Final Report Summary - BITES (Biodiversity Informatics and Technology Exchange for Snakebite management)
BITES: Biodiversity Informatics and Technology Exchange for Snakebite Management
bites.bangor.ac.uk
Contact: a.malhotra@bangor.ac.uk
Background: 45,900 deaths from snakebite are estimated to occur in India every year (Mohapatra et al. 2011, PLoS Negl Trop Dis 5(4):e1018) and it is a major occupational hazard for farmers and plantation workers (Alirol et al. 2010. PLoS Negl Trop Dis 4(1):e603). Life-saving antivenom is available to treat bite by the so-called “Big Four” species (comprising two elapid and two viper species), traditionally thought to cause the highest numbers of envenoming in India, but several additional species are known to contribute. In this project, we aimed to provide a comprehensive understanding of the diversity and distribution of venomous snakes and their toxins on which to build the rational design and distribution of antivenoms (Gutiérrez et al. 2006. PLoS Med 3(6):e150).
Identification of species boundaries and within-species variation in Indian venomous snakes
Blood and/or scale clips were obtained (where permits were available) from 21 venomous species including three species of cobra, four species of krait, one species of coral snake and 13 species of vipers from 12 states covering all major regions in mainland India. Permits were not obtained in a number of cases meaning no collection of samples was possible. While this limits the conclusions that can be drawn, we have obtained evidence that three out of four species against whose venom the polyvalent antivenom is made contain significant population substructure (with the exception of the spectacled cobra). The snakes from the three southern states of Karnataka, Tamil Nadu and Kerala are genetically distinguishable from states further north, including Maharashtra and Madhya Pradesh. However, finer scale mapping is required to distinguish the exact boundary between different populations, and to determine whether these sub-structured populations correspond to sub-specific or specific divisions.
While these genetically sub-structured populations may partly explain venom variation, it is unlikely to be the whole story since considerable venom variation is present even between populations within these broad regions (Sharma et al. 2014,Toxin Rev. 33:1-2), and the relatively crude level of genetic mapping is not sufficient to explain these smaller-scale variations. Unpublished data from independent research also suggests that the more southerly, relatively isolated, mountain ranges in Tamil Nadu and Kerala are centres of as yet unrecognised diversity, but unless collecting permits are made available for these states, this cannot be investigated further.
Other highlights from this part of the project include the first DNA and venom samples from the Indian population of the Central Asian Cobra Naja oxiana, and the first DNA and venom samples from any population of the Himalayan pitviper Gloydius himalayanus, both from Himachal Pradesh in north India. Preliminary analysis of the latter cast doubt on its current assignation to the genus Gloydius and suggest more than one species is present in India. The hitherto enigmatic pitviper species Cryptelytrops erythrurus was sampled in Mizoram on India’s far eastern border with Myanmar, were it is extremely common. Similarly, Cryptelytrops sepentrionalis was sampled in Himachal Pradesh, where in some regions it is responsible for over 70% of venomous snake bites. The data collected on these species will help to resolve species boundaries in the Cryptrelytrops genus (manuscript in preparation).
Our preliminary data also shows that the banded krait Bungarus fasciatus and the monocled cobra Naja kaouthia in India are distinct from the populations assigned to these taxa from further east. In both cases, the type specimens are from Bengal and so the names stay with the Indian populations, and will require new taxa to be described for the other populations. This also invalidates the suggestion that Thai Red Cross anti-venom may be suitable for use against these species in north-eastern India. There are also strong indications that the populations of the monocled cobra from the far northeast of India (Arunachal Pradesh and Mizoram) are specifically distinct from those further west (West Bengal, Bihar etc.). Thus, the project has yielded data that provide further rationale for adopting a region-specific approach to antivenom manufacture in India.
Investigation of the potential of trace DNA analysis from bite-site swabs for the diagnosis of snake species responsible to improve the epidemiological knowledge of snake bite incidence
Establishment of a DNA database is a necessary first step before this can be implemented. In addition, independent studies in neighbouring countries (http://slideplayer.com/slide/4439069/) and in India (unpublished abstract) have found a low rate of DNA recovery from swabs. We therefore are investigating an alternative approach which holds considerable promise for both epidemiological and bed-side diagnostic studies, nucleotide-based aptamers, in collaboration with researchers from the Centre for Bio-design, Translational Health Science and Technology Institute.
Application of proteomic and antivenomic tools to elucidate venom composition and variability, and establishing neutralizing ability of existing antivenoms from different manufacturers.
The delay in obtaining the required samples because of permit issues, and the limited funding generated for analyses from the exchange program, prevented a full proteomic study from being completed. We focussed our efforts on describing geographic variation in venom in cobras (Naja naja, Naja kaouthia and Naja oxiana), and testing the efficacy of toxin neutralization by antivenoms from two different manufacturers, Premium Serums and Vaccines Pvt. Ltd (PSVPL) and VINS Bioproducts Ltd. (VINS). PSVPL proved to have superior binding properties and good cross-specificity for the venom of the two other species of cobras compared to VINS, which is significant as VINS is far more readily available. This is similar to other studies which have recently compared products for the African market from the two companies. This will be extended to other species and manufacturer’s products as funds become available.
Using next-generation transcriptomics to validate non-invasive quantitative PCR amplification of stable mRNA in expressed venom for profiling venom variation
Collection permits for non-lethal sampling from snakes were difficult to obtain, and requesting harvesting of venom glands (a lethal procedure) would likely be refused: hence, we proceeded with the back-up option of using specimens obtainable outside India for transcriptomic studies. A specimen of Trimeresurus puniceus from Indonesia was euthanised in accordance with Home Office guidelines four days after milking (to stimulate the transcription process) and tissues were immediately harvested and stored in RNAlater® at -20ͦC. Total RNA was extracted, mRNA was purified and a cDNA library was constructed for subsequent Illumina sequencing. The resulting data is currently being analysed and prepared for publication by one of the exchange participants from Indian Institute of Sciences. However, his exchange period was too short to allow the further stages of this objective to be tackled (i.e. validation of PCR-profiling of mRNA directly from the venom).
Provision of training events and workshops, and establishing additional collaboration.
Two workshops were held: a) 5-day workshop on Snakebite Management at Mizoram University, Aizawl (27th June to 3rd July 2016), Aizawl, with around 30 participants, and b) 3-day workshop in December 2017, Centre for Cellular and Molecular Biology, Hyderabad on Snakebite Mitigation, with over 60 participants, including several early career researchers. We also trained PhD and Masters students in Mizoram in field techniques, including safe handling techniques and sampling protocols for venomous snakes. We also presented our work as seminars in December 2013 at the Toxinological Society of India’s (TSI) annual conference in Goa, in June 2015 at Shri Shiv Chhatrapati College, Junnar, Maharashtra and at Tezpur University, Assam, in June 2016 at Centre for Cellular and Molecular Biology (CCMB), Hyderabad, and in December 2017 at the TSI conference in Hyderabad and as part of a Continuing Medical Education course on snakebite in Shimla, Himachal Pradesh. MOUs were signed between Bangor University and Mizoram University and Sikkim University. Seven grant applications for further funding were written during the project (two were funded, two were rejected and the outcome of three others are still not known) with partners at Mizoram University, CCMB Hyderabad, Premium Serums and Vaccines Pvt. Ltd., and several NGOs involved in snakebite mitigation and snake conservation.
bites.bangor.ac.uk
Contact: a.malhotra@bangor.ac.uk
Background: 45,900 deaths from snakebite are estimated to occur in India every year (Mohapatra et al. 2011, PLoS Negl Trop Dis 5(4):e1018) and it is a major occupational hazard for farmers and plantation workers (Alirol et al. 2010. PLoS Negl Trop Dis 4(1):e603). Life-saving antivenom is available to treat bite by the so-called “Big Four” species (comprising two elapid and two viper species), traditionally thought to cause the highest numbers of envenoming in India, but several additional species are known to contribute. In this project, we aimed to provide a comprehensive understanding of the diversity and distribution of venomous snakes and their toxins on which to build the rational design and distribution of antivenoms (Gutiérrez et al. 2006. PLoS Med 3(6):e150).
Identification of species boundaries and within-species variation in Indian venomous snakes
Blood and/or scale clips were obtained (where permits were available) from 21 venomous species including three species of cobra, four species of krait, one species of coral snake and 13 species of vipers from 12 states covering all major regions in mainland India. Permits were not obtained in a number of cases meaning no collection of samples was possible. While this limits the conclusions that can be drawn, we have obtained evidence that three out of four species against whose venom the polyvalent antivenom is made contain significant population substructure (with the exception of the spectacled cobra). The snakes from the three southern states of Karnataka, Tamil Nadu and Kerala are genetically distinguishable from states further north, including Maharashtra and Madhya Pradesh. However, finer scale mapping is required to distinguish the exact boundary between different populations, and to determine whether these sub-structured populations correspond to sub-specific or specific divisions.
While these genetically sub-structured populations may partly explain venom variation, it is unlikely to be the whole story since considerable venom variation is present even between populations within these broad regions (Sharma et al. 2014,Toxin Rev. 33:1-2), and the relatively crude level of genetic mapping is not sufficient to explain these smaller-scale variations. Unpublished data from independent research also suggests that the more southerly, relatively isolated, mountain ranges in Tamil Nadu and Kerala are centres of as yet unrecognised diversity, but unless collecting permits are made available for these states, this cannot be investigated further.
Other highlights from this part of the project include the first DNA and venom samples from the Indian population of the Central Asian Cobra Naja oxiana, and the first DNA and venom samples from any population of the Himalayan pitviper Gloydius himalayanus, both from Himachal Pradesh in north India. Preliminary analysis of the latter cast doubt on its current assignation to the genus Gloydius and suggest more than one species is present in India. The hitherto enigmatic pitviper species Cryptelytrops erythrurus was sampled in Mizoram on India’s far eastern border with Myanmar, were it is extremely common. Similarly, Cryptelytrops sepentrionalis was sampled in Himachal Pradesh, where in some regions it is responsible for over 70% of venomous snake bites. The data collected on these species will help to resolve species boundaries in the Cryptrelytrops genus (manuscript in preparation).
Our preliminary data also shows that the banded krait Bungarus fasciatus and the monocled cobra Naja kaouthia in India are distinct from the populations assigned to these taxa from further east. In both cases, the type specimens are from Bengal and so the names stay with the Indian populations, and will require new taxa to be described for the other populations. This also invalidates the suggestion that Thai Red Cross anti-venom may be suitable for use against these species in north-eastern India. There are also strong indications that the populations of the monocled cobra from the far northeast of India (Arunachal Pradesh and Mizoram) are specifically distinct from those further west (West Bengal, Bihar etc.). Thus, the project has yielded data that provide further rationale for adopting a region-specific approach to antivenom manufacture in India.
Investigation of the potential of trace DNA analysis from bite-site swabs for the diagnosis of snake species responsible to improve the epidemiological knowledge of snake bite incidence
Establishment of a DNA database is a necessary first step before this can be implemented. In addition, independent studies in neighbouring countries (http://slideplayer.com/slide/4439069/) and in India (unpublished abstract) have found a low rate of DNA recovery from swabs. We therefore are investigating an alternative approach which holds considerable promise for both epidemiological and bed-side diagnostic studies, nucleotide-based aptamers, in collaboration with researchers from the Centre for Bio-design, Translational Health Science and Technology Institute.
Application of proteomic and antivenomic tools to elucidate venom composition and variability, and establishing neutralizing ability of existing antivenoms from different manufacturers.
The delay in obtaining the required samples because of permit issues, and the limited funding generated for analyses from the exchange program, prevented a full proteomic study from being completed. We focussed our efforts on describing geographic variation in venom in cobras (Naja naja, Naja kaouthia and Naja oxiana), and testing the efficacy of toxin neutralization by antivenoms from two different manufacturers, Premium Serums and Vaccines Pvt. Ltd (PSVPL) and VINS Bioproducts Ltd. (VINS). PSVPL proved to have superior binding properties and good cross-specificity for the venom of the two other species of cobras compared to VINS, which is significant as VINS is far more readily available. This is similar to other studies which have recently compared products for the African market from the two companies. This will be extended to other species and manufacturer’s products as funds become available.
Using next-generation transcriptomics to validate non-invasive quantitative PCR amplification of stable mRNA in expressed venom for profiling venom variation
Collection permits for non-lethal sampling from snakes were difficult to obtain, and requesting harvesting of venom glands (a lethal procedure) would likely be refused: hence, we proceeded with the back-up option of using specimens obtainable outside India for transcriptomic studies. A specimen of Trimeresurus puniceus from Indonesia was euthanised in accordance with Home Office guidelines four days after milking (to stimulate the transcription process) and tissues were immediately harvested and stored in RNAlater® at -20ͦC. Total RNA was extracted, mRNA was purified and a cDNA library was constructed for subsequent Illumina sequencing. The resulting data is currently being analysed and prepared for publication by one of the exchange participants from Indian Institute of Sciences. However, his exchange period was too short to allow the further stages of this objective to be tackled (i.e. validation of PCR-profiling of mRNA directly from the venom).
Provision of training events and workshops, and establishing additional collaboration.
Two workshops were held: a) 5-day workshop on Snakebite Management at Mizoram University, Aizawl (27th June to 3rd July 2016), Aizawl, with around 30 participants, and b) 3-day workshop in December 2017, Centre for Cellular and Molecular Biology, Hyderabad on Snakebite Mitigation, with over 60 participants, including several early career researchers. We also trained PhD and Masters students in Mizoram in field techniques, including safe handling techniques and sampling protocols for venomous snakes. We also presented our work as seminars in December 2013 at the Toxinological Society of India’s (TSI) annual conference in Goa, in June 2015 at Shri Shiv Chhatrapati College, Junnar, Maharashtra and at Tezpur University, Assam, in June 2016 at Centre for Cellular and Molecular Biology (CCMB), Hyderabad, and in December 2017 at the TSI conference in Hyderabad and as part of a Continuing Medical Education course on snakebite in Shimla, Himachal Pradesh. MOUs were signed between Bangor University and Mizoram University and Sikkim University. Seven grant applications for further funding were written during the project (two were funded, two were rejected and the outcome of three others are still not known) with partners at Mizoram University, CCMB Hyderabad, Premium Serums and Vaccines Pvt. Ltd., and several NGOs involved in snakebite mitigation and snake conservation.