Effect of a rattlesnake venom (Crotalus viridis helleri) on bone marrow

Crotalus viridis helleri venom produces a fall in peripheral platelet count in rats but does not appear to damage the precursors in the bone marrow. The major response is a myeloid hyperplasia.     

Cardiovascular failure produced by a peptide from the venom of the southern Pacific rattlesnake, Crotalus viridis helleri.

R. C. Schaeffer, Jr., T. R. Pattabhiraman, R. W. Carlson, F. E. Russell and M. H. Weil. Cardiovascular failure produced by a peptide from the venom of the Southern Pacific rattlesnake, Crotalus viridis helleri. Toxicon17, 447–453, 1979.—Hemodynamic, metabolic and respiratory effects of a 30 min i.v. infusion of crude venom (1·4 mg/kg) and three venom components (Peptide I, 0·5 mg/kg; Protein I, 1·2 mg/kg, Protein II, 3·4 mg/kg) were studied in 24 sedated rats (270–309 g). Venom shock, characterized by hypotension, lactacidemia, hemoconcentration, hypoproteinemia and death was observed in animals given the crude venom or Peptide I. Just prior to death, respiratory distress was observed in most animals that died. Hemolysis and hematuria were observed in the animals given Protein I or Protein II. These data suggest that the increase in vascular permeability to protein and red blood cells induced by the crude venom can, for the most part, be attributed to the peptide. In addition, Protein I and Protein II appear to account for the hemolytic activity. The toxic effects of the venom components appear to be synergistic.     

Classification of myonecrosis induced by snake venoms: venoms from the prairie rattlesnake (Crotalus viridis viridis), western diamondback rattlesnake (Crotalus atrox) and the Indian cobra (Naja naja naja)

The pathogenesis of myonecrosis induced by three different snake venoms was studied by light microscopic examination of skeletal muscle tissue taken at time periods ranging from 0.25 hr to 4 weeks after an intramuscular injection of the venom into mice. It was possible to identify different types of myonecrosis based on the abnormal morphologic states of the damaged cells. The types of myonecrosis observed correlated with the types of components present in the venom injected. Venoms containing direct acting toxins such as myotoxin a or phospholipase A2 induced specific types of myonecrosis. Also, venoms containing hemorrhagic toxins produced a type of myonecrosis similar to that induced by pure hemorrhagic toxins. The pathogenesis of each type of myonecrosis could be divided into the same four phases based on the pathologic states of the affected cells and the time after injection. During the 'early phase' (0.25-3 hr) affected muscle cells were in several different pathologic states reflecting the types of components present in the venom injected. During the 'intermediate phase' (6-24 hr) the pathologic state of the damaged cells had changed and depending on the venom new states might be present. By the 'late phase' (48-96 hr) all damaged cells have reached a common pathologic state of necrosis. The 'final phase' (1-4 weeks) is characterized by regeneration (partial or complete) of muscle cells. Although the number of different types of myonecrosis depended on the type of venom injected, i.e. Naja naja naja venom produced only two different types whereas Crotalus atrox venom produced at least four different types, cells of each tpe of myonecrosis progressed through the same four phases. In studies of the myotoxicity of snake venoms it is important to examine tissues taken during the early and intermediate phases to obtain accurate and useful information on the types of myonecrosis caused by the venom.     

Antiserum to myotoxin from prairie rattlesnake (Crotalus viridis viridis) venom.

C. L. Ownby, W. M. Woods and G. V. Odell. Antiserum to myotoxin from prairie rattlesnake (Crotalus viridis viridis) venom. Toxicon17, 373–380. 1979.—A myotoxic component was isolated from rattlesnake (Crotalus viridis viridis) venom and an antibody to it was produced in rabbits. Gel-filtration and cation exchange chromatography were used to fractionate the crude venom and two components were shown to be locally myotoxic using an in vivo assay. One of these components, fraction 3 of the Sephadex cation exchange column, was shown to be homogeneous by electrophoresis. This purified myotoxin was injected into rabbits, and when the resulting antiserum was reacted with the myotoxin in agar-gel double-diffusion plates, one precipitin line was formed. Anti-myotoxin serum reacted with only 2 of 14 crude venoms forming one precipitin line to both C. v. viridis and C. durissus terrificus. The line which formed against these two crude venoms was identical to the one that formed against myotoxin indicating the presence in C. d. terrificus venom of a component immunologically very similar to myotoxin from C. v. viridis venom. It is postulated that this component might be crotamine, a known myotoxic component in C. d. terrificus venom. Antibodies to myotoxin could not be detected in Wyeth's polyvalent Crotalidae antivenin although at least one antibody to crude C. v. viridis venom was present. It is possible that antiserum to pure myotoxin could prevent the local myonecrosis induced by the pure myotoxin, crude C. v. viridis venom and perhaps by other crotalid venoms.     

Multiple myotoxin sequences from the venom of a single prairie rattlesnake (Crotalus viridis viridis)

Multiple myotoxin a sequences have been determined from the venom of a single adult male prairie rattlesnake (Crotalus viridis viridis). This is the first time such individual variation has been reported for this toxin class and the number of isoforms suggest that myotoxin a is the product of a duplicated locus.     

Neutralization of venoms from two Southern Pacific Rattlesnakes (Crotalus helleri) with commercial antivenoms and endothermic animal sera.

The Southern Pacific Rattlesnake (Crotalus helleri) is found in southwestern California (USA), southward through north Baja California (MX) into the northern part of southern Baja California (MX). In this study, the venoms from two Southern Pacific Rattlesnakes were characterized. The two venoms were different in color, concentration, and enzyme activities. Two commercial antivenoms neutralized both C. helleri venoms differently. Antivipmyn (Fab2H) and CroFab (FabO) neutralized both venoms but had different ED50. Four times more Fab2H antivenom was required to neutralize the C. helleri venom No. 011-084-009 than the venom from the snake No. 010-367-284. The hemorrhagic activity of two C. helleri venoms were neutralized differently by endothermic animal sera having a natural resistance to hemorrhagic activity of snake venoms. Opossums and Mexican ground squirrel sera did not neutralize the hemorrhagic activity of the venom No. 010-367-284. The sera of gray woodrats and hispid cotton rats neutralized all hemorrhagins in both C. helleri venoms. This is the first reported case in which opossum serum has not neutralized hemorrhagic activity of pit viper venom. Differences in the compositions of C. helleri venoms and their ability to be neutralized may help explain why snakebites are a difficult medical problem to treat and why effective polyvalent antivenoms are difficult to produce.     

Several isoforms of crotoxin are present in individual venoms from the South American rattlesnake Crotalus durissus terrificus

Crotalus durissus terrificus venoms collected either from individual snakes or from a large number of animals (more than 30) have been fractionated by high performance liquid chromatography on gel-filtration and ion exchange columns. The chromatographic patterns obtained with individual venom samples indicated that each Crotalus durissus terrificus snake synthesizes five to ten different crotoxin isoforms in widely variable relative proportions. Furthermore, the heterogeneity of venom samples collected from a large number of snakes did not appear significantly larger than that observed with venoms obtained from individual snakes. The comparison of the chromatographic patterns that we obtained with the various (individual and pooled) venoms allowed us to identify about 15 crotoxin isoforms, which may result from the expression of isogenes, since two amino acid variants have been reported to occur at several positions in the sequence of crotoxin component B. These observations confirm the existence of numerous molecular isoforms of crotoxin and suggest that an individual Crotalus durissus terrificus snake possesses several genes coding for the various crotoxin isoforms. The heterogeneity of venom samples collected from a large number of animals is explained, in a large measure, by the complexity of the venom obtained from the individual snakes.     

The histochemistry of the venom glands of the rattlesnake Crotalus viridis helleri. I. Lipid and non-specific esterase

Crotalus viridis helleri venom glands were exposed to histochemical reactions for lipid and non-specific esterase activities in unmilked snakes and at 1, 4, 8 and 15 days following milking. Lipid and non-specific esterase were found in homologous areas during all stages analyzed. These components were concentrated in the basal region of the secretory cells and smaller amounts were scattered throughout the cytoplasm of the cells. Supporting connective tissue septa contained heavy concentrations of lipid and non-specific esterase activities.     

Characterization of the biological and immunological properties of fractions of prairie rattlesnake (Crotalus viridis viridis) venom

C. L. and T. R. . Characterization of the biological and immunological properties of fractions of prairie rattlesnake (Crotalus viridis viridis) venom. Toxicon 25, 1329 – 1342, 1987. — Prairie rattlesnake (Crotalus viridis viridis) venom was separated using liquid column chromatography. The fractions were tested for biological activity in mice and for immunological reactivity against polyvalent (Crotalidae) antivenom and a monovalent antivenom to the crude venom. Several of the basic fractions and most of the non-basic fractions had hemorrhagic activity. Six of eight basic fractions had direct myotoxic activity, two of the basic fractions produced edema 30 min after injection, and two were lethal. Polyvalent antivenom contained few antibodies to the fractions of this venom, reacting with only two of the basic fractions. Monovalent antivenom formed mulitiple precipitin bands with almost all of the fractions. These results clearly demonstrate that most of the venom components are antigenic and immunogenic. Immunodiffusion using the monovalent antivenom demonstrated that C. v. viridis venom contains many antigens common to 10 other crotaline venoms. One of the hemorrhagic components was present in five of the other venoms tested, one of the direct myotoxic components was present in three other venoms, and one of the lethal components was common to two other venoms. Another highly active hemorrhagic component was common to all of the venoms tested except that of Trimeresurus flavoviridis.     

Rapid decline in blood antimyotoxin levels in the presence of myotoxin A from prairie rattlesnake (Crotalus viridis viridis) venom

The disappearance of antibodies to myotoxin a from the bloodstream in mice was measured with a specific ELISA over a ninety-six hour period in the presence and absence of myotoxin a. A gradual disappearance of antibodies to myotoxin in the absence of toxin was observed during the 96 hr sampling period, although antibodies were still detectable at 96 hrs. However, in the presence of myotoxin a very rapid decrease of antimyotoxin occurred. When antiserum injection was followed by myotoxin injection (5 min. later) the decline in antibodies was immediate and no antibodies could be detected 30 min. after the antiserum injection. When antiserum was injected either immediately or 30 min. after toxin, antibody levels declined sharply and were non-detectable 1 hr. after antiserum injection. These results have clinical significance since they indicate that antimyotoxin can still bind the toxin, even when administration of the antiserum is delayed for 30 min. after injection of the toxin. Multiple injections of antiserum may be required to maintain a neutralizing level of antiserum.     

In vivo ability of antimyotoxin a serum plus polyvalent (Crotalidae) antivenom to neutralize prairie rattlesnake (Crotalus viridis viridis) venom

A mixture of antimyotoxin a serum and polyvalent (Crotalidae) antivenom was injected i.v. in mice either 5 min before or 5 min, 30 min, 1 hr or 3 hr after i.m. injection of venom. Neutralization of the local myotoxicity of a sublethal dose (1.5 micrograms/g) of C. v. viridis venom occurred if the antisera were injected 5 min before or 5 or 30 min after venom, but not if injected 1 or 3 hr after the venom. Hemorrhage was neutralized when the mixture was injected either 5 min before or 5 min after injection of venom, but not when injected 30 min after injection of venom. Previous results showed that the mixture of antisera neutralized the same amount of venom (1.5 micrograms/g) when mixed with the venom prior to injection. Thus it is not possible with these two antisera to neutralize myonecrosis if the time interval between injections is greater than 30 min.     

Antigenic relationships of fractionated western diamondback rattlesnake (Crotalus atrox) hemorrhagic toxins and other rattlesnake venoms as indicated by monoclonal antibodies

Seven hemorrhagic factors have been isolated from Crotalus atrox venom, but their antigenic relationships have not been well studied. In this study, two different monoclonal antibodies, C. atrox peak 8 (CA-P-8) and C. atrox subclone 5 (CA-5+), were produced against two C. atrox venom hemorrhagic fractions and used in an ELISA (enzyme-linked immunosorbent assay) to determine if the hemorrhagic factors in C. atrox venom are antigenically related. The same ELISA test was used to determine cross-reactivity of seven other crude Crotalidae venoms. The two monoclonal antibodies were tested for their ability to neutralize each hemorrhagic HPLC fraction separated from C. atrox venom. C. atrox venom was fractionated into 22 fractions using HPLC analytical DEAE ion exchange. Fractions 4-17 were hemorrhagic. The CA-P-8 monoclonal antibody reacted strongly with hemorrhagic fraction 8; CA-5+ had a broader reactivity and reacted with several HPLC hemorrhagic and non-hemorrhagic fractions. Crude venoms of C. adamanteus, C. scutulatus scutulatus and C. viridis lutosus reacted with CA-P-8, while C. viridis lutosus, C. viridis oreganus, C. scutulatus scutulatus and C. horridus horridus reacted with CA-5+. C. molossus molossus and C. lepidus lepidus did not react with CA-P-8 and CA-5+. Hemorrhagic HPLC fractions 6, 7, 8, were completely neutralized by monoclonal antibody CA-P-8; fraction 9 was partially neutralized. The present study indicated that some C. atrox venom HPLC hemorrhagic fractions have both common and unique epitopes. Antigenic determinants were also found to be shared among different Crotalus species.(ABSTRACT TRUNCATED AT 250 WORDS)     

Physiological and immunological properties of small myotoxins from the venom of the midget faded rattlesnake (Crotalus viridis concolor)

Myotoxins from C. v. concolor venom were isolated by gel filtration. This crude myotoxin peak was subfractionated into either two or four subfractions by cation exchange FPLC, depending upon the source of the venom. When injected at 2 micrograms/g, crude concolor myotoxin caused vacuolation of mouse muscle cells typical of myotoxin a from C. v. viridis and crotamine from C. d. terrificus. All four subfractions showed qualitatively identical myotoxin activity. In double immunodiffusion studies, myotoxin a antiserum produced lines of identity when reacted with myotoxin a, crude concolor myotoxin and the four concolor subfractions. A second batch of material showed two major components when subfractionated by cation exchange FPLC. The more basic of these two components displayed approximately twice the i.v. lethality of the more acidic component. The LD50 for the basic component lies between 0.625 and 0.75 microgram/g while that of the acidic component falls between 1.00 and 1.25 micrograms/g.     

The effects of myotoxin from midget faded rattlesnake (Crotalus viridis concolor) venom on neonatal rat myotubes in cell culture

Neonatal rat myoblasts were isolated and grown in culture until they fused into multinucleated myotubes. A small percentage of the myotubes showed spontaneous contractions when maintained in Dulbecco's Modified Eagle's Medium with 10% fetal bovine serum. Incubation of mature myotubes (at least 3 days after fusion) with myotoxin II from Crotalus viridis concolor venom at a concentration as low as 18.5 nM caused a marked increase in the number of myotubes demonstrating contractile activity. The increase was apparent within 24 hr of myotoxin application. The response of the myotubes appeared to be specific since, of the proteins tested, only native myotoxins caused the increase in contractile activity. This tissue culture system offers a rapid screening assay that requires less time and fewer animals than the assays currently in use for determining myotoxic activity.     

The midget faded rattlesnake (Crotalus Viridis Concolor) venom: Lethal toxicity and individual variability

The midget faded rattlesnake (Crotalus viridis concolor) venom: lethal toxicity and individual variability. Toxicon15, 129–133, 1977.—Crotalus viridis concolor venoms were collected from captive specimens from northeastern Utah and tested for lethal potency in mice and for protein content by polyacrylamide gel electrophoresis. We found C. v. concolor potency (i.p.-ld₅₀-0·25 μg per g) comparable to Crotalus durisses terrificus (i.p.-ld₅₀-0·25 μg) and Crotalus scutulatus scutulatus (California variety) (i.p.-ld₅₀-0·24 μg per g) venoms. Individual C. v. concolor venoms show a wide variability both in lethality and protein pattern. The venom appears to be one of the most lethal Crotalid venoms in the New World.     

The distribution among ophidian venoms of a toxin isolated from the venom of the Mojave rattlesnake (Crotalus scutulatus scutulatus)

A toxin analogous to Mojave toxin or protein K' was isolated from venom of the Mojave rattlesnake (Crotalus s. scutulatus) by anion exchange and gel permeation chromatography. This toxin has an apparent native molecular weight of 20,000-22,000, a subunit molecular weight of 14,000 and a pI of 4.9-5.0. The i.p. LD50 is 0.094 mg/kg for mice. A wide variety of ophidian venoms (crotaline, viperine, elapid, hydrophid and colubrid) were examined for the presence of this toxin using Ouchterlony, immunoelectrophoresis, ELISA and Western transfer. High concentrations were found in 4 of 6 C. scutulatus venom samples, 2 of 3 C. durissus samples and samples from C. viridis concolor and C. tigris. A moderate concentration was found in 1 of 3 C. durissus samples and low to trace concentrations in 1 C. durissus sample, 1 C. scutulatus sample, 2 of 12 C. atrox samples and a Trimeresurus flavoviridis sample, the latter being the only instance of detection of the toxin in a snake other than a rattlesnake. The toxin appears in at least two phylogenetic lines of rattlesnakes, and its geographic distribution in North American rattlesnake species resembles a mosaic.     

Resistance of California ground squirrels (Spermophilus beecheyi) to the venom of the northern Pacific rattlesnake (Crotalus viridis oreganus): a study of adaptive variation

Recent studies have documented natural resistance to snake venom in a number of diverse mammalian species. The present paper documents for the first time variation in such resistance within one single species, the California ground squirrel (Spermophilus beecheyi). This species is a frequent prey of the northern Pacific rattlesnake (Crotalus viridis oreganus) in certain habitats. Venom resistance was tested directly in two populations of ground squirrels by injection of 1-40 mg/kg venom doses. One population was obtained from a habitat with a high rattlesnake density; the other population came from a rattlesnake-free habitat. Dramatic differences in the response to venom between these populations were manifested, based on a variety of criteria, such as mortality, necrosis and healing time. Resistance to venom was also examined by LD50 tests in groups of mice pre-injected with ground squirrel sera from three rattlesnake-adapted California populations and a non-adapted Arctic population (S. parryii) from snake-free central Alaska. The California ground squirrel sera were 3.3-5.3 times more effective in the in vivo neutralization of venom than the sera from Arctic ground squirrels. Moreover, the level of protection by the sera as reflected by the LD50 values was highly correlated (P less than 0.005) with the level of in vitro squirrel serum-venom binding as quantified by radioimmunoassay (RIA). A subsequent RIA revealed that binding levels of sera from 14 California ground squirrel populations correlated significantly (P less than 0.025) with local rattlesnakes densities; i.e. sera pools from populations sympatric with rattlesnakes exhibited the highest binding, whereas populations living in habitats where rattlesnakes are rare or absent typically exhibited the lowest binding levels, several of which approximated the Arctic control. Taken together, these results demonstrate intraspecific variation that is probably the result of differential natural selection due to northern Pacific rattlesnakes. This intraspecific variation should be taken into consideration when testing for natural resistance in wild-caught species.     

Mojave toxin in venom of Crotalus helleri (Southern Pacific Rattlesnake): molecular and geographic characterization.

Mojave toxin (MT) was detected in five of 25 Crotalus helleri (Southern Pacific rattlesnake) sampled using anti-MT antibodies and nucleotide sequence analysis. All of the venoms that were positive for MT were collected from Mt San Jacinto in Riverside Co., California. Since this population is geographically isolated from C. scutulatus scutulatus (Mojave rattlesnake), it is unlikely that this finding is due to recent hybridization. MT concentration differences between C. helleri and C. s. scutulatus reflected the presence of 'isoforms' of the toxin in the venom. Whereas C. s. scutulatus generally has several isoforms of the toxin (detected by Western blotting), only one 'isoform' that focused at pI 5.1 was detected in C. helleri. Both acidic and basic subunits of MT sequences were obtained from C. helleri DNA with primers specific for MT, but only from snakes that had MT in their venom. The sequence identity of the C. helleri acidic subunit to the C. s. scutulatus subunit was 84.9%, whereas the sequence identity of the C. helleri basic subunit was 97% to the C. s. scutulatus basic subunit. Using casein, fibrin, and hide powder azure as substrates, assays for proteolytic activity suggested that C. helleri possesses several different types of metalloproteinases in their venom. However, proteolytic activity was not detected, or present in reduced amounts, in specimens having MT. Clinical neurotoxicity following envenomation by certain populations of C. helleri may be due to MT.