Crotalid Snakes Research Articles

Pit Viper Envenomation in Two Pregnant Bitches.

Snake envenomation is relatively common in small animals, particularly in endemic areas. Effects and outcomes of envenomation during pregnancy are poorly described in humans and more so in veterinary patients. Two young pregnant female dogs presented to a university teaching hospital with a history of acute soft tissue swelling and bleeding. History, physical examination findings, and diagnostics were consistent with envenomation by crotalid snakes. Medical management of one of the dogs included administration of antivenin. Both dogs survived envenomation with minimal complications and went on to whelp without complications, and all fetuses survived. This is the first description of the management of pit viper envenomation in pregnant dogs.

Inflammatory Effects of Bothrops Phospholipases A2: Mechanisms Involved in Biosynthesis of Lipid Mediators and Lipid Accumulation.

Phospholipases A2s (PLA2s) constitute one of the major protein groups present in the venoms of viperid and crotalid snakes. Snake venom PLA2s (svPLA2s) exhibit a remarkable functional diversity, as they have been described to induce a myriad of toxic effects. Local inflammation is an important characteristic of snakebite envenomation inflicted by viperid and crotalid species and diverse svPLA2s have been studied for their proinflammatory properties. Moreover, based on their molecular, structural, and functional properties, the viperid svPLA2s are classified into the group IIA secreted PLA2s, which encompasses mammalian inflammatory sPLA2s. Thus, research on svPLA2s has attained paramount importance for better understanding the role of this class of enzymes in snake envenomation and the participation of GIIA sPLA2s in pathophysiological conditions and for the development of new therapeutic agents. In this review, we highlight studies that have identified the inflammatory activities of svPLA2s, in particular, those from Bothrops genus snakes, which are major medically important snakes in Latin America, and we describe recent advances in our collective understanding of the mechanisms underlying their inflammatory effects. We also discuss studies that dissect the action of these venom enzymes in inflammatory cells focusing on molecular mechanisms and signaling pathways involved in the biosynthesis of lipid mediators and lipid accumulation in immunocompetent cells.

Nature as a Model for Technical Sensors

Nature has developed a stunning diversity of sensory systems. Humans and many animals mainly rely on visual information. In addition, they may use acoustic, olfactory, and tactile cues for object detection and spatial orientation. Beyond these sensory systems a large variety of highly specialized sensors have evolved. For instance, some buprestid beetles use infrared organs for the detection of forest fires. The infrared sensors of boid and crotalid snakes are used for prey detection at night. For object detection and spatial orientation many species of nocturnal fish employ active electrolocation. This review describes certain aspects of the detection and processing of infrared and electrosensory information (Bleckmann et al 2004). We show that the study of natural exotic sensory systems can lead to discoveries that are useful for the construction of technical sensors and artificial control systems. Comparative studies of animal sensory systems have the power to uncover at least a small fraction of the gigantic untapped reservoir of natural solutions for perceptive problems.

Microfluidic photomechanic infrared receptors in a pyrophilous flat bug

Infrared (IR) receptors are so far known only in boid and crotalid snakes and in three genera of pyrophilous beetles that seek out forest fires. Pyrophilous insects can also be found in other orders, however, so it can be hypothesised that IR receptors also occur in some of these species. We investigated the pyrophilous Australian flat bug Aradus albicornis and found a small number of dome-shaped sensilla (diameter 13 microm) on the prothorax, which have previously not been described. Ultrastructural investigations revealed that the sensilla are characterised by a fluid-filled inner compartment enclosed in a round cuticular shell. The cuticular apparatus is innervated by the dendrite of a ciliary mechanoreceptor, which is fluidically coupled to the inner compartment. Electrophysiological recordings demonstrated that the sensilla respond to brief warming by red laser light or to broadband IR radiation. Depending on the radiation intensity (4.4-549 mW/cm(2) tested, threshold measured as 11.3 mW/cm(2)), first spike latencies varied between 3.4 and 7.5 ms. Thus, our findings demonstrate that A. albicornis most probably possesses photomechanic IR sensilla resembling the metathoracic IR sensilla of buprestid beetles of the genus Melanophila. In the Melanophila sensillum, IR radiation causes thermal expansion of a fluid, which rapidly deforms the dendritic membrane of a mechanosensory cell. The existence of photomechanic IR receptors in both beetles and bugs demonstrates a remarkable convergent evolution towards this particular biophysical transduction mechanism and suggests that it provides selective advantages over other possible solutions.

Ability of fucoidan to prevent muscle necrosis induced by snake venom myotoxins: Comparison of high- and low-molecular weight fractions

Fucoidan, a natural polysaccharide extracted from brown seaweed, inhibits the myotoxic phospholipases A 2 present in the venoms of crotalid snakes. This study evaluated the influence of molecular weight on the ability of fucoidan to prevent muscle necrosis when rapidly administered after injection of a purified myotoxin or crude venom of Bothrops asper, in a mouse model. It was hypothesized that smaller fucoidan fragments, being of higher diffusibility to tissues, might have a better neutralizing efficiency in vivo. Fucoidan was subjected to acid hydrolysis to obtain low-molecular weight fragments ( F L), or to gel filtration to isolate its high-molecular weight fraction ( F H). These two preparations were standardized to the same neutralizing potency by preincubation assays, and subsequently tested in vivo, by independent administration assays. Local i.m. administration of either F H or F L, immediately after i.m. injection of myotoxin II, prevented nearly 50% of muscle necrosis, albeit with no difference between the two preparations. Muscle necrosis was not reduced when either F H or F L was administered by i.v. route, immediately after i.m. toxin injection. When tested against crude venom, which contains several myotoxin isoforms, the immediate in situ i.m. injection of F H still inhibited myonecrosis by nearly one-half of the effect recorded in the untreated group, whereas F L was ineffective. It is concluded that, in this model, and in contrast to expectations, the use of smaller fucoidan fragments to prevent muscle damage induced by snake venom myotoxins is not advantageous, when compared with larger fucoidan molecules.

SALMONELLA SURVEILLANCE IN A COLLECTION OF RATTLESNAKES (CROTALUS SPP)

Over the past 15 yr, Salmonella enterica ssp. arizonae (IIIa) 56:z4,z23:- has repeatedly been isolated from individual Crotalus willardi rattlesnakes with progressively debilitating osteomyelitis at the Knoxville Zoological Gardens. In April 2004, the serotype was linked with a fatal case of septicemia in another Crotalus species in this collection. Although the association of IIIa 56:z4,z23:- with disease in this colony of C. willardi is well established, prior disease or isolation of this serotype outside of the C. willardi colony had not been documented previously, and the serotype's distribution throughout the remainder of the Crotalus collection had yet to be determined. Forty-one fecal samples were obtained from each individual (n = 36) or exhibit group (n = 5) of crotalid snakes, representing nine species, housed at the zoo. Salmonella spp. were isolated from every sample, with 21 different serotypes. The 21 serotypes were distributed among S. enterica ssp. I (24%), IIIa (9%), and IIIb (67%). Although not recovered in the primary study, S. arizonae 56:z4,z23:- was recovered from additional samples taken from two C. willardi willardi. Although the overall recovery rate of this serotype from feces has been low, it seems that its distribution among the Crotalus collection at Knoxville Zoological Gardens remains largely restricted to the C. willardi species.

Nature as a model for technical sensors

Nature has developed a stunning diversity of sensory systems. Humans and many animals mainly rely on visual information. In addition, they may use acoustic, olfactory, and tactile cues for object detection and spatial orientation. Beyond these sensory systems a large variety of highly specialized sensors have evolved. For instance, some buprestid beetles use infrared organs for the detection of forest fires. The infrared sensors of boid and crotalid snakes are used for prey detection at night. For object detection and spatial orientation many species of nocturnal fish employ active electrolocation. This review describes certain aspects of the detection and processing of infrared and electrosensory information. We show that the study of natural exotic sensory systems can lead to discoveries that are useful for the construction of technical sensors and artificial control systems. Comparative studies of animal sensory systems have the power to uncover at least a small fraction of the gigantic untapped reservoir of natural solutions for perceptive problems.

Comparative study of synthetic peptides corresponding to region 115–129 in Lys49 myotoxic phospholipases A 2 from snake venoms

Lys49 phospholipase A 2 homologues constitute a group of catalytically-inactive proteins, present in the venoms of many crotalid snakes, which induce myonecrosis. Current evidence supports the mapping of their toxic site to the C-terminal region, where amino acids comprised within the sequence 115–129 appear to play a central role in toxicity. This study evaluated the possible toxic effects of several synthetic peptides corresponding to the sequence 115–129 of different Lys49 myotoxins, using in vitro cytotoxicity and in vivo myotoxicity assays. Peptides varied widely in their activities, ranging from fully toxic to harmless. Thus, the toxic actions of Lys49 myotoxins cannot always be reproduced by their free peptides 115–129. Peptides from Agkistrodon p. piscivorus (AppK) and A. contortrix laticinctus Lys49 myotoxins exerted both cytotoxicity and myotoxicity. Random scrambling of peptide AppK resulted in complete loss of toxicity, demonstrating that its specific sequence of residues, rather than their simple presence or frequency, confers its ability to damage muscle. Peptide AppK synthesized with d-amino acids retained both activities of the natural l-enantiomer, suggesting that its mechanism of action does not involve the recognition of a proteic receptor/acceptor site on muscle cells, but possibly the binding to other structures, such as negatively-charged membrane phospholipids.

Morphology of a thermosensitive multipolar neuron in the infrared organ of Merimna atrata (Coleoptera, Buprestidae)

Two pairs of infrared (IR) organs are situated ventrolaterally on the second and third abdominal sternites of the Australian fire beetle Merimna atrata (Buprestidae). In ventral view, each IR organ has a round IR absorbing area under which a sensory complex is attached to the epidermis. The main component of the complex is a single large multipolar neuron and its mass of highly branched dendrites. All parts of this neuron are enveloped in glial cells. The proximal primary dendrites, which arise from the soma, finally branch into several hundred tightly packed terminal dendrites, which contain many mitochondria. We term this unusual morphology of the dendritic region a terminal dendritic mass (TDM). Additionally, two chordotonal organs were found in each sensory complex. Their somata are integrated in the complex and the dendrites extend to the periphery of the absorbing area. The bauplan of the dendritic region is reminiscent of the thermosensitive trigeminal nerve fibers innervating the absorbing structures in the IR receptors in boid and crotalid snakes. Because this multipolar neuron also functions as a thermoreceptor, another example of a functional analogy between insect and vertebrate sensory systems could be demonstrated.

SCID mice as a tool for evaluation of heteroxenous life cycle pattern of Caryospora (Apicomplexa, Eimeriidae) species

Adult severe combined immunodeficient (SCID) mice were inoculated with oocysts of 13 different Caryospora (Protozoa, Apicomplexa) species isolated from the faeces of 10 reptilian and three raptorial bird hosts in attempt to test heteroxenous life cycle pattern. Only three reptilian isolates originated from viperid snakes, namely from Calloselasma rhodostoma, Atheris nitschei and Vipera ursinii induced lethal dermal caryosporosis in SCID mice. Neither clinical signs nor developmental stages were observed in mice infected with further nine caryosporan isolates originated from other reptilian and raptorial bird hosts. Results of this study confirmed that SCID mice represent a useful tool for evaluation of heteroxenous life cycle pattern of caryosporan coccidia and that only the Caryospora species from viperid and crotalid snakes produce dermal caryosporosis in mice

Recurrent Coagulopathy After Antivenom Treatment of Crotalid Snakebite

We studied whether recurrence of coagulopathy, defined as the return of a coagulation abnormality after initial normalization, occurred after the use of antivenin (Crotalidae) polyvalent. A retrospective, blinded, descriptive analysis of 354 consecutive cases of North American crotalid snake envenomation was done. Inclusion criteria were documented clinical evidence of crotalid snakebite, presence of a coagulopathy (platelet count <150,000/mm3, prothrombin time above normal, or fibrinogen level <150 mg/dL), and treatment with antivenin (Crotalidae) polyvalent. Of 112 cases with a coagulopathy extending beyond 6 hours after envenomation, 31 had sufficient coagulopathy testing to detect recurrence. Fourteen of these patients (45%) had recurrence of coagulopathy, and two cases were severe (fibrinogen level 29 mg/dL; platelet count 36,000/mm3). Recurrence of coagulopathy after envenomation by North American crotalid snakes may occur after use of antivenin (Crotalidae) polyvalent and can result in severe coagulation abnormalities.

Isolation and characterization of myotoxin II from Atropoides ( Bothrops) nummifer snake venom, a new Lys49 phospholipase A 2 homologue

Myotoxic phospholipases A 2 of class II are commonly found in the venoms of crotalid snakes. As an approach to understanding their structure–activity relationship, diverse natural variants have been characterized biochemically and pharmacologically. This study describes a new myotoxic phospholipase A 2 homologue, isolated from the venom of Atropoides ( Bothrops) nummifer from Costa Rica. A. nummifer myotoxin II is a basic protein, with an apparent subunit molecular mass of 16 kDa, which migrates as a dimer in sodium dodecylsulfate-polyacrylamide gel electrophoresis under nonreducing conditions. It is strongly recognized by antibodies generated against Bothrops asper myotoxin II, by enzyme-immunoassay. The toxin induces rapid myonecrosis upon intramuscular injection in mice (evidenced by an early increase in plasma creatine kinase activity), and significant edema in the footpad assay. It also displays cytolytic activity upon cultured murine endothelial cells. The toxin (up to 50 μg) has no detectable phospholipase A 2 activity on egg yolk phospholipids, and does not show an anticoagulant effect on sheep platelet-poor plasma in vitro. N-terminal sequence determination (53 amino acid residues) demonstrated that A. nummifer myotoxin II is a new Lys49 variant of the family of myotoxic, class II phospholipases A 2. Sequence comparison with other phospholipases A 2 revealed Asn53 as a novel substitution. In addition, this myotoxin is the first Lys49 variant presenting Asn in its N-terminus. Consequently, these findings suggest that neither Ser1 or Lys53, usually found in this family of proteins, are essential amino acid residues for their myotoxic, cytolytic, or edema-inducing effects.

Snake envenomation and protective natural endogenous proteins: a mini review of the recent developments (1991–1997)

The properties of several factors — antihaemorrhagic, antineurotoxic, antimyotoxic — isolated from the blood serum or plasma of different animals are described with more emphasis placed on the structural differences and similarities among the factors of the snake ( Trimeresurus flavoviridis) and mammals ( Didelphis marsupialis and Herpestes edwardsii). Classification of antihaemorrhagic factors of snake and mammals according to structural homologies, and their effectiveness in neutralizing venom haemorrhagic activities in comparison with that of commercial antivenoms are also reviewed. The antineurotoxic factors isolated so far from the sera of viperid ( Vipera palestinae, Daboia r. siamensis), crotalid ( Crotalus d. terrificus, T. flavoviridis, Agkistrodon b. siniticus) and elapid ( Naja naja atra) snakes, as reviewed, are inhibitors of phospholipase A 2, and the amino acid sequences, particularly of those inhibitors from the sera of crotalid snakes, do not share significant sequence homology even within the same family Crotalidae. The amino acid sequences of antineurotoxic factors of the snake ( Crotalus d. terrificus) also are not homologous to those of the antihaemorrhagic factors from the blood of the snake ( T. flavoviridis) or mammals ( Didelphis virginiana, Herpestes edwardsii). The mechanism of action of antihaemorrhagic and antineurotoxic factors is briefly discussed as well as the possibility that crotalids and viperids might possess both of those endogenous neutralizing factors in their blood. Some recent findings on the antimyotoxic factors from the snake serum or plasma with inhibition properties against PLA 2 activity and myotoxicity of venoms or toxins are also shortly reviewed.

Isolation of a venom factor devoid of proteolytic activity from Taiwan habu (Trimeresurus mucrosquamatus): N-terminal sequence homology and no functional similarity to factors IX/X-binding proteins and botrocetin.

One novel venom factor was isolated and purified from the venom of Taiwan habu (Trimeresurus mucrosquamatus) using two consecutive anion-exchange and gel-filtration chromatographies followed by cation-exchange HPLC. Further characterization of the purified protein indicated that it lacks the proteolytic activity toward fibrinogen molecules, suggesting that this protein factor does not belong to the familes of metalloproteinases and thrombin-like serine proteases commonly found in the crude venoms of various crotalid snakes. The purified protein exists as a native dimeric protein of 26 kDa, consisting of two closely similar subunits of 16 and 13 kDa, held together by disulfide linkage. N-Terminal sequence analysis revealed that both chains are homologous to each other at the N-terminal fragment and also similar to the factors IX/X-binding protein isolated from Trimeresurus flavoviridis and botrocetin from Bothrops jararaca. This study points to the existence of one new two-chain venom factor without fibrinogenase activity from Taiwan habu, which, in contrast to botrocetin, promotes platelet agglutination even in the absence of von Willebrand factor. Unlike factors IX/X-binding proteins, it did not show affinity to coagulation factors IX and X in the presence of Ca2+ ion. It also shows no inhibition on thrombin, in contrast with bothrojaracin, a thrombin inhibitor isolated from Bothrops jararaca venom. We have therefore named this novel venom factor trimecetin to distinguish it from some structurally related venom factors present in various crotalid and viperid snakes.

Efficacy of four antivenoms against venoms of Ecuadorian crotalid snakes: results of WHO laboratory assays

Efficacy of four antivenoms against venoms of Ecuadorian crotalid snakes: results of WHO laboratory assays

Hemorrhagic action of the venoms of Central Asian snakes

Collective characteristics of the venoms belonging to various taxons of Central Asian snakes, demonstrating levels of toxicity and efficiency of hemorrhagic action, together with proteolytic, esterase, and phospholipase activities, have been obtained. A comparative analysis of the collective characteristics (“portraits”) of the venoms and their changes on degradative treatment has shown that the hemorrhagic action of the venoms of viperine and crotalid snakes is connected with metalloproteins. In contrast to the venoms of the elapids (cobras) the toxicity of the venoms of the viperine and crotalid snakes exhibits a dependence on the hemmorhagic effect and the activity of the metalloproteins causing it.

Amino acid sequence of a phospholipase A 2 from the venom of Trimeresurus gramineus (green habu snake)

Two phospholipases A 2, named phospholipases A 2-I and A 2-II, were purified to homogeneity from the venom of Trimeresurus gramineus (green habu snake). The complete amino acid sequence of phospholipase A 2-I was determined by sequencing the native protein and the peptides produced by enzymatic ( Achromobacter protease I, clostripain, and chymotrypsin) and chemical (hydroxylamine) cleavages of the S-pyridylethylated derivative of the protein. The protein consisted of 122 amino acid residues and was similar in sequence to phospholipases A 2 from the venoms of crotalid snakes which belong to the category of Group II. A most striking feature of this protein is that tyrosine at the 28th position which is common in phospholipases A 2 and is assumed to be a part of the Ca 2+-binding loop is replaced by phenylalanine. Such replacement is the first finding in Group II phospholipases A 2. Secondary structure compositions of phospholipase A 2-I are similar to those of Crotalus atrox phospholipase A 2. No appreciable Ca 2+-induced difference spectrum was observed, due probably to the absence of the effective chromophoric groups in the neighborhood of the Ca 2+ binding site although Ca 2+ is bound with affinity similar to that for T. flavoviridis phospholipase A 2.

Hemorrhagic Toxins from Snake Venoms

AbstractOne of the more dramatic consequences of envenomation by crotalid and viperid snakes is the occurrence of hemorrage. In cases where the envenomation is less severe, the hemorrhagic is generally observed to be localized at the site of the bite. However, hemorrhage can be found disseminated through a substantial area of the involved extremity. In cases where the envenomation is severe, bleeding in organs such as heart, lungs, kidneys and brain may also occur. From the biochemical investigations on these toxins over the past 30 years, the nature of the venom toxins and their mechanism of activity are now becoming clear. Virtually all of the hemorrhagic toxins isolated and characterized thus far have been determined to be metalloproteinases. In this review we discuss the history of the isolation and characterization of these toxins in an attempt to clarify some of the confusion surrounding these toxins and their biochemical activities. We also survey the data available on the natural and synthetic inh...

The Roles of Vision and the Chemical Senses in Predatory Behavior of the Skink, Scincella lateralis

-S-cincella lateralis responds to both visual and chemical cues of prey. A visual stimulus is associated with an increased rate of tongue flicking, orientation to the prey, and attack behavior. Chemical cues are of reduced importance when the visual cue (movement) is present. However, when the visual stimulus of prey is removed, the rate of tongue flicking increases, and is significantly greater in the presence of a prey extract than to a dead prey item or a water control. When prey are non-moving or dead, chemical cues enable the lizard to distinguish potential prey from inanimate objects. A previous study on the relative roles of vision and the chemical senses in prey detection by Scincella lateralis indicate that visual cues stimulate an increased rate of tongue flicking (RTF) and predatory behavior. Chemical cues of normal concentrations (one prey item) do not stimulate an increased RTF or predatory behavior, but if the chemical cue is sufficiently strong an increase in tongue flicking is observed (Nicoletto, 1986). Therefore it appears likely that visual cues are required to release a predatory response and in the absence of visual cues only a super normal chemical stimulus can elicit such behavior. The interaction between vision and the chemical senses in mediating predation is known in snakes (see Burghardt, 1970, 1980; Chiszar and Scudder, 1980 for reviews). There are at least two basic predatory repertoires employed by snakes. Natricene snakes use chemical cues to locate, identify, track, and attack prey (Burghardt, 1970, 1980; Chiszar et al., 1981). Visual cues elevate the rate of tongue flicking and cause orientation, but fail to elicit attack behavior (Burghardt, 1970; Chiszar et al., 1981). Crotalid snakes use visual and thermal cues to locate, identify, and attack prey (Chiszar et al., 1980, 1981; Chiszar and Scudder, 1980), and use chemical cues to track the wounded prey (Chiszar and Scudder, 1980; Golan et al., 1982; Scudder et al., 1983). Thus, both natricenes and crotalids use visual and chemical cues during predation, but the way these cues interact to mediate predatory behavior is different. The objective of this study was to investigate the interaction between visual and chemical cues in mediating the predatory behavior of S. lateralis. This experiment tests the hypothesis that visual cues initiate a sequence of predatory behavior, which will persist in the bsence of visual stimuli if appropriate chemical cues are present. MATERIALS AND METHODS One hundred freshly caught adult S. lateralis (mean snout-vent length = 42.3 mm, standard error = 0.319 mm) were purchased from Snake Farm in La Place, Louisiana in May 1983. Lizards were maintained in three 61 W by 122 L by 61 H cm terraria for two weeks prior to testing. These cages contained a sand and sawdust substrate with pieces of plywood and branches to provide shelter and basking sites. Illumination was provided by Vita-lights and a single 75 w heat lamp per cage permitted thermoregulation. To insure maximum activity during the testing period, the lizards were phase shifted from a natural This content downloaded from 157.55.39.100 on Thu, 25 Aug 2016 05:29:16 UTC All use subject to http://about.jstor.org/terms

Venom of the mamushiAgkistrodon halys halys. III. Fractionation of DEAE-Sephadex A-50

The composition and properties of the venoms of Central Asian snakes are being widely studied, but the venom of the mamushi has scarcely been investigated. Nevertheless, in the Central Asian fauna the numerous family of crotalid snakes is represented only by the genus AgkisDrodon. We have continued a systematic investigation of mamushi venom undertaken in connection with its influence on the blood-clotting reaction [1-3]. In the present paper the composition of the protein-peptide fraction of this venom is characterized from the results of its chromatography on DEAE-Sephadex A-50.