Variaciones de caracteres morfológicos dentro de la especie Bothrops alternatus / Morphological characters variations within the Bothrops alternatus species

Resumen El género Bothrops (yarará) es responsable de más del 95% de los accidentes por ofidios venenosos en Argentina. La mayoría de éstos son notificados en provincias del Nordeste y Noroeste. Bothrops alternatus, es la especie más frecuentementehallada, siendo el vipérido de gran talla más meridional del mundo y una de las especies de mayor importancia sanitaria en Argentina, pudiendo hallarse en relación a núcleos urbanos. Presentan en el dorso figuras arriñonadas características, que remedan atubos de teléfono o porotos unidos, completos o incompletos, hasta la forma fusionada. Presenta foseta loreal, órgano termore-ceptor ubicado entre el ojo y la fosa nasal. Su dentición es solenoglifa, dientes inoculadores huecos, con conducto cerrado, fijosa un hueso maxilar móvil. Si bien se describen variaciones entre animales, se considera una especie monotípica. Su identificaciónes importante dada la abundancia de especímenes y de hallazgos en regiones periurbanas.

Abstract The Bothrops genus is responsible for more than 95% of ophidian accidents in Argentina, most of these are reported in the Northeast and Northwest provinces. Bothrops alternatus, is the most frequent species of the genus Bothrops (yarará), themost southern large-scale viperid in the world and one of the most important species in Argentina, being able to be found in rela-tion to urban nucleus. Their characteristic features are kidney-like figures in the skin, that mimic phone tubes or beans, completeor incomplete, until the merged form. It presents a loreal pit, thermoreceptor organ located between the eye and the nostril. Itsdentition is solenoglyphous, hollow inoculating teeth, with closed duct, fixed to a mobile maxillary bone. Although variationsbetween animals are described, it is considered a monotypic species. The identification of this snake is important due their abun-dance in around urban regions and its frequency of finding.

Hemostatic evaluation of rabbits envenomed with Bothrops alternatus treated with anti-bothropic serum, desmopressin and tranexamin acid / Avaliação hemostática de coelhos envenenados com Bothrops alternatus e tratados com soro antibotrópico, desmopressina e ácido tranexâmico

In Brazil, snakes from the Bothrops genus are responsible for thousands of accidents, and their venoms are mainly composed of proteolytic enzymes. Although the antibothropic serum produced by the Brazilian Institutes is remarkably efficient, more studies are necessary, especially in veterinary medicine. The venom contain enzymes and non-enzymatic proteins that interfere with hemostasis leading to hemorrhage or even thrombosis. Possible treatment associations with known bothropic antivenom were the reason for the development of the present study. The aim of this study was to evaluate hemostasis alterations caused by Bothrops alternatus venom in rabbits followed by treatments with anti-bothropic serum, tranexamic acid and desmopressin. Twenty New Zealand rabbits were distributed into five groups (n=4) that were experimentally envenomed with 150mcg/kg of B. alternatus venom via intramuscular injection and treated as follow: Group 1 (G1) was the positive control and received venom and PBS/BSA; Group 2 (G2) was treated with tranexamic acid; Group 3 (G3) with desmopressin; Group 4 (G4) with tranexamic acid and anti-bothropic serum; and Group 5 (G5) with anti-bothropic serum and desmopressin. Blood samples were collected before venom administration, and one, four, eight and 12 hours after, for Partial activated partial thromboplastin time, Prothrombin Time, Thrombin Time and fibrinogen evaluation. Thrombin generation (TG) test was carried out with a pool of samples from final times (8 and 12h). At the end of 12h, all animals were euthanized and necropsy was conducted. Samples from muscle tissue, heart, lungs and kidney were analyzed. Classic coagulation tests showed no significant differences amongst groups and times. However, TG indicated that the venom causes a hypocoagulability state, which was not reversed by proposed treatments. Histology showed muscle inflammation, hemorrhage and necrosis, as well as hemorrhage in other tissues with no differences amongst groups. B. alternatus envenomation causes hypocoagulability detected by TG assay, but not through classical coagulation tests. The use of tranexamic acid and desmopressin for hemostasis stabilization after inoculation of the venom did not show advantage in coagulation restoration.(AU)

No Brasil, as serpentes do gênero Bothrops são responsáveis por milhares de acidentes, e seus venenos são compostos principalmente de enzimas proteolíticas. Embora o soro antiofídico produzido pelos institutos brasileiros seja notavelmente eficiente, mais estudos são necessários, especialmente na medicina veterinária. O veneno contem enzimas e proteínas não-enzimáticas que interferem com a hemostasia levando a hemorragias ou trombose. A associação de outros tratamentos ao soro antibotrópico foi a razão para o desenvolvimento do presente estudo. O objetivo deste estudo foi avaliar as alterações da hemostasia causadas pelo veneno de Bothrops alternatus em coelhos, após tratamento com soro antibotrópico, ácido tranexâmico e desmopressina. Vinte coelhos da Nova Zelândia foram distribuídos em cinco grupos (n = 4) que foram submetidos a experimentos com 150mcg/kg de veneno de B. alternatus por injeção intramuscular. O Grupo 1 (G1) foi o controle positivo e recebeu veneno e PBS / BSA, enquanto o Grupo 2 (G2) foi tratado com ácido tranexâmico, o Grupo 3 (G3) com desmopressina, o Grupo 4 (G4) com ácido tranexâmico e soro antibotrópico, e o Grupo 5 (G5) com soro antibotrópico e desmopressina. As amostras de sangue foram coletadas antes da administração do veneno, e uma, quatro, oito e 12 horas após os tratamentos para realização de tempo de tromboplastina parcial ativada parcial (TTPa), tempo de protrombina (TP), tempo de trombina (TT) e mensuração de fibrinogênio. Para o ensaio de geração de trombina (TG) foi realizado com um pool de amostras nos tempos finais (8 e 12h). Ao final das 12h, todos os animais foram sacrificados e a necropsia foi realizada. Amostras de tecido muscular, coração, pulmões e rins foram analisadas. Os testes TTPa, TP, TT e fibrinogênio não mostraram diferenças significativas entre os grupos e os tempos. No entanto, o TG indicou que o veneno causa um estado de hipocoagulabilidade, que não foi revertido pelos tratamentos propostos. Na histologia, foram observadas inflamação muscular, hemorragia e necrose, além de hemorragia em outros tecidos, sem diferenças entre os grupos. O envenenamento por B. alternatus causa hipocoagulabilidade detectada mais precocemente pelo teste de geração de trombina. O uso de ácido tranexâmico e desmopressina para estabilização da hemostasia após a inoculação do veneno não mostrou vantagem na restauração da coagulação.(AU)

BaltDC: purification, characterization and infrared spectroscopy of an antiplatelet DC protein isolated from Bothrops alternatus snake venom

Abstract Background: Snake venoms are a complex mixture of proteins, organic and inorganic compounds. Some of these proteins, enzymatic or non-enzymatic ones, are able to interact with platelet receptors, causing hemostatic disorders. The possible therapeutic potential of toxins with antiplatelet properties may arouse interest in the pharmacological areas. The present study aimed to purify and characterize an antiplatelet DC protein from Bothrops alternatus snake venom. Methods: The protein, called BaltDC (DC protein from B. alternatus snake venom), was purified by a combination of ion-exchange chromatography on DEAE-Sephacel column and gel filtration on Sephadex G-75. The molecular mass was estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE). The amino acid sequence of the N-terminal region was carried out by Edman degradation method. Platelet aggregation assays were performed in human platelet-rich plasma (PRP). Infrared (IR) spectroscopy was used in order to elucidate the interactions between BaltDC and platelet membrane. Results: BaltDC ran as a single protein band on SDS-PAGE and showed apparent molecular mass of 32 kDa under reducing or non-reducing conditions. The N-terminal region of the purified protein revealed the amino acid sequence IISPPVCGNELLEVGEECDCGTPENCQNECCDA, which showed identity with other snake venom metalloproteinases (SVMPs). BaltDC was devoid of proteolytic, hemorrhagic, defibrinating or coagulant activities, but it showed a specific inhibitory effect on platelet aggregation induced by ristocetin and epinephrine in PRP. IR analysis spectra strongly suggests that PO 3 2 groups, present in BaltDC, form hydrogen bonds with the PO 2 groups present in the non-lipid portion of the membrane platelets. Conclusions: BaltDC may be of medical interest since it was able to inhibit platelet aggregation.

BaltDC: purification, characterization and infrared spectroscopy of an antiplatelet DC protein isolated from Bothrops alternatus snake venom

Background: Snake venoms are a complex mixture of proteins, organic and inorganic compounds. Some of these proteins, enzymatic or non-enzymatic ones, are able to interact with platelet receptors, causing hemostatic disorders. The possible therapeutic potential of toxins with antiplatelet properties may arouse interest in the pharmacological areas. The present study aimed to purify and characterize an antiplatelet DC protein from Bothrops alternatus snake venom. Methods: The protein, called BaltDC (DC protein from B. alternatus snake venom), was purified by a combination of ion-exchange chromatography on DEAE-Sephacel column and gel filtration on Sephadex G-75. The molecular mass was estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE). The amino acid sequence of the N-terminal region was carried out by Edman degradation method. Platelet aggregation assays were performed in human platelet-rich plasma (PRP). Infrared (IR) spectroscopy was used in order to elucidate the interactions between BaltDC and platelet membrane. Results: BaltDC ran as a single protein band on SDS-PAGE and showed apparent molecular mass of 32 kDa under reducing or non-reducing conditions. The N-terminal region of the purified protein revealed the amino acid sequence IISPPVCGNELLEVGEECDCGTPENCQNECCDA, which showed identity with other snake venom metalloproteinases (SVMPs). BaltDC was devoid of proteolytic, hemorrhagic, defibrinating or coagulant activities, but it showed a specific inhibitory effect on platelet aggregation induced by ristocetin and epinephrine in PRP. IR analysis spectra strongly suggests that PO 3 2 − groups, present in BaltDC, form hydrogen bonds with the PO 2 − groups present in the non-lipid portion of the membrane platelets. Conclusions: BaltDC may be of medical interest since it was able to inhibit platelet aggregation.(AU)

Estudio morfológico e histoquímico de las glándulas cefálicas de Bothrops alternatus (Ophidia, Viperidae) / Morphological and histochemical study of the cephalic glands of bothrops alternatus (Ophidia, Viperidae)

El objetivo del presente trabajo fue estudiar la histología de las glándulas cefálicas de la víbora de la cruz (Bothrops alternatus), identificar los tipos celulares y caracterizar histoquimicamente el producto de secreción. Con esta finalidad fueron usados ejemplares machos adultos de B. alternatus. Después del sacrificio, las glándulas cefálicas fueron fijadas en formol neutro al 10 por ciento e incluidas en parafina. Los cortes, de 6 µm de espesor, fueron teñidos con hematoxilina y eosina, tricrómico de Masson y con técnicas histoquímicas para identificación de mucopolisacáridos y proteínas. Los resultados histológicos demostraron que las glándulas linguales, de veneno y accesorias son de tipo tubular, las glándulas labiales y premaxilar son tubuloacinares y la glándula de Harder es tubuloacinar compuesta. Las células mucosas de los adenómeros y conductos secretores de las glándulas linguales, labiales y premaxilar, así como las células de los conductos de la glándula de Harder, secretan sulfosialomucinas y mucopolisacáridos neutros. Las células mucoserosas de las glándulas linguales, labiales y premaxilar sintetizan mucopolisacáridos neutros, ácido siálico y radicales tirosina, cistina, cisteína, arginina y triptófano. Las células seromucosas de las glándulas de veneno, accesorias y de Harder producen polisacáridos neutros y radicales cistina, cisteína y triptófano, mientras que la glándula de Harder también sintetiza tirosina.

The aim of this paper was to studythe histology of the cephalic glands of the snake Bothropsalternatus, identifying the cell types and characterize histochemically the secretion product. For this purpose we used male adult specimens of B. alternatus. After slaughter, the cephalic gland was fixed in 10 percent neutral formalin and embedded in paraffin-wax. Histological sections, 6 µm thick, were stained with hematoxylin and eosin, Masson trichrome and histochemical techniques to identify mucosubstances and proteins. Histologically they were identified tubuloacinar composed glands (Harderian gland), tubuloacinar glands (upper and lower lip and premaxillary glands) and tubular (supralingual, sublingual, venom, and accessory glands). The mucous cells of the acini of the upper and lower lip glands and of the premaxillary gland, the secretory ducts of these glands and ducts of the Harderian gland secrete sulfosialomucins and neutral mucosubstances. Mucoserous cells of upper and lower lip glands and premaxillary gland secrete neutral mucosubstances, sialic acid and protein radicals tyrosine, cystine, cysteine, arginine and tryptophane. Seromucous cells of venom, accessories and Harderian glands secrete neutral mucosubstances and protein radicals cystine, cysteine and tryptophan, whereas the seromucous cells of the Harderian gland also secrete the tyrosine radical.

Neutralización heteróloga e interacción de amanitinas con venenos de Bothropsy Crotalus y de abeja / Heterologous neutralization and interaction of toxic amanitins with Bothrops and Crotalus venoms, and honey bee venom / Neutralização heteróloga e interação das amanitinas tóxicas com veneno de Bothrops e Crotalus e veneno de abelha

En el presente trabajo se examinó la interacción de las amanitinas de Amanita phalloides (Basidiomycetes) con los venenos de las serpientes Bothrops neuwiedi diporus ("yarará pequeña"), B. alternatus ("yarará grande"), Crotalus durissus terrificus ("serpiente de cascabel") y de la abeja mielera Apis mellifera. Se aplicaron las técnicas de Ouchterlony, inmunotransferencia, electroforesis rocket y electroforesis en gel de poliacrilamida a los anti-venenos y anti-toxinas obtenidos por inmunización en caballos y/o en conejos. Los anti­sueros de serpientes y las amanitinas reaccionaron en forma cruzada, así como el veneno de abeja y las amanitinas. Cuando los venenos de Bothrops neuwiedii diporus y Crotalus durissus terrificus se preincubaron con las amanitinas y se analizaron por electroforesis en gel de poliacrilamida-dodecilsulfato de sodio (dodecylsulfate-polyacrylamide gel electrophoresis: SDS-PAGE), algunas bandas de proteínas desaparecieron y otras se redujeron notablemente. Estos resultados revelan por primera vez la interacción y la degradación de las proteínas de los venenos de serpientes por las amanitinas. Por otra parte, la modificación del tiempo de coagulación de la sangre humana, debida a los venenos, se corrigió con los ciclopéptidos de Amanita. Estos resultados también se informan por primera vez en este trabajo. La presencia de polipéptidos tóxicos en los venenos de serpientes y abejas, así como en A. phalloides y la reactividad cruzada demostradas en este trabajo, sugieren la existencia de epítopos comunes a todos ellos. Teniendo en cuenta estas reacciones, el uso de anti-venenos heterólogos parece ser de utilidad en el tratamiento del envenenamiento.

In the present work, the interaction of the amanitins of Amanita phalloides (Basidiomycetes) with the venoms of Bothrops neuwiedi diporus ("small yarará snake"), B. alternatus ("big yarará"), Crotalus durissus terrificus ("rattlesnake"), and honey bee Apis mellifera was examined. Ouchterlony, immunotransfer, rocket-electrophoresis, and polyacrylamide gel electrophoresis techniques were applied to anti-venoms and anti-toxins obtained by immunization in horses and/or in rabbits. Snake antisera and amanitins cross-reacted as well as bee venom and amanitins. When venoms of Bothrops neuwiedii diporus and Crotalus durissus terrificus were preincubated with amanitins and analysed by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), some protein bands disappeared and others were significantly reduced. These results reveal for the first time the interaction and degradation of proteins in snake venoms by amanitins. Moreover, the modification of the human blood clotting time due to snake venoms was corrected by the Amanita cyclopeptides. These results are also reported for the first time in this work. The occurrence of toxic polypeptides in the snake and bee venoms as well as in A. phalloides, and the cross-reactivity demostrated herein, suggest the occurrence of epitopes common to all of them. Taking into account these reactions,the use of heterologous anti-venoms seems to be of value in envenomation treatment.

No presente trabalho foi examinada a interação das amanitinas de Amanita phalloides (Basidiomy­cetes) com os venenos das serpentes Bothrops neuwiedi diporus ("jararaca-cruzeira"), B. alternatus ("urutu"), Crotalus durissus terrificus ("serpente cascavel") e da abelha-europeia Apis mellifera. Foram aplicadas as técnicas de Ouchterlony, imunotransferência, eletroforese rocket e eletroforese em gel de poliacrilamida aos anti-venenos e anti-toxinas obtidos por imunização em cavalos e/ou em coelhos. Os anti-soros de serpentes e as amanitinas reagiram em forma cruzada, bem como o veneno de abelha e as amanitinas. Quando os venenos de Bothrops neuwiedii diporus e Crotalus durissus terrificus foram incubados previamente com as amanitinas e foram analisados por eletroforese em gel de poliacrilamida-dodecilsulfato de sódio (dodecylsulfate-polyacrylamide gel electrophoresis: SDS-PAGE), algumas faixas de proteínas desapareceram e outras se reduziram notavelmente. Estes resultados revelam por primeira vez a interação e a degradação das proteínas dos venenos de serpentes pelas amanitinas. Por outra parte, a modificação do tempo de coagulação do sangue humano, devido aos venenos, se corrigiu com os ciclopeptídeos de Amanita. Estes resultados também se informam por primeira vez neste trabalho. A presença de polipeptídeos tóxicos nos venenos de serpentes e abelhas, bem como em A. phalloides e a reatividade cruzada demonstradas neste trabalho, sugerem a existência de epítopos comuns a todos eles. Levando em consideração estas reações, o uso de anti-venenos heterólogos parece ser de utilidade no tratamento do envenenamento.

Perfil hematológico de bovinos experimentalmente inoculados com veneno de Bothrops alternatus / mentally inoculatede experimentally inoculated / with Bothrops alternatus venom

Apesar de ser alto o número de acidentes ofídicos em bovinos, as alterações hematológicas e bioquímicas séricas sãopouco estudadas, especialmente nas três primeiras semanas após o envenenamento. Diante deste contexto, objetivou-senesta pesquisa caracterizar o perfil hematológico e bioquímico sérico de bovinos após a inoculação experimental do venenode Bothrops alternatus. Cinco novilhas mestiças de peso corporal entre 188 e 278kg e idade de 12 a 18 meses receberamna altura média da face cranial do membro anterior direito, entre as articulações umerorradioulnar e do carpo, por viaintramuscular superficial, 0,15mg/kg de veneno de Bothrops alternatus diluído em salina. Coletou-se sangue de todos osanimais antes (tempo zero) e as 3, 8, 24, 48, 72, 96, 168, 240, 405 e 576 horas após inoculação do veneno para avaliaçãodo hemograma, coagulograma e dosagem de proteína total, albumina, globulinas, ureia, creatinina, creatina fosfotransferase,aspartato aminotransferase e gama glutamiltransferase. O veneno de B. alternatus causou intoxicação grave nos bovinos,e foi letal para dois animais às 53 horas e 78 horas. As alterações hematológicas observadas foram: anemia normocíticanormocrômica, leucocitose com neutrofilia com desvio para esquerda regenerativo, linfocitose, aumentos dos tempos decoagulação, de protrombina e de tromboplastina parcial ativada, trombocitopenia e elevação da ureia e das enzimas CPT eAST.

Despite the high number of Bothrops envenomation in cattle, the hematological and biochemical changes are poorly known mainly in the first few weeks after the poisoning. In this context, the aim of this study was to evaluate the bovine blood profile after B. alternatus envenomation. Five crossbred heifers were inoculated with B. alternatus venom (0.15mg/kg) diluted in saline solution, by intramuscular injection, in the mean height of the cranial side of right forelimb, between the joints umerradio-ulnar and carpus. The blood was collected before the beginning of the experiment (zero time) and at 3, 8, 24, 48, 72, 96, 168, 240, 405 e 576 hours after inoculation, in order to evaluate the hemogram, coagulogram, total protein, albumine, globulins, urea, creatinine, creatinine phosphokinase (CPT) and aspartate aminotransferase. The B. alternatus venon caused a serious envenomation in catlle and was lethal for two animals at 53 and 78 hours. The blood profile alteratons were normocitic and normocromic anaemia, leucocitosis with neutrophilia, linfocitosis, coagulation time increased, trombocytopenia and elevation in the urea, CPT and AST enzymes.

Cytotoxicity and morphological analysis of cell death induced by Bothrops venoms from the northeast of Argentina

Bothrops snake venoms have been proved toxic to a variety of cell types, in both in vivo and in vitro models. Studies on the pharmacological actions of Bothrops venoms from Argentina are relatively secarce and the direct action of the crude venoms has not been assessed using cell culture models. In this work, we investigated the cytotoxicity of crude venoms from B. alternatus and B. diporus in a skeletal muscle (C2C12) cell line, which is commonly used as a model for studying the myotoxic action of snake venom. Both venoms (1.25-50 miug/mL) induced an early and significant decrease in cell viability. The cytotoxic concentration 50 (CC50), determined three hours after exposure, revealed that B. diporus venom was significantly more cytotoxic (CC50: 2 miug/mL) than B. aftematus (CC50: 5.8 miug/mL). To investigate the cell death mechanism involved, myoblast cells were examined by phase contrast microscopy and after acridine orange and ethidium bromide fluorescence staining, respectively. Our data clearly demonstrated that an apoptotic mediated this cell line destruction. The current study aimed to provide new information on the citotoxicity meohanisms of Argentine Bothrops snake venoms on a skeletal muscle cell line

ALT-C, a disintegrin-like Cys-rich protein from Bothrops alternatus, increases skeletal myoblast viability

ALT-C, an ECD motif (glutamic acid, cysteine, aspartic acid) disintegrin from Bothrops alternatus snake venom, induces alfa2beta1 integrin-mediated signaling and neutrophil chemotaxis. In vitro, in human umbilical vein endothelial cells (HUVEC), ALT-C induces cell proliferation, thus showing an interesting potential for tissue regeneration studies. This work aimed to evaluate the influence of ALT-C in myoblast viability and differentiation. Myoblasts were obtained from hind limb muscles of 3 to 4-day old Wistar rats. The cells were incubated with ALT-C at different concentrations and incubation periods were followed by total RNA isolation. cDNA synthesis and real time polymerase chain reaction (PCR) were performed with primers of myoD as well as of both (slow and fast) myosin heavy chain isoforms (MHC). ECD-disintegrin increased myoblast viability in a dose-dependent way, mostly with 50 to 100 nM concentrations, and such effect was more prevalent after 48 hours. No changes in gene expression of both MHC isoforms were observed in ALT-C-treated cells. MyoD expression was not detected, which suggests that myoblasts were in mature stages. Protease activity and cytokine array tested in a medium of 50 nM ALT-C-treated cells after 48 hours were not different from controls. In conclusion, it was shown that myoblats are sensitive to ALT-C indicating an integrin-mediated intracellular signaling that increases cell viability.

Aspectos clínico-patológicos e laboratoriais do envenenamento experimental por Bothrops alternatus em bovinos / Clinic and pathological and laboratory aspects of experimental poisoning by Bothrops alternatus venom in cattle

Esse estudo teve como objetivo determinar as alterações clínico-patológicas e os achados laboratoriais em bovinos inoculados com a peçonha de Bothrops alternatus, no intuito de fornecer subsídios para o estabelecimento do diagnóstico e do diagnóstico diferencial, bem como esclarecer pontos obscuros da literatura pertinente. O veneno liofilizado foi diluído em 1 ml de solução fisiológica e administrado a cinco bovinos, por via subcutânea, nas doses de 0,0625, 0,125 e 0,25 mg/kg e a dois outros, por via intramuscular, nas doses de 0,25 e 0,45 mg/kg. Seis bovinos foram a óbito e um que recebeu a dose de 0,0625mg/kg, por via subcutânea, recuperou-se. Os sinais clínicos tiveram início entre 25 minutos a 5 horas 30 minutos após a inoculação. O período de evolução variou de 7 horas 18 minutos a 66 horas 12 minutos. Um animal recuperou-se após 92 horas. O quadro clínico, independentemente das doses, caracterizou-se por aumento de volume (hemorragia/hematoma) no local da inoculação, tempo de sangramento aumentado, mucosas hipocoradas e apatia. O exame laboratorial revelou progressiva anemia normocítica normocrômica, trombocitopenia, redução de fibrinogênio e proteínas plasmáticas totais, hematócrito e hemoglobina diminuídos, além de leve aumento dos níveis de creatinaquinase e desidrogenase lática. Á necropsia, havia, a partir do local da inoculação, extensos hematomas e áreas de hemorragia no tecido celular subcutâneo dos animais que receberam o veneno por via subcutânea; nos animais inoculados por via intramuscular, adicionalmente, havia hemorragia intramuscular. O endocárdio esquerdo apresentava extensas hemorragias e verificaram-se petéquias na serosa do rúmen e do omaso e na mucosa do abomaso e da vesícula biliar. Em cinco animais, o cólon, reto e região perirrenal estavam envoltos por coágulos de sangue. Ao exame histológico observou-se, além do quadro hemorragíparo, necrose muscular coagulativa, acompanhada de hemorragia, no entorno do...

The aim of this study was to determine the clinical-pathological alterations and laboratory findings in cattle inoculated with Bothrops alternatus venom, with the intention of providing information for the establishment of diagnosis and differential diagnosis procedures, as well as to elucidate some obscurities observed in the pertinent literature. The lyophilized venom was diluted in 1 ml of physiologic solution. It was administered to 5 bovines by the subcutaneous route at doses of 0.0625, 0.125 and 0.25mg/kg body weight, and to 2 bovines by the intramuscular route at doses of 0.25 e 0.45mg/kg. Six bovines died and the only animal that survived, who had subcutaneously received the venom at a dose of 0.0625mg/kg, recovered. The first clinical signs were observed from 25min to 5h30min after the inoculation. The clinical evolution time varied from 7 hours 18 minutes to 92 hours. Regardless of the dose, the clinical picture was characterized by swelling (hemorrhage/hematoma) at the site of inoculation, increase in bleeding time and capillary refill time, paleness of mucous membranes and apathy. The laboratory exams revealed progressive normocytic normochromic anemia, thrombocytopenia, reduction in fibrinogen and total plasma proteins, decreased hematocrit and hemoglobin, and slight increase in creatine kinase and lactate dehydrogenase. When subjected to necropsy, the animals that received the venom through the subcutaneous route showed large hematomas and hemorrhagic areas in the subcutaneous tissue, extending from the site of inoculation. The animals in which inoculation was performed intramuscularly exhibited, in addition, intramuscular hemorrhage. The left endocardium showed extensive hemorrhagic lesions, and petechiae were found on the serosae of rumen, omasum, abomasum and gall bladder. The colon, rectum and perirenal areas were surrounded by clotted blood in three animals. Besides hemorrhage, the histological examination revealed...

Perfil clínico e imunológico de bovinos experimentalmente inoculados com veneno bruto e iodado de Bothrops alternatus / Clinical and immunological characteristics in cattle experimentally inoculated with crude and iodinated Bothrops alternatus venom

Dez novilhas mestiças, distribuídas em dois grupos experimentais (n=5) receberam na altura média da face cranial do membro anterior direito, entre as articulações umerorradioulnar e do carpo, por via intramuscular superficial, 0,15mg/kg de veneno de Bothrops alternatus bruto ou iodado. Todos os animais foram avaliados clinicamente antes - tempo zero - e às 6 e 10h, no 2º, 3º, 4º, 5º, 8º, 11º, 18º e 25º dias após a inoculação dos venenos. Dois animais do grupo que recebeu veneno bruto foram a óbito às 53h e 78h e os sobreviventes apresentaram apatia, letargia, anorexia, postura indicativa de dor, melena, petéquias e sufusões nas mucosas, aumento do tempo de preenchimento capilar, enfartamento ganglionar regional, aumento das freqüências respiratória e cardíaca, redução da freqüência de pulsação arterial periférica, elevação da temperatura retal e diminuição da movimentação ruminal. No local da inoculação do veneno bruto houve sangramento e ulceração dérmica, além de aumento significativo na circunferência e dobra da pele do membro inoculado, revelando formação de edema. Todos os animais também foram avaliados imunologicamente no 17º, 24º, 31º, 45º, 60º e 180º dia. Somente os que receberam veneno bruto produziram anticorpos, detectados até o 45º dia. Os que receberam veneno botrópico iodado apresentaram alterações gerais e locais de menor intensidade, porém sem produção de IgG nos tempos pesquisados, demonstrando que a iodação alterou a composição bioquímica do veneno, diminuindo sua toxicidade e imunogenicidade.

The effects of bothropic envenomation in 10 crossbred heifers, randomly divided into two groups, that received 0.15mg/kg of body weight of Bothrops alternatus crude or iodinated venom were studied. Behavior; attitude; appetite; defecation; urination; mucous membranes; capillary perfusion time; lymph nodes; respiratory, cardiac and pulse frequencies; rectal temperature and rumination frequency diameter and skin fold of the foreleg on the inoculation site were observed before(zero time) and at 6 and 10h and on the 2nd, 3rd, 4th, 5th, 8th, 11th, 18th and 25th day after venom inoculation. Two cattle of Bothrops crude venom group, died at 53 and 78h and the surviving animals showed apathy, lethargy, anorexia, pain indicative attitudes, melena, hemorrhagic spots and suffusions on mucous membranes, increased capillary perfusion time, enlarged regional lymph nodes, increased respiratory and cardiac frequencies, decreased peripheric arterial pulse frequency, elevated rectal temperature and decreased of ruminal movements. Bleeding, necrotic point and increase (P< 0.05) diameter and skin fold of the foreleg were identified on the inoculated site, confirming local edema. All the animals were evaluated for Bothrops alternatus venom specific IgG on the 17th, 24th, 31st, 45th, 60th and 180th day. Only the animals receiving crude venom produced IgG specific until the 45th day. The animals inoculated with iodinated venom showed general alterations and minor local effects and did not produce specific IgG, indicating that the iodination decreased both toxicity and immunogenicity response.

Purification and functional characterization of two fibrinogenolytic enzymes from Bothrops alternatus venom

Two fibrinogenolytic enzymes, Bothrops alternatus metalloprotease isoform (BaltMP)-I and II, were purified from Bothrops alternatus venom using Diethylaminoethyl (DEAE) Sephacel, Sephadex G-75 and Heparin-Agarose column chromatography. Purified BaltMP-I and II ran as single protein bands on analytical polyacrylamide gel electrophoresis and showed molecular weights of 29000 and 36000, respectively, under reducing conditions in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). BaltMP-II, but not BaltMP-I, displayed blood-clotting activity in bovine plasma, which was about 10-fold higher than that of the crude venom. Both enzymes were proteolytically active against bovine fibrinogen as substrate. When fibrinogen and each enzyme were incubated at 37°C, at a ratio of 1:100 (w/w), BaltMP-II cleaved preferentially the Aalpha -chain and more slowly the Bbeta -chain. The action of BaltMP-I was similar, but lower. None of the proteases degraded the gamma-chain of fibrinogen. The fibrinogenolytic activity of the enzymes was inhibited by 1,10-phenanthroline, suggesting they are metalloproteases. Since both enzymes were found to cause defibrinogenation when intraperitoneally (i.p.) administered to mice, they can be of medical interest as a therapeutic agent in the treatment and prevention of arterial thrombosis.(AU)