Compositional and toxicological investigation of pooled venom from farm-raised Naja atra

Background: Naja atra is a venomous snake species medically relevant in China. In the current study, we evaluated the composition and toxicological profile of venom collected from farm-raised N. atra. Methods: Venom was collected from third-generation captive bred N. atra on a snake farm in Hunan Province, China. The venom was analyzed using sodium dodecyl sulfate polyacrylamide gel electrophoresis and nano-liquid chromatography with electrospray ionization tandem mass spectrometry. In addition, hemolytic activity, median lethal dose, serum biochemical and histopathological parameters were accessed. Results: N. atra venom proteome was dominated by phospholipase A2 (46.5%) and three-finger toxins (41.4 %), and a set of common low relative abundance proteins, including cysteine-rich secretory proteins (4.7%), NGF-beta (2.4%), snake venom metalloproteinase (1.5%), glutathione peroxidase (0.6%), vespryn (0.3%), and 5ʹ-nucleotidases (0.2%) were also found. Furthermore, the venom exhibited direct hemolytic activity, neurotoxicity, myotoxicity, and high lethal potency in mice, with a subcutaneous median lethal dose of 1.02 mg/kg. Histopathological analysis and serum biochemical tests revealed that venom caused acute hepatic, pulmonary and renal injury in mice. Conclusion: This study revealed the composition and toxicity of venom collected from farm-raised N. atra, thereby providing a reference for the analysis of venom samples collected from captive-born venomous snakes in the future.(AU)

Anticancer potential of nanogold conjugated toxin GNP-NN-32 from Naja naja venom

Background: Cancer is the second most common fatal disease in the world, behind cardiovascular disorders in the first place. It accounts for around 0.3 million deaths per year in India due to the lack of proper diagnostic facilities, prevention and treatment. Current therapeutic methods do not provide adequate protection and affect normal cells along with cancerous ones. Thus, there is a need for some alternative therapeutic strategy, preferably from natural products, which have been traditionally used for treatment of various diseases in the country. Methods: In this study, we have conjugated purified NN-32 toxin from Naja naja venom with gold nanoparticles and its anticancer potential was evaluated against human breast cancer cell lines. UV-Vis spectroscopy, dynamic light scattering, transmission electron microscopy, atomic force microscopy and zeta potential analysis were the techniques used for characterization of GNP-NN-32. Results: GNP-NN-32 showed dose- and time-dependent cytotoxicity against breast cancer cell lines (MCF-7 and MDA-MB-231). NN-32 and GNP-NN-32 induced apoptosis in both breast cancer cell lines. The results of CFSE cell proliferation study revealed that NN-32 and GNP-NN-32 arrested cell division in both MCF-7 and MDA-MB-231 cell lines resulting in inhibition of proliferation of these cancer cells. Conclusion: GNP-NN-32 showed an anticancer potential against human breast cancer cell lines. Analysis of detailed chemical characterization along with its cytotoxic property might help to perceive a new dimension of the anti-cancer potential of GNP-NN-32 that will enhance its biomedical function in near future.(AU)

Molecular phylogenetics of Black Cobra (Naja naja) in Pakistan

Background: Snakes are found on every continent in the world except Antarctica, and on smaller land masses. Being ecologically important, they also cause a large number of bites, leading to millions of deaths. Mitochondrial and nuclear gene sequences are being used to identify, characterize, and infer genetic biodiversity among different snake species. Furthermore, phylogenetics helps in inferring the relationships and evolutionary histories among these species. Black cobra is one of the four most venomous snakes in Pakistan. Four mitochondrial (ND4, Cytochrome b, 12S rRNA, and 16S rRNA) and four nuclear (C-mos, RAG-1, BDNF, and NT3) genes were used to trace diversity and infer the phylogenetic relationship of black cobra in Pakistan. Results: Almost similar phylogenies were obtained through maximum likelihood and Bayesian inference, showing two species of cobra in Pakistan, namely, black cobra (Naja naja) and brown cobra (Naja oxiana). All Naja species were divided into three clades: black cobra (N. naja) and brown cobra (N. oxiana) cladding with different species of Naja; N. naja (Pakistan) cladding with N. naja from Nepal; and N. oxiana showed close relationship with Naja kaouthia from Thailand and Naja siamensis from Thailand. Conclusion: It was confirmed genetically that there are two cobra species in Pakistan, i.e., black and brown cobras. This study will help in not only genetic conservation but also developing anti-venom against snake species.

Hematological and plasma biochemical parameters in a wild population of Naja naja (Linnaeus, 1758) in Sri Lanka

Background Hematological studies of any animal species comprise an important diagnostic method in veterinary medicine and an essential tool for the conservation of species. In Sri Lanka, this essential technique has been ignored in studies of many species including reptiles. The aim of the present work was to establish a reference range of hematological values and morphological characterization of wild spectacled cobras (Naja naja) in Sri Lanka in order to provide a diagnostic tool in the assessment of health condition in reptiles and to diagnose diseases in wild populations. Methods Blood samples were collected from the ventral caudal vein of 30 wild-caught Naja naja (18 males and 12 females). Hematological analyses were performed using manual standard methods. Results Several hematological parameters were examined and their mean values were: red blood cell count 0.581 ± 0.035 × 106/μL in males; 0.4950 ± 0.0408 × 106/μL in females; white blood cell count 12.45 ± 1.32 × 103/μL in males; 11.98 ± 1.62 × 103/μL in females; PCV (%) in males was 30.11 ± 1.93 and in females was 23.41 ± 1.67; hemoglobin (g/dL) was 7.6 ± 0.89 in males and 6.62 ± 1.49 in females; plasma protein (g/dL) was 5.11 ± 0.75 in males and 3.25 ± 0.74 in females; whereas cholesterol (mg/mL) was 4.09 ± 0.12 in males and 3.78 ± 0.42 in females. There were no significant differences in hematological parameters between the two genders except for erythrocyte count, thrombocyte count, hematocrit, hemoglobin, plasma protein, percentage of azurophil and heterophil. Intracellular parasites were not found in any of the studied specimens. Conclusion Hematological and plasma biochemical parameters indicated a difference between geographically isolated populations and some values were significantly different between the two genders. These hematological results provide a reference range for Sri Lankan population of adult Naja naja.(AU)

Preparation and neutralization efficacy of IgY antibodies raised against Deinagkistrodon acutus venom

Background The five-paced pit viper (Deinagkistrodon acutus), endemic to China and northern Vietnam, is responsible for most snakebites in the Chinese territory. Antivenom produced from horses is the main treatment for snakebites, but it may cause numerous clinical side effects and have other disadvantages involved in their production such as the welfare of animals. The present study was conducted aiming to develop an alternative antibody (IgY) from the egg yolk of leghorn chickens immunized with snake venom. Methods IgY from the egg yolk of white leghorn chickens previously immunized intramuscularly with D. acutus venom was extracted by water, precipitated by ammonium sulfate and purified by affinity chromatographic system. IgY was identified by SDS-PAGE, ELISA and Western blot. Finally, IgY neutralization assays to test its efficacy against hemorrhagic, edema-forming and myotoxic activities of D. acutus venom were conducted on mice. Results For the first time, IgY antibodies against D. acutus venom were raised successfully in egg yolk of chickens injected with D. acutus venom multiple times. By three steps, including caprylic acid extraction, ammonium sulfate precipitation and affinity chromatography, IgY antibodies were isolated and purified from egg yolk, which exhibited a single protein band on SDS-PAGE and two bands (about 65 kDa and 35 kDa, respectively) under reducing conditions, and presented a high titer (1:40,000) tested by ELISA. Immunoblot analysis confirmed that these IgY were polyclonal antibodies since they bound to components of D. acutus venom. Furthermore, immunodiffusion assay showed that anti-D. acutus venom IgY cross-reacted with the venoms of Trimeresurus albolabris and D. saxatilis Emelianov, but did not react to the venoms of Bungarus multicinctus and Naja atra. In the neutralizing lethal assay, the median effective dose of anti-D. acutus venom IgY was 14.14 mg/kg of mouse body weight under the challenge dose (3 LD50 of D. acutus venom). In neutralizing the hemorrhagic, edema-forming and myotoxic activities of D. acutus venom, IgY showed the characteristic dose-dependent neutralization effects against all these toxic activities of D. acutus venom. Conclusion Anti-D. acutus venom IgY antibodies with high purity and titer were for the first time raised successfully in egg yolk of chickens immunized with D. acutus venom. They were effective in neutralizing the lethal effects, and the hemorrhagic, edema-forming and myotoxic acitivities of D. acutus venom. IgY could be an effective source to develop a treatment against snake bites in humans or animals in the future.(AU)

Mad, bad and dangerous to know: the biochemistry, ecology and evolution of slow loris venom

Only seven types of mammals are known to be venomous, including slow lorises (Nycticebus spp.). Despite the evolutionary significance of this unique adaptation amongst Nycticebus, the structure and function of slow loris venom is only just beginning to be understood. Here we review what is known about the chemical structure of slow loris venom. Research on a handful of captive samples from three of eight slow loris species reveals that the protein within slow loris venom resembles the disulphide-bridged heterodimeric structure of Fel-d1, more commonly known as cat allergen. In a comparison of N. pygmaeus and N. coucang, 212 and 68 compounds were found, respectively. Venom is activated by combining the oil from the brachial arm gland with saliva, and can cause death in small mammals and anaphylactic shock and death in humans. We examine four hypotheses for the function of slow loris venom. The least evidence is found for the hypothesis that loris venom evolved to kill prey. Although the venom's primary function in nature seems to be as a defense against parasites and conspecifics, it may also serve to thwart olfactory-orientated predators. Combined with numerous other serpentine features of slow lorises, including extra vertebra in the spine leading to snake-like movement, serpentine aggressive vocalisations, a long dark dorsal stripe and the venom itself, we propose that venom may have evolved to mimic cobras (Naja sp.). During the Miocene when both slow lorises and cobras migrated throughout Southeast Asia, the evolution of venom may have been an adaptive strategy against predators used by slow lorises as a form of Müllerian mimicry with spectacled cobras.(AU)

Cytotoxicity of Southeast Asian snake venoms

Cytotoxicity of venoms from eleven medically important snakes found in Southeast Asia (Naja kaouthia, Naja siamensis, Naja sumatrana, Ophiophagus hannah, Bungarus candidus, Bungarus fasciatus, Enhydrina schistosa, Calloselasma rhodostoma, Trimeresurus purpureomaculatus and Tropidolaemus sumatranus) was determined, based on the MTS cytotoxicity assay, which determines the survival of viable cells in monolayer MDCK and Vero cell cultures upon exposure to the snake venoms. Snake venom toxicity was expressed as the venom dose that killed 50% of the cells (CTC50) under the assay conditions. Venoms of C. rhodostoma (2.6 µg/mL, 1.4 µg/mL) and O. hannah were the most cytotoxic (3.8 µg/mL, 1.7 µg/mL) whereas N. siamensis venom showed the least cytotoxicity (51.9 µg/mL, 45.7 µg/mL) against Vero and MDCK cells, respectively. All the viper venoms showed higher cytotoxic potency towards both Vero and MDCK cell lines, in comparison to krait and cobra venoms. E. schistosa did not cause cytotoxicity towards MDCK or Vero cells at the tested concentrations. The cytotoxicity correlates well with the known differences in the composition of venoms from cobras, kraits, vipers and sea snakes.(AU)

Efficacy of tannins from Mimosa pudica and tannic acid in neutralizing cobra (Naja kaouthia) venom

In the present study, the effectiveness of Mimosa pudica tannins (MPT) in neutralizing the lethality of Naja kaouthia venom was compared with commercially derived tannins. Preincubation of MPT with N. kaouthia venom maintained 100 percent survival of mice after 24 hours. The mouse group in which there was no preincubation, no protection against the effects of the venom was observed. M. pudica tannin was found to be more effective in neutralizing the lethality of N. kaouthia venom when compared to commercial tannic acid. Two protein spots were missing in the two-dimensional gel electrophoresis (2-DE) of the MPT treated mouse indicating the down-regulation of venom proteins. The results from this study indicated that tannins obtained from M. pudica are better than tannic acid in neutralizing the lethality of N. kaouthia venom in vitro. However, further investigations are required to establish that M. pudica has potential for treating N. kaouthia snakebites.(AU)

Proteomic characterization of the thermostable toxins from Naja naja venom

Naja naja snake venom presents abundant thermostable peptides. Many of them possess useful pharmacological activity that may be employed for drug development. For the proteomic characterization of such toxins, in the present study, Naja naja venom solution was heated up to 100°C for 10, 30, 60, 120, 180 and 300 minutes and protein fractions of non-heated and heated venom were analyzed by two-dimensional nano-liquid chromatography coupled online with tandem mass spectrometry. After heating for 300 minutes, a total of 32 peptides were still detected in the supernatant. The identified peptides belong to the following groups: cardiotoxins, neurotoxins and cytotoxins. It was found that thermostable peptides are able to preserve their analgesic activity after a long heating time in formalin test. Mice injected with 15 µg/g of 60-minute heated venom or with 25 µg/g of 300-minute heated venom revealed even a better analgesic activity than those treated with lidocaine.(AU)

Venomous snakebites and antivenom treatment according to a protocol for pediatric patients in taiwan

Children bitten by venomous snakes comprise emergency cases that present high morbidity and mortality, especially in some regions of Asia and Africa. Reports on clinical features and management of envenomated children are scarce. This observational study implemented a protocol for antivenom use in pediatric snakebite victims in Taiwan, between 1994 and 2007. The present work investigated demographics, clinical features and effects of antivenom therapy and the complications and prognosis for snakebites. A total of 55 children and adolescents, with a median age of 9.9 years (ranging from 2 to 18 years), was identified. Forty-five patients (82 percent) were bitten between May and September. Thirty-five patients (64 percent) received antivenom therapy, 28 of them (80 percent) within two hours after being bitten. No fatalities occurred and only five of 35 patients (14 percent) had major morbidity when treated according to the protocol. Thirty-one snakes (56 percent) were identified and 12 of them (38 percent) were Naja atra. This study indicates that a protocol for children affected by snakebites is valuable for guiding emergency physicians to treat these patients immediately. Further strategies are required to reduce the morbidity rate that occurs as a result of Naja atra bite.(AU)

Appropriate antivenom doses for six types of envenomations caused by snakes in taiwan

Six of the 15 species of venomous snakes found in Taiwan are responsible for most of the clinically significant envenomations in the country. These species are: Trimeresurus mucrosquamatus, Trimeresurus stejnegeri, Naja atra, Bungarus multicinctus, Deinagkistrodon acutus and Daboia russelii siamensis, which together can be subdivided into three groups based on their venom effects. Primary treatment consists of rapid administration of appropriate antivenoms. The present study aimed to identify a proper dose of antivenom for each snake group as well as to describe hemorrhagic, neurotoxic, and mixed effects of their venoms. A retrospective chart review identified 72 snakebite cases referred to an emergency department. Data on epidemiology, examination findings, snake identification, treatment, antivenom dose and complications were collected. After excluding 14 patients, data from 58 victims were analyzed. Most studied cases were male (86 percent). Significantly higher doses of antivenom were administered against neurotoxic envenomations (mean dose: three vials) compared with the other two (p < 0.05). Moreover, patients affected by neurotoxic bites were more likely to develop blurred vision and other complications (p < 0.05). Multivariate logistic regression analysis indicated that neurotoxic envenomation was a risk factor for complications (OR: 8.84, 95 percent CI: 1.06-73.73). Neurotoxic envenomations and complication occurrence were positively correlated with antivenom dosage. In conclusion, patients affected by neurotoxic envenomations received higher doses of antivenom than others whereas incidence of complications was associated with higher antivenom doses.

Characterization of an antibacterial peptide from indian cobra (Naja naja) venom

Due to the development of antibiotic resistance in microorganisms, antimicrobial peptides from natural sources have attracted attention in recent times. Several antimicrobial peptides have been isolated from a wide range of animal sources, particularly snake venoms. Naja naja venom showed antibacterial as well as direct and indirect hemolytic activities, and an antibacterial peptide was purified through gel permeation and ion exchange chromatography. Its molecular mass was 2491Da, which was determined using Matrix Assisted Laser Desorption/Ionization-Time-of-Flight (MALDI-TOF) mass spectrometry and the amino acids sequence of the N-terminus was DEQSTHGAYVWKL. The purified peptide showed potent antibacterial activity against Gram-negative and Gram-positive bacterial strains like Escherichia coli, Pseudomonas aeruginosa and Vibrio cholerae, and Staphylococcus aureus, Enterococcus faecalis, Streptococcus pneumoniae, Streptococcus pyogenes, Bacillus subtilis, respectively. The most potent activity was towards Gram-negative bacteria. Activity was retained at concentrations as low as 100µg/ml. Minimum inhibitory concentrations (MIC; in mg) of Naja Antibacterial Peptide (NAP) and known antibiotics against Gram-positive and Gram-negative bacteria were determined using microdilution susceptibility test in sterile 96-well microdilution plates. However, the peptide did not show direct or indirect hemolytic activity.(AU)