Juan Carlos Vidal

A study on the venom yield of venomous snake species from Argentina

A study on the venom yield of snakes from Argentina over a three year period was carried out on adult specimens of Bothrops alternatus (n = 74); Bothrops neuwiedii (n = 127); Bothrops ammodytoides (n = 30); Bothrops moojeni (n = 14); Bothrops jararaca (n = 14); B. jararacussu (n = 6); Crotalus durissus terrificus (n = 120) and Micrurus spp. (n = 6) as well as with 12 specimens of newborn C. d. terrificus kept in captivity. While for each species there was a positive correlation between venom yield and number of snakes milked, the correlation with the snakes body weights after individual milkings was even better, suggesting that the size of the snakes is more important in determining the venom yield than the number of snakes milked or the specimens sex. Individual milkings indicated that, in addition to the snake size, when the amount of venom is normalized per 100 g body weight there is a species specific difference in venom yield. It follows the order B. jararacussu > B. moojeni approximately = B. jararaca approximately = B. alternatus > B. neuwiedii> Micrurus spp approximately = B. ammodytoides> C. d. terrificus. Although the venom yield per 100 g body weight of newborn C. d. terrificus specimens is 2-fold higher than that of adults, no correlation was observed between venom yield and body weight.

Hemorrhagic activity of Bothrops venoms determined by two different methods and relationship with proteolytic activity on gelatin and lethality

The changes in hemorrhagic activity, proteolytic activity on gelatin and the lethal potency of four Bothrops venoms treated at different pH values or with EDTA were studied. Venoms from B. alternatus, B. jararaca, B. moojeni and B. neuwiedii of Argentina were preincubated at pH 5.8, 5.1 or 3.8 or with EDTA and the hemorrhagic activity expressed as size of the hemorrhagic lesion or as the amount of hemoglobin extracted, the proteolytic activity on gelatin and the lethal potency were determined. Although the MHDs recorded in rats were 19-56 fold higher than those recorded in mice, the A(550) extracted per gram of hemorrhagic haloes was very similar in rats or mice independent of the venom dose. Inhibition of proteolytic activity after preincubation at pH 5.1 or 3.8, agrees with the decreased amount of hemoglobin extracted from the hemorrhagic haloes, and with the increase in mean survival time after the i.p. injection to mice. Preincubation with EDTA resulted in 80% inhibition of hemorrhagic activity of B. jararaca venom and complete inhibition with the other Bothrops venoms tested. Measurement of the amount of hemoglobin extracted gives significant information in comparative studies, not available by measurement of the size of hemorrhagic haloes.

A comparison of different methods to assess the hemorrhagic activity of Bothrops venoms.

The hemorrhagic activity of Bothrops (B.) alternatus, B. ammodytoides, B. jararaca, B. jararacussu, B. moojeni and B. neuwiedii venoms from specimens captured in Argentina was assayed after i.d. injection to mice. The hemorrhagic haloes produced by each venom had different color intensities, although no significant differences were observed by measurement of the average diameters or the weight of the excised hemorrhagic haloes. Conversely, important differences were found by measuring the amount of hemoglobin extracted from excised hemorrhagic haloes of similar size produced by different venoms. The relationship between the amount of hemoglobin extracted and the weight of the excised hemorrhagic haloes was linear, with a slope (hemoglobin released per gram of hemorrhagic halo) characteristic for each venom, and proportional to the potency. On this basis, the activity of B. alternatus, B. ammodytoides and B. jararaca is similar, about 1.5 times higher than that of B. jararacussu and B. moojeni venoms and threefold higher than that of B. neuwiedii venom. Thus, measurement of the of hemoglobin released provides additional information in comparative studies, and may be used to assess the antihemorrhagic potency of antivenoms.

Hematological values of some Bothrops species (Ophidia - Crotalidae) in captivity

Blood samples of 50 healthy specimens from each of the following species: Bothrops alternatus, Bothrops jararacussu, Bothrops moojeni, and Bothrops neuwiedi diporus all kept in captivity were taken to determine the hematocrit (PCV) value, red blood cell count (RBC), total leukocyte (WBC) and differential leukocyte count, thrombocyte count, mean corpuscular volume (MCV), hemoglobin concentration (HbC), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC). These hematological parameters were compared to those obtained from other Bothrops species. PCV values, RBC, hemoglobin, WBC count, and differential leukocyte count are within the range of values reported for other Bothrops species, while the thrombocyte count was significantly lower. All the hematological parameters obtained from the four studied Bothrops species were higher than those described for B. ammodytoides.

THE IMMUNOCHEMICAL REACTIVITY AND NEUTRALIZING CAPACITY OF POLYVALENT Vipera (EUROPEAN) ANTIVENOM ON ENZYMATIC AND TOXIC ACTIVITIES IN THE VENOMS OF CROTALIDS FROM ARGENTINA

The immunochemical reactivity and neutralizing capacity of polyvalent Vipera antivenom (Vipera ammodytes, Vipera aspis, Vipera berus, Vipera lebetina, and Vipera xanthina) were tested on the enzymatic and biological activities of Crotalus durissus terrificus and the following Bothrops venoms from Argentina (Bothrops alternatus, Bothrops ammodytoides, Bothrops neuwiedii, Bothrops jararaca, Bothrops jararacussu, and Bothrops moojeni). The Vipera antivenom reacted weakly when tested by double immunoprecipitation (DIP) and reacted with all the venoms when tested by ELISA. Several components in all the venoms studied were recognized in Western blots. Vipera antivenom deactivated to different degrees in vitro procoagulant, (indirect) hemolytic, and proteolytic activities in all the venoms studied. Preincubation of Bothrops alternatus venom with Vipera antivenom neutralized a lethal potency of 4.5 LD50 in mice with an ED50 of 1.25 ± 0.25 ml per mg of venom, and with 1.0 ml/mg inhibited 54% of the hemorragic activity and 48% of necrotic activity. Vipera antivenom (2.0 ml per mg toxin) inhibited the phospholipase A2 activity of purified crotoxin and decreased its lethal potency by 60%, while the neutralizing capacity on the lethal potency of crude Crotalus durissus terrificus venom was poor even at a level of 5.0 ml/mg of venom.

Effect of chemical modification with p-bromophenacyl bromide on the enzymatic and lethal proper ties of phospholipase A2 from Bothrops alternatus (Víbora de la Cruz) venom

The effects on lethal potency and enzymatic activity were determined following alkylation, with p-bromophenacyl bromide, of the acidic toxic phospholipase A2 from Bothrops alternatus. The modified B. alternatus enzyme, which lost its enzymatic activity, retained considerable toxicity. Histopathologic studies on mice have demonstrated features similar to those of the native enzyme. However, the distribution of the damage was different and the survival time was longer. It is concluded that the enzyme activity is not important for the lethal action of the enzyme although it influences the distribution of the damage and survival time.

Some toxic and enzymatic activities of Bothrops ammodytoides (‘yarará ñata’) venom

Bothrops ammodytoides, the smallest representative of this genus, is found only in Argentina. Venom was extracted from thirty adult specimens (35-70 cm in length, 90-300 g in weight) captured in the Province of Buenos Aires and kept in captivity. Venom yield was 3-30 mg. SDS-PAGE showed strong bands at 14.0; 23-25; 45; 54 and 63 kDa and weak bands at 17.0; 30.0; 40.0 and 85.0 kDa. Toxic activities were: LD50 (intravenous, mice) 0.5+/-0.2 microg/g; minimal procoagulant dose on human plasma (MPD-P) 35+/-2 mg/l; and minimal defibrinogenating dose (MDD, mice) 6-12 microg. Hemorrhagic and/or necrotic activities appear to play a major role in lethality; minimal hemorrhagic dose (MHD, mice) is 10+/-2 microg/g and minimal necrotizing dose (MND, mice) is 38+/-5 microg. The LD50, MPD-P and MND are among the lowest in venoms from Bothrops species found in Argentina. B. ammodytoides venom exhibited high proteolytic and phospholipase A2 activities. Most of the B. ammodytoides venom components cross-react with Bivalent Bothropic antivenom (Instituto Nacional de Producción de Biológicos ANLIS Dr. G. Malbrin, against B. alternatus and B. neuwiedii venoms). One ml of antivenom neutralizes 1.2 mg of B. ammodytoides venom.

Blood biochemical profile of the South American rattlesnake (Crotalus durissus terrificus) in captivity

Blood samples were collected from 180 healthy specimens of the South American rattlesnake, Crotalus durissus terrificus, in captivity. All animals were in good clinical condition. Normal biochemical reference values were established for the following: total proteins, albumin, globulins, uric acid, creatinine, urea, glucose, triglycerides, cholesterol, total lipids, calcium, phosphorus, potassium, sodium, chloride, magnesium, GOT (AST), GPT (ALT), and alkaline phosphatase (ALP). Samples were obtained by venipuncture of the ventral tail vein. Values were compared with published data for Boidae, Elapidae, and Viperidae.