A fatal snakebite envenomation due to King cobra (Ophiophagus hannah) in the Eastern Visayas, Philippines.

This report details a documented case of fatal King cobra (Ophiophagus hannah) envenomation in the Philippines. A 46-year-old woman from a mountainous town in Leyte was bitten on her left thigh by a snake. Despite receiving prompt medical attention, including administration of fluids and oxygen, she went into arrest and succumbed within 2.5 hours of the bite. Inadequate pre-hospital care, including endotracheal intubation and assisted ventilation, highlights a notable gap in emergency medical services. Photographic evidence, verified by a herpetologist, confirmed the involvement of a King cobra, with venom presenting with a swift and lethal systemic effect that led to the patient's demise, despite minimal local manifestations. This incident accentuates the urgent need for accessible, effective antivenom and improved snakebite management protocols in the Philippines. It also calls for heightened awareness and preparedness among pre-hospital healthcare providers and the public, alongside advocating for more research into snakebite envenomation.

House-warming: Wild king cobra nests have thermal regimes that positively affect hatching success and hatchling size.

Incubation temperature in nests of oviparous reptiles affects reproductive success indicators, including hatching time and success, offspring size, fitness, and behaviour. The female king cobra builds an above ground nest to incubate and protect its eggs. However, it is not clear how thermal regimes inside king cobra nests respond to external environmental temperature regimes, especially in subtropical regions that witness high diel and seasonal temperature fluctuations. To better understand the relationship between inside nest temperatures and hatching outcomes for this snake, we monitored the thermal regimes of 25 natural king cobra nests in the subtropical forests of the Western Himalayas in Uttarakhand state, northern India. We hypothesized that inside nest temperatures would be higher than outside (ambient) temperatures and that thermal regimes inside nests would affect hatching success and hatchling size. Internal and external temperatures at nest sites were measured every hour until hatching, via automatic data loggers. We then calculated hatching success of eggs and measured hatchling length and weight. Mean inside nest temperatures were consistently higher by about 3.0 °C than outside environmental temperatures. External temperature reduced with increasing elevation of nest sites and was the best determinant of inside nest temperature, which had a smaller range of variability. Physical characteristics of nests (size and leaf materials used) did not influence nest temperature significantly, but nest size was positively related to clutch size. Mean inside nest temperature was the best predictor of hatching success. Average daily minimum nest temperature, which indicates a possible lower threshold for thermal tolerance by eggs, was also correlated positively with hatching success. Mean daily maximum temperature was a significant predictor of mean length of hatchlings, but not of mean hatchling weight. Our study provides unequivocal evidence for the critical thermal benefits of king cobra nests for increased reproductive success, in subtropical environments with lower and sharply fluctuating temperature regimes.

Preclinical assessment of VPEAV, a new trivalent antivenom for elapid snakebite envenoming in the Philippines: Proteomics, immunoreactivity and toxicity neutralization.

Snakebite envenoming is an important neglected tropical disease. Antivenom supply, however, remains limited in many parts of the world. This study aimed to examine the protein composition, immunoreactivity and neutralization efficacy of a new antivenom product (VINS Philippine Elapid Antivenoms, VPEAV) developed for the treatment of snakebite envenoming caused by the Philippine Cobra (Naja philippinensis), Samar Cobra (Naja samarensis) and King Cobra (Ophiophagus hannah). Size-exclusion chromatography, sodium-dodecyl sulfate-polyacrylamide gel electrophoresis and tandem mass spectrometry showed that VPEAV consisted of F(ab)'2 (∼90% of total antivenom proteins) with minimal protein impurities. Indirect ELISA showed varying immunoreactivity of VPEAV toward the different venoms (EC50 = 4-16 µg/ml), indicating distinct venom antigenicity between the species. In mice, the neutralization potency of VPEAV against the King Cobra venom was moderate (potency, P = 2.6 mg/ml, defined as the amount of venom completely neutralized per unit volume of antivenom). The potency was significantly lower against the N. philippinensis and N. samarensis venoms (P = 0.18-0.30 mg/ml), implying a higher dose may be needed for effective neutralization of the Naja venoms. Together, the findings suggest the potential and limitation of VPEAV in neutralizing the venom toxicity of the three Philippine elapid snakes.

King or royal family? Testing for species boundaries in the King Cobra, Ophiophagus hannah (Cantor, 1836), using morphology and multilocus DNA analyses.

In widespread species, the diverse ecological conditions in which the populations occur, and the presence of many potential geographical barriers through their range are expected to have created ample opportunities for the evolution of distinct, often cryptic lineages. In this work, we tested for species boundaries in one such widespread species, the king cobra, Ophiophagus hannah (Cantor, 1836), a largely tropical elapid snake distributed across the Oriental realm. Based on extensive geographical sampling across most of the range of the species, we initially tested for candidate species (CS) using Maximum-Likelihood analysis of mitochondrial genes. We then tested the resulting CS using both morphological data and sequences of three single-copy nuclear genes. We used snapclust to determine the optimal number of clusters in the nuclear dataset, and Bayesian Phylogenetics and Phylogeography (BPP) to test for likely species status. We used non-metric multidimensional scaling (nMDS) analysis for discerning morphological separation. We recovered four independently evolving, geographically separated lineages that we consider Confirmed Candidate Species: (1) Western Ghats lineage; (2) Indo-Chinese lineage (3) Indo-Malayan lineage; (4) Luzon Island lineage, in the Philippine Archipelago. We discuss patterns of lineage divergence, particularly in the context of low morphological divergence, and the conservation implications of recognizing several endemic king cobra lineages.

Envenoming by king cobras (Ophiophagus hannah) in Vietnam with cardiac complications and necrotizing fasciitis.

King Cobra (Ophiophagus hannah) bite is well-known for its potentially fatal neurotoxicity. However, fatalities still occur, despite specific antivenom and respiratory support. Cardiovascular disturbances, which have attracted little attention in published reports of O. hannah envenoming, could contribute to fatality. We present two cases of confirmed O. hannah envenoming in Southern Vietnam in which there were cardiac abnormalities including arrhythmias and electrocardiographic changes, as well as elevated markers of myocardial damage. Cardiac pacing was required. One patient developed critical multi-organ dysfunctions partly explained by extensive necrotizing fasciitis/myositis originating from an Aeromonas sobria wound infection. This resulted in rhabdomyolysis, disseminated intravascular coagulation and acute kidney injury. Specific antivenom reversed neurotoxic effects of envenoming. Additional therapeutic interventions included antibiotics, surgical debridement, continuous renal replacement therapy and therapeutic plasma exchange. Both patients eventually made full recoveries. Apart from the critical problem of rapidly evolving and severe neurotoxicity, our case reports also emphasises the risk of cardiotoxic envenoming, and the complications of an overwhelming secondary bacterial wound infection. We suggest a practical approach to diagnosis and management.

King cobra bite - Can early decompression prevent digital amputation?

King cobra bites are extremely rare in the western world. These bites can be fatal due to the large volume of the venom injected. We report a case of digital ischaemia from a King cobra bite in a young man who was working in a zoo in Netherlands. He was protected from systemic envenomation as he was wearing a protective glove. However, his right index finger developed subsequent gangrene and he underwent a ray amputation. The current literature on the management of snake bites to hands is reviewed and the role of early decompression discussed.

Suppression of cardiomyocyte functions by ß-CTX isolated from the Thai king cobra (Ophiophagus hannah) venom via an alternative method.

BACKGROUND: Beta-cardiotoxin (ß-CTX), the three-finger toxin isolated from king cobra (Ophiophagus hannah) venom, possesses ß-blocker activity as indicated by its negative chronotropy and its binding property to both ß-1 and ß-2 adrenergic receptors and has been proposed as a novel ß-blocker candidate. Previously, ß-CTX was isolated and purified by FPLC. Here, we present an alternative method to purify this toxin. In addition, we tested its cytotoxicity against different mammalian muscle cell types and determined the impact on cardiac function in isolated cardiac myocyte so as to provide insights into the pharmacological action of this protein. METHODS: ß-CTX was isolated from the crude venom of the Thai king cobra using reverse-phased and cation exchange HPLC. In vitro cellular viability MTT assays were performed on mouse myoblast (C2C12), rat smooth muscle (A7r5), and rat cardiac myoblast (H9c2) cells. Cell shortening and calcium transient dynamics were recorded on isolated rat cardiac myocytes over a range of ß-CTX concentration. RESULTS: Purified ß-CTX was recovered from crude venom (0.53% w/w). MTT assays revealed 50% cytotoxicity on A7r5 cells at 9.41 ± 1.14 µM (n = 3), but no cytotoxicity on C2C12 and H9c2 cells up to 114.09 µM. ß-CTX suppressed the extend of rat cardiac cell shortening in a dose-dependent manner; the half-maximal inhibition concentration was 95.97 ± 50.10 nM (n = 3). In addition, the rates of cell shortening and re-lengthening were decreased in ß-CTX treated myocytes concomitant with a prolongation of the intracellular calcium transient decay, indicating depression of cardiac contractility secondary to altered cardiac calcium homeostasis. CONCLUSION: We present an alternative purification method for ß-CTX from king cobra venom. We reveal cytotoxicity towards smooth muscle and depression of cardiac contractility by this protein. These data are useful to aid future development of pharmacological agents derived from ß-CTX.

Geographical variations in king cobra (Ophiophagus hannah) venom from Thailand, Malaysia, Indonesia and China: On venom lethality, antivenom immunoreactivity and in vivo neutralization.

The wide distribution of king cobra (Ophiophagus hannah), a medically important venomous snake in Asia could be associated with geographical variation in the toxicity and antigenicity of the venom. This study investigated the lethality of king cobra venoms (KCV) from four geographical locales (Malaysia, Thailand, Indonesia, China), and the immunological binding as well as in vivo neutralization activities of three antivenom products (Thai Ophiophagus hannah monovalent antivenom, OHMAV; Indonesian Serum Anti Bisa Ular, SABU; Chinese Naja atra monovalent antivenom, NAMAV) toward the venoms. The Indonesian and Chinese KCV were more lethal (median lethal dose, LD50 ~0.5 µg/g) than those from Malaysia and Thailand (LD50 ~1.0 µg/g). The antivenoms, composed of F(ab)'2, were variably immunoreactive toward the KCV from all locales, with OHMAV exhibited the highest immunological binding activity. In mice, OHMAV neutralized the neurotoxic lethality of Thai KCV most effectively (normalized potency = 118 mg venom neutralized per g antivenom) followed by Malaysian, Indonesian and Chinese KCV. In comparison, the hetero-specific SABU was remarkably less potent by at least 6 to10 folds, whereas NAMAV appeared to be non-effective. The finding supports that a specific king cobra antivenom is needed for the effective treatment of king cobra envenomation in each region.

Suppression of cardiomyocyte functions by ß-CTX isolated from the Thai king cobra (Ophiophagus hannah) venom via an alternative method

Beta-cardiotoxin (ß-CTX), the three-finger toxin isolated from king cobra (Ophiophagus hannah) venom, possesses ß-blocker activity as indicated by its negative chronotropy and its binding property to both ß-1 and ß-2 adrenergic receptors and has been proposed as a novel ß-blocker candidate. Previously, ß-CTX was isolated and purified by FPLC. Here, we present an alternative method to purify this toxin. In addition, we tested its cytotoxicity against different mammalian muscle cell types and determined the impact on cardiac function in isolated cardiac myocyte so as to provide insights into the pharmacological action of this protein. Methods: ß-CTX was isolated from the crude venom of the Thai king cobra using reverse-phased and cation exchange HPLC. In vitro cellular viability MTT assays were performed on mouse myoblast (C2C12), rat smooth muscle (A7r5), and rat cardiac myoblast (H9c2) cells. Cell shortening and calcium transient dynamics were recorded on isolated rat cardiac myocytes over a range of ß-CTX concentration. Results: Purified ß-CTX was recovered from crude venom (0.53% w/w). MTT assays revealed 50% cytotoxicity on A7r5 cells at 9.41 ± 1.14 µM (n = 3), but no cytotoxicity on C2C12 and H9c2 cells up to 114.09 µM. ß-CTX suppressed the extend of rat cardiac cell shortening in a dose-dependent manner; the half-maximal inhibition concentration was 95.97 ± 50.10 nM (n = 3). In addition, the rates of cell shortening and re-lengthening were decreased in ß-CTX treated myocytes concomitant with a prolongation of the intracellular calcium transient decay, indicating depression of cardiac contractility secondary to altered cardiac calcium homeostasis. Conclusion: We present an alternative purification method for ß-CTX from king cobra venom. We reveal cytotoxicity towards smooth muscle and depression of cardiac contractility by this protein. These data are useful to aid future development of pharmacological agents derived from ß-CTX.(AU)

Genome-wide mining of microsatellites in king cobra (Ophiophagus hannah) and cross-species development of tetranucleotide SSR markers in Chinese cobra (Naja atra).

The complete genome sequence provides the opportunity for genome-wide and coding region analysis of SSRs in the king cobra and for cross-species identification of microsatellite markers in the Chinese cobra. In the Ophiophagus hannah genome, tetranucleotide repeats (38.03%) were the most abundant category, followed by dinucleotides (23.03%), pentanucleotides (13.07%), mononucleotides (11.78%), trinucleotides (11.49%) and hexanucleotides (2.6%). Twenty predominant motifs in the O. hannah genome were (A)n (C)n, (AC)n, (AG)n, (AT)n, (AGG)n, (AAT)n, (AAG)n, (AAC)n, (ATG)n, (ATAG)n, (AAGG)n, (ATCT)n, (CCTT)n, (ATTT)n, (AAAT)n, (AATAG)n, (ATTCT)n, (ATATGT)n, (AGATAT)n. In total, 4344 SSRs were found in coding sequences (CDSs). Tetranucleotides (52.79%) were the most abundant microsatellite type in CDS, followed by trinucleotides (28.50%), dinucleotides (11.02%), pentanucleotides (4.42%), mononucleotides (1.77%), and hexanucleotides (1.50%). A total of 984 CDSs containing microsatellites were assigned 11152 Gene Ontology (GO) functional terms. Gene Ontology (GO) analysis demonstrated that cellular process, cell and binding were the most frequent GO terms in biological process, cellular component and molecular function, respectively. Thirty-two novel highly polymorphic (PIC > 0.5) SSR markers for Naja atra were developed from cross-species amplification based on the tetranucleotide microsatellite sequences in the king cobra genome. The number of alleles (NA) per locus had between 3 and 11 alleles with an average of 6.5, the polymorphism information content (PIC) value ranged from 0.521 to 0.858 (average = 0.707), the observed heterozygosity (Ho) of 32 microsatellite loci ranged from 0.292 to 0.875 (mean = 0.678), the expected heterozygosity (HE) ranged from 0.561 to 0.889 (average = 0.761), and 3 microsatellite loci exhibited statistically significant departure from Hardy-Weinberg equilibrium (HWE) after Bonferroni correction (p < 0.003).

TREATMENT OF CRYPTOSPORIDIUM SERPENTIS INFECTION IN A KING COBRA ( OPHIOPHAGUS HANNAH) WITH PAROMOMYCIN.

Feces collected from a wild-caught, young adult king cobra ( Ophiophagus hannah) were repeatedly positive for Cryptosporidium on both direct immunofluorescent antibody (DFA) and polymerase chain reaction (PCR), and sequencing identified the organism as Cryptosporidium serpentis. Infection was subclinical, as the snake was in good body condition and active, and readily consumed dead rats that were scented with snake skin. A course of paromomycin, inserted in feeder rats, was initiated at 360 mg/kg, orally, twice weekly for 6 wk. Feces collected at the end of treatment were negative for Cryptosporidium on PCR, as were feces collected 3 wk, 6 mo, 12 mo, and 18 mo later. At higher dosages, paromomycin may prove useful and may be curative for early gastric and intestinal cryptosporidiosis in squamate reptiles.

Snake Genome Sequencing: Results and Future Prospects.

Snake genome sequencing is in its infancy-very much behind the progress made in sequencing the genomes of humans, model organisms and pathogens relevant to biomedical research, and agricultural species. We provide here an overview of some of the snake genome projects in progress, and discuss the biological findings, with special emphasis on toxinology, from the small number of draft snake genomes already published. We discuss the future of snake genomics, pointing out that new sequencing technologies will help overcome the problem of repetitive sequences in assembling snake genomes. Genome sequences are also likely to be valuable in examining the clustering of toxin genes on the chromosomes, in designing recombinant antivenoms and in studying the epigenetic regulation of toxin gene expression.

Venom Proteomics of Indonesian King Cobra, Ophiophagus hannah: Integrating Top-Down and Bottom-Up Approaches.

We report on the first application of top-down mass spectrometry in snake venomics. De novo sequence tags generated by, and ProSight Lite supported analysis of, combined collisional based dissotiations (CID and HCD) recorded in a hybrid LTQ Orbitrap instrument in data-dependent mode identified a number of proteins from different toxin families, namely, 11 three-finger toxins (7-7.9 kDa), a Kunitz-type inhibitor (6.3 kDa), ohanin (11.9 kDa), a novel phospholipase A2 molecule (13.8 kDa), and the cysteine-rich secretory protein (CRISP) ophanin (25 kDa) from Indonesian king cobra venom. Complementary bottom-up MS/MS analyses contributed to the completion of a locus-resolved venom phenotypic map for Ophiophagus hannah, the world's longest venomous snake and a species of medical concern across its wide distribution range in forests from India to Southeast Asia. Its venom composition, comprising 32-35 proteins/peptides from 10 protein families, is dominated by α-neurotoxins and convincingly explains the main neurotoxic effects of human envenoming caused by king cobra bite. The integration of efficient chromatographic separation of the venom's components and locus-resolved toxin identification through top-down and bottom-up MS/MS-based species-specific database searching and de novo sequencing holds promise that the future will be bright for the field of venom research.

Establishing a leaf proteome reference map for Ginkgo biloba provides insight into potential ethnobotanical uses.

Although ginkgo (Maidenhair tree, Ginkgo biloba L.) is an ancient medicinal and ornamental tree, there has not previously been any systematic proteomic study of the leaves. Herein we describe results from the initial study identifying abundant ginkgo leaf proteins and present a gel reference map. Proteins were isolated from fully developed mature leaves in biological triplicate and analyzed by two-dimensional electrophoresis plus tandem mass spectrometry. Using this approach, we were able to reproducibly quantify 190 abundant protein spots, from which 157 proteins were identified. Most of identified proteins are associated with the energy and protein destination/storage categories. The reference map provides a basis for understanding the accumulation of flavonoids and other phenolic compounds in mature leaves (e.g., identification of chalcone synthase, the first committed enzyme in flavonoid biosynthesis). We additionally detected several proteins of as yet unknown function. These proteins comprise a pool of potential targets that might be useful in nontraditional medical applications.

Functional proteomic approach to discover geographic variations of king cobra venoms from Southeast Asia and China.

This study deciphers the geographic variations of king cobra (Ophiophagus hannah) venom using functional proteomics. Pooled samples of king cobra venom (abbreviated as Ohv) were obtained from Indonesia, Malaysia, Thailand, and two provinces of China, namely Guangxi and Hainan. Using two animal models to test and compare the lethal effects, we found that the Chinese Ohvs were more fatal to mice, while the Southeast Asian Ohvs were more fatal to lizards (Eutropis multifasciata). Various phospholipases A2 (PLA2s), three-finger toxins (3FTxs) and Kunitz-type inhibitors were purified from these Ohvs and compared. Besides the two Chinese Ohv PLA2s with known sequences, eight novel PLA2s were identified from the five Ohv samples and their antiplatelet activities were compared. While two 3FTxs (namely oh-55 and oh-27) were common in all the Ohvs, different sets of 3FTx markers were present in the Chinese and Southeast Asian Ohvs. All the Ohvs contain the Kunitz inhibitor, OH-TCI, while only the Chinese Ohvs contain the inhibitor variant, Oh11-1. Relative to the Chinese Ohvs which contained more phospholipases, the Southeast Asian Ohvs had higher metalloproteinase, acetylcholine esterase, and alkaline phosphatase activities. BIOLOGICAL SIGNIFICANCE: Remarkable variations in five king cobra geographic samples reveal fast evolution and dynamic translational regulation of the venom which probably adapted to different prey ecology as testified by the lethal tests on mice and lizards. Our results predict possible variations of the king cobra envenoming to human and the importance of using local antivenin for snakebite treatment.

Purification and antibacterial activities of an L-amino acid oxidase from king cobra (Ophiophagus hannah) venom

Some constituents of snake venom have been found to display a variety of biological activities. The antibacterial property of snake venom, in particular, has gathered increasing scientific interest due to antibiotic resistance. In the present study, king cobra venom was screened against three strains of Staphylococcus aureus [including methicillin-resistant Staphylococcus aureus (MRSA)], three other species of gram-positive bacteria and six gram-negative bacteria. King cobra venom was active against all the 12 bacteria tested, and was most effective against Staphylococcus spp. (S. aureus and S. epidermidis). Subsequently, an antibacterial protein from king cobra venom was purified by gel filtration, anion exchange and heparin chromatography. Mass spectrometry analysis confirmed that the protein was king cobra L-amino acid oxidase (Oh-LAAO). SDS-PAGE showed that the protein has an estimated molecular weight of 68 kDa and 70 kDa under reducing and non-reducing conditions, respectively. The minimum inhibitory concentrations (MIC) of Oh-LAAO for all the 12 bacteria were obtained using radial diffusion assay method. Oh-LAAO had the lowest MIC value of 7.5 µg/mL against S. aureus ATCC 25923 and ATCC 29213, MRSA ATCC 43300, and S. epidermidis ATCC 12228. Therefore, the LAAO enzyme from king cobra venom may be useful as an antimicrobial agent.(AU)

King cobra egg yolk antibody from the egg yolks of immunized hens / 中国药理学通报

Aim To develop King cobra antivenom from the egg yolks of immunized hens,and study dynamic expression of IgY in egg yolks.Methods chickens(white Leghorn) were immunized with detoxicated King cobra venom by formaldehyde ,Egg yolk antibody (IgY) was isotated by thiophilic interaction chromatography and identified by SDS-PAGE. Activity of IgY was evaluated by enzyme-linked immunoserbent assay(ELISA) and Double immuodiffusion. Protein was measured using folin-lowry method. Results Specific antivenom could be detected in the yolk laid by the hens 9 d after immunization, At the 60th day after primary immunization ,ELISA titers reached 1∶ 100 000,and 97.5 mg IgY?ml -1 yolk was obtained from thiophilic interaction chromatography. IgY was highly specific, No cross reactivity was observed among IgY and agkistrodon acutus venom and vipera venom ;Little cross reactivity was shown with cobra genus venoms.Conclusion King cobra IgY was obtained and purified from the egg yolks of immunized hens,The dynamic expression of IgY was manitered during the course of immunization,Further investigation is needed.

A preliminary study of egg yolk antibody for kingcobra venom antigens detection / 中国药理学通报

Aim To discuss whether specific Egg yolk antibody(IgY) can be used for snake venom antigens detection.Methods Chickens(white Leghorn) were immunized with detoxicated king cobra venom by formaldehyde.Egg yolk antibody were isolated from egg yolk,and labeled with horse radish peroxidase(HRP).Snake venom antigens samples,including king cobra venom,cobra venom,bungrarus fasciatus venom,bungrarus multicinctus venom,agkistrodon actus venom and Guangdong viper venom,were detected using ELISA,and sensitivity,precision and specificity were tested,respectively.Results At about 32 ?g?L~(-1),the king cobra antigens were detected using the method;Linear relation was better(r=0.963) when the concentration of kingcobra venom was within 32～750?g?L~(-1).The method had good specificity and no cross reactivity was observed among the reagents and agkistrodon acutus guenther venom and vipera russelli siamensis smith venom;little cross reactivity was shown with bungarus multicinctus blyth venom and bungarus fasciatus chmeider venom;cross reactivity was obvious with cobra venom;the average intra-assay coefficient of variation(CV) was 1%～3%,and the inter-assay CV was within 8%.Conclusions The study indicates that IgY can be good reagents for snake venom antigens detecton,and the study provides foundation for the development of the diagnosis kits of snakebites.