A Guide to the Clinical Management of Vipera Snakebite in Italy.

The genus Vipera encompasses most species of medically significant venomous snakes of Europe, with Italy harbouring four of them. Envenomation by European vipers can result in severe consequences, but underreporting and the absence of standardised clinical protocols hinder effective snakebite management. This study provides an updated, detailed set of guidelines for the management and treatment of Vipera snakebite tailored for Italian clinicians. It includes taxonomic keys for snake identification, insights into viper venom composition, and recommendations for clinical management. Emphasis is placed on quick and reliable identification of medically relevant snake species, along with appropriate first aid measures. Criteria for antivenom administration are outlined, as well as indications on managing potential side effects. While the protocol is specific to Italy, its methodology can potentially be adapted for other European countries, depending on local resources. The promotion of comprehensive data collection and collaboration among Poison Control Centres is advocated to optimise envenomation management protocols and improve the reporting of epidemiological data concerning snakebite at the country level.

Investigating Snake-Venom-Induced Dermonecrosis and Inflammation Using an Ex Vivo Human Skin Model.

Snakebite envenoming is a neglected tropical disease that causes >100,000 deaths and >400,000 cases of morbidity annually. Despite the use of mouse models, severe local envenoming, defined by morbidity-causing local tissue necrosis, remains poorly understood, and human-tissue responses are ill-defined. Here, for the first time, an ex vivo, non-perfused human skin model was used to investigate temporal histopathological and immunological changes following subcutaneous injections of venoms from medically important African vipers (Echis ocellatus and Bitis arietans) and cobras (Naja nigricollis and N. haje). Histological analysis of venom-injected ex vivo human skin biopsies revealed morphological changes in the epidermis (ballooning degeneration, erosion, and ulceration) comparable to clinical signs of local envenoming. Immunostaining of these biopsies confirmed cell apoptosis consistent with the onset of necrosis. RNA sequencing, multiplex bead arrays, and ELISAs demonstrated that venom-injected human skin biopsies exhibited higher rates of transcription and expression of chemokines (CXCL5, MIP1-ALPHA, RANTES, MCP-1, and MIG), cytokines (IL-1ß, IL-1RA, G-CSF/CSF-3, and GM-CSF), and growth factors (VEGF-A, FGF, and HGF) in comparison to non-injected biopsies. To investigate the efficacy of antivenom, SAIMR Echis monovalent or SAIMR polyvalent antivenom was injected one hour following E. ocellatus or N. nigricollis venom treatment, respectively, and although antivenom did not prevent venom-induced dermal tissue damage, it did reduce all pro-inflammatory chemokines, cytokines, and growth factors to normal levels after 48 h. This ex vivo skin model could be useful for studies evaluating the progression of local envenoming and the efficacy of snakebite treatments.

Variability in antivenom neutralization of Mexican viperid snake venoms.

BACKGROUND: Each year, 3,800 cases of snakebite envenomation are reported in Mexico, resulting in 35 fatalities. The only scientifically validated treatment for snakebites in Mexico is the use of antivenoms. Currently, two antivenoms are available in the market, with one in the developmental phase. These antivenoms, produced in horses, consist of F(ab')2 fragments generated using venoms from various species as immunogens. While previous studies primarily focused on neutralizing the venom of the Crotalus species, our study aims to assess the neutralization capacity of different antivenom batches against pit vipers from various genera in Mexico. METHODOLOGY: We conducted various biological and biochemical tests to characterize the venoms. Additionally, we performed neutralization tests using all three antivenoms to evaluate their effectiveness against lethal activity and their ability to neutralize proteolytic and fibrinogenolytic activities. RESULTS: Our results reveal significant differences in protein content and neutralizing capacity among different antivenoms and even between different batches of the same product. Notably, the venom of Crotalus atrox is poorly neutralized by all evaluated batches despite being the primary cause of envenomation in the country's northern region. Furthermore, even at the highest tested concentrations, no antivenom could neutralize the lethality of Metlapilcoatlus nummifer and Porthidium yucatanicum venoms. These findings highlight crucial areas for improving existing antivenoms and developing new products. CONCLUSION: Our research reveals variations in protein content and neutralizing potency among antivenoms, emphasizing the need for consistency in venom characteristics as immunogens. While Birmex neutralizes more LD50 per vial, Antivipmyn excels in specific neutralization. The inability of antivenoms to neutralize certain venoms, especially M. nummifer and P. yucatanicum, highlights crucial improvement opportunities, given the medical significance of these species.

Expression, Purification and Characterization of Recombinant Disintegrin from Gloydius Brevicaudus Venom in Escherichia Coli.

Disintegrins, a family of snake venom protein, which are capable of modulating the activity of integrins that play a fundamental role in the regulation of many physiological and pathological processes. The main purpose of this study is to obtain the recombinant disintegrin (r-DI) and evaluate its biological activity. In this study, we explored a high-level expression prokaryotic system and purification strategy for r-DI. Then, r-DI was treated to assay effects on cell growth, migration, and invasion. The affinity for the interactions of r-DI with integrin was determined using Surface plasmon resonance (SPR) analyses. The r-DI can be expressed in Escherichia coli and purified by one-step chromatography. The r-DI can inhibit B16F10 cells proliferation, migration, and invasion. Also, we found that r-DI could interact with the integrin αIIbß3 (GPIIb/IIIa). The r-DI can be expressed, purified, characterized through functional assays, and can also maintain strong biological activities. Thus, this study showed potential therapeutic effects of r-DI for further functional and structural studies.

Phylogenomic insights into the diversity and evolution of Palearctic vipers.

Despite decades of molecular research, phylogenetic relationships in Palearctic vipers (genus Vipera) still essentially rely on a few loci, such as mitochondrial barcoding genes. Here we examined the diversity and evolution of Vipera with ddRAD-seq data from 33 representative species and subspecies. Phylogenomic analyses of âˆ¼ 1.1 Mb recovered nine major clades corresponding to known species/species complexes which are generally consistent with the mitochondrial phylogeny, albeit with a few deep discrepancies that highlight past hybridization events. The most spectacular case is the Italian-endemic V. walser, which is grouped with the alpine genetic diversity of V. berus in the nuclear tree despite carrying a divergent mitogenome related to the Caucasian V. kaznakovi complex. Clustering analyses of SNPs suggest potential admixture between diverged Iberian taxa (V. aspis zinnikeri and V. seoanei), and confirm that the Anatolian V. pontica corresponds to occasional hybrids between V. (ammodytes) meridionalis and V. kaznakovi. Finally, all analyzed lineages of the V. berus complex (including V. walser and V. barani) form vast areas of admixture and may be delimited as subspecies. Our study sets grounds for future taxonomic and phylogeographic surveys on Palearctic vipers, a group of prime interest for toxinological, ecological, biogeographic and conservation research.

A longitudinal study of the blood and urine metabolome of Vipera berus envenomated dogs.

Envenomation of dogs by the common European adder (Vipera berus) is associated with high morbidity. The cytotoxic venom of Vipera berus contains enzymes with the potential to cause acute kidney injury, among other insults, however robust biomarkers for such effects are lacking. A prospective observational follow-up study of naturally envenomated dogs and controls was conducted to fill knowledge gaps regarding canine Vipera berus envenomation, attempt to identify novel biomarkers of envenomation and related kidney injury, and elucidate potential long-term effects. Blood and urine samples were analyzed with a global metabolomics approach using liquid chromatography-mass spectrometry, uncovering numerous features significantly different between cases and controls. After data processing and feature annotation, eight features in blood and 24 features in urine were investigated in order to elucidate their biological relevance. Several of these are associated with AKI, while some may also originate from disturbed fatty acid ß-oxidation and soft tissue damage. A metabolite found in both blood and a venom reference sample may represent identification of a venom component in case dogs. Our findings suggest that envenomated dogs treated according to current best practice are unlikely to suffer permanent injury.

Peripheral Nerve Block to Treat Pain Caused by Daboia palaestinae Envenomation.

Snakebite pain can be challenging to control. We describe our experience managing intolerable pain after conventional treatment failed. A 35-year-old man, presented after a viper snakebite, suffering from intolerable pain in the affected extremity. He had no significant past medical history. All attempts to control the pain conventionally were unsuccessful. Treatment with a supraclavicular nerve block resulted in immediate relief. After the block receded, only a dull pain remained, which later disappeared without recurrence. This experience illustrates the need for personalized pain treatment to avoid subsequent complications.

Phylogenomics of Psammodynastes and Buhoma (Elapoidea: Serpentes), with the description of a new Asian snake family.

Asian mock vipers of the genus Psammodynastes and African forest snakes of the genus Buhoma are two genera belonging to the snake superfamily Elapoidea. The phylogenetic placements of Psammodynastes and Buhoma within Elapoidea has been extremely unstable which has resulted in their uncertain and debated taxonomy. We used ultraconserved elements and traditional nuclear and mitochondrial markers to infer the phylogenetic relationships of these two genera with other elapoids. Psammodynastes, for which a reference genome has been sequenced, were found, with strong branch support, to be a relatively early diverging split within Elapoidea that is sister to a clade consisting of Elapidae, Micrelapidae and Lamprophiidae. Hence, we allocate Psammodynastes to its own family, Psammodynastidae new family. However, the phylogenetic position of Buhoma could not be resolved with a high degree of confidence. Attempts to identify the possible sources of conflict in the rapid radiation of elapoid snakes suggest that both hybridisation/introgression during the rapid diversification, including possible ghost introgression, as well as incomplete lineage sorting likely have had a confounding role. The usual practice of combining mitochondrial loci with nuclear genomic data appears to mislead phylogeny reconstructions in rapid radiation scenarios, especially in the absence of genome scale data.

Paranannizziopsis spp. Infection in Wild Vipers, Europe.

We describe the detection of Paranannizziopsis sp. fungus in a wild population of vipers in Europe. Fungal infections were severe, and 1 animal likely died from infection. Surveillance efforts are needed to better understand the threat of this pathogen to snake conservation.

The use of rotational thromboelastometry to guide management following Bitis nasicornis envenoming.

A man in his thirties presented following Bitis nasicornis envenoming. His coagulation was assessed using rotational thromboelastometry (ROTEM). It identified a subtle abnormality, not detected using standard laboratory assessments of coagulation, and influenced ongoing management. The abnormality resolved following treatment with antivenom. There are few documented cases of using ROTEM to assess patients following haemotoxic envenoming. This case highlights some of the potential benefits and limitations of doing so.

Pit Viper Envenomation in Two Pregnant Bitches.

Snake envenomation is relatively common in small animals, particularly in endemic areas. Effects and outcomes of envenomation during pregnancy are poorly described in humans and more so in veterinary patients. Two young pregnant female dogs presented to a university teaching hospital with a history of acute soft tissue swelling and bleeding. History, physical examination findings, and diagnostics were consistent with envenomation by crotalid snakes. Medical management of one of the dogs included administration of antivenin. Both dogs survived envenomation with minimal complications and went on to whelp without complications, and all fetuses survived. This is the first description of the management of pit viper envenomation in pregnant dogs.

Reproductive cycles of two island pitvipers species (Serpentes, Viperidae) determined by ultrasonography and radiography.

We used ultrasonography and radiography to assess the sexual organs and characterize the reproductive cycle of captive golden lancehead (Bothrops insularis) and Alcatrazes lancehead (B. alcatraz), two endangered island snake species in Brazil. We assessed 46- individuals of golden lancehead and 12 of Alcatrazes lancehead kept in captivity between 2014 and 2020. Follicular development was similar between species, but follicles in Alcatrazes lancehead were smaller than in the golden lanceheads. Female golden lanceheads produced 24 live young, seven stillborn and 73 undeveloped eggs. Parturition of live young occurred between midsummer (February) and early autumn and gestation averaged 8 months. Female Alcatrazes lanceheads produced four live young in midsummer, and one undeveloped egg in early autumn. Males and females of both species have seasonal and biennial reproductive cycles. Sperm storage in both sexes is essential to coordinate male and female cycles. The data obtained with golden lancehead and Alcatrazes lancehead in captivity, demonstrate a degree of conservatism, following data from other Bothrops.

Serpentirhabdias mexicanus n. sp. (Nematoda: Rhabdiasidae), a parasitic lungworm of the nauyaca viper Bothrops asper (Serpentes: Viperidae) in the Mexican Neotropics.

Serpentirhabdias mexicanus n. sp. (Nematoda: Rhabdiasidae) is described from the lung of the nauyaca viper Bothrops asper in Puebla State, central Mexico. This new species is the fifth of the genus described having onchia. Among the species included in this group, the new species is morphologically closest to S. viperidicus and S. atroxi. However, it differs from both species mainly by having only one excretory gland (compared to two present in S. viperidicus and S. atroxi). In addition, S. mexicanus n. sp. can be separated of S. viperidicus by tail length, shape of vulval lips, geographic distribution and host species and from S. atroxi by body length, number of papillae in the cephalic region, as well as the host species and geographic distribution. In the present study, we propose the new species based on morphological, host spectrum and genetic evidence. Phylogenetic analysis indicated Serpentirhabdias as a monophyletic group, with two subgroups that are congruent with the presence/absence of onchia in the esophagostome, host association and other relevant morphological characters.

Isolation and characterization of polymorphic microsatellite loci for the three Iberian vipers, Vipera aspis, V. Latastei and V. seoanei by Illumina MiSeq sequencing.

BACKGROUND: European vipers (genus Vipera) are a well-studied taxonomic group, but the low resolution of nuclear sanger-sequenced regions has precluded thorough studies at systematic, ecological, evolutionary and conservation levels. In this study, we developed novel microsatellite markers for the three Iberian vipers, Vipera aspis, V. latastei and V. seoanei, and assessed their polymorphism in north-central Iberian populations. METHODS AND RESULTS: Genomic libraries were developed for each species using an Illumina Miseq sequencing approach. From the 70 primer pairs initially tested, 48 amplified reliably and were polymorphic within species. Cross-species transferability was achieved for 31 microsatellites loci in the three target species and four additional loci that were transferable to one species only. The 48 loci amplified in average seven alleles, and detected average expected and observed heterozygosities of 0.7 and 0.55, in the three genotyped populations/species (26 V. aspis, 20 V. latastei and 10 V. seoanei). CONCLUSIONS: Our study provides a selection of 48 polymorphic microsatellite markers that will contribute significantly to current knowledge on genetic diversity, gene flow, population structure, demographic dynamics, systematics, reproduction and heritability in these species, and potentially in other congeneric taxa.

Distinct regulatory networks control toxin gene expression in elapid and viperid snakes.

BACKGROUND: Venom systems are ideal models to study genetic regulatory mechanisms that underpin evolutionary novelty. Snake venom glands are thought to share a common origin, but there are major distinctions between venom toxins from the medically significant snake families Elapidae and Viperidae, and toxin gene regulatory investigations in elapid snakes have been limited. Here, we used high-throughput RNA-sequencing to profile gene expression and microRNAs between active (milked) and resting (unmilked) venom glands in an elapid (Eastern Brown Snake, Pseudonaja textilis), in addition to comparative genomics, to identify cis- and trans-acting regulation of venom production in an elapid in comparison to viperids (Crotalus viridis and C. tigris). RESULTS: Although there is conservation in high-level mechanistic pathways regulating venom production (unfolded protein response, Notch signaling and cholesterol homeostasis), there are differences in the regulation of histone methylation enzymes, transcription factors, and microRNAs in venom glands from these two snake families. Histone methyltransferases and transcription factor (TF) specificity protein 1 (Sp1) were highly upregulated in the milked elapid venom gland in comparison to the viperids, whereas nuclear factor I (NFI) TFs were upregulated after viperid venom milking. Sp1 and NFI cis-regulatory elements were common to toxin gene promoter regions, but many unique elements were also present between elapid and viperid toxins. The presence of Sp1 binding sites across multiple elapid toxin gene promoter regions that have been experimentally determined to regulate expression, in addition to upregulation of Sp1 after venom milking, suggests this transcription factor is involved in elapid toxin expression. microRNA profiles were distinctive between milked and unmilked venom glands for both snake families, and microRNAs were predicted to target a diversity of toxin transcripts in the elapid P. textilis venom gland, but only snake venom metalloproteinase transcripts in the viperid C. viridis venom gland. These results suggest differences in toxin gene posttranscriptional regulation between the elapid P. textilis and viperid C. viridis. CONCLUSIONS: Our comparative transcriptomic and genomic analyses between toxin genes and isoforms in elapid and viperid snakes suggests independent toxin regulation between these two snake families, demonstrating multiple different regulatory mechanisms underpin a venomous phenotype.

Preparation, Characterization, and Anticancer Activity Assessment of Chitosan/TPP Nanoparticles Loaded with Echis carinatus Venom.

AIMS AND BACKGROUND: Echis carinatus venom is a toxic substance naturally produced by special glands in this snake species. Alongside various toxic properties, this venom has been used for its therapeutic effects, which are applicable in treating various cancers (liver, breast, etc.). OBJECTIVE: Nanotechnology-based drug delivery systems are suitable for protecting Echis carinatus venom against destruction and unwanted absorption. They can manage its controlled transfer and absorption, significantly reducing side effects. METHODS: In the present study, chitosan nanoparticles were prepared using the ionotropic gelation method with emulsion cross-linking. The venom's encapsulation efficiency, loading capacity, and release rate were calculated at certain time points. Moreover, the nanoparticles' optimal formulation and cytotoxic effects were determined using the MTT assay. RESULTS: The optimized nanoparticle formulation increases cell death induction in various cancerous cell lines. Moreover, chitosan nanoparticles loaded with Echis carinatus venom had a significant rate of cytotoxicity against cancer cells. CONCLUSION: It is proposed that this formulation may act as a suitable candidate for more extensive assessments of cancer treatment using nanotechnology-based drug delivery systems.

An agonist of CXCR4 induces a rapid recovery from the neurotoxic effects of Vipera ammodytes and Vipera aspis venoms.

People bitten by Alpine vipers are usually treated with antivenom antisera to prevent the noxious consequences caused by the injected venom. However, this treatment suffers from a number of drawbacks and additional therapies are necessary. The venoms of Vipera ammodytes and of Vipera aspis are neurotoxic and cause muscle paralysis by inducing neurodegeneration of motor axon terminals because they contain a presynaptic acting sPLA2 neurotoxin. We have recently found that any type of damage to motor axons is followed by the expression and activation of the intercellular signaling axis consisting of the CXCR4 receptor present on the membrane of the axon stump and of its ligand, the chemokine CXCL12 released by activated terminal Schwann cells. We show here that also V. ammodytes and V. aspis venoms cause the expression of the CXCL12-CXCR4 axis. We also show that a small molecule agonist of CXCR4, dubbed NUCC-390, induces a rapid regeneration of the motor axon terminal with functional recovery of the neuromuscular junction. These findings qualify NUCC-390 as a promising novel therapeutics capable of improving the recovery from the paralysis caused by the snakebite of the two neurotoxic Alpine vipers.

Changes in attachment and metabolic activity of rat neonatal cardiomyocytes and nonmyocytes caused by Macrovipera lebetina obtusa venom.

The Caucasian viper Macrovipera lebetina obtusa (MLO) is one of the most prevalent and venomous snakes in the Caucasus and the surrounding regions, yet the effects of MLO venom on cardiac function remain largely unknown. We examined the influence of MLO venom (crude and with inhibited metalloproteinases and phospholipase A2) on attachment and metabolic activity of rat neonatal cardiomyocytes (CM) and nonmyocytes (nCM), assessed at 1 and 24 h. After exposing both CM and nCM to varying concentrations of MLO venom, we observed immediate cytotoxic effects at a concentration of 100 µg/ml, causing detachment from the culture substrate. At lower MLO venom concentrations both cell types detached in a dose-dependent manner. Inhibition of MLO venom metalloproteinases significantly improved CM and nCM attachment after 1-hour exposure. At 24-hour exposure to metalloproteinases inhibited venom statistically significant enhancement was observed only in nCM attachment. However, metabolic activity of CM and nCM did not decrease upon exposure to the lower dose of the venom. Moreover, we demonstrated that metalloproteinases and phospholipases A2 are not the components of the MLO venom that change metabolic activity of both CM and nCM. These results provide a valuable platform to study the impact of MLO venom on prey cardiac function. They also call for further exploration of individual venom components for pharmaceutical purposes.

Hidden in the sand: Phylogenomics unravel an unexpected evolutionary history for the desert-adapted vipers of the genus Cerastes.

The desert vipers of the genus Cerastes are a small clade of medically important venomous snakes within the family Viperidae. According to published morphological and molecular studies, the group is comprised by four species: two morphologically similar and phylogenetically sister taxa, the African horned viper (Cerastes cerastes) and the Arabian horned viper (Cerastes gasperettii); a more distantly related species, the Saharan sand viper (Cerastes vipera), and the enigmatic Böhme's sand viper (Cerastes boehmei), only known from a single specimen in captivity allegedly captured in Central Tunisia. In this study, we sequenced one mitochondrial marker (COI) as well as genome-wide data (ddRAD sequencing) from 28 and 41 samples, respectively, covering the entire distribution range of the genus to explore the population genomics, phylogenomic relationships and introgression patterns within the genus Cerastes. Additionally, and to provide insights into the mode of diversification of the group, we carried out niche overlap analyses considering climatic and habitat variables. Both nuclear phylogenomic reconstructions and population structure analyses have unveiled an unexpected evolutionary history for the genus Cerastes, which sharply contradicts the morphological similarities and previously published mitochondrial approaches. Cerastes cerastes and C. vipera are recovered as sister taxa whilst C. gasperettii is a sister taxon to the clade formed by these two species. We found a relatively high niche overlap (OI > 0.7) in both climatic and habitat variables between C. cerastes and C. vipera, contradicting a potential scenario of sympatric speciation. These results are in line with the introgression found between the northwestern African populations of C. cerastes and C. vipera. Finally, our genomic data confirms the existence of a lineage of C. cerastes in Arabia. All these results highlight the importance of genome-wide data over few genetic markers to study the evolutionary history of species.

Structural, biochemical and immunochemical characterization of an acidic phospholipase A2 from Lachesis acrochorda (Viperidae: Crotalinae) venom.

Viperids of the genus Lachesis, also known as bushmasters, are capable of injecting great amounts of venom that cause severe envenomation incidents. Since phospholipases type A2 are mainly involved in edema and myonecrosis within the snakebite sites, in this work, the isolation, amino acid sequence and biochemical characterization of the first phospholipase type A2 from the venom of Lachesis acrochorda, named Lacro_PLA2, is described. Lacro_PLA2 is an acidic aspartic 49 calcium-dependent phospholipase A2 with 93% similarity to the L. stenophrys phospholipase. Lacro_PLA2 has a molecular mass of 13,969.7 Da and an experimental isoelectric point around 5.3. A combination of N-terminal Edman degradation and MS/MS spectrometry analyses revealed that Lacro_PLA2 contains 122 residues including 14 cysteines that form 7 disulfide bridges. A predicted 3D model shows a high resemblance to other viperid phospholipases. Nevertheless, immunochemical and phospholipase neutralization tests revealed a notorious level of immunorecognition of the isolated protein by two polyclonal antibodies from viperids from different genus, which suggest that Lacro_PLA2 resembles more to bothropic phospholipases. Lacro_PLA2 also showed significantly high edema activity when was injected into mice; so, it could be an alternative antigen in the development of antibodies against toxins of this group of viperids, seeking to improve commercial polyclonal antivenoms.