In vitro activity of phospholipase A 2 and of peptides from Crotalus durissus terrificus venom against amastigote and promastigote forms of Leishmania (L) infantum chagasi

Background American visceral leishmaniasis is caused by the intracellular parasiteLeishmania (L.) infantum chagasi, and transmitted by the sand fly Lutzomyia longipalpis. Since treatment is based on classical chemotherapeutics with significant side effects, the search for new drugs remains the greatest global challenge. Thus, this in vitro study aimed to evaluate the leishmanicidal effect ofCrotalus durissus terrificus venom fractions on promastigote and amastigote forms of Leishmania (L.) infantum chagasi. Methods Phospholipase A 2 (PLA 2 ) and a pool of peptide fraction (<3 kDa) were purified from Crotalusvenom. Furthermore, promastigotes and peritoneal macrophages of mice infected by amastigotes were exposed to serial dilutions of the PLA 2 and peptides at intervals varying between 1.5625 μg/mL and 200 μg/mL. Both showed activity against promastigotes that varied according to the tested concentration and the time of incubation (24, 48 and 72 h). Results MTT assay for promastigotes showed IC 50 of 52.07 μg/mL for PLA2 and 16.98 μg/mL for the peptide fraction of the venom. The cytotoxicity assessment in peritoneal macrophages showed IC50 of 98 μg/mL and 16.98 μg/mL for PLA 2 and peptide by MTT assay, respectively. In peritoneal macrophages infected by Leishmania (L.) infantum chagasi amastigotes, the PLA 2 stimulated growth of parasites, and at higher doses reduced growth by 23 %. The peptide fraction prevented 43 % of the intracellular parasite growth at a dose of 16.98 μg/mL, demonstrating the toxicity of this dose to macrophages. Both fractions stimulated H 2 O 2 production by macrophages but only PLA 2 was able to stimulate NO production. Conclusion We have demonstrated the in vitro leishmanicidal activity of the PLA2 and peptide fraction ofCrotalus venom. The results encourage further studies to describe the metabolic pathways involved in cell death, as well as the prospecting of molecules with antiparasitic activity present in the peptide fraction of Crotalus durissus terrificus venom.(AU)

Envenenamento crotálico em cães / Crotalic envenomation in dogs

No Brasil, foram notificados no ano de 2011 30.826 casos de acidentes ofídicos em seres humanos, sendo que destes, 71,9% foram serpentes do gênero Bothrops. Todavia, dados do Serviço de Toxicologia do Hospital João XXIII (Belo Horizonte, MG) reportam que a maior parte dos acidentes por serpentes são decorrentes da espécie Caudisona durissa (Crotalus durissus) (ANDRADE FILHO et al., 2013). Do mesmo modo, no Hospital Veterinário da Escola de Veterinária da Universidade Federal de Minas Gerais (UFMG), o percentual de atendimentos decorrentes de picadas com serpentes da espécie C. durissa no ano de 2012 até maio de 2013 foi de 90% (foi atendido um total de 10 animais envenenados por serpentes neste período e apenas um animal foi serpente Bothrops). Os diagnósticos caninos foram realizados com base na identificação da serpente, nas manifestações clínicas e achados laboratoriais.

Aspectos clínico-patológicos e laboratoriais do envenenamento crotálico experimental em equinos / Clinical and pathological aspects of experimental crotalic envenoming in horses

Descrevem-se os quadros clínico-patológicos e laboratoriais de equinos inoculados experimentalmente com a peçonha de Caudisona durissa terrificus (Crotalus durissus terrificus na antiga nomenclatura), com a finalidade de fornecer subsídios que favoreçam a compreensão desse tipo de acidente ofídico em equinos. O veneno liofilizado foi diluído em 1ml de solução salina a 0,9% e inoculado por via subcutânea em cinco equinos, nas doses de 0,12mg/kg (um animal), 0,066mg/kg (dois animais) e 0,03mg/kg (dois animais). O veneno causou a morte do equino que recebeu a dose de 0,12mg/kg e de um dos dois que receberam a dose de 0,066mg/kg, com evolução de 27h27min e 52h29min, respectivamente. O segundo animal que recebeu a dose de 0,066mg/kg também adoeceu, mas recuperou-se após 12 dias da inoculação. A dose de 0,03mg/kg determinou quadros não fatais do envenenamento, com período de evolução que variou entre 6 e 10 dias. O quadro clínico caracterizou-se por considerável aumento de volume no local de inoculação (escápula) que se estendeu por todo o membro, apatia e cabeça baixa, alterações locomotoras evidenciadas pelo arrastar das pinças no solo, decúbito e dificuldade para levantar, redução dos reflexos auricular, palatal, do lábio superior e de ameaça, e aumento das frequências cardíaca e respiratória. Os exames laboratoriais revelaram leucocitose por neutrofilia e linfocitose em apenas dois animais. Houve aumento das enzimas creatina quinase (CK), dehidrogenase láctica (DHL) e da ureia, e também redução nos níveis séricos de cálcio, fósforo e magnésio. O tempo de tromboplastina parcial ativada (TTPA) aumentou nos equinos que morreram. Os achados de necropsia foram edema do tecido subcutâneo em todo o membro em que foi aplicado o veneno, sufusões no epicárdio dos ventrículos cardíacos esquerdo e direito, e bexiga com áreas hemorrágicas em grande parte da mucosa. Ao exame histopatológico observaram-se fígado com moderada vacuolização difusa, afetando mais a zona intermediária do lóbulo hepático, leve dilatação dos sinusoides hepáticos em algumas áreas e rim com leve dilatação dos túbulos uriníferos, principalmente no córtex.

The clinic-pathological picture and laboratory findings in horses experimentally inoculated with the venom of Caudisona durissa terrificus (Crotalus durissus terrificus, according to the former nomenclature) are described. The purpose of this study was to contribute to the understanding of this type of snake accident in horses. The lyophilized venom was diluted into 1ml of a 0.9% saline solution and was inoculated subcutaneously into five horses, at the doses of 0.12mg/kg (one horse), 0.066mg/kg (two horses) and 0.03mg/kg (two horses). The venom caused death of the horse that had received 0.12mg/kg, and of one horse of the two that had received the dose of 0.066mg/kg. The clinical course varied from 27h27min to 52h29min. The second horse inoculated with 0.066mg/kg recovered within 12 days after inoculation. The dose of 0.03mg/kg had a course of 6 to 10 days, but did not cause fatal envenomation. The clinical picture in the horses was characterized by swelling of the inoculation site (shoulder) that spread to the whole leg, by apathy and lowered head, locomotory alterations shown by dragging of the hoves on the ground, decubitus and difficulty to get up, reduction of auricular, palatal, upperlip and threat reflexes, and increase of heart and breathing frequency. The laboratory examination revealed leukocytosis and lymphocytosis in two horses. There was increase of the creatine-kinase (CK), lactic dehydrogenase (DHL) and urea, and reduction in the seric levels of calcium, phosphorus and magnesium. The activated partial tromboplastina time (TTPA) increased in the horses that died. Postmortem findings were edema of the subcutaneous tissue of the whole leg into which the venom was inoculated, suffusions in the epicard of left and right heart ventricles, and bladder with hemorrhagic areas in its mucosa. Histopatologic examination revealed the liver parenchyma with diffuse moderate vacuolation affecting predominantly the intermediate area of the hepatic lobe, and slight dilation of the sinusoides in some areas, and slight dilation of the kidney tubules mainly in the cortex.

Crotamine: A Small Basic Polypeptide Myotoxin from Rattlesnake Venom with Cell-Penetrating Properties

Crotamine, a low molecular weight cationic polypeptide from the venom of the South American rattlesnake Crotalus durissus terrificus is a natural cell-penetrating peptide with functional versatility. The presence of nine lysine residues and three disulfide bonds renders crotamine highly compact, stable and positively charged. Topologically, crotamine adopts an ancient â-defensin fold that is found in diverse families of endogenous and venom polypeptides dedicated to host defense. Crotamine is unique among several classes of bioactive peptides because it possesses both cell penetrating and antimicrobial activities and selective biological action toward some cell types at a given cell cycle phase. Because it can rapidly and efficiently translocate into actively proliferating cells, crotamine is being investigated for labeling highly replicating cells and for use as a chemotherapeutic adjuvant. Peptides derived from crotamine, nucleolar targeting peptides (NrTPs), have been designed and are being studied. NrTPs retain some crotamine properties, such as efficient cellular uptake and preferential nuclear localization whereas they improve upon other properties. For example, NrTPs are smaller than crotamine, show higher preferential nucleolar localization, and better facilitate ZIP-code localization of therapeutic proteins.

Aspectos clínico-patológicos e laboratoriais do envenenamento crotálico experimental em bovinos / Clinical and pathological aspects of experimental Crotalus poisoning in cattle

Reproduziu-se experimentalmente o envenenamento crotálico, através da inoculação, por via subcutânea, do veneno de Crotalus durissus terrificus (cascavel sul-americana) em dez bovinos mestiços. Dois animais foram utilizados como controle. O bovino que recebeu dose de 0,03mg/kg de peso corporal, morreu 7h40min após a inoculação. A dose de 0,015mg/kg causou a morte em quatro de sete bovinos inoculados, enquanto os dois animais que receberam 0,0075mg/kg adoeceram discretamente e se recuperaram. Os sintomas tiveram início entre 1h30min e 13h45min após a inoculação. A evolução oscilou entre 5h25min e 45h para os animais que morreram e entre 33h15min e 17 dias entre os animais que se recuperaram. Os principais sinais nervosos observados foram diminuição da resposta aos estímulos externos, reflexos hipotônicos, arrastar dos cascos no solo, aparente apatia, paralisia do globo ocular e da língua, decúbito esternal e lateral. Verificaram-se também adipsia e, por vezes, petéquias nas mucosas vaginal e conjuntival. Houve discreto a moderado aumento do tempo de sangramento e moderado aumento do tempo de tromboplastina parcial ativada. Houve moderada leucocitose com neutrofilia, linfopenia relativa, eosinopenia, monocitose e discreto aumento do número de bastões. Foi evidenciado significativo aumento dos níveis séricos de creatinaquinase, contudo, não foram observadas alterações significativas através da urinálise. À necropsia constataram-se edema quase imperceptível no local da inoculação, discretas petéquias e sufusões no epicárdio, omento, vesícula biliar e mucosa da bexiga em alguns dos animais envenenados experimentalmente. Os exames histopatológicos revelaram necrose (hialinização) de grupos de miócitos ou em miócitos isolados em dez diferentes músculos esqueléticos examinados, próximos ou distantes do local de inoculação em todos os animais necropsiados. Concluí-se que o envenenamento por Crotalus Sul-americanas em bovinos não cursa com mioglobinúria...

Crotalus poisoning was experimentally reproduced by subcutaneous inoculation of Crotalus durissus terrificus (South American rattlesnake) venom into 10 clinically healthy mixed bred 12 to 36-month-old cattle, weighing 125 to 449 kg. Two animals were used as controls. The animal that received a dose of 0.03mg/kg body weight died 7h40min after inoculation. A 0.015mg/kg dose provoked death in 4 out of 7 young oxen. Two animals given 0.0075mg/kg became slightly sick and recovered. Onset of symptoms occurred from 1h30min to 13h45min after inoculation. The clinical course varied from 5h25min to 45h for animals that died, and from 33h15min to 17 days for animals that recovered. The main nervous signs observed were diminished response to external stimuli, hypotonic reflexes, dragging of the hooves, apathy, difficulties in moving around obstacles, ocular globe paralysis, lateral and sternal decubitus, and tongue paralysis. Adipsia and sometimes petechiae in the conjunctival and vaginal mucosa were observed. A slight to moderate increase in bleeding time was noted in 6 animals, and a moderate increase in partial thromboplastin time was found in 7 others. Moderate leukocytosis with neutrophilia, relative lymphopenia, eosinopenia, and monocytosis was found. There was a significant increase in creatine kinase serum levels of a ten-fold order. No significant alterations were revealed by urinalysis. Necropsy revealed minimal edema at the inoculation site, few petechiae and equimoses in the epicardium, omentum, biliary vesicle and bladder mucosa of some animals. Histopathological examination revealed necrosis (hyalinization) of groups or isolated myocytes in different muscles examined, both near and far from the inoculation site, in all animals. The diagnosis of Crotalus poisoning and its differentiation from diseases causing paralysis and muscular necrosis in cattle in Brazil are discussed.

Nucleotide sequence of crotamine isoform precursors from a single South American rattlesnake (Crotalus durissus terrificus)

A cDNA phage library was constructed from venom glands of a single adult specimen of crotamine-plus Crotalus durissus terrificus (South American rattlesnake) captured in a known region. Fifteen crotamine positive clones were isolated using a PCR-based screening protocol and sequenced. These complete cDNAs clones were grouped for maximal alignment into six distinct nucleotide sequences. The crotamine cDNAs, with 340-360 bases, encompass open reading frame of 198 nucleotides with 5' and 3' untranslated regions of variable size, signal peptide sequence, one crotamine isoform message, and putative poly(A+) signal. Of these six different crotamine cDNA precursors, two predict the identical amino acid sequence previously described by , and the other four a crotamine isoform precursor where the Leucine residue at position 19 is replaced by isoleucine by a single base change. On the other hand, nucleotide variation was observed in the 5' and 3' untranslated regions, with one interesting variant containing an 18 base pair deletion at the 5' untranslated region which results in the usual ATG initiator being replaced by the rarely used GUG start codon.Comparison by Northern blot analysis of poly(A+) RNA from venom glands of a crotamine-plus specimen to total and poly(A+) RNA from a crotamine-minus snake indicated that crotamine transcripts were not expressed in the crotamine-minus specimen.