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A comprehensive investigation, incorporating both morphological and molecular analyses, has unveiled the existence of a hitherto unknown nematode species, Paracapillaria (Ophidiocapillaria) siamensis sp. nov., residing in the intestine of the monocled cobra, Naja kaouthia, in the central region of Thailand. This study integrates morphological characteristics, morphometric examination, scanning electron microscopy and molecular phylogenetic analysis (COI, 18S rRNA and ITS1 genes). The findings place the newly described species within the subgenus Ophidiocapillaria, elucidating its distinctive characteristics, including a frame-like proximal spicule shape, approximate lengths of 19 000 and 22 500 μm with approximate widths of 90 and 130 μm for males and females, 39‒45 stichocytes, elevated lips without protrusion, a dorsal bacillary band stripe with an irregular pattern of bacillary cells and evidence of intestinal infection. These features serve to differentiate it from other species within the same subgenus, notably Paracapillaria (Ophidiocapillaria) najae De, 1998, a species coexisting P. siamensis sp. nov. in the monocled cobra from the same locality. This study addresses the co-infection of the novel species and P. najae within the same snake host, marking the second documented instance of a paracapillariid species in the monocled cobra within the family Elapidae. The genetic characterization supports the formal recognition of P. siamensis sp. nov. as a distinct species, thereby underscoring its taxonomic differentiation within the Capillariidae family. This research identifies and characterizes the new nematode species, contributing valuable insights into the taxonomy of this nematode.

Snakebite is a neglected public health issue, with many scientific and medical issues to be solved. Cobras are among the most common venomous snakes in Myanmar and are responsible for a considerable number of severe snakebite envenoming. There are three species of cobra (Naja kaouthia, Naja mandalayensis and Ophiophagus hannah) in Myanmar. The study aims to characterize the N. kaouthia and N. mandalayensis venoms and to investigate the efficacy of anti-cobra antivenom (BPI) against the two venoms. Protein components and fibrinogenolytic activity were determined by SDS-PAGE. Enzymatic activities for PLA2, protease and acetylcholinesterase were determined by spectrophotometric method. Anticoagulant activity was determined by recalcification time of citrated human plasma. Myotoxicity, necrotizing activity, median lethal dose (LD50) and median effective dose (ED50) were determined by WHO recommended methods. The SDS-PAGE displayed the proteins and enzymes containing in two venoms were different. N. kaouthia venom exhibited more in PLA2, acetylcholinesterase, anticoagulant, fibrinogenolytic and necrotizing activities than N. mandalayensis venom. N. mandalayensis venom had more protease activity and myotoxicity than N. kaouthia venom. The median lethal dose (LD50) of N. kaouthia and N. mandalayensis venom was 4.33 μg/mouse and 5.04 μg/mouse respectively. Both venoms induced fibrinogen Aα chain degradation in 30 min (N. kaouthia) and in 6 h (N. mandalayensis). The same median effective dose (ED50) (19.56 μg/mouse) showed that anti-NK antivenom can neutralize against lethal effect of N. mandalayensis venom. It can also neutralize the protease activity, anticoagulant activity and fibrinogenolytic activity of both venoms. Immunodiffusion and immunoblotting studies showed that the antivenom recognized its homologous venom (N. kaouthia) and cross-reacted against the heterologous venom (N. mandalayensis). The anti-NK antivenom is suitable to use for N. mandalayensis bite if monospecific antivenom is not available.

The parasitic nematode Paracapillaria (Ophidiocapillaria) najae De, 1998, found in the Indian cobra Naja naja is redescribed and re-illustrated in the present study. The monocled cobra Naja kaouthia was discovered to be a new host for this parasite in central Thailand. A comprehensive description extending the morphological and molecular characteristics of the parasites is provided to aid species recognition in future studies. The morphometric characters of 41 parasites collected from 5 cobra specimens are compared with those described in the original studies. Phylogenetic analyses using mitochondrial cytochrome c oxidase subunit 1 and nuclear 18S ribosomal RNA genes were performed to provide novel information on the systematics of P. najae. Similar characteristics were observed in the examined nematode samples, despite being found in different hosts, confirming their identity as P. najae. The molecular genetic results support the species status of P. najae, indicating P. najae is well defined and separated from other related nematode species in the family Capillariidae. Morphological descriptions, genetic sequences, evolutionary relationships among capillariids and new host and distribution records of P. najae are discussed. Paracapillaria najae specimens found in the Thai cobra had some morphological variation, and sexual size dimorphism was also indicated. Paracapillaria najae was found to infect various cobra host species and appeared to be common throughout the Oriental regions, consistent with its hosts' distribution.

The impacts of commonly used and often carelessly discarded plastics are well reported in marine animals but have been poorly reported in the terrestrial animals. Here, we report for the first time two cases of dead N. kaouthia from two different habitat types due to consumption of plastic bag and fishing net. We performed a postmortem and recorded observations for both cases, confirmed the cause of death as plastic ingestion and briefly describe the clinical signs and how plastic consumption causes death in N. kaouthia. Here, we highlight how improper waste management impacts each individual's health and sometimes causes painful death in snakes. Our observation indicates that plastic ingestion in snakes is an underestimated cause of death that occurs where the snake's foraging habitat is polluted. We recommend creating awareness, proper waste management, and use of biodegradable/reusable bags can avoid wildlife mortalities in non-protected areas.

This study investigated the clinical characteristics, treatments, and outcomes of envenomation involving cobra species in Thailand (Naja kaouthia, Naja siamensis, and Naja sumatrana). Data of patients who had been bitten by a cobra or inoculated via the eyes/skin in 2018–2021 were obtained from the Ramathibodi Poison Center. There were 1045 patients admitted during the 4-year study period (bite, n = 539; ocular/dermal inoculation, n = 506). Almost all patients with ocular/dermal inoculation had eye involvement and ocular injuries, but none had neurological effects. Most of the patients bitten by a cobra had local effects (69.0%) and neurological signs and symptoms (55.7%). The median interval between the bite and the onset of neurological symptoms was 1 h (range, 10 min to 24 h). Accordingly, patients should be observed closely in hospitals for at least 24 h after a bite. Intubation with ventilator support was required in 45.5% of patients and for a median duration of 1.1 days. Antivenom was administered in 63.5% of cases. There were nine deaths, most of which resulted from severe infection. Neurological effects and intubation were significantly more common after a monocled cobra bite than after a spitting cobra bite. The administration of antivenom with good supportive care, including the appropriate management of complications, especially wound infection, might decrease fatality. [ABSTRACT FROM AUTHOR]

Snake envenoming is caused by many biological species, rather than a single infectious agent, each with a multiplicity of toxins in their venom. Hence, developing effective treatments is challenging, especially in biodiverse and biogeographically complex countries such as India. The present study represents the first genus-wide proteomics analysis of venom composition across Naja species (N. naja, N. oxiana, and N. kaouthia) found in mainland India. Venom proteomes were consistent between individuals from the same localities in terms of the toxin families present, but not in the relative abundance of those in the venom. There appears to be more compositional variation among N. naja from different locations than among N. kaouthia. Immunoblotting and in vitro neutralization assays indicated cross-reactivity with Indian polyvalent antivenom, in which antibodies raised against N. naja are present. However, we observed ineffective neutralization of PLA2 activities of N. naja venoms from locations distant from the source of immunizing venoms. Antivenom immunoprofiling by antivenomics revealed differential antigenicity of venoms from N. kaouthia and N. oxiana, and poor reactivity towards 3FTxs and PLA2s. Moreover, there was considerable variation between antivenoms from different manufacturers. These data indicate that improvements to antivenom manufacturing in India are highly desirable. [ABSTRACT FROM AUTHOR]

Simple Summary: Cobras (Naja Laurenti, 1768) are a group of well-known highly venomous snakes, which cause numerous cases of snakebites every year, especially in South Asia and Southern China. Taxonomic framework is essential for the medical treatment of snake bites and accurate antivenin development. However, the taxonomy of Asian cobras is still puzzling, especially for the widespread species Monocled Cobra (N. kaouthia Lesson, 1831). This study provided new materials and understanding for the taxonomy of this species by combining mitochondrial phylogenetic analysis and morphological comparisons based on samples from a vast area in Asia. The results showed that the Chinese population of N. kaouthia represents a new species. This study also provided new data for N. atra and designated a neotype for it. Furthermore, the subspecies N. naja polyocellata was resurrected and recognized as a full species, N. polyocellatacomb. nov., and the subspecies N. sumatrana miolepis was also resurrected. This study highlighted the necessity to evaluate the effectiveness of cobra antivenin based on a comprehensive taxonomic framework. Taxonomic frameworks for medically important species such as cobras (genus Naja Laurenti, 1768; Squamata, Elapidae) are essential for the medical treatment of snake bites and accurate antivenin development. In this paper, we described the former N. kaouthia populations recorded from China as a new species and designated a neotype for N. atra-based morphological and mitochondrial phylogenetic analysis. The new species N. fuxisp. nov. was morphologically diagnosed from N. kaouthia by (1) regular single narrow crossband present on the middle and posterior parts of the dorsum (3–15, 7.9 ± 2.7, n = 32) and the dorsal surface of the tail (1–6, 4.2 ± 1.1, n = 32) of both adults and juveniles, buff-colored with dark fringes on both edges, vs. South Asian populations (n = 39) and Southeast Asian populations (n = 35) without cross bands, with irregular cross bands or multiple light-colored crossbands pairs, or densely woven lines; (2) small scales between the posterior chin shields, usually three (40%) or two (37%), rarely four (13%), or one (10%) (n = 30) vs. mostly one (81%) and rarely two (19%) (n = 28); (3) ventrals 179–205 (195.4 ± 6.7, n = 33) vs. South Asian populations 179–199 (188.7 ± 5.9, n = 12); Southeast Asian populations 168–186 (177.8 ± 4.9, n = 18). Phylogenetically, the new species forms an independent sister clade to the clade including N. atra, N. kaouthia, N. oxiana and N. sagittifera. Furthermore, the subspecies N. naja polyocellata should be resurrected and recognized as a full species, N. polyocellatacomb. nov., and the subspecies N. sumatrana miolepis should be resurrected. [ABSTRACT FROM AUTHOR]

This study investigated the clinical characteristics, treatments, and outcomes of envenomation involving cobra species in Thailand (Naja kaouthia, Naja siamensis, and Naja sumatrana). Data of patients who had been bitten by a cobra or inoculated via the eyes/skin in 2018–2021 were obtained from the Ramathibodi Poison Center. There were 1045 patients admitted during the 4-year study period (bite, n = 539; ocular/dermal inoculation, n = 506). Almost all patients with ocular/dermal inoculation had eye involvement and ocular injuries, but none had neurological effects. Most of the patients bitten by a cobra had local effects (69.0%) and neurological signs and symptoms (55.7%). The median interval between the bite and the onset of neurological symptoms was 1 h (range, 10 min to 24 h). Accordingly, patients should be observed closely in hospitals for at least 24 h after a bite. Intubation with ventilator support was required in 45.5% of patients and for a median duration of 1.1 days. Antivenom was administered in 63.5% of cases. There were nine deaths, most of which resulted from severe infection. Neurological effects and intubation were significantly more common after a monocled cobra bite than after a spitting cobra bite. The administration of antivenom with good supportive care, including the appropriate management of complications, especially wound infection, might decrease fatality.

Introduction: Snake venom is a combination of various proteins and peptides that cause diverse biological effects on multiple organ systems. In elapid venom, three-finger toxins are the most abundant type of toxin. Although toxins share similarities in their structure, they are known for their capability to cause a myriad of toxic actions such as neurotoxicity, cardiotoxicity, and cytotoxicity. Unfortunately, many of these toxins are not fully characterized especially on their binding affinity and selectivity towards receptors and their effect to the organ system. Materials and method: Therefore, this work was conducted to compare the binding properties of selected three-finger toxins (3FTxs) from cobras (Naja sumatrana and Naja kaouthia) and mangrove catsnake (Boiga dendrophila) towards human and bird nicotinic acetylcholine receptors (?3?2, ?4?2, ?7) using computational approaches. Results: The results show that all toxins bind to the orthosteric site, which is located outside the extracellular domain of ? subunit for all receptors in both species. Interaction between receptors and toxins occurs by the formation of hydrogen bond, ionic bond, and hydrophobic contact with important residues involved in their binding pocket. Conclusion: Based on the data, the toxins showed different binding affinities towards nicotinic acetylcholine receptors in different species. Differences in the binding affinity towards different species could have a significant impact on the functional characterization of venom caused by these toxins and toxins with nearly similar sequences.

Each year, thousands of people fall victim to envenomings caused by cobras. These incidents often result in death due to paralysis caused by α‐neurotoxins from the three‐finger toxin (3FTx) family, which are abundant in elapid venoms. Due to their small size, 3FTxs are among the snake toxins that are most poorly neutralized by current antivenoms, which are based on polyclonal antibodies of equine or ovine origin. While antivenoms have saved countless lives since their development in the late 18th century, an opportunity now exists to improve snakebite envenoming therapy via the application of new biotechnological methods, particularly by developing monoclonal antibodies against poorly neutralized α‐neurotoxins. Here, we describe the use of phage‐displayed synthetic antibody libraries and the development and characterization of six synthetic antibodies built on a human IgG framework and developed against α‐cobratoxin – the most abundant long‐chain α‐neurotoxin from Naja kaouthia venom. The synthetic antibodies exhibited sub‐nanomolar affinities to α‐cobratoxin and neutralized the curare‐mimetic effect of the toxin in vitro. These results demonstrate that phage display technology based on synthetic repertoires can be used to rapidly develop human antibodies with drug‐grade potencies as inhibitors of venom toxins. [ABSTRACT FROM AUTHOR]

Taxonomic frameworks for medically important species such as cobras (genus Naja Laurenti, 1768; Squamata, Elapidae) are essential for the medical treatment of snake bites and accurate antivenin development. In this paper, we described the former N. kaouthia populations recorded from China as a new species and designated a neotype for N. atra-based morphological and mitochondrial phylogenetic analysis. The new species N. fuxisp. nov. was morphologically diagnosed from N. kaouthia by (1) regular single narrow crossband present on the middle and posterior parts of the dorsum (3–15, 7.9 ± 2.7, n = 32) and the dorsal surface of the tail (1–6, 4.2 ± 1.1, n = 32) of both adults and juveniles, buff-colored with dark fringes on both edges, vs. South Asian populations (n = 39) and Southeast Asian populations (n = 35) without cross bands, with irregular cross bands or multiple light-colored crossbands pairs, or densely woven lines; (2) small scales between the posterior chin shields, usually three (40%) or two (37%), rarely four (13%), or one (10%) (n = 30) vs. mostly one (81%) and rarely two (19%) (n = 28); (3) ventrals 179–205 (195.4 ± 6.7, n = 33) vs. South Asian populations 179–199 (188.7 ± 5.9, n = 12); Southeast Asian populations 168–186 (177.8 ± 4.9, n = 18). Phylogenetically, the new species forms an independent sister clade to the clade including N. atra, N. kaouthia, N. oxiana and N. sagittifera. Furthermore, the subspecies N. naja polyocellata should be resurrected and recognized as a full species, N. polyocellatacomb. nov., and the subspecies N. sumatrana miolepis should be resurrected.

Background: Bites from nonnative snakes are uncommon, accounting for 1.1% of envenomations reported to poison centers between 2015 and 2018. Here we discuss two monocled cobra (Naja kaouthia) envenomations resulting in respiratory failure.Case Reports: A 30-year-old man and a 40-year-old man were bitten by their captive monocled cobras. At the first hospital, the first patient was mildly hypotensive, transiently bradycardic, and confused. He was intubated for respiratory distress. He was hypertensive to 211/119 mm Hg upon arrival to the second hospital. In the Emergency Department, cobra antivenom was administered. He was admitted to the medical intensive care unit (MICU) and had an additional bradycardic episode that corrected with atropine. He was extubated after 35 h. He was observed for an additional 9 h prior to going home, where he recovered without incident. The second patient developed abdominal pain, blurry vision, and dyspnea within 90 min of the bite. He was intubated at the first hospital. At the second hospital he received cobra antivenom and was admitted to the MICU. He was extubated after 9 h and discharged the following day with no further symptoms. WHY SHOULD AN EMERGENCY PHYSICIAN BE AWARE OF THIS?: Envenomations after N. kaouthia bites are characterized by local tissue injury and various neurotoxic effects. Nonspecific signs and symptoms are common. Hematologic toxicity and cardiovascular manifestations are uncommon. Antivenom is the specific treatment for snake envenomation, but only certain antivenoms are indicated for N. kaouthia. Cholinesterase inhibitors may reduce toxicity from postsynaptic alpha toxins by increasing acetylcholine concentrations. [ABSTRACT FROM AUTHOR]

Snakebite is a neglected public health issue, with many scientific and medical issues to be solved. Cobras are among the most common venomous snakes in Myanmar and are responsible for a considerable number of severe snakebite envenoming. There are three species of cobra ( Naja kaouthia , Naja mandalayensis and Ophiophagus hannah ) in Myanmar. The study aims to characterize the N. kaouthia and N. mandalayensis venoms and to investigate the efficacy of anti-cobra antivenom (BPI) against the two venoms. Protein components and fibrinogenolytic activity were determined by SDS-PAGE. Enzymatic activities for PLA 2 , protease and acetylcholinesterase were determined by spectrophotometric method. Anticoagulant activity was determined by recalcification time of citrated human plasma. Myotoxicity, necrotizing activity, median lethal dose (LD 50 ) and median effective dose (ED 50 ) were determined by WHO recommended methods. The SDS-PAGE displayed the proteins and enzymes containing in two venoms were different. N. kaouthia venom exhibited more in PLA 2 , acetylcholinesterase, anticoagulant, fibrinogenolytic and necrotizing activities than N. mandalayensis venom. N. mandalayensis venom had more protease activity and myotoxicity than N. kaouthia venom. The median lethal dose (LD 50 ) of N. kaouthia and N. mandalayensis venom was 4.33 μg/mouse and 5.04 μg/mouse respectively. Both venoms induced fibrinogen Aα chain degradation in 30 min ( N. kaouthia ) and in 6 h ( N. mandalayensis ). The same median effective dose (ED 50 ) (19.56 μg/mouse) showed that anti-NK antivenom can neutralize against lethal effect of N. mandalayensis venom. It can also neutralize the protease activity, anticoagulant activity and fibrinogenolytic activity of both venoms. Immunodiffusion and immunoblotting studies showed that the antivenom recognized its homologous venom ( N. kaouthia ) and cross-reacted against the heterologous venom ( N. mandalayensis ). The anti-NK antivenom is suitable to use for N. mandalayensis bite if monospecific antivenom is not available., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Mya Nila Win reports financial support was provided by 10.13039/100009647Ministry of Health, Myanmar., (© 2024 Published by Elsevier Ltd.)

While snakebite is a severe problem for several countries throughout the world the Indian subcontinent has witnessed the highest global incidence of snakebite which represents an occupational health hazard. Belonging to the family Elapidae, the Indian spectacled cobra (Naja naja), is one of the members of the "Big Four" venomous snakes found throughout the Indian subcontinent. Indian monocled cobra Naja kaouthia is prevalent in eastern and north-eastern India as well as in Bangladesh, Nepal, and Bhutan. Species-specific and geographical differences in venom composition of two major Indian cobras (N. naja vs. N. kaouthia) may limit the effectiveness of antivenom treatment. Therefore, exploring the venom proteomes of N. naja and N. kaouthia from different geographical locales of the Indian subcontinent, and the effect of this variation on antivenom efficacy is warranted. Proteomic analyses have demonstrated that although eastern Indian N. naja and N. kaouthia venoms have similarities, species-specific and geographical variation is evident by differences in their venom toxicity values and pathobiological effects in experimental animals. This is the major reason as to why a polyvalent antivenom against N. naja venom exhibited only partial neutralization of enzymatic activity and immunological cross-reactivity against cobra venoms from other regions of India. This suggests that addition of polyclonal antibodies against N. kaouthia venom to polyantivenom, or preparation/administration of region-specific polyantivenom will provide better treatment of cobra bites in the Indian subcontinent. • Indian spectacled cobra (Naja naja) is one of the medically important venomous snakes of the Indian subcontinent. • Indian monocle cobra (Naja kaouthia) is prevalent in eastern, north-eastern India, Nepal, and Bangladesh. • The zoogeographic and species-specific differences in venom composition of Indian cobras are discussed. • These differences result in variation in lethality of cobra venoms across the Indian subcontinent. • Necessity of production of region-specific commercial antivenom against cobra venoms is highlighted. [ABSTRACT FROM AUTHOR]

Naja kaouthia is one of the most prevalent medically important snakes of North East India and Bangladesh responsible for most of the bite cases. In this study, an attempt was made to decipher venom variation of Naja kaouthia venom from North East India and Bangladesh. Using multidimensional methods including reverse phase HPLC, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (1D-PAGE) and two-dimensional gel electrophoresis (2D-PAGE), the quantitative differences in venom composition have been revealed. Moreover, tested in-vitro biochemical and biological activities also exhibited differences which could be due to venom variability. Furthermore, neutralization efficacy of commercially available Indian polyvalent antivenoms (Vins, Bharat Serum, Haffkine) was evaluated and the results displayed significant differences in neutralizing efficacy between the antivenoms. Immunoblotting experiments showed antivenom molecules cross reacted with high molecular mass components while poorly reacted towards low molecular mass proteins. Immuno-depletion study demonstrated that Vins polyvalent antivenom was poor in immunocapturing the venom proteins of both North East Indian and Bangladesh origin Naja kaouthia at the ratio of 1:16 (venom: antivenom). Image 1 • North East Indian and Bangladesh origin Naja kaouthia venom demonstrates variation. • Neutralization of venom toxic effects was analysed using three Indian polyvalent antivenoms. • Haffkeine polyvalent antivenom performed better in neutralizing toxic effects compared to Vins and Bharat Serum. • Antivenomics study revealed poor immunocapturing ability of Naja kaouthia venom proteins by Vins polyvalent antivenom. [ABSTRACT FROM AUTHOR]

Background The monocled cobra (Naja kaouthia) is a medically important venomous snake in Southeast Asia. Its venom has been shown to vary geographically in relation to venom composition and neurotoxic activity, indicating vast diversity of the toxin genes within the species. To investigate the polygenic trait of the venom and its locale-specific variation, we profiled and compared the venom gland transcriptomes of N. kaouthia from Malaysia (NK-M) and Thailand (NK-T) applying next-generation sequencing (NGS) technology. Methods The transcriptomes were sequenced on the Illumina HiSeq platform, assembled and followed by transcript clustering and annotations for gene expression and function. Pairwise or multiple sequence alignments were conducted on the toxin genes expressed. Substitution rates were studied for the major toxins co-expressed in NK-M and NK-T. Results and discussion The toxin transcripts showed high redundancy (41–82% of the total mRNA expression) and comprised 23 gene families expressed in NK-M and NK-T, respectively (22 gene families were co-expressed). Among the venom genes, three-finger toxins (3FTxs) predominated in the expression, with multiple sequences noted. Comparative analysis and selection study revealed that 3FTxs are genetically conserved between the geographical specimens whilst demonstrating distinct differential expression patterns, implying gene up-regulation for selected principal toxins, or alternatively, enhanced transcript degradation or lack of transcription of certain traits. One of the striking features that elucidates the inter-geographical venom variation is the up-regulation of α-neurotoxins (constitutes ∼80.0% of toxin’s fragments per kilobase of exon model per million mapped reads (FPKM)), particularly the long-chain α-elapitoxin-Nk2a (48.3%) in NK-T but only 1.7% was noted in NK-M. Instead, short neurotoxin isoforms were up-regulated in NK-M (46.4%). Another distinct transcriptional pattern observed is the exclusively and abundantly expressed cytotoxin CTX-3 in NK-T. The findings suggested correlation with the geographical variation in proteome and toxicity of the venom, and support the call for optimising antivenom production and use in the region. Besides, the current study uncovered full and partial sequences of numerous toxin genes from N. kaouthia which have not been reported hitherto; these include N. kaouthia-specific l-amino acid oxidase (LAAO), snake venom serine protease (SVSP), cystatin, acetylcholinesterase (AChE), hyaluronidase (HYA), waprin, phospholipase B (PLB), aminopeptidase (AP), neprilysin, etc. Taken together, the findings further enrich the snake toxin database and provide deeper insights into the genetic diversity of cobra venom toxins.

Venomous snakebites are an important health problem in tropical and subtropical countries. King cobra (Ophiophagus hannah) is the largest venomous snake found in South and Southeast Asia. In this study, the O. hannah venom proteome and the venom components cross-reactive to N. kaouthia monospecific antivenin were studied. O. hannah venom consisted of 14 different protein families, including three finger toxins, phospholipases, cysteine-rich secretory proteins, cobra venom factor, muscarinic toxin, L-amino acid oxidase, hypothetical proteins, low cysteine protein, phosphodiesterase, proteases, vespryn toxin, Kunitz, growth factor activators and others (coagulation factor, endonuclease, 5’-nucleotidase). N. kaouthia antivenin recognized several functionally different O. hannah venom proteins and mediated paratherapeutic efficacy by rescuing the O. hannah envenomed mice from lethality. An engineered human ScFv specific to N. kaouthia long neurotoxin (NkLN-HuScFv) cross-neutralized the O. hannah venom and extricated the O. hannah envenomed mice from death in a dose escalation manner. Homology modeling and molecular docking revealed that NkLN-HuScFv interacted with residues in loops 2 and 3 of the neurotoxins of both snake species, which are important for neuronal acetylcholine receptor binding. The data of this study are useful for snakebite treatment when and where the polyspecific antivenin is not available. Because the supply of horse-derived antivenin is limited and the preparation may cause some adverse effects in recipients, a cocktail of recombinant human ScFvs for various toxic venom components shared by different venomous snakes, exemplified by the in vitro produced NkLN-HuScFv in this study, should contribute to a possible future route for an improved alternative to the antivenins.

Research on nanoparticles has created interest among the biomedical scientists. Nanoparticle conjugation aims to target drug delivery, increase drug efficacy and imaging for better diagnosis. Toxicity profile of the nanoconjugated molecules has not been studied well. In this communication, the toxicity profile of snake venom cytotoxin (NKCT1), an antileukemic protein toxin, was evaluated after its conjugation with gold nanoparticle (GNP-NKCT1). Gold nanoparticle conjugation with NKCT1 was done with NaBH4 reduction method. The conjugated product GNP-NKCT1 was found less toxic than NKCT1 on isolated rat lymphocyte, mice peritoneal macrophage, in culture, which was evident from the MTT/Trypan blue assay. Peritoneal mast cell degranulation was in the order of NKCT1 > GNP-NKCT1. The in vitro cardiotoxicity and neurotoxicity were increased in case of NKCT1 than GNP-NKCT1. On isolated kidney tissue, NKCT1 released significant amount of ALP and γ-GT than GNP-NKCT1. Gold nanoconjugation with NKCT1 also reduced the lethal activity in mice. In vivo acute/sub-chronic toxicity studies in mice showed significant increase in molecular markers due to NKCT1 treatment, which was reduced by gold nanoconjugation. Histopathology study showed decreased toxic effect of NKCT1 in kidney tissue after GNP conjugation. The present study confirmed that GNP conjugation significantly decreased the toxicity profile of NKCT1. Further studies are in progress to establish the molecular mechanism of GNP induced toxicity reduction.

In Southeast Asia, envenoming resulting from cobra snakebites is an important public health issue in many regions, and antivenom therapy is the standard treatment for the snakebite. Because these cobras share a close evolutionary history, the amino acid sequences of major venom components in different snakes are very similar. Therefore, either monovalent or polyvalent antivenoms may offer paraspecific protection against envenomation of humans by several different snakes. In Taiwan, a bivalent antivenom—freeze-dried neurotoxic antivenom (FNAV)—against Bungarus multicinctus and Naja atra is available. However, whether this antivenom is also capable of neutralizing the venom of other species of snakes is not known. Here, to expand the clinical application of Taiwanese FNAV, we used an animal model to evaluate the neutralizing ability of FNAV against the venoms of three common snakes in Southeast Asia, including two ‘true’ cobras Naja kaouthia (Thailand) and Naja siamensis (Thailand), and the king cobra Ophiophagus hannah (Indonesia). We further applied mass spectrometry (MS)-based proteomic techniques to characterize venom proteomes and identify FNAV-recognizable antigens in the venoms of these Asian snakes. Neutralization assays in a mouse model showed that FNAV effectively neutralized the lethality of N. kaouthia and N. siamensis venoms, but not O. hannah venom. MS-based venom protein identification results further revealed that FNAV strongly recognized three-finger toxin and phospholipase A2, the major protein components of N. kaouthia and N. siamensis venoms. The characterization of venom proteomes and identification of FNAV-recognizable venom antigens may help researchers to further develop more effective antivenom designed to block the toxicity of dominant toxic proteins, with the ultimate goal of achieving broadly therapeutic effects against these cobra snakebites. [ABSTRACT FROM AUTHOR]

We conducted an omics-analysis of the venom of Naja kaouthia from China. Proteomics analysis revealed six protein families [three-finger toxins (3-FTx), phospholipase A 2 (PLA 2 ), nerve growth factor, snake venom metalloproteinase (SVMP), cysteine-rich secretory protein and ohanin], and venom-gland transcriptomics analysis revealed 28 protein families from 79 unigenes. 3-FTx (56.5% in proteome/82.0% in transcriptome) and PLA 2 (26.9%/13.6%) were identified as the most abundant families in venom proteome and venom-gland transcriptome. Furthermore, N . kaouthia venom expressed strong lethality (i.p. LD 50 : 0.79 μg/g) and myotoxicity (CK: 5939 U/l) in mice, and showed notable activity in PLA 2 but weak activity in SVMP, l -amino acid oxidase or 5′ nucleotidase. Antivenomic assessment revealed that several venom components (nearly 17.5% of total venom) from N . kaouthia could not be thoroughly immunocaptured by commercial Naja atra antivenom. ELISA analysis revealed that there was no difference in the cross-reaction between N . kaouthia and N . atra venoms against the N . atra antivenom. The use of commercial N . atra antivenom in treatment of snakebites caused by N . kaouthia is reasonable, but design of novel antivenom with the attention on enhancing the immune response of non-immunocaptured components should be encouraged. Biological significance The venomics, antivenomics and venom-gland transcriptome of the monocoled cobra ( Naja kaouthia ) from China have been elucidated. Quantitative and qualitative differences are evident when venom proteomic and venom-gland transcriptomic profiles are compared. Two protein families (3-FTx and PLA 2 ) are found to be the predominated components in N . kaouthia venom, and considered as the major players in functional role of venom. Other protein families with relatively low abundance appear to be minor in the functional significance. Antivenomics and ELISA evaluation reveal that the N . kaouthia venom can be effectively immunorecognized by commercial N . atra antivenom, but still a small number of venom components could not be thoroughly immunocaptured. The findings indicate that exploring the precise composition of snake venom should be executed by an integrated omics-approach, and elucidating the venom composition is helpful in understanding composition-function relationships and will facilitate the clinical application of antivenoms. [ABSTRACT FROM AUTHOR]

Snakebite envenoming is a medical emergency requiring urgent and specific treatment. Unfortunately, snakebite diagnostics are scarce, time-consuming and lacking specificity. Hence, this study aimed to develop a simple, quick and specific snakebite diagnostic assay using animal antibodies. Anti-venom horse immunoglobulin G (IgG) and chicken immunoglobulin Y (IgY) were produced against the venoms of four major medically important snake species in Southeast Asia, i.e., the Monocled Cobra (Naja kaouthia), Malayan Krait (Bungarus candidus), Malayan Pit Viper (Calloselasma rhodostoma), and White-lipped Green Pit Viper (Trimeresurus albolabris). Different capture:detection configurations of double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) were constructed using both immunoglobulins, and the horse IgG:IgG-HRP configuration was found to be most selective and sensitive in detecting the corresponding venoms. The method was further streamlined to develop a rapid immunodetection assay, which is able to produce a visual color change within 30 min for discrimination between different snake species. The study shows it is feasible to develop a simple, quick and specific immunodiagnostic assay using horse IgG, which can be derived directly from antisera prepared for antivenom production. The proof-of-concept indicates it is a sustainable and affordable approach in keeping with on-going antivenom manufacturing activities for specific species in the region. • Anti-venom horse IgG and chicken IgY for four venomous snakes were produced. • Horse IgG:IgG-HRP configuration is selective and sensitive for venom detection. • This was modified into a rapid immunodetection assay for use in Southeast Asia. • Venom of different species were discriminated by a visual color change in 30 mins. • This is a proof-of-concept study for a simple and rapid immunodiagnostic assay. [ABSTRACT FROM AUTHOR]

Snake venom-metalloproteinases (SVMPs) are the primary factors that disturb hemostasis and cause hemorrhage in the venomous snake bitten subjects. Kaouthiagin is a unique SVMP that binds and cleaves von Willebrand factor (vWF) at a specific peptide bond leading to inhibition of platelet aggregation, which enhances the hemorrhage. Kaouthiagin is a low abundant venom component of Thai cobra (Naja kaouthia); thus, most horse-derived antivenins used for cobra bite treatment do not contain adequate anti-kaouthiagin. This study aimed to produce human single-chain antibody variable fragments (HuscFvs) that bind to and interfere with kaouthiagin activity for further clinical use. Kaouthiagin was purified from N. kaouthia-holovenom by a single-step gel-filtration chromatography. The purified venom component was used in phage-biopanning to select the kaouthiagin-bound HuscFv-displayed-phage clones from a HuscFv-phage display library. The selected phages were used to infect Escherichia coli bacteria. Soluble HuscFvs expressed by three phage-transformed-E. coli clones interfered with cobra kaouthiagin binding to human vWF. Computerized simulation indicated that HuscFv of two phage-transformed E. coli clones formed contact interface with kaouthiagin residues at or near catalytic site and effectively inhibited fibrinogenolytic activity of the kaouthiagin. The HuscFvs have therapeutic potential as an adjunct of antivenins in treatment of bleeding caused by venomous snakebites.

A 35 year old, male patient, bitten by Naja kaouthia with mild pain was admitted in Demow Government Community Health Centre. After 90 min post bite he developed neurotoxic symptoms. As per standard protocol, the patient was treated with 25 vials of antivenom and two doses of glycopyrrolate and neostigmine. Subsequently, he was seemingly devoid of any neurotoxic symptoms and showed signs of recovery. However, after 70 h, the neurotoxic symptoms recurred, and the patient was again treated with an additional 10 vials of ASV along with one dose of glycopyrrolate and neostigmine. Subsequently, the patient recovered completely from all the other symptoms of envenomation. This is the first report of recurrence of neurotoxic symptoms in a patient envenomed by Naja kaouthia in Assam, India and supports the need for greater attention and careful documentation of management of snakebite in the region. [Display omitted] • This is the first case report of recurrent neurotoxic symptoms observed in Naja kaouthia envenomation from Assam, India. • The patient recovered on treatment with 25 vials of antivenom and 2 doses of neostigmine when admitted to the hospital. • Neurotoxic symptoms recurred 70 h post bite and the patient was given another 10 vials antivenom and 1 dose neostigmine. • The patient became stable after 30 min of second dose of antivenom and was under observation for 72 h. • He was discharged after being asymptomatic on the 6th day. [ABSTRACT FROM AUTHOR]

Background & objectives: Increased severity of osteoarthritis (OA) and adverse side effects of its treatment led to the search for alternative therapies. It was previously reported that snake venom protein toxin Naja kaouthia cytotoxin 1 (NKCT1) and gold nanoparticle (GNP) individually have potential against excremental arthritis. In this study, we analyzed the protective activity of GNP conjugated protein toxin NKCT1 (GNP-NKCT1) against experimental OA. Methods: Gold nanoparticle conjugation with NKCT1 (GNP-NKCT1) was done and its physiochemical properties were studied. OA was induced in male albino rats by intra-articular injection of bacterial collagenase and treatment was done with NKCT1/GNP-NKCT1/standard drug (indomethacin). Physical parameter (ankle diameter), urinary markers (hydroxyproline, glucosamine, pyridinoline, deoxypyridinoline), serum and synovial membrane pro-inflammatory markers [tumour necrosis factoralpha (TNF-α), interleukin-1β (IL-1β), IL-17, vascular endothelial growth factor (VEGF)] and matrix metalloproteinase 1 (MMP1) were measured. Joint histopathology and scanning electron microscopy imaging of articular cartilage surface were also done. Results: Physical parameters, urinary markers, serum and synovial membrane pro-inflammatory makers and MMP1 were increased in arthritic rats and significantly restored after GNP-NKCT1/ NKCT1 treatment. Joint histopathology and scanning electron microscopy imaging of articular cartilage surface also indicated the protective effect of GNP-NKCT1 against inflammatory response and cartilage degradation in osteoarthritic rats. Interpretation & conclusions: In this study restoration of the arthritic markers and bone degradation by GNP-NKCT1 treatment indicated the anti-osteoarthritic property of GNP-NKCT1. Further studies need to be done to confirm these findings. [ABSTRACT FROM AUTHOR]

Background Gold nanoparticle (GNP) and snake venom protein toxin NKCT1 was conjugated as stated earlier (Bhowmik et al., 2013). The aim of this study was to explore the caspase dependent apoptotic pathway and autophagy inducing ability of gold nanoparticles tagged snake venom protein toxin NKCT1 (GNP-NKCT1) in human leukemic U937 and K562 cell line. Methods GNP-NKCT1 induced apoptosis in U937 and K562 cell line were assessed through mitochondrial membrane potential assay, ROS generation assay, caspase 3 pathways and western blotting. GNP-NKCT1 induced autophagic pathway was detected through Akt, mTOR and PI3K expression by western blotting. Autophagic cell death also checked after addition of caspase 3 inhibitor and which also reconfirmed by western blotting of autophagic marker protein, lysosomal staining. Results Loss of mitochondrial membrane potential was occurred in both the leukemic cell line after induction by GNP-NKCT1 and treatment of which also exhibited high ROS generation. Caspase 3 expression of cell was also increased. With caspase 3 inhibitor, GNP-NKCT1 downregulated PI3K/Akt and mTOR expression and thus undergoing autophagic cell death. Lysosomal staining confirmed lysosomal enzyme involvement in the autophagic response. Up regulation of Atg 3, Atg12, Beclin 1, LC3-II protein and BIF-1 and down regulation of Atg4B were also showed by blotting. Conclusion The results demonstrated that conjugation of Gold nanoparticles with NKCT1 could induce an alternate cell death pathway other than apoptosis in the form of autophagy in leukemic cell. General significance This study might provide the understanding area of chemotherapeutic drug development from natural resources like snake venoms. [ABSTRACT FROM AUTHOR]

‘Three-finger’ toxin WTX from Naja kaouthia interacts with nicotinic and muscarinic acetylcholine receptors (nAChRs and mAChRs). Mutagenesis and competition experiments with 125 I-α-bungarotoxin revealed that Arg31 and Arg32 residues from the WTX loop II are important for binding to Torpedo californica and human α7 nAChRs. Computer modeling suggested that loop II occupies the orthosteric binding site at α7 nAChR. The similar toxin interface was previously described as a major determinant of allosteric interactions with mAChRs. [ABSTRACT FROM AUTHOR]

Venoms of snakes of the family Elapidae (cobras, kraits, mambas, and relatives) are predominantly composed of numerous phospholipases A 2 (PLA 2 s) and three-finger toxins (3FTxs), some of which are lethal while others are not significantly toxic. Currently, the only identified prey-specific toxins are several nonconventional 3FTxs, and given the large diversity of 3FTxs within Monocled Cobra ( Naja kaouthia ) venom, it was hypothesized that several 3FTxs, previously found to be non-toxic or weakly toxic 3FTxs in murine models, could potentially be toxic towards non-murine prey. Additionally, it was hypothesized that ontogenetic dietary shifts will be correlated with observable changes in specific 3FTx isoform abundance. Adult and juvenile N. kaouthia venom composition was investigated using ion-exchange FPLC, 1D and 2D SDS-PAGE, mass spectrometry, and various enzymatic and LD 50 assays. Alpha-cobratoxin (α-elapitoxin) was the only significantly toxic (LD 50 < 1 μg/g) 3FTx found in N. kaouthia venom and was equally toxic toward both lizard and mouse models. The abundance and diversity of 3FTxs and most enzyme activities did not vary between adult and juvenile cobra venoms; however, total venom PLA 2 activity and specific PLA 2 isoforms did vary, with juveniles lacking several of the least acidic PLA 2 s, and these differences could have both biological (related to predation) and clinical (antivenom efficacy) implications. Nevertheless, the ubiquitous presence of α-cobratoxin in both adult and juvenile cobra venoms, with high toxicity toward both reptiles and mammals, represents a venom compositional strategy wherein a single potent toxin effectively immobilizes a variety of prey types encountered across life history stages. [ABSTRACT FROM AUTHOR]

The Southeast Asian monocled cobras ( Naja kaouthia ) exhibit geographical variations in their venom proteomes, especially on the composition of neurotoxins. This study compared the neuromuscular depressant activity of the venoms of N. kaouthia from Malaysia (NK-M), Thailand (NK-T) and Vietnam (NK-V), and the neutralization of neurotoxicity by a monospecific antivenom. On chick biventer cervicis nerve-muscle preparation, all venoms abolished the indirect twitches, with NK-T venom being the most potent (shortest t 90 , time to 90% twitch inhibition), followed by NK-V and NK-M. Acetylcholine and carbachol failed to reverse the blockade, indicating irreversible/pseudo-irreversible post-synaptic neuromuscular blockade. KCl restored the twitches variably (NK-M preparation being the least responsive), consistent with different degree of muscle damage. The findings support that NK-T venom has the most abundant curarimimetic alpha-neurotoxins, while NK-M venom contains more tissue-damaging cytotoxins. Pre-incubation of tissue with N. kaouthia monovalent antivenom (NKMAV) prevented venom-induced twitch depression, with the NK-T preparation needing the largest antivenom dose. NKMAV added after the onset of neuromuscular depression could only halt the inhibitory progression but failed to restore full contraction. The findings highlight the urgency of early antivenom administration to sequester as much circulating neurotoxins as possible, thereby hastening toxin elimination from the circulation. In envenomed mice, NKMAV administered upon the first neurological sign neutralized the neurotoxic effect, with the slowest full recovery noticed in the NK-T group. This is consistent with the high abundance of neurotoxins in the NK-T venom, implying that a larger amount or repeated dosing of NKMAV may be required in NK-T envenomation. [ABSTRACT FROM AUTHOR]

Antivenom neutralization against cobra venoms is generally low in potency, presumably due to poor toxin-specific immunoreactivity. This study aimed to investigate the effectiveness of two elapid antivenoms to neutralize the principal toxins purified from the venoms of the Thai monocled cobra (Naja kaouthia, Nk-T) and the Malaysian beaked sea snake (Hydrophis schistosus, Hs-M). In mice, N. kaouthia Monovalent Antivenom (NKMAV) neutralization against Nk-T long neurotoxin (LNTX) and cytotoxin was moderate (potency of 2.89-6.44 mg toxin/g antivenom protein) but poor against the short neurotoxin (SNTX) (1.33 mg/g). Its cross-neutralization against Hs-M LNTX of Hs-M is compatible (0.18 mg/g) but much weaker against Hs-M SNTX (0.22 mg/g). Using CSL (Seqirus Limited) Sea Snake Antivenom (SSAV), we observed consistently weak neutralization of antivenom against SNTX of both species, suggesting that this is the limiting factor on the potency of antivenom neutralization against venoms containing SNTX. Nevertheless, SSAV outperformed NKMAV in neutralizing SNTXs of both species (0.61-2.49 mg/g). The superior efficacy of SSAV against SNTX is probably partly attributable to the high abundance of SNTX in sea snake venom used as immunogen in SSAV production. The findings indicate that improving the potency of cobra antivenom may be possible with a proper immunogen formulation that seeks to overcome the limitation on SNTX immunoreactivity. [ABSTRACT FROM AUTHOR]

ABSTRACT Snake venom three finger toxins (3FTxs) are a non-enzymatic family of venom proteins abundantly found in elapids. We have purified a 7579.5 ± 0.591 Da 3FTx named as Nk-3FTx from the venom of Naja kaouthia of North East India origin. The primary structure was determined by a combination of N-terminal sequencing and electrospray ionization liquid chromatography-mass spectrometry/mass spectrometry. Biochemical and biological characterization reveal that it is nontoxic to human cell lines and exhibit mild anticoagulant activity when tested on citrated human plasma. Nk-3FTx was found to affect the compound action potential (CAP) and nerve conduction velocity of isolated toad sciatic nerve. This is the first report of a non-conventional 3FTx from Naja kaouthia venom that reduces CAP for its neurotoxic effect. Further studies can be carried out to understand the mechanism of action and to explore its potential therapeutic application. [ABSTRACT FROM AUTHOR]

Weak toxin from Naja kaouthia (WTX) belongs to the group of nonconventional "three-finger" snake neurotoxins. It irreversibly inhibits nicotinic acetylcholine receptors and allosterically interacts with muscarinic acetylcholine receptors (mAChRs). Using site-directed mutagenesis, NMR spectroscopy, and computer modeling, we investigated there combinant mutant WTX analogue (rWTX) which, compared with the native toxin, has an additional N-terminal methionine residue. In comparison with the wild-type toxin, rWTX demonstrated an altered pharmacological profile, decreased binding of orthosteric antagonist N-methylscopolamine to human M1- and M2-mAChRs, and increased antagonist binding to M3-mAChR. Positively charged arginine residues located in the flexible loop II were found to be crucial for rWTX interactions with all types of mAChR. Computer modeling suggested that the rWTX loop II protrudes to the M1-mAChR allosteric ligand-binding site blocking the entrance to the orthosteric site. In contrast, toxin interacts with M3-mAChR by loop II without penetration into the allosteric site. Data obtained provide new structural insight into the target-specific allosteric regulation of mAChRs by "three-finger" snake neurotoxins. [ABSTRACT FROM AUTHOR]

The venom proteome of the monocled cobra, Naja kaouthia , from Thailand, was characterized by RP-HPLC, SDS-PAGE, and MALDI-TOF-TOF analyses, yielding 38 different proteins that were either identified or assigned to families. Estimation of relative protein abundances revealed that venom is dominated by three-finger toxins (77.5%; including 24.3% cytotoxins and 53.2% neurotoxins) and phospholipases A 2 (13.5%). It also contains lower proportions of components belonging to nerve growth factor, ohanin/vespryn, cysteine-rich secretory protein, C-type lectin/lectin-like, nucleotidase, phosphodiesterase, metalloproteinase, l -amino acid oxidase, cobra venom factor, and cytidyltransferase protein families. Small amounts of three nucleosides were also evidenced: adenosine, guanosine, and inosine. The most relevant lethal components, categorized by means of a ‘toxicity score’, were α-neurotoxins, followed by cytotoxins/cardiotoxins. IgGs isolated from a person who had repeatedly self-immunized with a variety of snake venoms were immunoprofiled by ELISA against all venom fractions. Stronger responses against larger toxins, but lower against the most critical α-neurotoxins were obtained. As expected, no neutralization potential against N. kaouthia venom was therefore detected. Combined, our results display a high level of venom complexity, unveil the most relevant toxins to be neutralized, and provide prospects of discovering human IgGs with toxin neutralizing abilities through use of phage display screening. [ABSTRACT FROM AUTHOR]

Previous studies showed that venoms of the monocled cobra, Naja kaouthia from Thailand and Malaysia are substantially different in their median lethal doses. The intraspecific venom variations of N. kaouthia , however, have not been fully elucidated. Here we investigated the venom proteomes of N. kaouthia from Malaysia (NK-M), Thailand (NK-T) and Vietnam (NK-V) through reverse-phase HPLC, SDS-PAGE and tandem mass spectrometry. The venom proteins comprise 13 toxin families, with three-finger toxins being the most abundant (63–77%) and the most varied (11–18 isoforms) among the three populations. NK-T has the highest content of neurotoxins (50%, predominantly long neurotoxins), followed by NK-V (29%, predominantly weak neurotoxins and some short neurotoxins), while NK-M has the least (18%, some weak neurotoxins but less short and long neurotoxins). On the other hand, cytotoxins constitute the main bulk of toxins in NK-M and NK-V venoms (up to 45% each), but less in NK-T venom (27%). The three venoms show different lethal potencies that generally reflect the proteomic findings. Despite the proteomic variations, the use of Thai monovalent and Neuro polyvalent antivenoms for N. kaouthia envenomation in the three regions is appropriate as the different venoms were neutralized by the antivenoms albeit at different degrees of effectiveness. Biological significance Biogeographical variations were observed in the venom proteome of monocled cobra ( Naja kaouthia ) from Malaysia, Thailand and Vietnam. The Thai N. kaouthia venom is particularly rich in long neurotoxins, while the Malaysian and Vietnamese specimens were predominated with cytotoxins. The differentially expressed toxin profile accounts for the discrepancy in the lethal dose of the venom from different populations. Commercially available Thai antivenoms (monovalent and polyvalent) were able to neutralize the three venoms at different effective doses, hence supporting their uses in the three regions. While dose adjustment according to geographical region seems possible, changes to standard recommended dosage should only be made if further study validates that the monocled cobras within a population do not exhibit remarkable inter-individual venom variation. [ABSTRACT FROM AUTHOR]