Crotamine in Crotalus durissus: distribution according to subspecies and geographic origin, in captivity or nature

Abstract Background: Crotalus durissus is considered one of the most important species of venomous snakes in Brazil, due to the high mortality of its snakebites. The venom of Crotalus durissus contains four main toxins: crotoxin, convulxin, gyroxin and crotamine. Venoms can vary in their crotamine content, being crotamine-negative or -positive. This heterogeneity is of great importance for producing antivenom, due to their different mechanisms of action. The possibility that antivenom produced by Butantan Institute might have a different immunorecognition capacity between crotamine-negative and crotamine-positive C. durissus venoms instigated us to investigate the differences between these two venom groups. Methods: The presence of crotamine was analyzed by SDS-PAGE, western blotting and ELISA, whereas comparison between the two types of venoms was carried out through HPLC, mass spectrometry analysis as well as assessment of antivenom lethality and efficacy. Results: The results showed a variation in the presence of crotamine among the subspecies and the geographic origin of snakes from nature, but not in captive snakes. Regarding differences between crotamine-positive and -negative venoms, some exclusive proteins are found in each pool and the crotamine-negative pool presented more phospholipase A2 than crotamine-positive pool. This variation could affect the time to death, but the lethal and effective dose were not affected. Conclusion: These differences between venom pools indicate the importance of using both, crotamine-positive and crotamine-negative venoms, to produce the antivenom.

研究背景：南美响尾蛇（Crotalus durissus）是巴西境内最为重要的有毒蛇类之一，其引发的蛇咬伤事件致死率极高。该蛇的蛇毒含有四种核心毒素：克罗毒素（crotoxin）、惊厥毒素（convulxin）、陀螺毒素（gyroxin）与响尾蛇毒素（crotamine）。不同蛇毒样品的响尾蛇毒素含量存在异质性，可分为响尾蛇毒素阴性与阳性两类。由于两类蛇毒的作用机制存在显著差异，这种异质性对抗蛇毒血清的生产具有关键意义。巴西布坦坦研究所（Butantan Institute）制备的抗蛇毒血清，在针对响尾蛇毒素阴性与阳性的南美响尾蛇蛇毒时，可能存在不同的免疫识别能力，这一推测促使我们开展本研究以探究两类蛇毒之间的具体差异。
研究方法：本研究通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）、蛋白质印迹法（western blotting）与酶联免疫吸附测定（ELISA）检测蛇毒中响尾蛇毒素的存在情况；同时采用高效液相色谱（HPLC）、质谱分析，以及抗蛇毒血清的致死性与中和效力评估，对两类蛇毒进行对比分析。
研究结果：结果显示，野生南美响尾蛇的蛇毒中响尾蛇毒素的存在情况会随亚种与地理分布的不同而存在差异，但人工饲养个体的蛇毒则无此特征。在两类蛇毒的对比中，各自的混合毒样均含有专属的蛋白质组分；且响尾蛇毒素阴性混合毒样的磷脂酶A2含量高于阳性混合毒样。这种组分差异可能会影响中毒动物的死亡时间，但并未改变蛇毒的半数致死量与抗蛇毒血清的有效中和剂量。
研究结论：两类蛇毒混合样间的上述组分差异表明，在制备抗蛇毒血清时，需同时使用响尾蛇毒素阳性与阴性的蛇毒原料，这一点具有重要的实践指导价值。