: Bogado, F.. Universidad Nacional del Nordeste. Facultad de Cs.veterinarias; ArgentinaFil: Núñez, S.. Universidad Nacional del Nordeste. Facultad de Cs.veterinarias; ArgentinaFil: Mussart, Norma Beatriz. Universidad Nacional del Nordeste. Facultad de Cs.veterinarias; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Leiva, L.. Universidad Nacional del Nordeste. Facultad de Ciencias Exactas Naturales y Agrimensura; ArgentinaFil: Acosta, Ofelia Cristina. Universidad Nacional del Nordeste. Facultad de Cs.veterinarias; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

An alternative method to isolate protease and phospholipase A2 toxins from snake venoms based on partitioning of aqueous two-phase systems

Snake venoms are rich sources of active proteins that have been employed in the diagnosis and treatment of health disorders and antivenom therapy. Developing countries demand fast economical downstream processes for the purification of this biomolecule type without requiring sophisticated equipment. We developed an alternative, simple and easy to scale-up method, able to purify simultaneously protease and phospholipase A2 toxins from Bothrops alternatus venom. It comprises a multiple-step partition procedure with polyethylene-glycol/phosphate aqueous two-phase systems followed by a gel filtration chromatographic step. Two single bands in SDS-polyacrylamide gel electrophoresis and increased proteolytic and phospholipase A2 specific activities evidence the homogeneity of the isolated proteins.Fil: Gomez, Gabriela Noemi. Universidad Nacional del Nordeste. Facultad de Ciencias Exactas Naturales y Agrimensura. Departamento de Bioquímica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste; ArgentinaFil: Nerli, Bibiana Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Acosta, Ofelia Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste; Argentina. Universidad Nacional del Nordeste; ArgentinaFil: Picó, Guillermo Alfredo. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Leiva, Laura Cristina Ana. Universidad Nacional del Nordeste; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste; Argentin

Extraction and Identification of Alkaloids of the <i>Ipomoea fistulosa</i> (Aguapei or Mandiyurá) of Argentina

The classical toxic effect produced by this Ipomoea fistulosa is due to the accumulation of oligosacharides in various tissues cell cytoplasm, mainly nervous, liver and lymphatic tissues, that leads to the cell vacuolitation. These effects are attributed to the presence of substances called swansonina and calisteginas in the vegetables, which produce inhibition of enzymes liposomales responsible for the metabolic carbohydrates. In natural conditions, the plant is not consumed by the animals. Its consumption occurs alone in determined times of year, for lack of sufficient pasture and it is more frequent in young animals. Since standard literature does not register any data on the chemical analysis of the kind Ipomoea, we report here the results on the extraction, isolation and spectroscopic identification of the present alkaloids in this plant.Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicada

Cross-neutralization of the coagulant activity of Crotalus durissus terrificus venom from the northeast of Argentina by bivalent bothropic antivenom

Cross-neutralization of Crotalus durissus terrificus venom coagulant activity was tested using bivalent horse antivenom against Bothrops alternatus and Bothrops diporus venoms. Our in vitro and in vivo experiments showed that bothropic antivenom neutralizes the thrombin-like activity of crotalic snake venom and this cross-reaction was demonstrated by immunoassays either with whole venom or a purified thrombin-like enzyme. These results suggest common antigenic properties and, consequently, similar molecular structure among venom thrombin-like enzymes. Besides, they provide information that could be further used in the development of new antivenom formulations.Fil: Rodríguez, Juan Pablo. Universidad Nacional del Nordeste. Facultad de Ciencias Exactas Naturales y Agrimensura. Departamento de Bioquímica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste; ArgentinaFil: Gay, Claudia Carolina. Universidad Nacional del Nordeste. Facultad de Ciencias Exactas Naturales y Agrimensura. Departamento de Bioquímica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste; ArgentinaFil: Fusco, Luciano Sebastian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste; Argentina. Universidad Nacional del Nordeste. Facultad de Ciencias Exactas Naturales y Agrimensura. Departamento de Bioquímica; ArgentinaFil: Gauna Pereira, María del Carmen. Universidad Nacional del Nordeste. Facultad de Ciencias Exactas Naturales y Agrimensura. Departamento de Bioquímica; ArgentinaFil: Acosta, Ofelia Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste; Argentina. Universidad Nacional del Nordeste; ArgentinaFil: Leiva, Laura Cristina Ana. Universidad Nacional del Nordeste. Facultad de Ciencias Exactas Naturales y Agrimensura. Departamento de Bioquímica; Argentin

Purification of a fragment obtained by autolysis of a PIIIb-SVMP from Bothrops alternatus venom

Snake Venom Metalloproteinases (SVMPs) represent 43.1% of the components in Bothrops alternatus venom and play an important role in envenomation. Disintegrins and disintegrin-like domains are released by proteolytic processing of PII and PIII classes of SVMPs respectively and are potent inhibitors of integrin–ligand interaction. Baltergin is a PIIIb-SVMP isolated from this venom and able to undergo autolysis in vitro, giving rise to a stable disintegrin-like/cystein-rich fragment (baltergin-DC). Conditions of baltergin autolysis were adjusted in order to carry out the purification of baltergin-DC and its effect on cell adhesion was studied. Autolysis was maximal at 37 °C and a pH range of 7.0–8.0. Baltergin-DC amino-terminal sequence begins with IISPPVCGNELLEVGEECDCGTPENCQNECCDAATC, which shows a high degree of homology with other disintegrin-like proteins. Baltergin and purified baltergin-DC were both able to inhibit C2C12 adhesion to fetal bovine serum (FBS) coated plates, indicating that a non-catalytic process is involved, probably mediated by binding to membrane integrins. Baltergin-DC, lacking proteolytic action, becomes an attractive molecule for future studies on blocking integrin–ligand interactions.Fil: Van de Velde, Andrea Carolina. Universidad Nacional del Nordeste. Facultad de Ciencias Exactas Naturales y Agrimensura. Departamento de Bioquímica. Laboratorio de Investigación en Proteínas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Química Básica y Aplicada del Nordeste Argentino. Universidad Nacional del Nordeste. Facultad de Ciencias Exactas Naturales y Agrimensura. Instituto de Química Básica y Aplicada del Nordeste Argentino; ArgentinaFil: Gay, Claudia Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Química Básica y Aplicada del Nordeste Argentino. Universidad Nacional del Nordeste. Facultad de Ciencias Exactas Naturales y Agrimensura. Instituto de Química Básica y Aplicada del Nordeste Argentino; Argentina. Universidad Nacional del Nordeste. Facultad de Ciencias Exactas Naturales y Agrimensura. Departamento de Bioquímica. Laboratorio de Investigación en Proteínas; ArgentinaFil: Olivera Moritz, Milene Nobrega de. Universidade Federal de São Carlos; BrasilFil: dos Santos, Patty Karina. Universidade Federal de São Carlos; BrasilFil: Bustillo, Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Química Básica y Aplicada del Nordeste Argentino. Universidad Nacional del Nordeste. Facultad de Ciencias Exactas Naturales y Agrimensura. Instituto de Química Básica y Aplicada del Nordeste Argentino; ArgentinaFil: Rodríguez, Juan Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Química Básica y Aplicada del Nordeste Argentino. Universidad Nacional del Nordeste. Facultad de Ciencias Exactas Naturales y Agrimensura. Instituto de Química Básica y Aplicada del Nordeste Argentino; ArgentinaFil: Acosta, Ofelia Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste; Argentina. Universidad Nacional del Nordeste. Facultad de Ciencias Veterinarias; ArgentinaFil: Biscoglio, Mirtha Josefa. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Sobreiro Selistre de Araujo, Heloisa. Universidade Federal de São Carlos; BrasilFil: Leiva, Laura Cristina Ana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Química Básica y Aplicada del Nordeste Argentino. Universidad Nacional del Nordeste. Facultad de Ciencias Exactas Naturales y Agrimensura. Instituto de Química Básica y Aplicada del Nordeste Argentino; Argentin

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 2 h 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.Fil: Peichoto, María Elisa. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste; Argentina. Universidad Nacional del Nordeste. Facultad de Ciencias Exactas y Naturales y Agrimensura; ArgentinaFil: Teibler, Gladys Pamela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste; Argentina. Universidad Nacional del Nordeste. Facultad de Ciencias Veterinarias; ArgentinaFil: Ruíz, Raquel. Universidad Nacional del Nordeste. Facultad de Ciencias Veterinarias; ArgentinaFil: Leiva, Laura Cristina Ana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste; Argentina. Universidad Nacional del Nordeste. Facultad de Ciencias Exactas y Naturales y Agrimensura; ArgentinaFil: Acosta, Ofelia Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste; Argentina. Universidad Nacional del Nordeste. Facultad de Ciencias Veterinarias; Argentin

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.Fil: Gay, Claudia Carolina. Universidad Nacional del Nordeste. Facultad de Ciencias Exactas Naturales y Agrimensura; Argentina. Universidad Nacional del Nordeste. Facultad de Cs.veterinarias; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Nordeste; ArgentinaFil: Maruñak, Silvana. Universidad Nacional del Nordeste. Facultad de Cs.veterinarias; ArgentinaFil: Teibler, Pamela. Universidad Nacional del Nordeste. Facultad de Cs.veterinarias; ArgentinaFil: Leiva, Laura. Universidad Nacional del Nordeste. Facultad de Ciencias Exactas Naturales y Agrimensura; ArgentinaFil: Acosta, Ofelia Cristina. Universidad Nacional del Nordeste. Facultad de Cs.veterinarias; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Nordeste; Argentin

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 Na2EDTA, 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 Aα-chain faster than the Bβ-chain, whereas γ-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 μg 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 α-monomer. Inhibition studies with Na2EDTA, 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.Fil: Peichoto, María Elisa. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste; Argentina. Universidad Nacional del Nordeste. Facultad de Ciencias Exactas y Naturales y Agrimensura; ArgentinaFil: Leiva, Laura Cristina Ana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste; Argentina. Universidad Nacional del Nordeste. Facultad de Ciencias Exactas y Naturales y Agrimensura; ArgentinaFil: Guaimás Moya, L. E.. Universidad Nacional del Nordeste. Facultad de Ciencias Veterinarias; ArgentinaFil: Rey, L.. Serpentario del Zoológico de la Ciudad de Corrientes; ArgentinaFil: Acosta, Ofelia Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste; Argentina. Universidad Nacional del Nordeste. Facultad de Ciencias Veterinarias; Argentin