Synergistic effect of serine protease inhibitors and a bothropic antivenom in reducing local hemorrhage and coagulopathy caused by Bothrops jararaca venom.

Snakebite accidents are a public health problem that affects the whole world, causing thousands of deaths and amputations each year. In Brazil, snakebite envenomations are caused mostly by snakes from the Bothrops genus. The local symptoms are characterized by pain, swelling, ecchymosis, and hemorrhages. Systemic disturbances can lead to necrosis and amputations. The present treatment consists of intravenous administration of bothropic antivenom, which is capable of reversing most of the systemic symptoms, while presenting limitations to treat the local effects, such as hemorrhage and to neutralize the snake venom serine protease (SVSP). In this context, we aimed to evaluate the activity of selective serine protease inhibitors (pepC and pepB) in combination with the bothropic antivenom in vivo. Further, we assessed their possible synergistic effect in the treatment of coagulopathy and hemorrhage induced by Bothrops jararaca venom. For this, we evaluated the in vivo activity in mouse models of local hemorrhage and a series of in vitro hemostasis assays. Our results showed that pepC and pepB, when combinated with the antivenom, increase its protective activity in vivo and decrease the hemostatic disturbances in vitro with high selectivity, possibly by inhibiting botropic proteases. These data suggest that the addition of serine protease inhibitor to the antivenom can improve its overall potential.

A functional and thromboelastometric-based micromethod for assessing crotoxin anticoagulant activity and antiserum relative potency against Crotalus durissus terrificus venom.

The assessment of the capacity of antivenoms to neutralize the lethal activity of snake venoms still relies on traditional rodent in vivo lethality assay. ED50 and LD50 assays require large quantities of venoms and antivenoms, and besides leading to animal suffering. Therefore, in vitro tests should be introduced for assessing antivenom neutralizing capacity in intermediary steps of antivenom production. This task is facilitated when one key lethal toxin is identified. A good example is crotoxin, a ß-neurotoxin phospholipase A2-like toxin that presents anticoagulant activity in vitro and is responsible for the lethality of venoms of Crotalus durissus snakes. By using rotational thromboelastometry, we reported recently one sensitive coagulation assay for assessing relative potency of the anti-bothropic serum in neutralizing procoagulant activity of Bothrops jararaca venom upon recalcified factor-XII-deficient chicken plasma samples (CPS). In this study, we stablished conditions for determining relative potency of four batches of the anti-crotalic serum (ACS) (antagonist) in inactivating crotoxin anticoagulant activity in CPS (target) simultaneously treated with one classical activator of coagulation (agonists). The correlation coefficient (r) between values related the ACS potency in inactivating both in vitro crotoxin anticoagulant activity and the in vivo lethality of whole venom (ED50) was 0.94 (p value < 0.05). In conclusion, slowness in spontaneous thrombin/fibrin generation even after recalcification elicit time lapse sufficient for elaboration of one dose-response curve to pro- or anti-coagulant agonists in CPS. We propose this methodology as an alternative and sensitive assay for assessing antivenom neutralizing ability in plasma of immunized horses as well as for in-process quality control.

An alternative micromethod to access the procoagulant activity of Bothrops jararaca venom and the efficacy of antivenom.

The assessment of the capacity of antivenoms to neutralize the lethal activity of snake venoms still relies largely on traditional rodent lethality assay (LD50). However, adequately validated in vitro tests should be introduced for assessing antivenom neutralizing capacity in plasma of immunized horses as well as for in-process quality control. The dynamic of fibrin formation in recalcified avian plasma samples is extremely slow, when compared to that presented by mammalian plasmas. In this study, we present one new coagulant assay, by performing dose-response curve after plotting the clotting time (CT) parameter of the ROTEM profile of recalcified chicken plasma samples (target) against semi-logarithmic doses of Bothrops jararaca venom (agonist), either in absence or in presence of the semi-logarithmic doses of anti-bothropic serum (ABS) (antagonist). The mean coagulant dose 50% (CD50) was defined as the quantity of venom (in µg) which reduces CT to 900 s, between minimum and maximum responses. The CT induced by 5CD50 of the venom was used as the control for calculating the effective dose (ED) of each batch of ABS. ED was defined as the ABS dose (nanoliters, nL) at which CT induced by one amount of venom corresponding to 5CD50 is displaced to the maximum threshold (1800 s). Five batches of the ABS, previously assayed for their lethality neutralizing activity (ED50) were assayed. The correlation coefficient (r) between both in vitro (ED) and in vivo (ED50) values was 0.87 (p value < 0.05). We propose this micro method as highly sensitive for characterization and quantification of possible procoagulant activity of small doses of snake venoms (nanograms) and for detecting small doses (nanoliters) of specific antibodies against this effect in little volume samples of biological fluids.

Maintaining rear-fanged snakes for venom production: an evaluation of mortality and survival rates for Philodryas olfersii and P. patagoniensis in captivity

This study evaluates the mortality and average survival rates of captive female Philodryas olfersii and Philodryas patagoniensis snakes maintained for venom production. Also, two factors likely to reduce captive survival were studied - body condition at admission and seasonality. Mortality peaks occurred during the second month in captivity. More than half the individuals were dead at the end of the third month. This suggests that the first three months in captivity are the most critical in terms of survival and adaptation. Females collected and admitted during spring and summer lived less time than those collected in autumn and winter. As gravidity and egg-laying occur during spring and summer, we suggest that the lower survival rates in these seasons may be due to high costs and stress involved in these reproductive events. Unexpectedly, body mass and body condition were poor predictors of survival in captivity. Our results have important implications in maintaining snakes for venom production. We propose some prophylactic measures to minimize the deleterious impacts of captivity during the adaptation period.(AU)

Sexual dimorphism in venom of Bothrops jararaca(Serpentes: Viperidae).

Bothrops jararaca is an abundant snake in Brazil, and its venom has been studied exhaustively. The species exhibits adult size dimorphism in which female are larger. We registered the growth in Snout-Vent Length and weight of one litter (with 11 females and 12 males). We compared growth curves and venom profile between male and female of B. jararaca in order to establish the relationship of those characters and sex. Their venoms were analyzed when they were 36 months old, concerning SDS PAGE, protein content, proteolytic, hyaluronidasic, phospholipasic, blood-clotting, edematogenic, hemorrhagic, myotoxic activities, and lethality. Differences in the growth curves of the females and the males were significantly different after the 12th month of age, with the females growing faster. Females produced five times more venom than males. The electrophoretic patterns were variable: the venom from males had more protein bands than females. Venom composition varied significantly between males and females. Venom from females is more potent for hyaluronidasic, hemorrhagic, and lethality activities, whereas venom from males is more potent for coagulant, phospholipasic, and myotoxic activities. The variability of proteolytic and edematogenic activities were not significant. The important sexual dimorphism in body size and mass, amount of venom produced, and venom composition in B. jararaca may reflect a divergence in niche partitioning.