Microbiological evaluation of different strategies for management of snakes in captivity

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Young ovine death during hyperimmunization: crotalic envenomation or copper toxicosis?

The unfavorable evolution of a young ovine during hyperimmunization process with Crotalus durissus terrificus venom was investigated in order to differentiate its origin between ophidic envenomation and copper toxicosis. Clinical, laboratory, necroscopic and histological exams as well as evaluation and measurement of heavy metals (copper) in the kidneys and in the liver were carried out. Blood counts revealed anemia and serological tests showed high levels of blood urea nitrogen, creatinine, aspartate aminotransferase, creatine phosphokinase, total bilirubin and indirect bilirubin; which indicates liver, kidney and skeletal muscle damages. At necropsy, the animal presented hepatopathy and nephropathy. Histological examination revealed renal and hepatic features that may imply copper intoxication. Copper levels were 237.8 mu g/g in the liver and 51.2 mu g/g in the kidneys. Although the amount of metal found in both organs was below the level that can cause death, according to the literature, anatomopathological signs were suggestive of copper intoxication. Therefore, the hypothesis of metal toxicosis during the hyperimmunization process became more consistent than the crotalic envenomation one.

Ação neuro-muscular do veneno crotálico: dados preliminares

Estudamos 6 pacientes, 2 cães e um coelho com intoxicação crotálica. Avaliamos a condução nervosa periférica sensitiva e motora, a transmissão neuromuscular e eletromiografias. As biópsias de músculo foram processadas por histoquímica. Os 6 pacientes apresentaram mononeuropatia sensitiva no nervo periférico adjacente ao local da inoculação do veneno e encontramos evidências histoquímicas de miopatia mitocondrial. Os defeitos da transmissão neuromuscular foram mínimos. A maioria dos autores admite que veneno crotálico determina síndrome miastênica. Nossos achados indicam que ptose palpebral, facies miastênico e fraqueza muscular observados após acidente crotálico, correspondem provavelmente a miopatia mitocondrial, muitas vezes transitória e reversível.

cDNA sequence and molecular modeling of a nerve growth factor from Bothrops jararacussu venomous gland

The complete nucleotide sequence of a nerve growth factor precursor from Bothrops jararacussu snake (Bj-NGF) was determined by DNA sequencing of a clone from cDNA library prepared from the poly(A) + RNA of the venom gland of B.jararacussu. cDNA encoding Bj-NGF precursor contained 723 bp in length, which encoded a prepro-NGF molecule with 241 amino acid residues. The mature Bj-NGF molecule was composed of I 18 amino acid residues with theoretical pI and molecular weight of 8.31 and 13,537, respectively. Its amino acid sequence showed 97%, 96%, 93%, 86%, 78%, 74%, 76%, 76% and 55% sequential similarities with NGFs from Crotalus durissus terrificus, Agkistrodon halys pallas, Daboia (Vipera) russelli russelli, Bungarus multicinctus, Naja sp., mouse, human, bovine and cat, respectively. Phylogenetic analyses based on the amino acid sequences of 15 NGFs separate the Elapidae family (Naja and Bungarus) from those Crotalidae snakes (Bothrops, Crotalus and Agkistrodon). The three-dimensional structure of mature Bj-NGF was modeled based on the crystal structure of the human NGF. The model reveals that the core of NGF, formed by a pair of P-sheets, is highly conserved and the major mutations are both at the three beta-hairpin loops and at the reverse turn. (C) 2002 Societe francaise de biochimie et biologic moleculaire/Editions scientifiques et medicales Elsevier SAS. All rights reserved.

Crotoxin: Novel activities for a classic beta-neurotoxin

Crotoxin, the main toxin of South American rattlesnake (Crotalus durissus terrificus) venom, was the first snake venom protein to be purified and crystallized. Crotoxin is a heterodimeric beta-neurotoxin that consists of a weakly toxic basic phospholipase A(2) and a nonenzymatic, non-toxic acidic component (crotapotin). The classic biological activities normally attributed to crotoxin include neurotoxicity, myotoxicity, nephrotoxicity and cardiotoxicity. However, numerous studies in recent years have shown that crotoxin also has immunomodulatory, anti-inflammatory, anti-microbial, anti-tumor and analgesic actions. In this review, we describe the historical background to the discovery of crotoxin and its main toxic activities and then discuss recent structure-function studies and investigations that have led to the identification of novel pharmacological activities for the toxin. (C) 2010 Elsevier Ltd. All rights reserved.

Identification of continuous interaction sites in PLA(2)-based protein complexes by peptide arrays

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Prevalence of Hepatozoon spp. (Apicomplexa, Hepatozoidae) among recently captured Brazilian snakes

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

The role of nitric oxide in regulation of the cardiovascular system in reptiles

The roles that nitric oxide (NO) plays in the cardiovascular system of reptiles are reviewed, with particular emphasis on its effects on central vascular blood flows in the systemic and pulmonary circulations. New data is presented that describes the effects on hemodynamic variables in varanid lizards of exogenously administered NO via the nitric oxide donor sodium nitroprusside (SNP) and, preliminary data on the effects of SNP inhibition of nitric oxide synthase (NOS) by L-nitroarginine methyl ester (L-NAME). Furthermore. on hemodynamic variables in the tegu lizard are presented. The findings are compared with previously published data from Our laboratory on three other species of reptiles: pythons (Skovgaard, N., Galli, G., Taylor, E.W., Conlon, J.M., Wang.. T., 2005. Hemodynamic effects of python neuropeptide gamma in the anesthetized python, Python regius. Regul. Pept. 18, 15-26), rattlesnakes (Galli, G., Skovgaard, N., Abe, A.S., Taylor, E.W., Wang, T., 2005. The role of nitric oxide in the regulation of the systemic and the pulmonary vasculature of the rattlesnake, Crotalus durissus terrificus. J. Comp. Physiol. 175B, 201-208) and turtles (Crossley, D.A., Wang, T., Altimiras, J., 2000. Role of nitric oxide in the systemic and pulmonary circulation of anesthetized turtles (Trachemys scripta). J. Exp. Zool. 286, 683-689). These five species of reptiles possess different combinations of division of the heart and structural complexity of the lungs. Comparison of their responses to NO donors and NOS inhibitors may reveal whether the potential contribution of NO to vascular tone correlates with pulmonary complexity and/or with blood pressure. All existing studies oil reptiles have clearly established a potential role for NO in regulating vascular tone in the systemic circulation and NO may be important for maintaining basal systemic vascular tone in varanid lizards, pythons and turtles, through a continuous release of NO. In contrast., the pulmonary circulation is less responsive to NO donors or NOS inhibitors, and it was only in pythons and varanid lizards that the lungs responded to SNP. Both species have a functionally separated heart, so it is possible that NO may exert a larger role in species with low pulmonary blood pressures, irrespective of lung complexity. (C) 2005 Elsevier B.V. All rights reserved.

Minimal volume regulation after shrinkage of red blood cells from five species of reptiles

Red blood cells (RBCs) from most vertebrates restore volume upon hypertonic shrinkage and the mechanisms underlying this regulatory volume increase (RVI) have been studied extensively in these cells. Despite the phylogenetically interesting position of reptiles, very little is known about their red cell function. The present study demonstrates that oxygenated RBCs in all major groups of reptiles exhibit no or a very reduced RVI upon -25% calculated hyperosmotic shrinkage. Thus, RBCs from the snakes Crotalus durissus and Python regius, the turtle Trachemys scripta and the alligator Alligator mississippiensis showed no statistically significant RVI within 120 min after shrinkage, while the lizard Tupinambis merianae showed 22% volume recovery after 120 min. Amiloride (10(-4) M) and bumetanide (10(-5) M) had no effect on the RVI in T merianae, indicating no involvement of the Na(+)/H(+) exchanger (NHE) or the Na(+)/K(+)/2Cl(-) co-transporter (NKCC) or insentive transporters. Deoxygenation of RBCs from A. mississippiensis and T merianae did not significantly affect RVI upon shrinkage. Deoxygenation per se of red blood cells from T merianae elicited a slow volume increase, but the mechanism was not characterized. It seems, therefore, that the RVI response based on NHE activation was lost among the early sauropsids that gave rise to modern reptiles and birds, while it was retained in mammals. An RVI response has then reappeared in birds, but based on activation of the NKCC. Alternatively, the absence of the RVI response may represent the most ancient condition, and could have evolved several times within vertebrates. (C) 2008 Elsevier B.V. All rights reserved.

Physiological importance of the coronary arterial blood supply to the rattlesnake heart

The reptilian heart consists of a thick inner spongy myocardium that derives its oxygen and nutrient supply directly from the blood within the ventricular cavity, which is surrounded by a thin outer compact layer supplied by coronary arteries. The functional importance of these coronary arteries remains unknown. In the present study we investigate the effects of permanent coronary artery occlusion in the South American rattlesnake (Crotalus durissus) on the ability to maintain heart rate and blood pressure at rest and during short term activity. We used colored silicone rubber (Microfil) to identify the coronary artery distribution and interarterial anastomoses. The coronary circulation was occluded and the snakes were then kept for 4 days at 30 degrees C. Microfil injections verified that virtually all coronary arteries had successfully been occluded, but also made visible an extensive coronary supply to the outer compact layer in untreated snakes. Electrocardiogram (ECG), blood pressure (P(sys)) and heart rate (f(H)) were measured at rest and during enforced activity at day 1 and 4. Four days after occlusion of the coronary circulation, the snakes could still maintain a P(sys) and f(H) of 5.2 +/- 0.2 kPa and 58.2 +/- 2.2 beats min(-1), respectively, during activity and the ECG was not affected. This was not different from sham-operated snakes. Thus, while the outer compact layer of the rattlesnake heart clearly has an extensive coronary supply, rattlesnakes sustain a high blood pressure and heart rate during activity without coronary artery blood supply.

Crotacetin, a novel snake venom C-type lectin homolog of convulxin, exhibits an unpredictable antimicrobial activity

Snake venom (sv) C-type lectins encompass a group of hemorrhagic toxins that are capable of interfering with blood stasis. A very well-studied svC-type lectin is the heterodimeric toxin, convulxin (CVX), from the venom of South American rattlesnake Crotalus durissus terrificus. CVX is able to activate platelets and induce their aggregation by acting via p62/GPVI collagen receptor. By using polymerase chain reaction homology screening, we have cloned several cDNA precursors of CVX subunit homologs. One of them, named crotacetin (CTC) beta-subunit, predicts a polypeptide with a topology very similar to the tridimensional conformations of other subunits of CVX-like snake toxins, as determined by computational analysis. Using gel permeation and reverse-phase high-performance liquid chromatography, CTC was purified from C. durissus venoms. CTC can be isolated from the venom of several C. durissus subspecies, but its quantitative predominance is in the venom of C. durissus cascavella. Functional analysis indicates that CTC induces platelet aggregation, and, importantly, exhibits an antimicrobial activity against Gram-positive and -negative bacteria, comparable with CVX.

Crystallization and preliminary X-ray crystallographic analysis of the heterodimeric crotoxin complex and the isolated subunits crotapotin and phospholipase A(2)

Crotoxin, a potent neurotoxin from the venom of the South American rattlesnake Crotalus durissus terrificus, exists as a heterodimer formed between a phospholipase A(2) and a catalytically inactive acidic phospholipase A(2) analogue (crotapotin). Large single crystals of the crotoxin complex and of the isolated subunits have been obtained. The crotoxin complex crystal belongs to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 38.2, b = 68.7, c = 84.2 angstrom, and diffracted to 1.75 angstrom resolution. The crystal of the phospholipase A(2) domain belongs to the hexagonal space group P6(1)22 (or its enantiomorph P6(5)22), with unit-cell parameters a = b = 38.7, c = 286.7 angstrom, and diffracted to 2.6 angstrom resolution. The crotapotin crystal diffracted to 2.3 angstrom resolution; however, the highly diffuse diffraction pattern did not permit unambiguous assignment of the unit-cell parameters.

Effects of 60Co gamma radiation on crotamine

Ionizing radiation can change the molecular structure and affect the biological properties of biomolecules. This has been employed to attenuate animal toxins. Crotamine is a strongly basic polypeptide (pI 10.3) from Crotalus durissus terrificus venom composed of 42 amino acid residues. It induces skeletal muscle spasms leading to a spastic paralysis of hind limbs in mice. The objective of the present study was to carry out a biochemical study and a toxic activity assay on native and irradiated crotamine. Crotamine was purified from C.d. terrificus venom by Sephadex G-100 gel filtration followed by ion-exchange chromatography, and irradiated at 2 mg/ml in 0.15 M NaCl with 2.0 kGy gamma radiation emitted by a 60Co source. The native and irradiated toxins were evaluated in terms of structure and toxic activity (LD50). Irradiation did not change the protein concentration, the electrophoretic profile or the primary structure of the protein although differences were shown by spectroscopic techniques. Gamma radiation reduced crotamine toxicity by 48.3%, but did not eliminate it.

Gabaergic-benzodiazepine system is involved in the crotoxin-induced anxiogenic effect

The behavioral effects of crotoxin (CTX), the major component of Crotalus durissus terrificus venom, were studied in rats submitted to the open field, holeboard, and social interaction tests. CTX (100, 250, and 500 mu g/kg, IP) was administered 2 h before the tests. In the open field, CTX reduced ambulation (250 mu g/kg) and rearing (250 and 500 mu g/kg) and increased grooming (100 and 250 mu g/kg) and freezing (250 mu g/kg). In the holeboard and social interaction, all the CTX doses evaluated decreased, respectively, head dip and head dipping, and social interaction time. The CTX-induced behavioral alterations could be attributed to its neuromuscular transmission blockade, but this possibility was ruled out because CTX (250 and 500 mu g/kg, IP, 2 h before the rotarod test) was unable to modify the rotarod performance of rats. The involvement of the benzodiazepine receptor in the CTX-induced behavioral alterations was investigated through the pretreatment (30 min before the tests, IP) of the animals with diazepam (1.2 mg/kg), or flumazenil (4 and 10 mg/kg). Both diazepam and flumazenil antagonized the CTX induced behavioral alterations in the open field, holeboard, and social interaction tests. This study demonstrated that: (1) CTX is an anxiogenic compound; and (2) the gabaergic-benzodiazepine system may play a role in the CTX-induced anxiogenic effect. (C) 1999 Elsevier B.V.