SNAKE VENOM PRODUCTION FOR ANTIVENOM IN INDIA by-DR. RAJESH KUMAR SINGH, (LIVESTOCK & POULTRY CONSULTANT), JAMSHEDPUR, JHARKHAND,INDIA
9431309542, rajeshsinghvet@gmail.com
India accounts for about half of all global snakebite deaths. The difficulty of treating snakebites starts with identifying the biting reptile. This is vital, since venom composition differs vastly between snake species. India has around 60 different species of poisonous snakes but most fatalities are caused by the ‘Big Four’ — the spectacled cobra (Naja naja), the common krait (Bungarus caerulus), the saw-scaled viper (Echis carinatus) and Russel’s viper (Daboia russelii). With some 50,000 citizens dying annually from snakebites, India is compelled to explore a range of effective methods to deal with the problem, which the WHO characterises as a neglected tropical disease.
Snakebite is an occupational hazard causing considerable morbidity and mortality worldwide, particularly so in tropical countries like India. An estimated 50,000 Indians die due to venomous snakebite every year, seventy percent of whom are males between the ages of 20 to 50 years. Along with the associated morbidity and mortality, snakebite leads to a significant financial burden on the victim, both by way of hospital bills and labour hours lost. Snakebite is also a cause for considerable psychological stress among survivors. Most snakebites are eminently treatable and curable. Given a concerted thrust from all concerned , this menace could surely be curtailed considerably over the next few years
Snakebite is a medically and socially significant issue in India, but the quality of treatment and reporting protocols need to be upgraded to international standards. There are currently seven pharmaceutical laboratories in India which produce antivenom against four medically important Indian snake species (cobra (Naja sp.), krait (Bungarus sp.), Russell’s viper (Daboia russelii) and saw-scaled viper (Echis carinatus sp.), the ‘big four’. Most venom for antivenom production is sourced from Chennai, South India. While the ‘big four’ are responsible for a majority of serious and fatal bites, the situation is actually much more complex.
The Anti-Venom————-
Parental administration of antivenom; the only specific antidote to snake venom is the cornerstone in management of snake bites . The antivenom for snake envenoming was introduced by Albert Calmette in 1895 and was quickly accepted without formal clinical trials. More than a century later, antivenoms are considered as essential drugs.
Antivenoms are derived from immunoglobulins, obtained and purified from the plasma of animals immunised with snake venoms. The toxins present in venoms, which are responsible for manifestations of envenoming are neutralized by the antivenom immunoglobulins .
An accurate selection of snake venoms is critical for the production of antivenoms that have capacity to cover the majority of cases of envenoming in a given geographical region, territory or country. As the composition of snake venoms is very complex and a high inter-species and intra-species variation has been documented, production of antigenic mixture to be used for antivenom production is a challenging task for manufacturers . The selection of most suitable snake venom for production of antivenom is based on the geographical area of interest, locally prevalent species of snakes and variability in the composition of snake venoms within the desired territory.
Monospecific vs. polyspecific antivenoms
There are two types of antivenom available namely, monospecific and polyspecific.
Monospecific antivenoms
These antivenoms are intended for use in envenoming due to a single species of snake or a few closely related species whose venoms show clinically effective cross-neutralization . It is practically possible only when there is a very high prevalence of a single species of snakes in the desired region, but most of the countries are inhabited by more than one medically relevant species of snakes, where use of polyspecific antivenoms is highly recommended.
Polyspecific antivenoms
The polyspecific antivenoms are produced by immunizing an animal with venoms of more than one species of snakes of high medical relevance to the concerned geographic area. Another methods of production includes i) immunizing individual animals with venom of a single species and then mixing the various hyper immune plasmas for fractionation and ii) mixing appropriate quantities of relevant purified antivenoms before formulation .
These polyspecific antivenoms should be promoted whenever feasible technically, as they offer clinical advantages like better usefulness than monospecific antivenoms. Their use reduces the need for identification of snakes prior to initiation of antivenom therapy and simplicity in logistics provides great advantages
Snake venom is a highly developed form of saliva, injected by the snake into its victim through hollow, modified fangs. Wear and tear are heavy on the fangs, which are soon blunted or wrenched out in the struggles of prey animals (or when being milked). But fresh fangs are always held in reserve; each poised to move into position when required.
The base of a functioning fang, and often the first reserve fang behind it as well , is penetrated by a duct that leads from a large gland behind the eye. These glands- one on either side of the head – are modified salivary glands surrounded by muscle which, when contracted, forces the clear or yellowish venom along the venom ducts and down through the fangs, squirting out under pressure as if from a pair of hypodermic syringes. Venom may be injected with each of a possible series of consecutive bites. Interestingly however, venom is not always injected.
Focused judgement and great dexterity are needed to obtain snake venom from the dangerous species of snakes found in India. Keepers position the snake’s fangs to penetrate a latex membrane stretched over a glass beaker. The beaker collects the venom, which is desiccated under vacuum or freeze-dried.
After drying, the venom crystals are carefully scraped from the beakers for weighing and packaging. Trained staff, who work with the venom in its various stages of processing, work extremely carefully with the venom to ensure it is not contaminated.
An inventory of dried venoms from a wide assortment of native and non-native snake species is maintained at all times. The venom of each species is unique, consisting of a combination of complex proteins, which act on the prey or bite victim in various ways.
Other components present in the venoms of certain species act to destroy blood cells, to cause blood clots or excessive bleeding, or to destroy tissue. Typical early symptoms of bites, where significant envenomation has occurred, include severe headache, nausea, vomiting, confusion, temporary loss of consciousness, fast pulse and tender lymph nodes. Later signs of envenomation may include drooping eyelids, voice change, double vision, difficulty in swallowing and intense abdominal pain, which may be followed by the vomiting of blood.
The horses are given increasing doses of venom over a period of time until they have built up sufficient antibodies to the venom. After this has occurred, antibodies are extracted from the blood, purified and reduced to a usable form – this becomes anti-venom.
The anti-venom taken from the horses are used to treat humans suffering from snake envenomation. Injected into the human bloodstream, the antibodies attack the venom, neutralising its effects. The dose of anti-venom given to a patient varies according to the species responsible for the bite and, when it can be ascertained, the amount of venom injected. The age and weight of the victim makes no difference to the dose of anti-venom required in the treatment.
How antivenom is made————-
To make life-saving antivenoms, scientists enlist the help of horses that live on specialized ranches. The scientists inject the animals with a tiny, harmless dose of venom, which causes their immune systems to produce antibodies—proteins that attack and disable the venom toxins. Then the scientists can collect the antibodies and use them to treat people who have been bitten or stung
1 A technician extracts and later purifies venom from the species for which scientists want to make an antivenom.
2.A ranch hand injects a small, harmless dose of venom into a horse. The toxins in the venom trigger the horse’s immune system to produce antibodies that neutralize those particular toxins.
3.Over the next year, the horse receives several booster shots with increasing amounts of venom. Eventually, the horse produces so many antibodies that it’s immune to the venom
4 A ranch hand draws blood from the horse. A machine extracts the plasma, the part of the blood that contains the antibodies. The rest of the blood is returned to the horse.
5 The plasma is sent to a lab, where chemists purify it and package it as a liquid or freezedried powder. It is then shipped in vials to hospital pharmacies
6.When a patient comes in with a bite or sting, doctors use an IV line to inject the antivenom into the patient’s veins. The antibodies circulate through the body and neutralize the toxin molecules.
Snake venom production for antivenom in India was done solely by the Haffkine Institute in Mumbai prior to the establishment of ISCICS outside Chennai12. There are currently seven laboratories producing antivenom in India with a total production capacity estimated at two million, 10 ml vials. Based solely on venom sales by the Irula Cooperative, requirements to fulfil this capacity are approximately 1,330 g each of N. naja and D. russelii and 133 g each of B. caeruleus and E. carinatus. However, these figures are subject to confirmation and requirements based on data from antivenom producers are significantly different. One data set indicates a requirement of about 2,260 g of N. naja, 1,508 g of D. russelii and 300 g each of E. carinatus and B. caeruleus. The current potency of Indian antivenoms is 0.60 mg/ ml for cobra, while prior to the 1950s, it was 4 mg/ml. In Russell’s viper venom, it was 2 mg/ml and is now a mere 0.45 mg/ml. When and why was this changed by the government antivenom potency regulators? This issue of antivenom potency needs urgent attention. Since the start of antivenom production in India over 100 years ago, conventional wisdom was that the ‘big four’ are responsible for the majority of serious bites. While this is still true at the generic level, current taxonomy now recognizes four species of cobras, eight species of kraits, one species of Russell’s viper and two subspecies of saw-scaled vipers. Also, considerable regional variation has been found in Russell’s viper venom which requires further study. There are growing indications from clinicians that antivenom produced from venoms of the ‘big four’, mainly sourced from Irula Cooperative, may not effectively neutralize envenomation by the ‘big four’ and related species in other parts of the country. Whether this is due to venom variation, bites by other species, low antivenom potency or a combination of these factors, needs to be determined. In addition, several of the 22 species of pit vipers in India43, a number of sea snakes and species such as the king cobra are capable of causing human and livestock disability and death. Though serious bites from most of these species are thought to be rare, snakebites which occur in more remote areas are often not reported. When bites occur from species other than the four used in India’s polyvalent antivenom production, clinicians are apt to use the available antivenom, even though there is a great likelihood that it is ineffectual. For example, a recent case of pit viper bite in the Himalayas was treated at a military hospital using 30 ampoules of polyvalent serum, which has no neutralizing effect on pit viper bites. Snakebite is responsible for tens of thousands of deaths and disabilities every year in India.
Reasons for such a high incidence of snakebite and resultant mortality in India include the following:——–
• High numbers of snake species of medical importance in agricultural areas. • Inadequate distribution/availability/publicity of antivenom serum (AVS). • Reliance on traditional and quack treatments. • Walking at night without light, no adequate footwear, sleeping on ground mats. • Lack of widely disseminated, standardized first aid and treatment protocols. • Geographic variation in venom composition. • Lack of knowledge about snake habits and behaviour. • Inadequate training of clinicians in dealing with snakebite
Socio demographic factors • Ever increasing population leading to greater encroachment thereby increasing the chances of human reptile contact and bites • inadequate infrastructure in villages, including lighting, sewerage systems, roads, in house water supply etc all of which co-contribute to bites specially at night • improper sanitation which i n t u r n i n c r e a s e s t h e r a t p o p u l a t i o n a n d t h e r e b y increases the likelihood of snake presence • poor transport facilities in the rural hinterland leading to an enormous and at times fatal delay in the shift of patients to a secondary or tertiary care centre Socio-Cultural • the fact that most village dwellers do not use protective foot wear (70% of bites are on the lower limbs) • habit of sleeping on the floor/ ground • presence of livestock near the house which in turn attracts rats • defecating in open fields, often after dark • i n c r e a s i n g a l c o h o l i s m , a significant number of male v i c t i m s b e i n g u n d e r t h e influence of alcohol when bitten Medical • l a c k o f a wa r e n e s s a m o n g v i c t i m s o f t h e i m m e d i a t e measures to be followed when bitten • Alternate forms of treatment practised in villages wherein the victim is first taken to a faith healer (quack), precious time being lost therein • Improper first aid measures immediately after the bite which increases the chances of systemic envenomation and additional complications • unavailability of the standard treatment, anti-snake venom (ASV) in rural centres, • r e l u c t a n c e o n t h e p a r t o f t h e p r i m a r y c a r e t a k e r a t the village centre to admit and treat snakebites fearing complications and reactions to ASV Legislative / Governmental • High cost of horse serum based ASV • absence of a centralized quality c o n t r o l o n t h e p r o c e s s o f manufacture of ASV as also its standardization • absence of regional or zonal pools of snake venom. The venom used for the manufacture of ASV for the entire country is from one or two sources limited to a small geographical area. Venom researchers have shown regional variation in venom constituents and chemical properties, intra-species, which is why it is mandatory that regional/zonal venom centres come up with the facilities to manufacture ASV for a particular region or zone • delay in initiation of ASV due to non-availability of kits for the early diagnosis of venomous snakebite • absence of a national protocol for the diagnosis and effective t r e a t m e n t o f v e n o m o u s snakebite • step motherly treatment meted out to the subject of snake bite in the medical curriculum as also in the governmental health policies
Venom production ——–
In India, all snakes are protected under the Wildlife Protection Act and as such, snakes cannot be collected or venom extracted without the permission of the state wildlife authorities11. There is no scientific study that adequately quantifies snake abundance (though the export of up to 10 million snake skins per year in the 1960s gives some indication), which has resulted in a conservative stance by the wildlife authorities in some states and a general reluctance to permit capture of large numbers of snakes for venom extraction to produce AVS.
Two antivenom producers have recently stopped production. • Serum Institute of India, Pune – Polyvalent for ‘big four’, lyophilized, average annual production >100,000 vials. Also lyophilized polyvalent for two species of African snakes combined (for reasons unknown) with Indian Daboia and Echis. Production of antivenom was stopped in 2008, reportedly in view of the stringent conditions which were implemented by the Committee for the Purpose of Control and Supervision of Experiments on Animals (India) CPCSEA. Venom source (India): Irula Cooperative. • Central Research Institute, Kasauli (Government of India) – Polyvalent for ‘big four’, lyophilized, average annual production was 25,000 vials. Production discontinued on 2007. Venom source: Irula Cooperative.
All Indian antivenom labs produce polyvalent serum of equine origin against the four most common and widely distributed medically important Indian snake species, referred to for brevity as the ‘big four’. It has been observed that 2010 prices for a 10 ml vial of Indian polyvalent AVS range from about INR 300 to 500 (US$ 6.50– 11.00), which is a fraction of the cost of a vial of CroFab antivenom in the USA (at over US$ 1900 per vial) or CSL antivenom in Australia (at US$ 1500 per vial)
There are several other producers of snake venom in India, but the status of their legality is questionable and some reportedly supply liquid or ‘light-bulb dried’ venom to antivenom producers. The WHO protocol for venom standards and production for the manufacture of antivenom is unfortunately, not yet implemented in India. In 2009, the Maharashtra State Forest Department announced plans, via a press release, to set up a Snake Venom Research and Extraction Centre in Nashik, utilizing the snakes caught by ‘snake rescuers’, often attached to local animal welfare bodies16. The current status of this initiative is unknown, but considering the popularity of snake rescue in many parts of India, this is an obvious potential source of snakes for venom production.
Venom and antivenom requirements for India———
It will be advantageous to ascertain exactly how much venom is required to produce an adequate quantity of antivenom for India in order that venom supply permits and protocols can be worked out. Based on a production breakdown provided by an antivenom producer (though subject to considerable variability depending on the immunization procedures used and other factors), production of 10,000 vials of antivenom requires approximately 2 g each of N. naja and D. russelii venom and 0.2 g each
of Bungarus caeruleus and Echis carinatus venom21. Production of 2,000,000 vials (estimated output for 2011/2012 based on responses from antivenom producers) would therefore require an annual production of at least 400 g each of N. naja and D. russelii venom and 40 g each of B. caeruleus and E. carinatus venom (see the next section). Using these estimates, it is inferred that the Irula Cooperative supplies only about half of India’s N. naja and D. russelii venom requirements, but almost all of its B. caeruleus and E. carinatus venom requirements
In a somewhat complex ‘snakes-of-medical-importance’ scenario, there is an urgent need to address the following issues to improve the situation.—————-
• Venom/antivenom research to establish venom toxicity, antivenom potency, minimum effective dose of antivenoms, cross-reactivity of antivenom among species and the important area of geographic variation of venoms. • Venom production in sufficient quantities and to supply the demand, under WHO protocol to produce a high standard of venom with immediate attention to proven and likely geographic variations. • To achieve the previous point it is suggested that India’s largest venom producer, the ISCICS be reconstituted as a multi-state cooperative under the central government so that snake venom for the production of antivenom can be collected from as wide a geographic area as possible in recognition of the fact that there is considerable regional variation in the composition of venoms and that there are species other than the ‘big four’ responsible for serious bites. • Field studies on the distribution and abundance of the medically important snakes to guide antivenom manufacture (regionally specific, monovalent, bivalent, polyvalent) and effects on local populations, if any, on capture of large numbers for antivenom production. • Designing a protocol acceptable to wildlife authorities for the capture of sufficient numbers of snakes for India’s antivenom needs, safety standards for venom extraction and humane treatment of captured snakes. • Education and awareness campaign to publicize use and effectiveness of antivenom as opposed to local, quack remedies. • Enforcement of stringent laws against bogus snakebite ‘treatments’ and appropriate public awareness against these practices. • Inducing state and central government health agencies to ensure wider availability of antivenom on a subsidized/free distribution basis for the rural poor via primary health centres and other rural health facilities. • Antivenom production to supply India’s needs under WHO protocol including additional species if venom research and clinical data proves their medical significance. Producing a pan-Asian antivenom – a high potency antivenom designed for several countries across the South Asian region, produced in large volumes and dispensed in single dose vials is a worthy goal. • Training of clinicians in correct treatment and management of serious snakebites.
To effectively implement these strategies, it has been suggested to the Ministry of Health, Government of India that it convenes a series of regional and central meetings of the key stakeholders including the following • Venom producers • Venom researchers • Antivenom producers • Clinicians with snakebite experience • State and central health authorities • Environment/wildlife authorities • WHO experts • Herpetologists with local experience.
Mitigation of Snakebite in South Asia — Some Suggestions for a Way Forward———
1. I m p r o v e m e n t o f v e n o m production protocol to the WHO standard by the Irula Snake-catchers Co-operative, I n d i a ’ s p r i m a r y v e n o m p r o d u c e r f o r a n t i – v e n o m production. 2. Production of venom from different geographic regions in India to solve the problem of geographic variation by amalgamating the Irula Cooperative into a Multi-State Co-operative. 3. Improvement of Indian antivenoms by raising the titre to the levels of anti-venom produced in the 1950s. Then the standard (apparently set by Government of India) was 4.0mg/mL for D. russelii venom and 2.0mg/ mL for N.naja venom. Today the standard is a fraction of what it was: 0.6mg/mL for D.russelii and N.naja. What this means is that much more antivenom needs to be bought and used by a patient. Complaints of allergic reactions to Indian anti-venoms also need critical attention. 4. Improvement of Indian antivenoms by using methods developed at the Instituto Clodomiro Picado in Costa R i c a u s i n g c a p r y l i c a c i d fractionation of plasma. 5. Standardizing and translating into local languages an accepted p r o t o c o l f o r S N A K E B I T E FIRST-AID AND TREATMENT with attention and discussion given to the more controversial and much debated subjects such as anti-venom dosages, use of neostigmine and a long list of other things regularly argued about by clinicians and others. 6. Guaranteed stocking of antivenom where it is needed, at Primary Health Centres in areas with high incidence of snakebite, with adequate training in its administration. 7. A n d p e r h a p s o n e o f t h e m o s t i m p o r t a n t m e a s u r e s to be taken: Development of well-publicized, all-India s n a k e b i t e h o t l i n e s , w i t h appropriate regional/language representations by doctors willing to receive calls at all hours to give instant advice on snakebite. 8. Bringing ASV into the list of essential medicines in states thereby helping reduce the cost. The Government must provide anti-venom free of charge to rural India 9. Development of a national protocol for the treatment of venomous snakebite 10. Make snakebite a notifiable d i s e a s e a n d c a r r y o u t e p i d e m i o l o g i c a l s t u d i e s in states where venomous s n a k e b i t e i s a s i g n i f i c a n t problem so as to be able to arrive at an accurate mortality and morbidity figure. 11. Venom to be made available to research laboratories to foster snake venom and genetic research on snake species 12. To analyse the LD50 of regional variants of the Big 4, namely Naja kouthia, Echis sochureki etc, study their toxicity profile and degree of neutralisation (ED50) by the standard ASV now available 13. T o i n c l u d e t r e a t m e n t o f venomous snakebite in the subject of Internal Medicine in the medical curriculum, and encourage research and conferences on this much neglected subject.
Snakebite in India has remained a much neglected subject in spite of the significant mortality and morbidity it causes. If diagnosis and treatment be given in a timely fashion the victim can go back to a productive life. Given the fact that most (70%) of bitten are males and the bread winners of the family adds an extra dimension to this problem. Snakebite was and still remains a problem that can easily be tackled given a sustained effort towards that goal involving all concerned, namely herpetologists, clinicians, basic scientists, industry and most importantly, the government. A number of state governments pay Rs.100,000 to the family in case of death due to snakebite. If 50,000 families of victims were to claim the same, it would drain the exchequer of Rs 500 crores annually. We believe that a small fraction of the above mentioned amount would be enough to mitigate the problem in India, to a large extent, once and for all. We are sure that with the advances that India has made on all fronts here too we can be successful in bringing down the annual death rate to a fraction of what it is in the coming years
