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TEST OF THE VENOM OF BUNGARUS CAERULEUS:AN ATTEMPT TO OBSERVE LETHAL DOSE


Anju Dhir
Lecturer (Microbiology), Shimla- 171012. Himachal Pradesh, India

Abstract

Snakes are the limbless vertebrates and belong to the class of living creatures known as reptiles. More than 2,000 different types of snakes exist and nearly 400 are known to be venomous. More than 40,000 people die every year from snake bite in India. The snakes have always been a potential hazard to the farmers and farm workers. The annual mortality rate from snake bite is more in India i.e. 46,000, out of this about 12,000 die because of snake bite. Among the most venomous snakes of South East Asia Indian common krait is less studied, so in the present study the toxicity of this snake venom was observed through different routes of inoculation like subcutaneous, intramuscular, intraperitoneal, intracerebral and intravenous route of inoculation.

Objective: The study is an attempt to see one of the many characters of venom of Indian common Krait i.e. toxicity.  It provides initial information on the mode of toxic action of the venom.

Method: Experimental Method was used. The toxicity of common krait was measured in the present study as LD50 or the lethal dose 50% test. It is used theoretically as an indication of the standard toxicity value for any chemical under observation.

Results: The common krait venom was found most toxic through intracerebral route and least toxic through subcutaneous route.

Conclusion: The greater toxicity through intracerebral route provides an indication that this venom is affecting the central nervous system and is neurotoxic in nature.

Keywords: Toxicity, Indian common krait venom, LD50, intracerebral, basis for classification.  

INTRODUCTION

The snakes have always been a potential hazard to the farmers and farm workers. Injuries and deaths due to snake bites occur in most part of the world, and especially in the tropics, where they may represent a major health problem. Snakes are the limbless vertebrates and belong to the class of living creatures known as reptiles. These reptiles came into existence long before the mammals and birds were born on this earth. Fossil remains of snakes have been found in the later cretaceous and early tertiary periods in the world’s history. Herpetologists agree that the snakes and lizards share a common ancestor having labial glands on both jaws. The venomous snakes have a pair of venom glands which are lying on either side of the skull beneath the skin, situated just under and behind the eyes. The venom glands may be true salivary glands with or without digestive enzymes. Without the venom it may be very difficult or even impossible for snakes to obtain their food.

Snakes are scattered all over the world. They like heat, it means life to them, and therefore the snakes are most abundant in the tropical and semi tropical regions of the globe.  In South East Asia, the mortality is high. In the Maharashtra state of India more than 1000 deaths per year occur due to snake bite 1. More than 2,000 different types of snakes exist and nearly 400 are known to be venomous.

The annual mortality rate from snake bite is more in India i.e. 46,000, out of this about 12,000 die because of snake bite 2. The common four species responsible for these casualties belong to two families of snakes: elapidae and viperidae. The venom of elapids is neurotoxic and that of viperidae is cardiotoxic.

The common krait and cobra belong to elapidae family. There are twelve species of krait, out of which ten are found in India. The colour of Indian common krait is shining blue black with white bands. It is nocturnal and very timid and it never attacks unless disturbed. This snake does not produce hissing sound instead a shrill noise could be heard at times. Indian Common krait has small fangs so bite is either painless or very little discomfort is caused. The common krait bite leaves indistinct marks. Local effects are negligible despite the presence of serious systemic envenoming 3, 4.

In the present study an effort has been made to observe toxicity of this timid but potentially very poisonous snake. The toxicity of common krait was measured in the present study as LD50 or the lethal dose 50% test. The amount of substance that kills half of animals under experiment gives LD50. It is used theoretically as an indication of the standard toxicity value for each chemical under observation 5.

Venom: The proteins are the main components of snake venom and can be grouped as:-
1)The proteins with toxic principles.
2) The proteins with enzymatic activities.
3) The proteins with no known biological activities.

As little or no pain occurs from krait bite this could give false reassurance to the victim. Typically victim complains of severe abdominal cramps. The patient shows the signs of ptosis, glossopharyngeal palsy, blurring vision, opthalmoplagia, dilated pupils, respiratory failure, drowsiness, headache and coma. The venom of Indian common krait is mainly neurotoxic and it induces muscle paralysis. The venom of B. caeruleus or Indian common krait contains postsynaptic and presynaptically acting neurotoxins, which was proved by Chang 6.

AIMS & OBJECTIVES

In 1980, a coordination committee on venoms and antivenom sera was conceived by W.H.O. and the progress on the characterisation of venoms has been published in the form of an offset (W.H.O., 1981) 7. In this publication also, not much has been discussed on the characterisation of common krait venom.

The common krait venom had not been included in the list of the international preparations of reference pools of snake venoms for characterisation. Therefore this study was undertaken to observe the toxic character of common krait venom which is one of the potentially dangerous snakes of south East Asia. Data from this study may:

  1. Serve as the basis for classification of venom
  2. Provide initial information on the mode of toxic action
  3. Help arrive at a dose of this venom
  4. Help in dose determination in various animal species.
  5. Help in determining LD50 values in various indices of potential types of venom activity

Therefore, it is an attempt to see one of the many characters of venom of Indian common Krait that is toxicity.

MATERIALS & METHOD 

Material
1) Venom: The common krait (B. caeruleus) venom used in this study was obtained from Central Research Institute Kasauli. The venom was dissolved in sterile normal saline. The solution was centrifuged at 1500 rpm for 10 minutes and then passed through membrane filter of pore size 0.22 micron. The sterile solution was then used in the experiments.

2) Laboratory mice of 18-20 gm were taken as experimental animal. They were obtained from random breeding in a closed colony.

Methodology

Type of Study: Experimental Method was used. The toxicity of common krait was observed in the present study as LD50 or the lethal dose 50% test. It involves dosing selected laboratory animals with the venom/toxin either by mouth (force feeding), by injection, via the skin or by inhalation. It is usually an initial step in the assessment of toxic characteristics of venom. The amount of substance that kills half of animals under experiment gives LD50. It is used theoretically as an indication of the standard toxicity value for a chemical under observation 8, 9.   Signs observed during toxicity test included tremors, arching and rolling, anaesthesia, muscle spasm, sedation and hypnosis 1,3. After the test the animal was left in the cage with free access to food and water. After 24 hrs the results were noted.

Toxicity of the venom under test was tested in mice by preparing a series of dilutions with 25% increment and injecting 0.2 ml into each of a group of six albino mice weighing 18-20 g. Readings in terms of deaths and survivals were noted after 24 hrs. LD50 was calculated according to the Reed and Munch 8. The venom dose has been transformed to log (dose) and percent mortality to probit mortality.  Each toxicity test through different routes has been repeated for six times. In making calculations it is assumed that the doses used are equally placed on the logarithmic scale, and 50% end point falls somewhere in the middle of the range of dilutions.  

Table: Toxicity test of common krait venom through different routes of inoculation

Route of Inoculation Volume of Inoculation                               LD50 of venom (µg )

     

Average LD50 in µg                                                             
01 02 03 04 05 06  
  S/C      0.5 20.190 21.360 21.50 21.760 22.090 22.250 21.5250
    I/M      0.5 15.070 15.820 17.120 18.840 18.840 19.360 17.4400
    I/P      0.5 5.113 6.224 6.470 6.500 7.050 7.775 6.5220
    I/V      0.2 1.638 1.781 1.813 2.048 2.242 2.355 1.9785
    I/C      0.03 0.300 0.334 0.384 0.394 0.414 0.450 0.379

 

The toxicity of Common Krait venom was observed in mice through different routes of inoculations and the results are being presented below. The bar diagram has been plotted by taking log dose on Y-axis and the number of test on x-axis. Toxicity test of Common Krait venom through each route of inoculation has been repeated six times and the comparative results have been presented below:

Figure: Comparative results of toxicity through different routes of inoculations.

[Abbreviations: s/c: subcutaneous; i/m: intramuscular; i/p: intraperitoneal; i/v: intravenous; i/c: intracerebral]

The result of above table shows the toxicity of common krait venom through different routes of inoculation. The bar diagram in figure given above shows the comparative results of all five routes of inoculation. Very high dose of venom is required in s/c route. The common krait venom was found most toxic through intracerebral route and least toxic through subcutaneous route. The order of toxicity is:

Intracerebral > intravenous > intraperitoneal > intramuscular > subcutaneous

This order of toxicity clearly indicates the nature of this venom that is neurotoxic.

 DISCUSSION

Scanty results on the toxicity of this snake venom have been described in literature. Underwood has observed this venom to be more toxic in mice through intravenous route and LD50 for a 20 g mouse has been calculated as 1.0 µg 9 which is in accordance with the present study. The active principle mainly responsible for the toxicity of common krait venom is the neurotoxin with low molecular weight protein which blocks the neuromuscular transmission postsynaptically. Because of low molecular weight, the venom can diffuse into vital target organs much faster, causing quick action. It has been observed in the present study that the mice injected intravenously showed progressive symptoms of weakness followed by paralysis of skeletal muscles and death occurred due to respiratory failure/paralysis. Here the toxicity of common krait was observed as LD50 or the lethal dose 50% test. It is used theoretically as an indication of the standard toxicity value for any chemical under observation. It can be seen from the Table-1 that the krait venom was most toxic through intracerebral route.

The greater toxicity through intracerebral route also provides an indication that this venom is affecting the central nervous system. This also suggests that the venom is neurotoxic in nature. It was quite difficult to control death by intracerebral route. The symptoms in the human patients of common krait bite are similar but in addition there is profuse salivation, ptosis and flaccid paralysis. A victim of common krait venom was personally observed by the author.  Death in these patients also definitely occurs due to respiratory failure. Death of an experimental animal is not necessarily due to a single component and results may vary with species of animal. Even route of inoculation changes the results, like in the intraperitoneal route the toxicity is less as many drugs get detoxified by the liver 1.

CONCLUSION

The present study indicates the neurotoxic behaviour of common krait venom. As LD50 is being estimated in mice so it cannot give us the exact measure in case of humans. Therefore LD50 in mice obviously does not give us the clear picture. But it is a baseline comparison and has merits, although standards may differ from one to another laboratory 10.  It can also serve as the basis for classification of venom. To some extent it provides initial information on the mode of toxic action based upon the signs and symptoms in experimental animal. Although lethality of venom may also differ from species to species of the snake and also on the geographical location.

REFERENCES 

  • N. Ghosh, “Toxicity studies in fundamentals of experimental pharmacology”, Scientific Book Agency, Calcutta 1984; pp. 153-158.
  • Mahapatra, “Snake bite kills 46K in India yearly’. December 2011. [internet (Cited Dec.1, 2015; The Times of India.
  • Englemann, Wolf–Eberhard, “Snakes: Biology, Behaviour and relationship to man”, Leipzig Eng. Version NY,USA: Leipzig Publishing, Eng.Version published by Exeter Books 1982; 51, ISBN 0-89673-110-3.
  • The Ten Deadliest Snakes in the World. [Internet (Cited Dec. 4, 2015)] Available from-www.ijapbr.com>location > the ten deadliest snakes in the world
  • Shetty Akhila, Shyamjith, Deepa, M. C. Alwar, “Acute toxicity studies and determination of lethal dose”, Current Science, 2014; 93(7): 917-920.
  • C. Chang, C.Y. Lee, Huang, “Studies of the presynaptic effects of bungarotoxin on neuromuscular transmission”, J. Pharmacology Exp. Ther. 1973; 184: 339.
  • Progress in the characterisation of Venoms and Standardisation of Antivenoms, W.H.O. Geneva. 1981, W.H.O. offset Publication No. 58.
  • J. Reed, H.C. Muench, “A simple method of estimating 50% and points”, American Journal of Hygiene, 1938; 24: 493.
  • Underwood, “Toxins from B. caeruleus venom from Venomous Snakes in India”, Handbook Exptl. Pharmcol 1979; 52:202.
  • Turner, “Acute toxicity: The determination of LD50”, Screening Methods in Pharmacology, Academic Press New York, 1965; 300