Vipera Ammodytes Research Articles

Preclinical Assessment of a New Polyvalent Antivenom (Inoserp Europe) against Several Species of the Subfamily Viperinae.

The European continent is inhabited by medically important venomous Viperinae snakes. Vipera ammodytes, Vipera berus, and Vipera aspis cause the greatest public health problems in Europe, but there are other equally significant snakes in specific regions of the continent. Immunotherapy is indicated for patients with systemic envenoming, of which there are approximately 4000 annual cases in Europe, and was suggested as an indication for young children and pregnant women, even if they do not have systemic symptoms. In the present study, the safety and venom-neutralizing efficacy of Inoserp Europe—a new F(ab’)2 polyvalent antivenom, designed to treat envenoming by snakes in the Eurasian region—were evaluated. In accordance with World Health Organization recommendations, several quality control parameters were applied to evaluate the safety of this antivenom. The venom-neutralizing efficacy of the antivenom was evaluated in mice and the results showed it had appropriate neutralizing potency against the venoms of several species of Vipera, Montivipera, and Macrovipera. Paraspecificity of the antivenom was demonstrated as well, since it neutralized venoms of species not included in the immunization schemes and contains satisfactory levels of total proteins and F(ab’)2 fragment concentration. Therefore, this new polyvalent antivenom could be effective in the treatment of snake envenoming in Europe, including Western Russia and Turkey.

Contribution to the phylogeography of the nose-horned viper (Vipera ammodytes, Linnaeus, 1758) in the Central Balkan Peninsula

Seven genetic clades have been recognized within the species Vipera ammodytes (nose-horned viper); however, the precise phylogenetic position of many Balkan populations is unknown. We used Bayesian analysis of the mtDNA sequences from the 16S rRNA mtDNA gene obtained from 47 individuals (26 novel samples sequenced in this study and 21 sequences available from GenBank). Our results show that sampled nose-horned vipers from localities in Serbia are clustered within three clades: the northeastern (23 individuals), the northwestern (two individuals) and the southeastern (one individual). Results revealed an overlapping distribution of the northeastern and the northwestern clades in two populations. We have revealed that the northeastern clade extends further south than previously suggested, to the Ohrid/Prespa lakes in North Macedonia. Our findings contribute to the knowledge of the genetic diversity of this species in Serbia and help to clarify the geographical distributions of mtDNA-defined clades. [Projects of the Serbian Ministry of Education, Science and Technological Development, Grant no. 173025 and Grant no. TR37009]

The First Intrinsic Tenase Complex Inhibitor with Serine Protease Structure Offers a New Perspective in Anticoagulant Therapy.

Components of the intrinsic blood coagulation pathway, among them factor VIIIa (FVIIIa), have been recognized as suitable therapeutic targets to treat venous thromboembolism, pathological process behind two very serious cardiovascular diseases, deep vein thrombosis and pulmonary embolism. Here, we describe a unique glycoprotein from the nose-horned viper (Vipera ammodytes ammodytes [Vaa]) venom, Vaa serine proteinase homolog 1 (VaaSPH-1), structurally a serine protease but without an enzymatic activity and expressing potent anticoagulant action in human blood. We demonstrated that one of its targets in the blood coagulation system is FVIIIa of the intrinsic tenase complex, where it antagonizes the binding of FIXa. Anticoagulants with such characteristics are intensively sought, as they would be much safer for medical application as the contemporary drugs, which frequently induce excessive bleeding and other complications. VaaSPH-1 is unlikely to be orally available for chronic usage as it has molecular mass of 35 kDa. However, it represents a very promising template to design low molecular mass FVIIIa-directed anticoagulant substances, based on structural features of the interaction surface between VaaSPH-1 and FVIIIa. To this end, we constructed a three-dimensional model of VaaSPH-1 bound to FVIIIa. The model exposes the 157-loop and the preceding α-helix as the most appropriate structural elements of VaaSPH-1 to be considered as a guideline to synthesize small FVIIIa-binding molecules, potential new generation of anticoagulants.

Effect of Vipera ammodytes ammodytes Snake Venom on the Human Cytokine Network

Local inflammation is a well-known symptom of envenomation by snakes of the family Viperidae, attributed primarily to the phospholipase A2s, metalloproteinases and L-amino acid oxidases contained in their venom. The inflammatory effect of snake venoms has been associated with a marked increase of the cytokines IL-1β, IL-6, IL-8, IL-10 and TNF-α. To determine the impact of Vipera ammodytes ammodytes snake venom on the expression of inflammation-related genes, we incubated human U937 monocyte cells with dilutions of snake venom. Gene expression was quantified for 28 different genes using a TaqMan® Array Human Cytokine Network 96-well Plate in a RT-qPCR system. Our results have demonstrated that 1.0 μg/mL Vipera ammodytes ammodytes venom solution induces a notable change in the expression of several cytokine network genes. Among the upregulated genes, there were several that encode interleukins, interferons, and tumor necrosis factors. We further report the downregulation of three interleukin-related genes. Our findings come as supportive information for the known complex effect of snake venoms on the human cytokine network. It also provides relevant new information regarding the expression of genes that have not been previously associated with the effect of snake venoms.

Cytotoxicity of Silica Nanoparticles with Transcaucasian Nose-Horned Viper, Vipera ammodytes transcaucasiana, Venom on U87MG and SHSY5Y Neuronal Cancer Cells.

Highly bioactive compounds of the snake venom make them particular sources for anticancer agent development. They contain very rich peptide-protein structures. Therefore, they are very susceptible to environmental conditions such as temperature, pH, and light. In this study, Vipera ammodytes transcaucasiana venom was encapsulated in PAMAM-G4 dendrimer by sol-gel method in order to prevent degradation of venom contents from the environmental conditions. For this purpose, nanoparticles were prepared by sol-gel methodology and SEM analyses were performed. U87MG and SHSY5Y neuronal cancer cell lines were treated with different concentrations of venom-containing nanoparticles and cytotoxicity was determined by MTT assay. IC50 values of nanoparticles with snake venom were calculated as 37.24 and 44.64μg/ml for U87MG and SHSY5Y cells, respectively. The IC50 values of nanoparticles with snake venom were calculated as 10.07 and 7.9μg/ml for U87MG and SHSY5Y cells, respectively. As a result, nanoparticles with V. a. transcaucasiana venom showed remarkably high cytotoxicity. Encapsulation efficiency of nanoparticles with 1mg/ml snake venom was determined as %67 via BCA™ protein analysis. In conclusion, this method is found to be convenient and useful for encapsulating snake venom as well as being suitable for drug delivery systems.

Contribution to the herpetofauna of Serbia: Distribution of reptiles in Kosovo and Metohija province

Kosovo and Metohija have already been recognized as regions with the highest diversity of reptiles in Serbia, where 92% (22 of 24) of existing reptile species can be found (Tomović et al., 2015a). First comprehensive contribution to herpetofauna of Kosovo and Metohija was provided by late Professor Gojko Pasuljević. In this study we present a complete dataset of distribution records for 13 most common reptile species in Kosovo and Metohija, including published and new distribution data compiled, and provide standardized 10 x 10 km UTM maps for these data. Results of this study include 1013 distribution records (278 new and 735 published data) for the following reptiles: Testudo hermanni, Ablepharus kitaibelii, Anguis fragilis, Lacerta agilis, Lacerta viridis, Podarcis muralis, Podarcis tauricus, Coronella austriaca, Dolichophis caspius, Natrix natrix, Natrix tessellata, Zamenis longissimus and Vipera ammodytes. The most widely distributed species, which occupy more than 50 UTM 10 x 10 km squares are: Podarcis muralis and Vipera ammodytes. Species with limited distribution which occupy less than 20 UTM 10 x 10 km are: Dolichophis caspius and Lacerta agilis. The largest numbers of new or confirmed literature data are recorded for: Anguis fragilis, Testudo hermanni and Vipera ammodytes. Having in mind that after 50 years, this is the first comprehensive study of the distribution of reptiles in Kosovo and Metohija exclusively, the presented faunistic data are far from being complete. Further systematic studies should provide a more complete insight into the distribution of herpetofauna of this province of Serbia.

Vipera ammodytes bites treated with antivenom ViperaTAb: a case series with pharmacokinetic evaluation

Context: In clinical practice it is difficult to differentiate between V. berus and V. ammodytes venomous bites. In the past this was not a concern, but due to the current shortage in Viperfav™ and European viper venom antiserum availability, V. a. ammodytes venomous bites have recently been treated with ViperaTAb®, which is a pharmaceutical formulation containing a monospecific ovine Fab fragments against the venom of V. berus.Objective: To evaluate ViperaTAb® in V. a. ammodytes envenomations.Materials and methods: This is a prospective case series of three consecutive patients envenomed by V. a. ammodytes snakebite treated with ViperaTAb®. V. ammodytes venom, neurotoxic ammodytoxins, and Fab fragment levels were determined in serum samples and a pharmacokinetic analysis of the antivenom Fab fragments was carried out.Results: Three patients bitten by V. a. ammodytes with extensive local swelling, neurological symptoms and recurrent thrombocytopenia were treated with ViperaTAb®. V. ammodytes venom was detected in serum of all three patients. Ammodytoxins were detected in the serum of only the most severely envenomed patient who developed neurological symptoms. In the presented moderate cases, a dose of 8 mL of ViperaTAb® reduced swelling and improved systemic effects, such as thrombocytopenia. However, this dose of ViperaTAb® was not effective in the most severely envenomed patient with the highest serum values of V. ammodytes venom. In this case ViperaTAb® did not stop local swelling and it had no effect on neurological signs. ViperaTAb®’s systemic clearance, distribution and elimination half-lives were 4.3–13.4 mL/h/kg, 1.2–3.2 h and 14.1–55.4 h, respectively.Conclusions: In patients envenomed by V. a. ammodytes venom, ViperaTAb® reduces moderate swelling and temporarily improves systemic effects, except neurological symptoms. ViperaTAb® application induces a decrement of V. ammodytes venom level in the blood, but did not affect serum concentration of neurotoxic ammodytoxins in the one patient with measurable concentrations.

Screening of cytotoxic, anti-angiogenic, anti-tumorogenic and antimicrobial activities of Anatolian Vipera ammodytes (Nose-horned viper) venom

Abstract Objective In the present study, we aimed to screen the cytotoxic, antimicrobial, anti-angiogenic and anti-tumorogenic activities of Anatolian Vipera ammodytes (Nose-horned Viper) crude venom. Material and methods The cytotoxicity was screened against PC3, HeLa, CaCo-2, U-87MG, MCF-7 and Vero cells by using MTT assay. The antimicrobial activity on Escherichia coli ATCC 25922, E. coli 0157:H7, Enterococcus faecalis 29212, Enterococcus faecium DSM 13590, Staphylococcus aureus ATCC 25923, Staphylococcus epidermidis ATCC 12228, Salmonella typhimirium CCM 5445, Proteus vulgaris ATCC 6957, Bacillus cereus ATCC 7064 and Candida albicans ATCC 10239 was assayed by determining the minimum inhibitory concentration using the broth dilution method. Anti-angiogenic and anti-tumorogenic activity was assessed by using chick chorioallantoic membrane (CAM) assay. Results The IC50 value of V. ammodytes venom on cultured cells varied from 1.8 to 7.0 μg/mL after 48 h treatment. Venom showed antimicrobial activity on P. vulgaris, S. aureus, S. epidermidis, E. faecium and C. albicans (the highest activity). The venom exhibited dose-dependent anti-angiogenic activity on CAM model at 2 and 10 μg/mL doses with scores of 1.1 and 2.0, respectively. Conclusion The results of the present study contributed to the knowledge of the biological activities of Anatolian V. ammodytes venom and showed its potential for further bioactivity guided characterization studies.

Cardiotoxic effects of the Vipera ammodytes ammodytes venom fractions in the isolated perfused rat heart

The nose-horned viper (Vipera ammodytes ammodytes) is the most venomous European snake. Its venom is known as haematotoxic, myotoxic and neurotoxic but it exerts also cardiotoxic effects. To further explore the cardiotoxicity of the venom we separated it into four fractions by gel filtration chromatography. Three fractions that contain polypeptides (A, B, and C) were tested for their effects on isolated rat heart. Heart rate (HR), incidence of arrhythmias (atrioventricular (AV) blocks, ventricular tachycardia, ventricular fibrillation, and asystolia), coronary flow (CF), systolic, developed and diastolic left ventricular pressure (LVP) were measured before, during, and after the application of venom fractions in three different concentrations. Fraction A, containing proteins of 60–100 kDa, displayed no effect on the rat heart. Fractions B and C disturbed heart functioning in similar way, but with different potency that was higher by the latter. This was manifested by significant decrease of HR and CF, the increase of diastolic, and the decrease of systolic and developed LVPs. All hearts treated with fraction C in the final CF concentrations 22.5 and 37.5 μg/mL suffered rapid and irreversible asystolia without AV blockade. They underwent also ventricular fibrillation and ventricular tachycardia. Fraction B affected hearts only at the highest dose inducing asystolia in all hearts, ventricular fibrillation in 80% and ventricular tachycardia in 70% of the hearts. Venom fraction C induced 71% of all recorded heart rhythm disturbances, significantly more than fraction B, which induced 29%. Most abundant proteins in fraction C were secreted phospholipases A2 among which the venom component acting on the heart is most probably to be looked for.

Venomics of Vipera berus berus to explain differences in pathology elicited by Vipera ammodytes ammodytes envenomation: Therapeutic implications

Vipera berus berus (Vbb) is the most widely distributed and Vipera ammodytes ammodytes (Vaa) the most venomous viper in Europe. In particular areas of the Old continent their toxic bites constitute a considerable public health problem. To make the current envenomation therapy more effective we have analysed the proteome of Vbb venom and compared it with that of Vaa. We found the proteome of Vbb to be much less complex and to contain smaller levels of particularly snaclecs and sPLA2s. Snaclecs are probably responsible for thrombocytopenia. The neurotoxic sPLA2s, ammodytoxins, are responsible for the most specific feature of the Vaa venom poisoning − induction of signs of neurotoxicity in patients. These molecules were not found in Vbb venom. Both venoms induce haemorrhage and coagulopathy in man. As Vaa and Vbb venoms possess homologous P-III snake venom metalloproteinases, the main haemorrhagic factors, the severity of the haemorrhage is dictated by concentration and specific activity of these molecules. The much greater anticoagulant effect of Vaa venom than that of Vbb venom lies in its higher extrinsic pathway coagulation factor-proteolysing activity and content of ammodytoxins which block the prothrombinase complex formation. Biological significanceEnvenomations by venomous snakes constitute a considerable public health problem worldwide, and also in Europe. In the submitted work we analysed the venom proteome of Vipera berus berus (Vbb), the most widely distributed venomous snake in Europe and compared it with the venom proteome of the most venomous viper in Europe, Vipera ammodytes ammodytes (Vaa). We have offered a possible explanation, at the molecular level, for the differences in clinical pictures inflicted by the Vbb and Vaa venoms. We have provided an explanation for the effectiveness of treatment of Vbb envenomation by Vaa antiserum and explained why full protection of Vaa venom poisoning by Vbb antiserum should not be always expected, especially not in cases of severe poisoning. The latter makes a strong case for Vaa antiserum production as we are faced with its shortage due to ceasing of production of two most frequently used products.

Comparative assay of Vipera ammodytes antivenom potency

The finding of the most appropriate way to assess precisely the antivenom efficacy represents one of the major issues for antivenom standardization and success increasing of antivenom therapy. The efficacy of experimental Vipera ammodytes antivenom raised in sheep was determined using in vivo mouse lethality test, respectively, L-aminoacid oxidase, total proteinase and phospholipase A2 antienzymatic effectiveness. The values gained for the antivenom potency depend on the method of measure. So, some of the most toxic venom proteins own phospholipase A2 activity and provide the highest antivenom potency (lowest effective dose) values by antienzymatic assay method. This value is similar with total antiproteolytic antivenom potency value, but almost three times higher than value obtained by L-aminoacid oxidase (low toxic viper venom protein) antienzymatic assay method.

Cloacoscopy in the Horned Viper (Vipera ammodytes)

This study illustrates the main morphological structures of the cloaca of six adult horned vipers Vipera ammodytes (3.3). The coprodeum is characterized by smooth and thin mucosa, with moderate vasculature. In the centre of the coprodeum the anal sphincter is located, which separates the coprodeum from the colon. Structures of the urodeum are divided by two highly developed septa in female vipers. The urodeum in female vipers is characterized by distinct chambers, which become visible immediately behind the exit of the sphincter of the coprodeum. Vaginal pouches leading to the entrance of the oviduct are visible. The male urodeum is characterized by two distinct structures. In the upper part of the urodeum, the coprodeum sphincter surrounded by a gently folded mucosa, and the urethral papillae surface on a large mucosal protrusion emerging from the dorsal urodeum are visible in male horned vipers. The mucosal protrusion that hosts the urethral papillae can be found in two different appearances, closed or open. The proctodeum is a tubular and muscular structure that leads to the vent. Cloacoscopy is as a safe and effective method for cloaca exploration in both sexes of the horned viper. This technique may be used to assist artificial insemination in the horned viper in the future.

The dynamics of coexistence: habitat sharing versus segregation patterns among three sympatric montane vipers

Contact zones of closely related and ecologically similar species constitute rare opportunities to study the evolutionary consequences of past speciation processes. They represent natural laboratories in which strong competition could lead to the exclusion of one species, or the various species may switch into distinct ecological niches. Alternatively, if reproductive isolation has not yet been achieved, they may hybridize. We elucidate the degree of taxon integrity by comparing genetics and habitat use of three similar-sized congeneric viper species, Vipera ammodytes, Vipera aspis, and Vipera berus, of Nadiza Valley in western Slovenia. No hybridization was detected for either mitochondrial or nuclear genomes. Similarly, external intermediacy by a single prestudy viper (probably V. ammodytes 9 V. aspis) indicates that hybridization occasionally occurs, but should be very rare. Populations of the three related viperids are partially allopatric in Nadiza Valley, but they also coexist in a narrow contact zone in the montane grassland along the south-exposed slope of Mount Stol (1673 m a.s.l.). Here, the three species that occupy areas in or near patches of rocky microhabitats (e.g. stone piles, slides, and walls) live in syntopy. However, fine-scale measurements of structural components show partial habitat segregation, in which V. berus becomes more dominant at elevations above 1400 m and occupies mostly the mountain ridge and north-exposed slopes of Mount Stol, V. aspis occurs below 1300 m and is the only species to inhabit stoneless patches of grass and bushes around 1000 m and lower, and V. ammodytes occurs at all elevations up to 1500 m, but is restricted to a rocky microhabitat. We suggest that a high degree of microstructure divergence, slightly different environmental niches, and a generally favourable habitat for all three viper species, keep the pressure for mis-mating and hybridization low, although mechanisms such as reduced hybrid inferiority and temporal mating segregation cannot yet be excluded.

Structural and biochemical characterisation of VaF1, a P-IIIa fibrinogenolytic metalloproteinase from Vipera ammodytes ammodytes venom.

A high molecular mass metalloproteinase with α-fibrinogenolytic activity, termed VaF1, was purified from nose-horned viper (Vipera ammodytes ammodytes) venom. Subcutaneous injection of 9 μg of VaF1 did not induce bleeding in rats. Nevertheless, in vitro it degraded collagen IV, nidogen and fibronectin, components of the extracellular matrix, although with low efficacy and narrow specificity. VaF1 would be expected to exert anti-coagulant action, due to its hydrolysis of fibrinogen, factor X, prothrombin and plasminogen, plasma proteins involved in blood coagulation. The enzyme is a single-chain glycoprotein with a molecular mass of 49.7 kDa, as determined by mass spectrometry, and multiple isoelectric points centred at pH 5.8. The complete amino acid sequence of the precursor of VaF1 was deduced by cloning and sequencing its cDNA. Composed of metalloproteinase, disintegrin-like and cysteine-rich domains, VaF1 is a typical P-IIIa subclass snake venom metalloproteinase. Although it possesses a collagen-binding sequence in its disintegrin-like domain, VaF1 displayed no effect on collagen-induced platelet aggregation in vitro. Two consensus N-glycosylation sites are present in the sequence of VaF1, however, the extent of its glycosylation is low, only 5.2% of the total molecular mass. Interestingly, in standard experimental conditions VaF1 is not recognised by antiserum against the whole venom, so it can contribute to post-serotherapy complications, such as ineffective blood coagulation, in the envenomed patient.

<p><strong>TWO FANGS GOOD, A HUNDRED LEGS BETTER: JUVENILE VIPER DEVOURED BY AN ADULT CENTIPEDE IT HAD INGESTED</strong></p>

On May 14th 2013, on the island of Golem Grad (Prespa Lake, FYR of Macedonia) a juvenile female nose-horned viper (Vipera ammodytes) was found dead, with head of a Scolopendra cingulata protruding through the body wall of its lower abdomen. The authors discuss the possibility that the snake had swallowed the centipede alive, and that, paradoxically, the prey has eaten its way through the snake, almost reaching its freedom.

New locality records for four rare species of vipers (Reptilia: Viperidae) in Turkey

We report new localities and range extensions for four vipers from Turkey. The distribution of Montivipera wagneri is extended substantially southwards into Muş province, showing that this species is not endemic to the Aras valley as previously stated in the literature. Similarly, an unverified photograph of Montivipera albizona from Erzincan province, at its currently known eastern limit, was confirmed with new material, and its range is extended substantially southwards to the Anamos (Nur) Mountains in Hatay province. Smaller range extensions are reported for Vipera ammodytes transcaucasiana and V. darevskii.

Production and assessment of ovine antisera for the manufacture of a veterinary adder antivenom

Medically important venomous snakes in Western Europe are Vipera ammodytes, Vipera aspis, Vipera berus and Vipera latastei. Envenomation of dogs and other animals by these snakes receives limited attention despite...

Snake venom poisoning in the Plovdiv region from 2004 to 2012.

Envenomation by poisons of biological origin is very common globally in the tropical and subtropical areas mainly, where the biological diversity of the species clearly leads to evolution of highly toxic species. The weather warming trend in Bulgaria, whether cyclic or permanent, allows for a change in the biological response of reptiles and insects inhabiting the temperate zone by a possible migration of biological species from the subtropical zone towards the temperate zone because of the new environmental conditions. There are very few studies on snake bite envenoming in Bulgaria. The AIM of the study was to find the incidence of the acute accidental intoxication (AAI) caused by snake venom in adult individuals in a large region of Bulgaria between 2004 and 2012 and characterises it by number, type, main clinical features, course and socio-demographic parameters of the victims so that preventive measures can be taken, wherever necessary. We studied retrospectively all 68 cases of AAI caused by snake venom in adult individuals (> 18 years old) hospitalized in the Clinic of Toxicology in St. George University Hospital, Plovdiv over the period from 2004 to 2012 by 23 quantitative and qualitative parameters. We found that the average annual incidence of snake venom AAI in adult population in the region of Plovdiv was relatively low for the specified period (9.5 per 100000 residents); the snake venom AAI increases or decreases every other year, with no clearly delineated trend for now. The prevalence of envenomation by poisons of biological origin increased from 2.3% in 1990-1998 to 9.5-10.33% between 2007 and 2012. The main sociodemographic characteristics of snake bite victims are similar to those in other Balkan and Central European countries. The clinical response to poisons of biological origin is generally identical with the response to the viper (Vipera ammodytes)--mild to medium intensity with predominantly local toxic syndrome. The algorithm of Clinical Pathway 293 (CP) is effective and conducive to the reduction of duration of the morbid condition. There are, however, still aspects of it that can be optimised.

Age-dependent variations in the venom proteins of Vipera kaznakovi Nikolsky, 1909 and Vipera ammodytes (Linnaeus, 1758) (Ophidia: Viperidae)

Polyacrylamide disc gel electrophoresis and densitometry analysis were used to analyze venom extracts of Vipera kaznakovi (16.5, 30, and 55 cm) and Vipera ammodytes (28.5, 36.7, and 51.5 cm) specimens of 3 different lengths. V. kaznakovi specimens were collected from Hopa (Artvin Province); 2 V. ammodytes specimens were from Ağva (İstanbul Province) and the longest one was from Yuvacık (Kocaeli Province). The electropherograms of the venom protein samples showed age-dependent qualitative and quantitative variations.

Paraspecificity of Vipera a. ammodytes-specific antivenom towards Montivipera raddei and Macrovipera lebetina obtusa venoms

Antivenom raised against the venom of nose-horned viper, Vipera ammodytes (V. a.) ammodytes (European viper venom antiserum, Zagreb antivenom), contains neutralising equine F(ab′)2 fragments that are clinically successful against homologous venom, but also against the venoms of several others medically important European snakes due to its paraspecific action. In this work we demonstrated that Zagreb antivenom is preclinically effective in neutralising lethal toxicity and hemorrhagicity of venoms of Armenian mountain snakes – Montivipera raddei and Macrovipera lebetina obtusa as well. In order to better understand the biochemical basis of the observed paraspecificity, the ability of anti-V. a. ammodytes serum to recognise and neutralise proteinases of the two venoms was also investigated. Anti-V. a. ammodytes serum showed surprisingly low capacity to inhibit metalloproteinases of both venoms included in the study, probably due to weak immunorecognition of their P-I representatives. Also, it completely failed to abolish enzymatic action of serine proteinases from Macrovipera lebetina obtusa venom. Relevance of such finding is yet to be established.