Bothrops envenomation in dogs: local and systemic manifestations

Background: Snakebite envenoming is a condition that affects humans and domestic animals worldwide. Identification of the snake species involved in the envenomation is infrequent. Bothrops envenomation presents typical clinicopathological features. This report describes epidemiological, clinical, and pathological data of 2 cases of Bothrops envenomation in dogs, including the first case of Bothrops moojeni snake striking a domestic animal in Brazil. Cases: Case 1. A dog was witnessed to have a Bothrops moojeni snakebite on a farm. In the first 24 h, acute lameness, pain, diffuse swelling, focal bleeding at the left forelimb, and increased whole-blood clotting time were observed in the envenomed dog. Polyvalent antivenom was administered in addition to fluid therapy, analgesics, corticosteroids, and antibiotics. On the 5th day, the animal presented spontaneous bleeding at the wound site, thrombocytopenia, and increased whole-blood clotting time. An additional dose of polyvalent antivenom was administered, and local treatment at the snakebite site was initiated. After 13 days, the dog showed no clinical or laboratory changes and recovered entirely. Case 2. A mongrel dog was taken for a necropsy to determine the cause of death. Grossly, major findings included swelling in the nasal plane that extended to the neck and dissecting hemorrhage in the subcutaneous tissue and adjacent musculature. Hemorrhages were observed in the heart, parietal pleura, left forelimb, lumbar region, and perirenal tissue. Marked necrosis and disruption of small blood vessels and lymphatics within the deep dermis and subcutaneous tissue were the main microscopic findings close to the snakebite site. Additionally, degeneration and necrosis of muscle fibers and dissecting hemorrhage were observed in the head and neck tissues surrounding the snakebite site. Kidneys showed marked interstitial hemorrhage and acute tubular nephrosis. Discussion: Bothrops envenoming is characterized by local (hemorrhage, dermonecrosis, and myonecrosis) and systemic (coagulative disorders, systemic hemorrhage, and acute kidney injury) changes due to the effect of the main venom components such as phospholipase A2 and metalloproteinases. These changes are hallmarks for the bothropic envenomation, supporting the diagnosis in cases 1 and 2. In case 1, the dog developed a Bothrops moojeni snakebite envenomation, but the immediate treatment with antivenom allowed a favorable outcome. In case 2, gross and microscopic findings supported the presumptive diagnosis of fatal bothropic envenomation. A marked local reaction such as swelling, pain, bleeding, bruising, and tissue necrosis was observed in case 1. In case 2, the most significant local changes were swelling and edema at the head and neck, hemorrhage in the subcutaneous tissue, and adjacent musculature. Systemic effects were observed clinically as spontaneous bleeding, thrombocytopenia, increased whole-blood clotting time (Case 1), systemic hemorrhages, and acute tubular nephrosis (Case 2). A proper treatment probably prevented the development of acute renal failure in Case 1. Herein, we show the first case of accidental snakebite envenomation by B. moojeni in a dog in Brazil. Information is scarce on the identification of venomous snake species striking domestic animals. Fast detection of well-determined clinical and pathological findings of Bothrops envenomation is essential for a correct diagnosis, therapeutics, and a good prognosis, even in cases with an unknown history.

P-I metalloproteinases and L-amino acid oxidases from Bothrops species inhibit angiogenesis

Snake venoms are composed of pharmacologically active proteins that are evolutionarily diverse, stable and specific to targets. Hence, venoms have been explored as a source of bioactive molecules in treating numerous diseases. Recent evidences suggest that snake venom proteins may affect the formation of new blood vessels. Excessive angiogenesis has been implicated in several pathologies including tumours, diabetic retinopathy, arthritis, inter alia. In the present study, we have examined the effects of P-I metalloproteinases isolated from Bothrops moojeni (BmMP-1) and Bothrops atrox (BaMP-1) and L-amino acid oxidases (LAAO) isolated from B. moojeni (BmLAAO) and B. atrox (BaLAAO) on biochemical and functional aspects of angiogenesis. Methods: P-I metalloproteinases and LAAO were purified from venom by molecular size exclusion and ion-exchange chromatography and subsequently confirmed using mass spectrometry. The P-I metalloproteinases were characterized by azocaseinolytic, fibrinogenolytic and gelatinase activity and LAAO activity was assessed by enzyme activity on L-amino acids. Influence of these proteins on apoptosis and cell cycle in endothelial cells was analysed by flow cytometry. The angiogenic activity was determined by in vitro 3D spheroid assay, Matrigel tube forming assay, and in vivo agarose plug transformation in mice. Results: P-I metalloproteinases exhibited azocaseinolytic activity, cleaved α and partially β chain of fibrinogen, and displayed catalytic activity on gelatin. LAAO showed differential activity on L-amino acids. Flow cytometry analysis indicated that both P-I metalloproteinases and LAAO arrested the cells in G0/G1 phase and further induced both necrosis and apoptosis in endothelial cells. In vitro, P-I metalloproteinases and LAAO exhibited significant anti-angiogenic properties in 3D spheroid and Matrigel models by reducing sprout outgrowth and tube formation. Using agarose plug transplants in mice harbouring P-I metalloproteinases and LAAO we demonstrated a marked disruption of vasculature at the periphery. Conclusion: Our research suggests that P-I metalloproteinases and LAAO exhibit anti-angiogenic properties in vitro and in vivo.(AU)

P-I metalloproteinases and L-amino acid oxidases from Bothrops species inhibit angiogenesis

Snake venoms are composed of pharmacologically active proteins that are evolutionarily diverse, stable and specific to targets. Hence, venoms have been explored as a source of bioactive molecules in treating numerous diseases. Recent evidences suggest that snake venom proteins may affect the formation of new blood vessels. Excessive angiogenesis has been implicated in several pathologies including tumours, diabetic retinopathy, arthritis, inter alia. In the present study, we have examined the effects of P-I metalloproteinases isolated from Bothrops moojeni (BmMP-1) and Bothrops atrox (BaMP-1) and L-amino acid oxidases (LAAO) isolated from B. moojeni (BmLAAO) and B. atrox (BaLAAO) on biochemical and functional aspects of angiogenesis. Methods: P-I metalloproteinases and LAAO were purified from venom by molecular size exclusion and ion-exchange chromatography and subsequently confirmed using mass spectrometry. The P-I metalloproteinases were characterized by azocaseinolytic, fibrinogenolytic and gelatinase activity and LAAO activity was assessed by enzyme activity on L-amino acids. Influence of these proteins on apoptosis and cell cycle in endothelial cells was analysed by flow cytometry. The angiogenic activity was determined by in vitro 3D spheroid assay, Matrigel tube forming assay, and in vivo agarose plug transformation in mice. Results: P-I metalloproteinases exhibited azocaseinolytic activity, cleaved α and partially β chain of fibrinogen, and displayed catalytic activity on gelatin. LAAO showed differential activity on L-amino acids. Flow cytometry analysis indicated that both P-I metalloproteinases and LAAO arrested the cells in G0/G1 phase and further induced both necrosis and apoptosis in endothelial cells. In vitro, P-I metalloproteinases and LAAO exhibited significant anti-angiogenic properties in 3D spheroid and Matrigel models by reducing sprout outgrowth and tube formation. Using agarose plug transplants in mice harbouring P-I metalloproteinases and LAAO we demonstrated a marked disruption of vasculature at the periphery. Conclusion: Our research suggests that P-I metalloproteinases and LAAO exhibit anti-angiogenic properties in vitro and in vivo.(AU)

Bothrops moojeni L-amino acid oxidase induces apoptosis and epigenetic modulation on Bcr-Abl+ cells

Resistance to apoptosis in chronic myeloid leukemia (CML) is associated with constitutive tyrosine kinase activity of the Bcr-Abl oncoprotein. The deregulated expression of apoptosis-related genes and alteration in epigenetic machinery may also contribute to apoptosis resistance in CML. Tyrosine kinase inhibitors target the Bcr-Abl oncoprotein and are used in CML treatment. The resistance of CML patients to tyrosine kinase inhibitors has guided the search for new compounds that may induce apoptosis in Bcr-Abl+ leukemic cells and improve the disease treatment. Methods: In the present study, we investigated whether the L-amino acid oxidase isolated from Bothrops moojeni snake venom (BmooLAAO-I) (i) was cytotoxic to Bcr-Abl+ cell lines (HL-60.Bcr-Abl, K562-S, and K562-R), HL-60 (acute promyelocytic leukemia) cells, the non-tumor cell line HEK-293, and peripheral blood mononuclear cells (PBMC); and (ii) affected epigenetic mechanisms, including DNA methylation and microRNAs expression in vitro. Results: BmooLAAO-I induced ROS production, apoptosis, and differential DNA methylation pattern of regulatory apoptosis genes. The toxin upregulated expression of the pro-apoptotic genes BID and FADD and downregulated DFFA expression in leukemic cell lines, as well as increased miR-16 expression - whose major predicted target is the anti-apoptotic gene BCL2 - in Bcr-Abl+ cells. Conclusion: BmooLAAO-I exerts selective antitumor action mediated by H2O2 release and induces apoptosis, and alterations in epigenetic mechanisms. These results support future investigations on the effect of BmooLAAO-I on in vivo models to determine its potential in CML therapy.(AU)

Bothrops moojeni L-amino acid oxidase induces apoptosis and epigenetic modulation on Bcr-Abl+ cells

Resistance to apoptosis in chronic myeloid leukemia (CML) is associated with constitutive tyrosine kinase activity of the Bcr-Abl oncoprotein. The deregulated expression of apoptosis-related genes and alteration in epigenetic machinery may also contribute to apoptosis resistance in CML. Tyrosine kinase inhibitors target the Bcr-Abl oncoprotein and are used in CML treatment. The resistance of CML patients to tyrosine kinase inhibitors has guided the search for new compounds that may induce apoptosis in Bcr-Abl+ leukemic cells and improve the disease treatment. Methods: In the present study, we investigated whether the L-amino acid oxidase isolated from Bothrops moojeni snake venom (BmooLAAO-I) (i) was cytotoxic to Bcr-Abl+ cell lines (HL-60.Bcr-Abl, K562-S, and K562-R), HL-60 (acute promyelocytic leukemia) cells, the non-tumor cell line HEK-293, and peripheral blood mononuclear cells (PBMC); and (ii) affected epigenetic mechanisms, including DNA methylation and microRNAs expression in vitro. Results: BmooLAAO-I induced ROS production, apoptosis, and differential DNA methylation pattern of regulatory apoptosis genes. The toxin upregulated expression of the pro-apoptotic genes BID and FADD and downregulated DFFA expression in leukemic cell lines, as well as increased miR-16 expression - whose major predicted target is the anti-apoptotic gene BCL2 - in Bcr-Abl+ cells. Conclusion: BmooLAAO-I exerts selective antitumor action mediated by H2O2 release and induces apoptosis, and alterations in epigenetic mechanisms. These results support future investigations on the effect of BmooLAAO-I on in vivo models to determine its potential in CML therapy.(AU)

Kinetic investigations and stability studies of two Bothrops L-amino acid oxidases

L-amino acid oxidases isolated from snake venoms (SV-LAAOs) are enzymes that have great therapeutic potential and are currently being investigated as tools for developing new strategies to treat various diseases, including cancer and bacterial infections. The main objective of this study was to make a brief evaluation of the enzymatic stability of two Bothrops LAAOs, one isolated from Bothrops jararacussu (BjussuLAAO-II) and the other from Bothrops moojeni (BmooLAAO-I) venoms. Methods and results: The enzymatic activity and stability of both LAAOs were evaluated by microplate colorimetric assays, for which BjussuLAAO-II and BmooLAAO-I were incubated with different L-amino acid substrates, in the presence of different ions, and at different pH ranges and temperatures. BjussuLAAO-II and BmooLAAO-I demonstrated higher affinity for hydrophobic amino acids, such as Phe and Leu. The two enzymes showed high enzymatic activity in a wide temperature range, from 25 to 75 °C, and presented optimum pH around 7.0. Additionally, Zn2+, Al3+, Cu2+ and Ni2+ ions negatively modulated the enzymatic activity of both LAAOs. As to stability, BjussuLAAO-II and BmooLAAO-I showed high enzymatic activity for 42 days stored at 4°C in neutral pH solution. Moreover, the glycan portions of both LAAOs were analyzed by capillary electrophoresis, which revealed that BjussuLAAO-II presented two main glycan portions with relative masses of 7.78 and 8.13 CGU, while BmooLAAO-I showed three portions of 7.58, 7.94 and 8.37 CGU. Conclusions: Our results showed that, when stored properly, BjussuLAAO-II and BmooLAAO-I present enzymatic stability over a long time period, which is very important to allow the use of these enzymes in pharmacological studies of great impact in the medical field.(AU)

Cytotoxic and pro-apoptotic action of MjTX-I, a phospholipase A2 isolated from Bothrops moojeni snake venom, towards leukemic cells

A leucemia mieloide crônica (LMC) é uma neoplasia mieloproliferativa BCR-ABL1 + marcada por aumento da mieloproliferação e presença de células leucêmicas resistentes à apoptose. A terapia de primeira linha atual para a LMC é a administração de inibidores da tirosina quinase, mesilato de imatinibe, dasatinibe ou nilotinibe. Embora eficaz no tratamento da LMC, alguns pacientes se tornaram resistentes a essa terapia, levando à progressão da doença e à morte. Assim, a descoberta de novos compostos para melhorar a terapia da LMC ainda é um desafio. Aqui, os destinatários se MjTX-I, uma fosfolipase A 2 isolado a partir de Bothrops moojeni de veneno de cobra, afecta a viabilidade de Bcr-Abl de mesilato de imatinib-resistente + linhas celulares. Métodos: Examinamos o efeito citotóxico e pró-apoptótico de MjTX-I em células K562-S e K562-R Bcr-Abl + e na linha de células HEK-293 não tumorais e células mononucleares de sangue periférico, usando o 3- (4, Brometo de 5-dimetiltiazol-2-il) -2,5-difeniltetrazólio e os métodos de solução fluorescente hipotônica, associados à detecção de ativação de caspases 3, 8 e 9 e clivagem de poli (ADP-ribose) polimerase (PARP). Também analisamos o potencial MjTX-I para modular a expressão de genes relacionados à apoptose em células K562-S e K562-R. Resultados: O MjTX-I diminuiu a viabilidade das células K562-S e K562-R em 60 a 65%, sem afetar a viabilidade das células não tumorais, ou seja, exerceu citotoxicidade seletiva para as linhagens celulares Bcr-Abl + . Em linhas de células leucêmicas, a toxina induziu apoptose, caspases 3, 8 e 9 ativadas, PARP clivada, expressão negativa do gene anti-apoptótico BCL-2 e expressão aumentada do gene pró-apoptótico BAD. Conclusão: O efeito antitumoral de MjTX-I está associado ao seu potencial para induzir apoptose e citotoxicidade em linhagens celulares positivas para Bcr-Abl sensíveis e resistentes ao mesilato de imatinibe, indicando que MjTX-I é um candidato promissor a fármaco para atualizar a terapia de LMC.(AU)

Cytotoxic and pro-apoptotic action of MjTX-I, a phospholipase A2 isolated from Bothrops moojeni snake venom, towards leukemic cells

Background: Chronic myeloid leukemia (CML) is a BCR-ABL1+ myeloproliferative neoplasm marked by increased myeloproliferation and presence of leukemic cells resistant to apoptosis. The current first-line therapy for CML is administration of the tyrosine kinase inhibitors imatinib mesylate, dasatinib or nilotinib. Although effective to treat CML, some patients have become resistant to this therapy, leading to disease progression and death. Thus, the discovery of new compounds to improve CML therapy is still challenging. Here we addressed whether MjTX-I, a phospholipase A2 isolated from Bothrops moojeni snake venom, affects the viability of imatinib mesylate-resistant Bcr-Abl+ cell lines. Methods: We examined the cytotoxic and pro-apoptotic effect of MjTX-I in K562-S and K562-R Bcr-Abl+ cells and in the non-tumor HEK-293 cell line and peripheral blood mononuclear cells, using the 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide and the hypotonic fluorescent solution methods, associated with detection of caspases 3, 8, and 9 activation and poly (ADP-ribose) polymerase (PARP) cleavage. We also analyzed the MjTX-I potential to modulate the expression of apoptosis-related genes in K562-S and K562-R cells. Results: MjTX-I decreased the viability of K562-S and K562-R cells by 60 to 65%, without affecting the viability of the non-tumor cells, i.e. it exerted selective cytotoxicity towards Bcr-Abl+ cell lines. In leukemic cell lines, the toxin induced apoptosis, activated caspases 3, 8, and 9, cleaved PARP, downregulated expression of the anti-apoptotic gene BCL-2, and upregulated expression of the pro-apoptotic gene BAD. Conclusion: The antitumor effect of MjTX-I is associated with its potential to induce apoptosis and cytotoxicity in Bcr-Abl positive cell lines sensitive and resistant to imatinib mesylate, indicating that MjTX-I is a promising candidate drug to upgrade the CML therapy.(AU)

Kinetic investigations and stability studies of two Bothrops L-amino acid oxidases

Background:L-amino acid oxidases isolated from snake venoms (SV-LAAOs) are enzymes that have great therapeutic potential and are currently being investigated as tools for developing new strategies to treat various diseases, including cancer and bacterial infections. The main objective of this study was to make a brief evaluation of the enzymatic stability of two Bothrops LAAOs, one isolated from Bothrops jararacussu (BjussuLAAO-II) and the other from Bothrops moojeni (BmooLAAO-I) venoms.Methods and results:The enzymatic activity and stability of both LAAOs were evaluated by microplate colorimetric assays, for which BjussuLAAO-II and BmooLAAO-I were incubated with different L-amino acid substrates, in the presence of different ions, and at different pH ranges and temperatures. BjussuLAAO-II and BmooLAAO-I demonstrated higher affinity for hydrophobic amino acids, such as Phe and Leu. The two enzymes showed high enzymatic activity in a wide temperature range, from 25 to 75 °C, and presented optimum pH around 7.0. Additionally, Zn2+, Al3+, Cu2+ and Ni2+ ions negatively modulated the enzymatic activity of both LAAOs. As to stability, BjussuLAAO-II and BmooLAAO-I showed high enzymatic activity for 42 days stored at 4°C in neutral pH solution. Moreover, the glycan portions of both LAAOs were analyzed by capillary electrophoresis, which revealed that BjussuLAAO-II presented two main glycan portions with relative masses of 7.78 and 8.13 CGU, while BmooLAAO-I showed three portions of 7.58, 7.94 and 8.37 CGU.Conclusions:Our results showed that, when stored properly, BjussuLAAO-II and BmooLAAO-I present enzymatic stability over a long time period, which is very important to allow the use of these enzymes in pharmacological studies of great impact in the medical field.(AU)

Pharmacological characterization of cnidarian extracts from the Caribbean Sea: evaluation of anti-snake venom and antitumor properties

Background: Cnidarians produce toxins, which are composed of different polypeptides that induce pharmacological effects of biotechnological interest, such as antitumor, antiophidic and anti-clotting activities. This study aimed to evaluate toxicological activities and potential as antitumor and antiophidic agents contained in total extracts from five cnidarians: Millepora alcicornis, Stichodactyla helianthus, Plexaura homomalla, Bartholomea annulata and Condylactis gigantea (total and body wall). Methods: The cnidarian extracts were evaluated by electrophoresis and for their phospholipase, proteolytic, hemorrhagic, coagulant, fibrinogenolytic, neuromuscular blocking, muscle-damaging, edema-inducing and cytotoxic activities. Results: All cnidarian extracts showed indirect hemolytic activity, but only S. helianthus induced direct hemolysis and neurotoxic effect. However, the hydrolysis of NBD-PC, a PLA2 substrate, was presented only by the C gigantea (body wall) and S. helianthus. The extracts from P. homomalla and S. helianthus induced edema, while only C gigantea and S. helianthus showed intensified myotoxic activity. The proteolytic activity upon casein and fibrinogen was presented mainly by B. annulata extract and all were unable to induce hemorrhage or fibrinogen coagulation. Cnidarian extracts were able to neutralize clotting induced by Bothrops jararacussu snake venom, except M. alcicornis. All cnidarian extracts were able to inhibit hemorrhagic activity induced by Bothrops moojeni venom. Only the C. gigantea (body wall) inhibited thrombin-induced coagulation. All cnidarian extracts showed antitumor effect against Jurkat cells, of which C. gigantea (body wall) and S. helianthus were the most active; however, only C. gigantea (body wall) and M. alcicornis were active against B16F10 cells...(AU)

Pharmacological characterization of cnidarian extracts from the Caribbean Sea: evaluation of anti-snake venom and antitumor properties

Background: Cnidarians produce toxins, which are composed of different polypeptides that induce pharmacological effects of biotechnological interest, such as antitumor, antiophidic and anti-clotting activities. This study aimed to evaluate toxicological activities and potential as antitumor and antiophidic agents contained in total extracts from five cnidarians: Millepora alcicornis, Stichodactyla helianthus, Plexaura homomalla, Bartholomea annulata and Condylactis gigantea (total and body wall). Methods: The cnidarian extracts were evaluated by electrophoresis and for their phospholipase, proteolytic, hemorrhagic, coagulant, fibrinogenolytic, neuromuscular blocking, muscle-damaging, edema-inducing and cytotoxic activities. Results: All cnidarian extracts showed indirect hemolytic activity, but only S. helianthus induced direct hemolysis and neurotoxic effect. However, the hydrolysis of NBD-PC, a PLA2 substrate, was presented only by the C gigantea (body wall) and S. helianthus. The extracts from P. homomalla and S. helianthus induced edema, while only C gigantea and S. helianthus showed intensified myotoxic activity. The proteolytic activity upon casein and fibrinogen was presented mainly by B. annulata extract and all were unable to induce hemorrhage or fibrinogen coagulation. Cnidarian extracts were able to neutralize clotting induced by Bothrops jararacussu snake venom, except M. alcicornis. All cnidarian extracts were able to inhibit hemorrhagic activity induced by Bothrops moojeni venom. Only the C. gigantea (body wall) inhibited thrombin-induced coagulation. All cnidarian extracts showed antitumor effect against Jurkat cells, of which C. gigantea (body wall) and S. helianthus were the most active; however, only C. gigantea (body wall) and M. alcicornis were active against B16F10 cells...

Antibacterial activity of different types of snake venom from the Viperidae family against Staphylococcus aureus / Atividade antibacteriana de diferentes tipos de veneno de serpentes da família Viperidae contra Staphylococcus aureus

Toxins and venoms produced by living organisms have exhibited a variety of biological activities against microorganisms. In this study, we tested seven snake venoms from the family Viperidae for antibacterial activity and the activities of reversal of antibiotic resistance and inhibition of biofilm formation against 22 clinical isolates of Staphylococcus aureus. Bothrops moojeni venom exhibited anti staphylococcal activity with the lowest mean value of minimum inhibitory concentration (MIC). Moreover, reversal of antibiotic resistance was observed for combinations of B. moojeni venom (½ x MIC) and norfloxacin or ampicillin (both ½ x MIC) for 86.4% and 50% of the isolates, respectively. B. moojeni venom alone at ½ MIC inhibited 90% of biofilm formation, whereas in combination with ciprofloxacin, both at ½ MIC, a reduction on the NorA efflux pump activity was observed. The detection of in vitro mutants colonies of S. aureus resistant to B. moojeni venom was low and they did not survive. A phospholipase A2 was purified from the venom of B. moojeni and displayed anti-staphylococcal activity when tested alone or in combination with ciprofloxacin. The results presented here will contribute to the search for new antimicrobial agents against resistant S. aureus.(AU)

Toxinas e venenos exibem uma variedade de atividades biológicas contra micro-organismos. Neste estudo, investigou-se a atividade de sete venenos de serpentes, da família Viperidae, sobre o crescimento de Staphylococcus aureus, na reversão fenotípica da resistência a antibióticos e inibição de formação de biofilme contra 22 isolados clínicos de S. aureus. O veneno de Bothrops moojeni apresentou a menor média de concentração inibitória mínima (CIM). Além disso, observou-se reversão da resistência a antibióticos para combinações do veneno de B. moojeni (½ x CIM) e norfloxacina ou ampicilina (ambos ½ x CIM) para 86,4% e 50% dos isolados, respectivamente. O veneno de B. moojeni na concentração de ½ CIM inibiu 90% de formação de biofilme, enquanto ele em combinação com ciprofloxacina, ambos na concentração de ½ CIM, diminuiu a atividade da bomba de efluxo NorA. A detecção in vitro de colônias mutantes de S. aureus resistente ao veneno de B. moojeni foi baixa e eles não sobreviveram. Uma fosfolipase A2 purificada a partir do veneno de B. moojeni exibiu atividade antibacteriana quando testada sozinha ou em combinação com ciprofloxacina. Os dados obtidos poderão contribuir para a pesquisa de novos agentes antimicrobianos contra S. aureus.(AU)

Caryospora bigenetica (Apicomplexa: Eimeriidae) in South America: new hosts and distribution records

The coccidian Caryospora bigenetica was first described in the snake Crotalus horridus (Viperidae) from United States of America. This study represents the first record of the occurrence of C. bigenetica in snakes in South America. Feces were sampled between November 2013 and May 2014 from 256 wild snakes maintained in scientific breeding facilities in the states of Mato Grosso do Sul (MS; n = 214) and Rio de Janeiro (RJ; n = 42), Brazil. Caryospora bigenetica was found in 14 (5.6%) snakes, all belonging to the family Viperidae. Ten Bothrops moojeni and two Crotalus durissus from MS were infected. The coccidian was also found in one C. durissus and in one Bothrops jararacussu from the state of RJ. The oocysts were spherical with a double wall, the exterior lightly mammillated, striations apparent in transverse view, 13.0 µm (12 14); polar granule fixed in the internal wall. Sporocysts oval or pyriform, 10.0 × 8.0 µm (9 11 × 8 9); Stieda body discoid; sub-Stieda body present; sporocyst residuum present, formed by a group of spheroid bodies between sporozoites. This study increases the number of viperid hosts of C. bigenetica and expands the geographical distribution to South America.

O coccídio Caryospora bigenetica foi descrito na serpente Crotalus horridus (Viperidae) nos Estados Unidos da América. Este estudo representa o primeiro registro da ocorrência de C. bigenetica em serpentes da América do Sul. Amostras de fezes foram obtidas, entre novembro de 2013 e maio de 2014, de 256 serpentes silvestres mantidas em um criatório científico nos Estados do Mato Grosso do Sul (MS; n = 214) e Rio de Janeiro (RJ; n = 42), Brasil. Caryospora bigenetica foi encontrada em 14 (5,6%) serpentes, todas pertencentes à família Viperidae. Dez Bothrops moojeni e duas Crotalus durissus de MS estavam infectadas. O coccídio também foi encontrado em uma C. durissus e uma Bothrops jararacussu do Estado do Rio de Janeiro. Os oocistos foram esféricos, com parede dupla, sendo a externa ligeiramente mamilonada, estriações aparentes transversalmente, 13.0 µm (12 14); grânulo polar junto à parede interna. Esporocisto oval ou piriforme, 10.0 × 8.0 µm (9 11 × 8 9); corpo de Stieda discóide; sub-Stieda presente; resíduo do esporocisto presente, formado por um grupo de corpos esféricos entre os esporozoítos. Este estudo aumenta o número de hospedeiros viperídeos de C. bigenetica e expande a distribuição geográfica para a América do Sul.

Caryospora bigenetica (Apicomplexa: Eimeriidae) in South America: new hosts and distribution records

The coccidian Caryospora bigenetica was first described in the snake Crotalus horridus (Viperidae) from United States of America. This study represents the first record of the occurrence of C. bigenetica in snakes in South America. Feces were sampled between November 2013 and May 2014 from 256 wild snakes maintained in scientific breeding facilities in the states of Mato Grosso do Sul (MS; n = 214) and Rio de Janeiro (RJ; n = 42), Brazil. Caryospora bigenetica was found in 14 (5.6%) snakes, all belonging to the family Viperidae. Ten Bothrops moojeni and two Crotalus durissus from MS were infected. The coccidian was also found in one C. durissus and in one Bothrops jararacussu from the state of RJ. The oocysts were spherical with a double wall, the exterior lightly mammillated, striations apparent in transverse view, 13.0 µm (12 14); polar granule fixed in the internal wall. Sporocysts oval or pyriform, 10.0 × 8.0 µm (9 11 × 8 9); Stieda body discoid; sub-Stieda body present; sporocyst residuum present, formed by a group of spheroid bodies between sporozoites. This study increases the number of viperid hosts of C. bigenetica and expands the geographical distribution to South America.

O coccídio Caryospora bigenetica foi descrito na serpente Crotalus horridus (Viperidae) nos Estados Unidos da América. Este estudo representa o primeiro registro da ocorrência de C. bigenetica em serpentes da América do Sul. Amostras de fezes foram obtidas, entre novembro de 2013 e maio de 2014, de 256 serpentes silvestres mantidas em um criatório científico nos Estados do Mato Grosso do Sul (MS; n = 214) e Rio de Janeiro (RJ; n = 42), Brasil. Caryospora bigenetica foi encontrada em 14 (5,6%) serpentes, todas pertencentes à família Viperidae. Dez Bothrops moojeni e duas Crotalus durissus de MS estavam infectadas. O coccídio também foi encontrado em uma C. durissus e uma Bothrops jararacussu do Estado do Rio de Janeiro. Os oocistos foram esféricos, com parede dupla, sendo a externa ligeiramente mamilonada, estriações aparentes transversalmente, 13.0 µm (12 14); grânulo polar junto à parede interna. Esporocisto oval ou piriforme, 10.0 × 8.0 µm (9 11 × 8 9); corpo de Stieda discóide; sub-Stieda presente; resíduo do esporocisto presente, formado por um grupo de corpos esféricos entre os esporozoítos. Este estudo aumenta o número de hospedeiros viperídeos de C. bigenetica e expande a distribuição geográfica para a América do Sul.

Caryospora bigenetica (Apicomplexa: Eimeriidae) in South America: new hosts and distribution records / Caryospora bigenetica (Apicomplexa: Eimeriidae) na América do Sul: Registro de novos hospedeiros e distribuição

The coccidian Caryospora bigenetica was first described in the snake Crotalus horridus (Viperidae) from United States of America. This study represents the first record of the occurrence of C. bigenetica in snakes in South America. Feces were sampled between November 2013 and May 2014 from 256 wild snakes maintained in scientific breeding facilities in the states of Mato Grosso do Sul (MS; n = 214) and Rio de Janeiro (RJ; n = 42), Brazil. Caryospora bigenetica was found in 14 (5.6%) snakes, all belonging to the family Viperidae. Ten Bothrops moojeni and two Crotalus durissus from MS were infected. The coccidian was also found in one C. durissus and in one Bothrops jararacussu from the state of RJ. The oocysts were spherical with a double wall, the exterior lightly mammillated, striations apparent in transverse view, 13.0 µm (12 14); polar granule fixed in the internal wall. Sporocysts oval or pyriform, 10.0 × 8.0 µm (9 11 × 8 9); Stieda body discoid; sub-Stieda body present; sporocyst residuum present, formed by a group of spheroid bodies between sporozoites. This study increases the number of viperid hosts of C. bigenetica and expands the geographical distribution to South America.(AU)

O coccídio Caryospora bigenetica foi descrito na serpente Crotalus horridus (Viperidae) nos Estados Unidos da América. Este estudo representa o primeiro registro da ocorrência de C. bigenetica em serpentes da América do Sul. Amostras de fezes foram obtidas, entre novembro de 2013 e maio de 2014, de 256 serpentes silvestres mantidas em um criatório científico nos Estados do Mato Grosso do Sul (MS; n = 214) e Rio de Janeiro (RJ; n = 42), Brasil. Caryospora bigenetica foi encontrada em 14 (5,6%) serpentes, todas pertencentes à família Viperidae. Dez Bothrops moojeni e duas Crotalus durissus de MS estavam infectadas. O coccídio também foi encontrado em uma C. durissus e uma Bothrops jararacussu do Estado do Rio de Janeiro. Os oocistos foram esféricos, com parede dupla, sendo a externa ligeiramente mamilonada, estriações aparentes transversalmente, 13.0 µm (12 14); grânulo polar junto à parede interna. Esporocisto oval ou piriforme, 10.0 × 8.0 µm (9 11 × 8 9); corpo de Stieda discóide; sub-Stieda presente; resíduo do esporocisto presente, formado por um grupo de corpos esféricos entre os esporozoítos. Este estudo aumenta o número de hospedeiros viperídeos de C. bigenetica e expande a distribuição geográfica para a América do Sul.(AU)

Preliminary assessment of Hedychium coronarium essential oil on fibrinogenolytic and coagulant activity induced by Bothrops and Lachesis snake venoms

The search for new inhibitors of snake venom toxins is essential to complement or even replace traditional antivenom therapy, especially in relation to compounds that neutralize the local effects of envenomations. Besides their possible use as alternative to traditional antivenom therapy, some plant species possess bioactive secondary metabolites including essential oils, which can be extracted from weeds that are considered substantial problems for agriculture, such as Hedychium coronarium. The essential oils of leaves and rhizomes from H. coronarium were extracted by hydrodistillation, and their potential inhibitory effects on the coagulant and fibrinogenolytic activities induced by the venoms of Lachesis muta, Bothrops atrox and Bothrops moojeni were analyzed. Citrated human plasma was used to evaluate the clotting time whereas changes in fibrinogen molecules were visualized by electrophoresis in polyacrylamide gel. The experimental design used for testing coagulation inhibition was randomized in a 3 × 2 factorial arrangement (concentration × essential oils), with three replications. The essential oils were compared since they were extracted from different organs of the same botanical species, H. coronarium. The results suggest that the oils interact with venom proteases and plasma constituents, since all oils evaluated, when previously incubated with venoms, were able to inhibit the clotting effect, with less inhibition when oils and plasma were preincubated prior to the addition of venoms. Thus, after extensive characterization of their pharmacological and toxicological effects, the essential oils can be used as an alternative to complement serum therapy, especially considering that these plant metabolites generally do not require specific formulations and may be used topically immediately after extraction.(AU)

Preliminary assessment of Hedychium coronarium essential oil on fibrinogenolytic and coagulant activity induced by Bothrops and Lachesis snake venoms

The search for new inhibitors of snake venom toxins is essential to complement or even replace traditional antivenom therapy, especially in relation to compounds that neutralize the local effects of envenomations. Besides their possible use as alternative to traditional antivenom therapy, some plant species possess bioactive secondary metabolites including essential oils, which can be extracted from weeds that are considered substantial problems for agriculture, such as Hedychium coronarium. The essential oils of leaves and rhizomes from H. coronarium were extracted by hydrodistillation, and their potential inhibitory effects on the coagulant and fibrinogenolytic activities induced by the venoms of Lachesis muta, Bothrops atrox and Bothrops moojeni were analyzed. Citrated human plasma was used to evaluate the clotting time whereas changes in fibrinogen molecules were visualized by electrophoresis in polyacrylamide gel. The experimental design used for testing coagulation inhibition was randomized in a 3 × 2 factorial arrangement (concentration × essential oils), with three replications. The essential oils were compared since they were extracted from different organs of the same botanical species, H. coronarium. The results suggest that the oils interact with venom proteases and plasma constituents, since all oils evaluated, when previously incubated with venoms, were able to inhibit the clotting effect, with less inhibition when oils and plasma were preincubated prior to the addition of venoms. Thus, after extensive characterization of their pharmacological and toxicological effects, the essential oils can be used as an alternative to complement serum therapy, especially considering that these plant metabolites generally do not require specific formulations and may be used topically immediately after extraction.

Monitoramento e Estudos moleculares de ovos de serpentes do gênero Bothrops

Os Squamatas são a única linhagem de répteis com espécies que dão à luz neonatos de vida livre (viviparidade), capazes de viver independente dos tecidos extraembrionários. Em muitas espécies de viperídeos a reprodução é sazonal, mas nem todas as fêmeas se reproduzem num dado ano. Também ocorrem variações intra e inter-específicas em relação ao acasalamento e atividade folicular, permitindo assim mudanças hormonais responsáveis por eventos fisiológicos. Rotineiramente é reportado o depósito, na natureza, de ovos inférteis e anormais tanto em espécies ovíparas quanto vivíparas de Squamatas. A atresia folicular é um processo degenerativo, hormonalmente controlado, pelo qual folículos ovarianos, em variados estágios de desenvolvimento e crescimento, de vertebrados mamíferos e não mamíferos, perdem sua integridade e são eliminados antes da ovulação. A apoptose, mecanismo fundamental de remoção de células germinativas, um programa de morte celular extremamente regulado e de grande eficiência. Com o objetivo monitorar e investigar o elevado número de ovos atrésicos liberados pelas serpentes do gênero Bothrops em cativeiro, investigamos possíveis processos de morte celular por apoptose com abordagens moleculares. Foram identificados os possíveis processos envolvidos na atresia de embriões de serpentes do gênero Bothrops. Os ovos atrésicos mostraram diferentes estadios de desenvolvimento, diferenciação e maturação dos precursores e de células-tronco envolvidos na organogênese destas espécies de serpente. A hipótese: ovo atrésico é fecundado?, neste estudo foi confirmada pela identificação de botões germinativos em diferentes fases de organogênese, que expressaram diferencialmente marcadores moleculares (CD-34, CD-44, CD-90, CD-117, Stro-1 e Caspase-3) envolvidos na cinética de crescimento e diferenciação celular, modulados pelo estresse oxidativo. Outros aspectos relevantes neste estudo envolvidos na atresia destes embriões foi o processo de partenogênese encontrado nas espécies B. moojeni, B. leucurus e B. erythromelas, nesta última foi inovador. B. insularis apresentou elevado número de ovos atrésicos não fecundados, a presença de intersexo entre fêmeas reprodutivas e as condições ambientais no cativeiro.

Squamatas are the only strain of reptiles with species that give birth to free-living neonates (viviparity), capable of living independently of extraembryonic tissues. In many Viperidae species reproduction is seasonal, but not all females reproduce in a given year. Intra and interspecific variations also occur in relation to mating and follicular activity, thus allowing hormonal changes responsible for physiological events. The routine deposit of infertile and abnormal eggs in both oviparous and viviparous Squamata species is routinely reported. Follicular atresia is a hormonally controlled degenerative process by which ovarian follicles, at various stages of development and growth, of mammalian and non-mammalian vertebrates, lose their integrity and are eliminated before ovulation. Apoptosis, a fundamental mechanism for germinal cell removal, a highly regulated and highly efficient cell death program. In order to monitor and investigate the high number of atretic eggs released by captive Bothrops snakes, we investigated possible apoptotic cell death processes with molecular approaches. Possible processes involved in the atresia of Bothrops snake embryos were identified. The atretic eggs showed different stages of development, differentiation and maturation of the precursors and stem cells involved in the organogenesis of these snake species. The hypothesis: Is atretic egg fertilized? In this study was confirmed by the identification of germinal discs in different stages of organogenesis, which differentially expressed molecular markers (CD-34, CD-44, CD-90, CD-117, Stro- 1 and Caspase-3) involved in growth kinetics and cell differentiation, modulated by oxidative stress. Other relevant aspects in this study involved in the atresia of these embryos was the parthenogenesis process found in B. moojeni, B. leucurus and B. erythromelas species, in the last one was innovative. B. insularis showed a high number of unfertilized atretic eggs, the presence of intersex between reproductive females and environmental conditions in captivity.

Circadian pattern of Bothrops moojeni in captivity (Serpentes: Viperidae)

Members of the subfamily Crotalinae are considered to be essentially nocturnal and most of the data about these snakes have been collected from the field. Information on how nutritional status affects the movement rate and activity patterns is a key point to elucidating the ecophysiology of snakes. In this study, we distributed 28 lancehead Bothrops moojeni into three groups under distinct feeding regimens after a month of fasting. Groups were divided as follows: ingestion of meals weighing (A) 40 percent, (B) 20 percent, or (C) 10 percent of the snake body mass. Groups were monitored for five days before and after food intake and the activity periods and movement rates were recorded. Our results show that B. moojeni is prevalently nocturnal, and the activity peak occurs in the first three hours of the scotophase. After feeding, a significant decrease in activity levels in groups A and B was detected. The current results corroborate previous field data that describe B. moojeni as a nocturnal species with low movement rates. The relationship between motion and the amount of food consumed by the snake may be associated with its hunting strategy.(AU)

Aspectos clinicopatológicos e laboratoriais do envenenamento experimental por Bothrops moojeni e Bothropoides neuwiedi em ovinos / Clinicopathological and laboratory aspects of experimental poisoning by Bothrops moojeni and Bothropoides neuwiedi snake venoms in sheep

O envenenamento ofídico espontâneo, ou acidente ofídico, é descrito como causa de morte em animais domésticos. No entanto, dados concretos relativos ao gênero e espécie de serpente envolvida, à evolução do quadro clínico, e às alterações clinicopatológicas desenvolvidas, são escassos. Assim sendo, este trabalho teve como objetivo determinar as alterações clinicopatológicas e laboratoriais provocadas pelo veneno de Bothrops moojeni e Bothropoides neuwiedi em ovinos no intuito de fornecer informações adicionais referentes a acidentes ofídicos em animais de produção, auxiliando o estabelecimento do diagnóstico dessa condição. Os venenos liofilizados foram diluídos em 1 ml de solução fisiológica e administrados a quatro ovinos por via subcutânea na face direita, nas doses de 0,41mg/kg e 0,82mg/kg do veneno de B. moojeni em dois ovinos, e de 1,0mg/kg do veneno de B. neuwiedi em dois ovinos. Apenas o ovino que recebeu a menor dose (0,41mg/kg) sobreviveu, apesar de ter desenvolvido quadro clínico muito severo e semelhante aos demais. Os sinais clínicos iniciaram nos primeiros 10 minutos após a inoculação em todos os ovinos. O período de evolução variou de dois a quatro dias. O quadro clínico dos quatro ovinos caracterizou-se por apatia, acentuado aumento de volume da face, da porção ventral do pescoço e do peito, leve aumento de volume da porção proximal dos membros anteriores, tempo de sangramento aumentado, taquicardia, mucosas pálidas e grande quantidade de sangue não digerido nas fezes. Ao exame laboratorial observou-se principalmente redução das proteínas plasmáticas e aumento de creatinaquinase em todos os ovinos. À necropsia, foram observados extensos hematomas nas áreas correspondentes ao aumento de volume subcutâneo. Observaram-se petéquias, equimoses e sufusões leves a moderadas na serosa de diversos órgãos e acúmulo de sangue em meio às fezes na porção final do reto. Além de hemorragias, a principal alteração histopatológica observada foi necrose das fibras musculares esqueléticas e da parede de vasos, nas áreas próximas à inoculação do veneno. Nos ovinos deste estudo o aumento de volume, observado na face, pescoço, peito e membros, era constituído por sangue.(AU)

Spontaneous envenoming by snake bite is described as a cause of death in domestic animals. However, there are just few information about the species of snake involved, course, and clinicopathological and laboratory findings. Thus, this research aimed to determine the clinicopathological and laboratory changes induced by Bothrops moojeni and Bothropoides neuwiedi snake venoms in sheep, in order to provide additional information regarding snakebites in farm animals and to help establish the diagnosis of this condition. The lyophilized snake venoms were dissolved in 1mL saline solution and administered subcutaneously into the right face of four sheep, at doses of 0.41mg/kg and 0.82mg/kg of B. moojeni venom for two sheep, and 1.0mg/kg of B. neuwiedi venom for two other sheep. Only the sheep which had received the lowest dose (0.41mg/kg) survived, but developed severe clinical signs, similar to the others. First clinical signs were observed about 10 minutes after inoculation in all sheep. The course varied from 2 to 4 days. The clinical findings in all sheep were characterized by apathy, marked swelling of the face, the ventral neck and esternal region, and mild swelling of the proximal portion of the forelimbs, as well as increased bleeding time, tachycardia, pale mucous membranes, and large quantity of undigested blood in the intestinal lumen. Laboratory exams showed mainly a reduction in serum protein and increased creatine kinase in all sheep. At necropsy, extensive hematomas were observed in the subcutaneous tissue of the swollen areas. Also petechiae, bruises and mild to moderate hemorrhagic suffusions on the serosa of various organs, and blood within the intestinal contents of the distal rectum were observed. In addition to hemorrhages, the main histopathological changes were necrosis of skeletal muscle fibers and blood vessel walls next to the inoculation site. The swollen areas on face, neck, sternum and limbs of the sheep were due the hematomas.(AU)