Toxicology, Agadatantra, Ayurveda

1. ANTISNAKE VENOM
Dr Anu Mariam Varghese
2nd yr MD Scholar
Dept of Agadatantra GAVC, TVM

2. CONTENTS
1. Prevalence of snake bite
2. Venom- its composition
3. Why ASV- Definition and Types
4. History
5. Preparation-collection, hyper immunization
6. Why horses as host animal
7. Lyophilisation
8. Centers of manufacture
9. Composition of ASV

3. 9. Indications
10.Administration and doses
11.Contraindication
12.Antivenom reactions
13.Preventions and treatment of antivenin reactions
14.Limitations
CONTENTS

4. WORLD WIDE PREVALENCE OF SNAKEBITE

5. • An estimated 5.4 million people are bitten each year with up to 2.7 million envenomation.
• Around 81 000 to 138 000 people die each year because of snake bites.
• India is the most heavily affected country in the world (at least 81,000 snake envenomations
and 11,000 fatalities ).
• India has witnessed an estimated 1.2 million deaths due to snakebites between 2001-2020.(a
new study, published in the journal eLife, claims 17-Jul-2020 dated)

6. SNAKE VENOM
Complex cocktails of numerous bioactive proteins
Secreted by special glands near the upper jaw of the snakes which are akin to
human parotid glands.

7. COMPOSTION OF VENOM
ENZYMES
Phospholipase A2
Phosphodiestrase
Phosphomomoestrases
L-amino acid oxidase
PROTEINS
Disintegrin
Ancrod
ORGANIC COMPOUNDS
Citrate
Nucleosides
Acetyl choline
PEPTIDES
Bradykinin- Potentiating peptides(BPP)

8. WHY ASV
• According to the WHO Guidelines, Antivenom is the only specific and scientifically proved
antidote to snake venom. A most important decision in the management of a snakebite victim
is, whether or not to administer antivenom.
DEFINITION
Antivenom is a immunoglobulin (usually pepsin refined F(ab’) fragment of whole
IgG) purified from the plasma of horse, mule or donkey( equine) or sheep( ovine) that has been
immunised with the venoms of one or more species of snake.

9. TYPES
1. Monovalent (monospecific) antivenom – neutralizes the venom of only one
species of snake.
2. Polyvalent (Polyspecific ) antivenom – neutralizes the venom of different
species of snakes, usually the most important species.
• In India only Polyvalent ASV is available using venom of Big 4 as they are
increasingly challenged.
• Monovalent variants are available in some countries like Australia, Thailand..

10. HISTORY
First introduced independently by Phisalix, Bertrand and Calmette in 1894
• They presents the antitoxic properties of the serum of rabbits and guinea –pigs
immunized against cobra and viper venoms respectively.
• First horse derived antivenom sera that he prepared clinically used in 1895 by Haffkine
in India and by Lepiney in VietNam and first successful ASV therapy latter reported in
patients in 1896.

11. PREPARATION
• VENOM COLLECTION (MILKING)
• HYPERIMMUNISATION
• COLLECTION OF SERUM
• LYOPHILIZATION

12. Venom is reconstituted as liquid and a very small quantity is introduced into
horse
Repeated injections given at periodic intervals ,dosage is increased gradually.
Horse’s immune system produces antibodies in its blood
At certain stage, horse’s blood is extracted and the blood serum contains
antibodies is separated and purified
This is Antivenin and final product is marketed in crystal form

13. COLLECTION AND STORAGE OF VENOM
• Venom is milked from the snake by mechanical pressure on the venom gland or
by electrical stimulation of straited muscles surrounding the gland.
• The interval between extractions varies from every 2 or 3 weeks to every 3
months.
• Period of quarantine from 6 to 12 weeks
• Venom have saliva and other impurities
purified by centrifuging and it is preserved
at -10⁰C

14. ADJUVANT
• Some snake venom cause local or systemic toxicity at the beginning of
immunization course.
• To avoid toxicity , inoculation is made with small dose of venom well-emulsified
in adjuvant such as Freund’s complete, bentonite etc..
• Adjuvant may be added to venom to modify the immune response by boosting
it.
• Liberate a higher amount of antibodies and a long lasting protection
• Minimizing the amount of injected foreign material(venom)

15. • 2% solution of Bentonite (an absorbent Aluminum phyllosilicate -
impure clay )
• Used to hold venom in tissue and slowly liberate antigen.
• Decrease acute toxicity of venom.
• Enhance immunogenicity of venom activity.

16. WHY HORSES AS HOST ANIMAL
• Horses are docile, thrive in most climates and yield a large volume of plasma.
• Easy to breed and handle.
• Amount of serum in one bleeding is larger than any of other animal.
• Antivenoms made from horse plasma have proven over time to have a satisfactory safety and
efficacy prolife.

17. HYPERIMMUNIZATION
• Antivenom is prepared by hyper immunizing horses against venom of four common
poisonous snakes- Big four.
1. Cobra
2. Common krait
3. Russel’s viper
4. Saw scaled viper
• Creates an immunological response that produces large numbers of neutralizing
antibodies against various components (toxins) of the venom.

18. BIG FOUR
Cobra
Naja naja
Russel’s Viper
Vipera russelli
Common krait
Bangarus caeruleus
Saw Scaled Viper
Echis carinatus

19. IN HORSES- HYPERIMMUNIZATION
• Areas to be immunized should thoroughly scrubbed with disinfectant, shaved and rubbed with
70 % ethanol.
• Site of immunization – close to major lymph nodes, preferably animal’s neck and back
• Route of injection – Subcutaneous
• Initial dose of each venom -1-4 mg/ horse, with a total combined volume of injection of about 2
ml
• Immunogen filled in a 1 –ml glass syringe with 18G needle.
• Subcutaneous injections of 100-200 Ül at each site, up to as may 8-12 sites.

20. • After 2 weeks booster injection with same venom
• Subsequent booster immunizations at 2 week intervals can made with higher dose 5-10mg
• Blood is drawn before each immunization .

21. COLLECTION OF PLASMA
Animals are bled by venipuncture from external jugular vein.
Area should shaved and disinfected In one bleeding session 13-15ml of
blood per kilogram body weight are collected. Blood is collected , ideally , in
disposable plastic bags containing sterile citrate anticoagulant. Put in
refrigerated room ( 2-8⁰ C) for plasma and blood cells separation procedure i.e validated
centrifugation or sedimentation procedure.

22. LYOPHILISATION (FREEZE DRYING)
• Plasma obtained is concentrated and purified.
• The serum is lyophilised by drying it from frozen state under high vacuum.
• A dehydration process typically used to preserve a perishable material or make the
material more convenient for transport
• In the presence of calcium and phosphoric acid.
Four stages are there
1. Freezing: Freeze-drying works by freezing the material

23. 3. Vacuum processing: reducing the surrounding pressure to allow the frozen water in
the material to sublimate directly from the solid phase to the gas phase.
4. Heating: to aid vaporization
3. Condensation: vaporized serum passed through low temperature condenser plates to
change vapor to powder.
• Antivenom is available in the form of lyophilised powder
• Unstable at room temperature, requires 0-4 degree
• It has the shelf life of 5 yrs.

24. CENTRES OF ASV MANUFACTURING
• Haffkine Biopharmaceuticals Ltd institute, Mumbai
• King institute of Preventive medicine , Chennai
• Serum institute of India, Pune
• Central research institute Kasauli
• Bharat Serums and Vaccines Ltd, Mumbai
• ‘The Irula Snake Catcher’ Industrial Cooperative Society, Vadnemeli, Tamil Nadu
• Vins Bioproduct Ltd , Hyderabad
• Biological ‘E’ Ltd, Hyderabad
• Bengal chemicals & pharmaceuticals , Calcutta

25. HAFFKINE INSTITUTE, MUMBAI KING INSTITUTE, CHENNAI
CENTRES OF ASV MANUFACTURING

26. SERUM INSTITUTE, PUNE VINS BIOPRODUCTS LIMITED, HYDERABAD

27. COMPOSITION
Each ml of ASV neutralizes the following quantities of standard venom tested in
mice by IV route.
 Cobra…………………………………………………...0.6 mg
 Common Krait……………………………………….…0.45mg
 Russell’s Viper………………………..………..………0.6mg
 Saw-scaled Viper.....................................................0.45mg
 Preservative: Cresol I.P NMT………………………....0.25%v/v
 Stabilizer : Gycine I.P, Excipients : Mannitol I.P and Sodium Chloride I.P

28. INDICATIONS
SYSTEMIC ENVENOMING
• Hemostatic abnormalities -Spontaneous systemic bleeding,
coagulopathy.
• Bleeding time > 20 minutes
• Thrombocyte < 10,000/cu mm
• Neurotoxic signs-Ptosis, paralysis, external opthalmoplegia
• CVS abnormalities- Hypotension, shock, cardiac arrhythmia,
abnormal ECG.
• Evidences of intravascular hemolysis.
• Acute kidney injury-Oliguria/Anuria
• Hyperkalemia
• Dark brown urine, Muscle aches etc
LOCAL ENVENOMING
• Local swelling involving more
than half of bitten limb.
• Rapid extension of swelling.
• Enlarged tender lymph node
draining the bitten limb

29. ADMINISTRATION
• The lyophilised powder is diluted in 500ml of distilled water or normal saline and infused over a
period of one hour.
• Skin test is conducted for detection of sensitivity ,A subcutaneous injection of a minute dose of
antivenin 0.1 ml dilute 1:10 )
• ASV is administered intravenously either diluted at the rate of not more than 1 ml per minute or is
diluted in 500ml of IV fluid( either sodium chloride injection or 5% Dextrose Injection) and
administered rapidly as tolerated over 1-2 hours.
• Antiserum, mix by gentle swirling rather than shaking to avoid foaming.

30. SENSITIVITY TEST
• 0.02 - 0.5 ml of 1:10 dilution of serum is injected intradermaly.
• If the reaction is positive
an urticarial wheel of 1cm diameter surrounded an erythema of about the
same width develops within 5-20 minutes.
• In non-allergic individuals usual dose is given.

31. DESENSITISATION
• 0.1 ml of Adrenaline in 1:1000 dilution is given subcutaneously.
• Then ASV is induced in increased doses at every 15 minutes
• Then repeat the same regimen with 1:10 dilution
• At last with undiluted ASV
Another method
intravenous administration of anti histamine followed by infusion of
adrenaline ( 1mg dissolved in 100ml saline ).

32. PROCEDURE
• Dissolve the antivenin in distilled water or normal saline
• Administer the appropriate dose as an infusion in 500ml of saline in 15-20 drops /minute
• The rate can be progressively increased so that infusion is completed in 1-2 hours
• Children requires the same dose
• ASV injection repeated after 1 hr , if the symptom persists
• Further doses repeated every 6 hrs till symptom disappears completely

33. DOSAGE
.
 Initial dose recommended in severe poisoning is 100 ml as in a moderately severe bite 60 mg of
venom injected (10 vials in the case of unidentified snake bites)
1. When local reaction only present 3-6 vials after test dose( one vial of ASV diluted in 200ml of
normal saline slowly), rest of dose is given as infusion in 20 minutes and observe for other
signs of envenomation.
2. When systemic envenomation is present 10 vials of ASV is given as infusion in 20-30
minutes and simultaneously start six vials of ASV in 5% glucose as a drip to be run in 4-6 hours.

34. Maximum dose of ASV
Mild envenomation ……….3 to 5 vials
Moderate envenomation……..5 to 10 vials
Severe envenomation ………10 to 20 vials (20 vials in case of neurotoxic bites and 30 vials for hemotoxic
bites)
Clotting time is to be repeated every four to six hour and ASV repeated if necessary
Better late than never is the policy in ASV therapy if signs of systemic envenomation are present even in
cases presenting late after a bite.
Dose in children exactly same as that of adults.

35. GENERAL MEASURES AFTER ASV
• Inject tetanus antitoxin or booster dose of tetanus toxoid.
• Broad spectrum of anti biotics used.
• Antihistamine & cortisone help in relieving symptoms.
• In severe poisoning infusion of normal saline or transfusion of blood or plasma
useful.
• Renal dialysis and peritoneal dialysis if needed.

36. ADMINISRATION CRITERIA
• ASV is a scarce, costly ,commodity and should only be administered when there
are definite signs of envenomation.
• Unbound, free flowing venom can only be neutralized when it is in the blood
stream or tissue fluid.
• If a patient has evidence to suggest systemic envenoming or severe local
envenoming then only ASV will be administered.

37. RESPONSES
GENERAL : Patient feels better partly placebo effect
SPONTANEOUS SYSTEMIC BLEEDING : usually stops within 15-30 min
BLOOD COAGULABILITY : usually restored in 3-9 hrs
IN SHOCKED PATIENTS : BP may increase first 30-60 min , arrhythmias may resolve
NEUROTOXIC ENVENOMING : of post synaptic type improve within 30 min after antivenom, with presynaptic toxins (kraits
and sea snakes) will not respond.
ACTIVE HAEMOLYSIS AND RHABDOMYOLYSIS : May cease within a few hrs and urine returns to its normal colour

38. CONTRAINDICATIONS
There is no absolute contraindication to ant venom treatment
• Have reacted to horse(equine) or sheep(ovine) serum in past
• With strong history of atopic diseases( esp. severe asthma).
• High risk of severe reactions.
High risk patients may be pretested empirically with subcutaneous epinephrine(adrenaline), IV
antihistamines and corticosteroid.
PREGNANCY IS NOT CONTRAINDICATED

39. ANTIVENOM REACTIONS
More than 10 % of patients will develop reactions as
1. Early anaphylactic reactions
• Usually within 10-180 mts of starting ASV
• Itch (often over scalp)
• Urticaria
• Fever, dry cough
• Nausea, Vomiting
• Abdominal colic, Diarrhea
• Tachycardia
• Shaking chills

40. Minority of these patients develop severe life threatening anaphylaxis, hypotension, bronchospasm
and angioedema .
• These are not IgE mediated type 1 hypersensitivity reactions, mechanism more likely are
Complement activation by IgE aggregate or residual fc fragments.
Direct stimulation of mast cells or basophils by antivenom protein.
2.Pyrogenic(endotoxin reactions)
• usually develops 1-2 hrs after Rx due to pyrogenic contamination during manufacturing process.
• Shaking chills(rigors)
• Fever
• Vasodilation
• Fall in BP
• Febrile convulsions precipitated in children

41. 3.Late(Serum sickness type) reactions
• Develop 1-12 days after treatment
• Fever
• Nausea, vomiting
• Diarrhea
• Itching, recurrent urticaria.
• Arthralgia, myalgia
• Lymphadenopathy
• Periarticular swelling
• Mononeuritis multiplex
• Proteinuria with immune complex nephritis
• Rarely encephalopathy

42. PREVENTIONS AND TREATMENT OF ANTIVENOM REACIONS
• Prophylatic drugs - Adrenaline, Antihistamine anti H1 blockers, Corticosteroids.
• Speed and dilution of Intravenous antivenom administration
(at the earliest signs of reaction)
 Administration must be temporarily suspended.
Rx for early anaphylactic and pyrogenic reactions- Epinephrine (adrenaline) IM,
0.5mg for adults and 0.1mg/kg body wt. for children.

43. LIMITATIONS
• Even among the same species of snakes, there are variations in the composition of venom
produced by individuals in different geographical areas .This places limitations on the efficacy
of antivenin.
• The polyvalent antivenin is not as effective as monovalent, thus necessitating larger dosages
results in pushing up the cost of treatment. And polyvalent type cause more adverse side effects
compared to a monovalent antivenin.
• Not effective against the bites of the King cobra, Hump-nosed pit vipers, sea snakes,
Sochurek’s saw scaled viper (Rajasthan).
• The Haffkine Anti Snake Venom (ASV) is thought to be not as effective in envenomed patients in
Burma and Sri Lanka as in India.

46. REFERENCES
• The essentials of forensic medicine & Toxicology – Dr K S Narayan Reddy & Dr O P Murty 34th
edition
• Comprehensive Medical Toxicology – V V Pillay 3rd edition , page no 1129-1134
• WHO Guidelines for the management of snake bites - Annex 5
• A Textbook of Agadatantra – Dr Sobha Bhat. K , page no 200-204
• Textbook of forensic medicine and toxicology- V V Pillai -17th edition
• Principles of forensic medicine including toxicology – Apurba Nandy
• Medical jurisprudence and Toxicology – Jaising P Modi

47. ANYTHING YOU CAN IMAGINE, YOU CAN CREATE