Sherman A. Minton

Geographic and ontogenic variation in venom of the western diamondback rattlesnake (Crotalus atrox)

Venom samples from western diamondback rattlesnakes (Crotalus atrox) from 13 localities in the United States were tested for i.v. and s.c. lethality for mice, protease activity, hemorrhagic activity, and the presence of Mojave toxin. Electrophoresis on polyacrylamide gel was used to compare protein composition. The neutralizing effect of two commercial antivenoms was evaluated against selected samples of venom. Venom of young snakes from north Texas was compared with that of adults from the same locality. Venom samples from the southwest portion of the range showed highest lethality, those from the northeast portion lowest. This trend was reversed with respect to protease activity. Hemorrhagic activity showed little geographic variation, but northern samples tended to be slightly higher. Differences in venom protein composition were evident between snakes from the eastern and western portions of the range. Mojave toxin in small to trace amounts was detected in two Arizona venom samples and one from west Texas. Antivenoms were relatively ineffective in neutralizing lethality. Venom of young snakes from north Texas was much more lethal by s.c. injection than that of adult snakes from any part of the range, but very low in protease activity. Hemorrhagic activity was about equal to that of adult snakes from the same region. Fifteen months later, lethality had declined almost five-fold, and protease activity had approached adult levels. There was a distinct change in protein composition. Mojave toxin was not detected in venoms of the young snakes.

Proteolytic, hemorrhagic and hemolytic activities of snake venoms

Proteolytic, hemorrhagic and hemolytic activities were tested on 47 different venoms from the Crotalidae, Viperidae, Elapidae, and Hydrophiidae families. Antihemorrhagic activity of crude opossum (Didelphis virginiana) and woodrat (Neotoma micropus) serum was tested against the venoms that presented hemorrhagic activity. All venoms showed proteolytic activity when non-specific substrates such as hide powder and collagen were used. Members of the Crotalidae family had the highest hide powder, chymotrypsin-like and hemorrhagic activity. However, members of the Elapidae family had the highest collagen activity. Hemolytic activity was present in 85% of the snake venoms tested. The crude opossum and woodrat serum neutralized the hemorrhagic activity of all the hemorrhagic venoms. Of particular interest is the poor correlation between the venom activities measured here and the phylogenetic position of the snake that possess them. This is particularly true at the genus and species level. Differences in activities were found among individuals of the same genus. The significance of these differences among venoms of closely related snakes is unknown. They do not seem to be adaptive, however little is known of the physiology and habits of most venomous snakes.

Present tests for detection of snake venom: Clinical applications

Immunologic tests for detection of snake venom and venom antibodies have important clinical applications. Enzyme-linked immunoassay (ELISA) and radioimmunoassay (RIA) provide adequate specificity and sensitivity. The former is much more widely used because it is inexpensive, relatively easy to perform, and uses stable reagents. Some ELISA systems will detect 0.5 ng of venom; however, a sensitivity of 10 to 100 ng is more usual. Minimum running time is 30 to 45 minutes; with longer times, greater sensitivity can be attained. Wound aspirate, serum, and urine are the most suitable materials for venom detection. ELISA has been used for clinical diagnosis of snakebite, to monitor antivenom dose, to study clinical syndromes associated with envenomation, to detect venom in forensic cases, and to evaluate first aid techniques. The indirect ELISA usually is used for detecting and titrating venom antibody. This is potentially useful in epidemiological studies of snakebite incidence, in evaluating potency and paraspecific activity of antivenoms, and in studying response to venom immunogens. Current ELISA systems involving snake venoms have low specificity, and most cannot reliably differentiate venoms of related snakes. Venom antibody detection assays are less satisfactory than those for venom; nonspecific reactions and cross-reactivity are unacceptably high. Methods for improvement of snake venom immunodiagnosis are discussed.

Bites by non-native venomous snakes in the United States

Fifty-four consultations regarding bites by venomous snakes not native to the United States are summarized. These are from a database of 164 consultations during the period 1977- 1995. At least 29 non-native snake species were involved with cobras making up -40% of the group. There was one fatality. A high percentage of venomous snakebites in the United States involve deliberate interaction with snakes. The proportion of bites by non-native species seems to be increasing. Some of the snake species involved are discussed, and some principles for management of these bites are given.

Colubrid Snake Venoms: Immunologic Relationships, Electrophoretic Patterns

Venom of colubrid snakes is secreted by Duvernoys gland which may or may not be associated with enlarged, grooved posterior maxillary teeth. Duvernoys secretion (=venom) was collected from 10 species of colubrid snakes, three with enlarged, grooved teeth and seven with ungrooved teeth. Oral secretions were also obtained from one colubrid and one boa which lack Duvernoys gland. These secretions were reacted with 14 commercial antivenoms in immunodiffusion

AN ENZYME-LINKED IMMUNOASSAY FOR DETECTION OF NORTH AMERICAN PIT VIPER VENOMS

We describe a method for immunodetection of North American pit viper venoms in clinical materials. Antibody-enzyme conjugates prepared against venoms of the western diamondback rattlesnake (Crotalus atrox), Mojave rattlesnake (Crotalus scutulatus), and copperhead (Agkistrodon contortrix) detect homologous venoms in concentrations of 0.1-.01 mcg/ml using a double antibody sandwich technique. Venoms of 10 additional species of U.S. pit vipers were detected in concentrations of 10 mcg/ml or less. Venoms of 4 species could not be detected at levels likely to be encountered in clinical situations. There are extensive cross-reactions between venoms of certain species, hence specific identification of a given venom cannot always be made. Venom usually can be detected at injection sites of experimental animals receiving intramuscular doses of 0.5-1.5 mg of venom but can rarely be detected in urine or plasma specimens. Venom was readily detected in specimens from experimental animals bitten by pit vipers of 6 species. The method is relatively rapid, simple, and inexpensive.

Comparative enzymatic study of HPLC-fractionated Crotalus venoms

1. Ten venoms of the genus Crotalus (Crotalus adamanteus, Crotalus atrox, Crotalus durissus durissus, Crotalus horridus horridus, Crotalus lepidus, Crotalus polystictus, Crotalus molossus molossus, Crotalus pusillus, Crotalus scutulatus scutulatus, venom B, and Crotalus viridis lutosus) were fractionated using HPLC anion and cation exchange chromatography. 2. HPLC venom fractions were tested for hemorrhagic, hemolytic, and proteolytic activities. 3. Crude Virginia opossum (Didelphis virginiana) serum neutralized the hemorrhagic activity of HPLC fractions.

Protease activity and lethal toxicity of venoms from some little known rattlesnakes

Information on yield, lethality, and protease activity is given for venoms of Crotalus exul, C. p. pricei, C. pusillus, C. w. willardi and Sistrurus ravus. Lethal toxicity of C. tigris venom (LD50 i.v. 0.056 mg/kg; s.c. 0.21 mg/kg) is the highest known for any rattlesnake venom. The lethal potency of C. pricei venom is high by i.v. but not by s.c. injection. Both these venoms lack protease activity. C. pusillus venom is lowest in lethality.

Serological Relationships among Some Colubrid Snakes

Sera of 22 species of colubrid snakes were studied by immunoelectrophoresis using antisera against serum proteins of Coluber constrictor, Elaphe vulpina, Natrix sipedon, Naja naja and Crotalus horridus to develop the preparations. Development of a snake serum with its homologous antiserum gives a pattern of seven or eight precipitin arcs. Some of these represent components widely shared among snakes, while others are more or less specific for closely related taxa. Two serological groups can be distinguished in our material. One consists of Elaphe (3 sp.), Lampropeltis (2 sp.) and Pituophis melanoleucus. The other group consists of Natrix (4 sp.), Thamnophis (2 sp.), and Regina septemvittata. Serologically these groups are as distinct from each other as they are from Naja and Crotalus. Coluber constrictor is somewhat intermediate between the two but closer to Elaphe. Three Old World colubrids, Coluber jugularis, C. ravergieri and Spalerosophis diadema, are serologically intermediate between Coluber constrictor and Elaphe. Malpolon monspessulanus and Psammophis schokari are also intermediate but slightly closer to Coluber. The affinities of Trimorphodon biscutatus are more nearly with Elaphe than with Coluber or Natrix. Leptodeira maculata and Heterodon platyrhinos do not fit the above groups nor do they appear to be closely related to one another.

Serological Relationships among Some Congeneric North American and Eurasian Colubrid Snakes

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