A comparative study of venomics of Naja naja from India and Sri Lanka, clinical manifestations and antivenomics of an Indian polyspecific antivenom
Graphical abstract
Introduction
Snakebite envenoming is an important public health problem in tropical developing world. There were about 421,000 to 1,841,000 snake envenomings each year, mostly in Asia, Africa and Latin America, with estimated fatalities of about 20,000 to 94,000 [1]. Although the high incidence of this health problem, it did not receive the proper attention until recently when WHO recognized snake envenoming as a neglected tropical disease [2].
Naja naja (Indian cobra) is found in some South Asian countries including Sri Lanka, India and Pakistan. It is responsible for high percentage of morbidity and mortality of snake envenomings in South Asia and is listed in WHO Category 1 medically important venomous snake in these countries.
N. naja is a member of the Elapidae family which is known to produce low molecular weight toxic proteins of the three finger toxin (3FT) family [3]. The highly lethal toxins of the elapids are postsynaptic neurotoxins which bind specifically and quasi-irreversibly with the nicotinic acethylcholine receptor at the muscle endplate [4]. This binding results in neuromuscular inhibition, muscle paralysis and respiratory failure [5]. The other subtype of the 3FT is the cytotoxins (CTX), also known as cardiotoxins, affects various cell types by their cytolytic action resulting in local tissue necrosis, cardiotoxicity, etc. [6], [7]. Furthermore CTX is known to act synergistically with phospholipase A2 (PLA2) in disrupting cell membrane [8], [9], [10]. Envenomation by N. naja usually results in local tissue necrosis which could lead to crippling disability and less often, in some neurotoxic signs and symptoms [11], [12], [13]. Biochemical study has shown the presence of CTX, PLA2 and other enzymes but not postsynaptic neurotoxins which are likely to be in the venom [14].
Intra-specific variations in venom compositions, both qualitative and quantitative, have been widely observed. Two populations of Bothrops asper in Costa Rica were shown to have different enzymatic and toxic activity profiles in their venoms [15]. Yang et al. [16] reported that the venoms of Notechis scutatus from various regions of Australia varied greatly in the quantities of notexin and notechis II-5. Calvete et al. [17] showed a difference in relative percentages of toxins and enzymes (PIII SVMP, PISVMP, PLA2, serine proteases, etc.) of Bothrops atrox from various regions of the Amazon. The venoms of Naja kaouthia from Thailand, Malaysia and Vietnam have been shown to exhibit varying toxin subtypes, pathological manifestation (cytolytic activity), LD50 and ED50 of a polyspecific antivenom (AV) [18]. Even within India, the venoms of N. naja from Eastern, Western, and Southern regions showed differences in PLA2, proteolytic, hemolytic, and hyaluronidase activities and lethal potency [19]. Thus, intra-specific variation in venom composition is a general feature of highly adaptable and widely distributed species [20] to suit their roles in predation and diet of the habitat [21]. The variations described were in snakes living in the same continuous land area. However, Sri Lanka is an island separated from the Indian subcontinent by the Gulf of Mannar and Palk Strait, thus it is expected that significant intra-specific variation in venom composition would exist.
The variation in venom composition is likely to bring about differing clinical manifestations. Thus, Mukherjee and Maity [22] showed that N. naja from 3 districts of West Bengal in India gave different clinical symptoms and pathological severities. Different systemic pathological effects on various vital organs of patients from envenomings by N. naja of Eastern, Western, and Southern regions were also noted [23]. Ranawaka et al. [24] reported that even with venoms displaying similar compositions, different clinical manifestations may be encountered. For example, numerous cases of neurotoxicity caused by Russell's viper from Sri Lanka were observed, fewer cases were reported from the same viper of Indian origin.
Various studies have shown that some polyspecific AVs produced in India against local species were occasionally found to be less effective against envenoming by Sri Lankan species [11], [25], [26], [27], [28], [29]. This observations suggested that the antigenic determinants on the corresponding toxins, even from the same species, differed and consequently the AV antibody raised against a toxin from one region failed to neutralize the corresponding ‘heterologous’ toxin. In this regard, Suzuki et al. [14] fractionated the venoms of N. naja found in South India and Sri Lanka and showed differences in amino acid sequences of the cytotoxins and PLA2s.
It is important, therefore, to study the venomics of the Indian N. naja (NNi) and Sri Lankan N. naja (NNsl) and to compare their toxin profiles. The information should be useful to the understanding of the intra-specific variation of these venoms and their clinical manifestations and management. Furthermore, the antivenomics study of the Indian AV should shed light on the cause of the difference in efficacy, and probably the appropriate dosage of the AV therapy. These studies could eventually lead to the production of more effective AV. The present communication reports the results of these studies on the NNi and NNsl venoms.
Section snippets
Snake venoms and antivenom
Pooled and lyophilized N. naja (India) venom, obtained from 17 snakes in Rajasthan and Gujurat of India, was purchased from Latoxan. N. naja (Sri Lanka) venom milked and pooled from several adult snakes was from Dr. CA Ariaratnam. The antivenom, produced by Vins Bioproduct Limited, Hyderabad, India was a kind gift from Professor NH Tan. It was a polyspecific equine lyophilized F(ab′)2 antivenom prepared against Indian cobra (N. naja), common krait (Bungarus caeruleus), Russell's viper (Daboia
Fractionation of the NNi and NNsl venoms
When the venoms of NNi and NNsl were separately subjected to RP-HPLC, the chromatographic profiles shown in Fig. 1 were obtained. NNi and NNsl venoms were separated into about 18 and 17 protein peaks, respectively. Each of the protein peaks was further subjected to 1D-SDS-PAGE. Most of the peaks were separated into more protein bands according to their molecular weights. Intense bands of highly abundant proteins at MW of about 6–7 KDa were found in HPLC peaks #2, 4, 9–12, and 14 of NNi while in
Conclusion
The proteomics data presented here revealed the intra-specific variations, both qualitative and quantitative, of venom protein compositions which should directly affect the immunogenicity and antigenicity of the venom proteins. Antivenomics study of an Indian antivenom has shown that although the antivenom antibodies could bind to all proteins of the Sri Lankan cobra, the binding affinities were significantly lower as compared to that towards the Indian cobra venom proteins. This finding could
Author contributions
Kitisak Sintiprungrat, Kamolwan Watcharatanyatip, W.D.S.T. Senevirathne and Kavi Ratanabanangkoon jointly designed the study; Kitisak Sintiprungrat, Kamolwan Watcharatanyatip, W.D.S.T. Senevirathne, and Papada Chaisuriya conducted the experiments and data analysis; Daranee Chokchaichamnankit and Chantragan Srisomsap identified the venom proteins by MS; Kavi Ratanabanangkoon wrote the manuscript; all authors read and approved the final version of manuscript.
Conflict of interest
The authors declared no conflict of interest.
Transparency document
Acknowledgements
This study was supported by a research grant from Chulabhorn Research Institute and a scholarship (Royal Project on International Academic Cooperation and Exchange Student Scholarship no. 13020112 Sandani to W.D.S.T. Senevirathne) from Chulabhorn Graduate Institute. We are grateful to Dr. Choo Hock Tan, Professor Nget Hong Tan and Dr. James M Dubbs, for valuable suggestions. The authors are grateful to Professor NH Tan for the kind gift of the Vins antivenom.
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These authors contributed equally to the research project.