Neutralization of Vipera and Macrovipera venoms by two experimental polyvalent antisera: A study of paraspecificity
Highlights
► We studied paraspecific neutralization of an array of venoms by experimental antisera. ► Vipera and Macrovipera venoms were of European and Asian origin. ► We found an important degree of intragenus paraspecific neutralization of lethality. ► The more polyvalent antiserum was generally more effective.
Introduction
The genus Vipera is widespread throughout Europe, Western and Central Asia. It is a genus in constant revision and recognizes some two dozen species and a number of subspecies (Barbanera et al., 2009, David and Ineich, 1999, Garrigues et al., 2005, Joger et al., 2007, Lenk et al., 2001, Stümpel and Joger, 2009, Thorpe et al., 2007, Ursenbacher et al., 2006, Ursenbacher et al., 2008, Wüster, 1998, Wüster et al., 2008). The genus Macrovipera extends from Eastern Europe to Western and Central Asia, as well as Mediterranean Africa (David and Ineich, 1999). Between 1999 and 2008, several genus-level name changes have occured, most notably the transfer of some species of Vipera and Macrovipera to the genera Daboia and Montivipera (usefully summarized in WHO, 2010, Reptile database, 2010, and references therein). The epidemiology of snake bite by several European Vipera is known, especially in West, North and Central Europe, whereas information on morbidity and mortality due to Vipera and Macrovipera in Eastern Europe and Asia is more patchy and scarce (Chippaux, 1998, Chippaux, 2006). Table 1 summarizes estimations of incidence of snake bites in Europe including Turkey. Although the incidence is not high, especially when comparing to the tropical and sub-tropical world, severe envenomations often require antivenom and there is little data for a number of European nations.
A few Vipera venoms have been studied in some depth, notably Vipera aspis, Vipera ammodytes ammodytes and Vipera berus, and several of their toxins and activities characterized (for example, see Calderón et al., 1993, Carlsson et al., 2004, Jan et al., 2007, Lang Balija et al., 2005, Ramazanova et al., 2008). However, less is known of Central Asian members of the genus (Bernadsky et al., 1986, Latifi, 1984, Sanz et al., 2008, Weinstein and Minton, 1984). Vipera and Macrovipera venoms have been described as strongly inflammatory and necrotizing, and in some instances Vipera bites result in manifest neurotoxicity (Ferquel et al., 2007, Westerström et al., 2010). Coagulopathy is rare, at least in V. berus bites (Warrell, 2005).
Paraspecificity (also known as cross-neutralization) refers to the capacity of an antivenom to neutralize the venom of species not included in the immunization scheme of the animals used for antivenom production at therapeutically useful doses, i.e. not excessively beyond those necessary for specific neutralization. It has been studied within some genera, and sometimes extends beyond a genus (Casasola et al., 2008, Christensen, 1959, Gutiérrez et al., 2010, Khow et al., 1997, Ramos-Cerrillo et al., 2008, Segura et al., 2010, Tan et al., 1994). Paraspecificity is determined by animal experimentation, notably by neutralization of venom lethality in mice, and extrapolation of these results to clinical envenomation is to be undertaken with caution (WHO, 2010). Nevertheless, systematic information of the bona fide spectrum of paraspecific neutralization of lethality may be of use to treating clinicians in cases where the offending snake is not identified, or in cases where the offending species is identified but not included in the immunization protocol; the severity of envenomation, the resources available and other considerations, i.e. the expected safety of the antivenom and the danger of sequelae even when symptomatic treatment would suffice to prevent death, must guide the choice to use antivenom in the absence of clinical validation of antivenom efficacy for particular species (WHO, 2010).
Paraspecific neutralization has not been studied in any detail in either Vipera or Macrovipera, and very little is known in general about the neutralization of some European species of Vipera such as Vipera latastei, Vipera seoanei or Vipera ammodytes montandoni or Asian Vipera such as Vipera (Mo.) raddei, Vipera (Mo.) latifii, Vipera (Mo.) bornmuelleri or Vipera lotievi. In this investigation we generated two polyvalent experimental equine antisera to study the paraspecific spectrum of protection afforded by each against a collection of 12 Vipera and 5 Macrovipera venoms. The aims of the study were to establish whether paraspecific neutralization exists, its extent and the potency of paraspecific versus specific neutralization within and between each genus.
Section snippets
Venoms
All venoms (Latoxan, Valence, France) were purchased as lyophilized solids and were certified as to the origin of the snakes. Before use, they were dissolved in sterile 0.15 M NaCl at a concentration of 2.5 mg/ml.
Antisera
The trivalent antiserum was raised by immunizing three horses with a mixture of equal quantities of V. ammodytes ammodytes, Vipera aspis aspis and Vipera berus berus venoms. The pentavalent antiserum was raised by immunization of three horses with a mixture consisting of equal
Lethal potency of Vipera and Macrovipera venoms
With the exception of the venoms of Vipera aspis and V. seoanei, all Vipera venoms were significantly more lethal than Macrovipera venoms. The most potent was that of V. (Mo). raddei (3.87 μg) and the one with lowest potency was that of V. aspis (15.6 μg). All LD50 values are summarized in Table 2. In the case of Macrovipera, the most lethal was that of Ma. l. cernovi (17.6 μg) and the least was that of Ma. l. obtusa (30.1 μg).
Hemorrhagic, procoagulant and antihemostatic effects of selected Vipera and Macrovipera venoms
As shown in Table 3, hemorrhage was induced in rats by all venoms
Discussion
In our hands Vipera venoms were significantly more lethal than Macrovipera venoms, with LD50 values which were low by comparison to many vipers studied to date. All venoms tested caused subcutaneous hemorrhage in rats, and all but the two subspecies of V. ammodytes (ammodytes and montandoni) were also hemorrhagic in mice. None of the venoms tested exhibited procoagulant effects on normal human plasma (indicating activation of thrombin and/or other coagulation factors) or bovine fibrinogen
Author contributions
A.A. and R.P.S. designed research. A.A., A.R.d.R, B.R.-C, I.G.A. and J.-P.C. conducted research. A.d.R., I.G.A., J.-P.C., L.O.-P. and R.P.S analyzed data. J.-P.C. and R.P.S. wrote the paper.
Conflict of Interest
None declared.
Acknowledgments
We wish to acknowledge the technical assistance of Carlos Olvera and Claudia Moctezuma. This project was partially funded by a collaborative research grant to R.P.S. and A.A. from Instituto Bioclon of Mexico and by a grant from FONCICyT-CONACyT (Mexico) 93608.
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