Biochemistry & Molecular Biology Journal Open Access

Venom; LD₅₀; Anti snake venom serum; ED₅₀

Introduction

There are almost 5.4 million venomous snake bites, about 2.5 million envenoming and over 125,000 deaths annually. As per global burden of snake bite incidents, (morbidity and mortality), South Asia is the most affected region. India has the highest number of deaths with 35,000–50,000 per year [1,2]. In Pakistan, 40,000 snake bites and 8000fatal cases per year [3] in Nepal, 1,000 deaths per year [4] by snake bite, in Sri Lanka, around 33,000 snake bite victims are reported annually [5]. The fatality rate is almost 20% [6]. Snakes are cold-blooded vertebrates and provoke a high number of human deaths due to envenoming characteristic [7].

In Pakistan, Echis carinatus (saw Scaled viper) is found in Thar and Cholistan desert, and Astola island of Makran in Baluchistan. It is almost 0.4 to 0.6-meter-long with flattened body and short tapered tail. It is active and can move rapidly in aside winding motion. Vipera russelli is found in Pakistan from India-Pakistan border to Indus Valley in Provinces of Sindh and Punjab at moist and cool places. Viper snakes distributed north to the Indus valley of Pakistan and Kashmir, regarded as one of Pakistan's deadliest snakes. Bungares caeruleus Sindanus (Krait) is about 1.0 to 1.8-meter-long with glossy appearance, flattened body and jerky movement. It is very active at night. Naja naja (Cobra) has two subspecies of almost 1.9 to 2.4-meter long. The hood appearance varies greatly and it is present in eastern Pakistan and Karachi at sea level [8].

The almost 3000 species of snake are found in all over the world and near about 600 among them are found venomous. These snakes inject modified saliva (venom) containing toxins into the body of their prey through their fangs cause bleeding, muscle paralysis, and tissue destruction (necrosis) around the bite site [9].

The assessment of snake venom median lethal doses (LD₅₀) is an important step for an accurate evaluation of the toxic activity of specific venom, and is also regularly used to select the relevant anti-venom batch, as well as to establish the neutralizing capacity of each vial. According to the WHO, venom lethality is expressed as median lethal dose (LD₅₀). The LD₅₀ value is defined as the amount of a substance (or venom) causing death of 50% injected mice [10]. The LD₅₀ for snake venom was first determined in mice by Meier and Theakston in 1986 [11]. According to their study the approximate LD₅₀ for Naja naja snake was determined in mice to be equal to 0.05 μg/g body weight.

The potency of anti-snake venom serum is expressed by its action of neutralizing the LD₅₀ of snake venom. The toxic effect of venom is expressed in term of its LD₅₀. This LD₅₀ also helped in the establishment of the effective titer of the anti-snake venom serum as median effective dose (ED₅₀). The median effective dose is the minimum amount of anti-snake venom serum to neutralize and protects 50% population of the mice injected [12].

The current study is based upon the calculation of LD₅₀ of venom of different poisonous snakes present in Pakistan and its evaluation in terms of LD₅₀. The findings from this investigation will help in the production of highly potent and cost effective anti-snake venom serum in Pakistan.

Procedure

The LD₅₀ was calculated by Reed & Munch method according to WHO guidelines. The calculation is carried by observing and calculating cumulative survival/death, proportionate of difference in dilution factor and mortality rate. The standardized LD₅₀ of venom is essential for the neutralization of antivenom i.e., for potency determination of each manufactured batch.

The study has been performed on following four venomous snakes found in Pakistan.

Echis carinatus (Saw Scaled Viper)

Bungares caeruleus (Sung Choor, Krait)

Vipera russelli (Russell’s viper, Dobia russuii)

Naja naja (Cobra or Sheesh Nag or Kala Nag)

Results

Each dilution was injected in group of eight mice both male and female 18 to 20 g weight. The dose of venom was inoculated intravenous in tail. The observations were noted after 24 hours of inoculation and LD₅₀ were calculated by Reed and Munch

Table 1 shows the mortality/survival rate of the mice after injecting specified amount of venom intravenously in mice.

Venom of the Snake	No. of doses	Dose in μg		No. of Mice injected	Weight of mice (g)	Route of inoculation/dose in ml	No. of Survival after 24Hrs.
Echis carinatus (Saw scaled viper)	1		32	8	18-20	Intravenous in Tail	0
	2		16	8			3
	3		8	8			5
	4		4	8			8
Vipera russelli (Russell’s viper)	1		20	8	18-20	Intravenous in Tail	0
	2		10	8			1
	3		5	8			6
	4		2.5	8			8
Bungares caeruleus Sindanus (Krait)	1		16	8	18-20	Intravenous in Tail	0
	2		8	8			3
	3		4	8			5
	4		2	8			8
Naja Naja (Cobra)	1		16	8	18-20	Intravenous in Tail	0
	2		8	8			3
	3		4	8			5
	4		2	8			8

Table 1: Determination of LD50 values through reed and munch method.

Table 2 shows that the approximate lethal dose (LD₅₀) of each species of the snakes in microgram per dose as well as in μg/g of body weight. The LD₅₀ of venom of Echis carinatus is 11.311 μg/dose(approx.0.5655 μg/g), Russell Viper is 6.643 μg/dose (approx.0.3321 μg/g), Krait is 5.656 μg/dose (approx.0.2828 μg/g), and Cobra 5.656 μg/dose (approx.0.2828 μg/g).

Venom of the Snake	Dose in μg as dilution				Weight of Mice (g)	Route of inoculation/dose in ml	No. of Survival after 24Hrs.				LD50 μg/dose	LD50 μg/g
Echis carinatus	32	16	8	4	18 – 20	Intravenous in Tail	0	3	5	8	11.311	0.5655
Russell viper	20	10	5	2.5	18 – 20	Intravenous in Tail	0	1	6	8	6.643	0.3321
Krait	16	8	4	2	18 – 20	Intravenous in Tail	0	3	5	8	5.656	0.2828
Cobra	16	8	4	2	18 – 20	Intravenous in Tail	0	3	5	8	5.656	0.2828

Table 2 LD50 values of different snake venom through intravenous mode of injection.

Discussion

This study is carried out to estimate approximate lethal dose (LD₅₀) of the four snakes venom i,e., Echis carinatus, Russell Viper, Krait and Cobra which are 11.311 μg/dose (0.5655 μg/ gm), 6.643 μg/dose (0.3321 μg/gm), 5.656 μg/dose (0.2828 μg/ gm), 5.656 μg/dose (0.2828 μg/gm).

In this study LD₅₀ value obtained for venom of Echis carinatus was 0.5655 μg/g which shows that its lethality is lesser as compared to Cobra, Krait and Russell viper. Now specific studies in Pakistan has been carried out to standardize the values of LD₅₀ for Ekis Carinatus venom throughout the country however results obtained by international researchers depicts Different value of LD₅₀ for instant a study reported by Christensen in 1979 demonstrate the LD₅₀ of Echis carinatus 22 μg/18-20 g of mice or 1.2 μg/gm through intravenous route [13,14] while according to Australian online Biodata of Snake Venom, the LD₅₀ of Echis carinatus multisquamatus (found at Iran) is 3.26 μg/g [15]. It is strongly felt there is dire need to do more work in this field because most of LD₅₀ value varies from study to study.

In the case of Russell Viper, the LD₅₀ obtained through I.V injection is 0.3321 μg/g, previously no specific study had been carried out to evaluate the lethality of local species of viper snake in Pakistan. Kankokar and Rao et al. characterize the venom of Indian Krait on the basis of its lethality and composition and its LD₅₀ was demonstrated to be 0.31 μg/g in mice through I.V route [16]. In another study performed by Meier and Theakston revealed that the lethality of venom of Russell viper varies with change in route of injection as their results predicts the LD₅₀ of 0.4 μg/g through Intraparietal (I.P) route, 0.75 μg/subcutaneous (S.C) route and 0.3 μg/g through intravenous(I.V) route [7,14]. This fact of high lethality through I.V route could be attributed to the size of venom molecules in Russell viper, such as anticoagulants, hemorrhagic compounds, edematous proteases and molecules with amidolytic and caseinolytic properties.

Bungarus caeruleus (Krait) is found in Peninsular India spreaded from Sindh (Pakistan), to the West Bengal plains [17]. In Pakistan, no specific work is done on local species of Bungarus caeruleus for the determination of LD₅₀. In our study the LD₅₀ value of its venom in mice model is 0.2828 μg/g which is contradictory to previous reports demonstrated Engelmann and Obst et al. their obtained LD₅₀ value is 0.169 μg/g through I.V mode of injection [18] Other findings obtained by Mirajkar and More et al. demonstrated that when crude venom administered intravenously cause lethality in mice with usual neurotoxic symptoms and resultant LD₅₀ value was 160 μg/kg or 0.16 μg/g [19]. According to geographical conditions and other factors, venom composition varies widely as demonstrated in values. In another study LD₅₀ of Krait species is reported to be 0.30 μg/g [14]. Further research and online data of LD₅₀ of Krait’s venom obtained from Australian Venom and Toxin database depicts the LD₅₀ value 0.169 μg/g which is also inconsistent to our results [15]. Bungarus candidus venom show high lethal activity with an I.V. LD₅₀ of 0.2828 μg/g while previous values collected from Sean Thomas biodata depicts the value as 0.169 μg/g. Although the value is inacceptable range in comparison to our results, the small difference is due to geographical variation and other factors. Another study carried out at Malaysia for the determination of LD₅₀ of crude venom of Bungarus candidus depicts the variation in the lethality of venom with an I.V. LD₅₀ of 0.11 μg/g [20].

Interestingly, the venom of Naja naja (cobra snake) is far more poisonous than viper venoms, in agreement with previously described values in literature. [10]. These results (Table 2) indicate small molecular weight of cobra venom (with molecular weights < 15 kDa). The venom toxicity of the potent Naja naja is resultant of low molecular weight (usually < 30 kDa) toxins, such small molecules have a shorter residence time at the site of inoculation and quick diffusion causing an instant bioavailability in the blood [21]. Riaz and Zaman et al. also determined lethal dose 50 (LD₅₀) of Naja naja in Pakistan by Reed and Munch method through intramuscular route (I.M) and the value obtained were 1.2 mg/kg or 1.2 μg/g which shows that lethality of snake venom is decreased through I.M route [22]. It is demonstrated that the route of injection of venom could affect the LD₅₀ values. Another study reveal this impact in which the LD₅₀ value of Naja naja venom has increased about four times when mode of injection was changed from I.V to I.M [10].

Our results of LD₅₀values for Pakistani Naja naja are in concordance with the recently reported study carried out at University of Malaya by Wong, Tan et al. They determined the subcutaneous and intravenous LD₅₀ values for Pakistani Naja naja in mice model. The LD₅₀value obtained through intravenous mode of injection was 0.22 μg/g which also favors our results. [23]. Diverse immunological properties of cobras from different geographical regions have fascinated researchers specially protein chemists to work for developing new antidotes.

All these findings show that variation in lethality and composition of snake venom is a ubiquitous phenomenon both interspecific and intraspecific. Venom variation can lead to severe consequences for snakebite victims by rendering the particular antibodies present in antivenom unproductive against heterologous toxins in venoms [24].

The LD₅₀ reflects the venom of the snakes which are found in Pakistan is of high quality and very much potent in terms of its toxicity and lethality [25]. This high-quality venom [26,27] provokes the production of highest quality of anti-snake venom serum.

Conclusion

In the present work, we determined experimentally the LD₅₀ values of reference snake venoms in mice, and evaluated the venom potency with relevant to previous literature [28-30]. The LD₅₀ values obtained from this study can effectively be utilized to develop potent anti-venom serum in Pakistan. The determination of LD₅₀ of each venom is important not only to produce potent antivenom serum but also for determination of neutralization capacity of each produced lot of antivenom serum before release for consumption. The results show that most of the venom obtained from same species with geographical barrier has different LD₅₀ values depicting the effect of geographical regions, route of injection and several other factors so it is the need of time for in-depth research in this area, to develop antivenom serum for specific species based upon their exact lethality. In addition, the LD₅₀ value of these assays must be noticed with caution since these are obtained using a mouse model.

Future Perspectives

The results of current study could persuade scientific community to explore further in-depth knowledge to identify the myotonic components and characterization of venom of deadliest snakes found in Pakistan, which may be of huge biodiscovery potential for the development of antivenom. Moreover, these findings indicate that there is dire need for effective antivenom in Pakistan based upon the lethality of local species. Sera Processing Lab. NIH, Pakistan is capable enough to produce polyvalent antivenom against the venom of local snakes and will fulfill the country demand of antivenom in near future by the expansion of its manufacturing capacity.

References

1. Warrell DA, Gutiérrez JM (2013) New approaches and technologies of venomics to meet the challenge of human envenoming by snakebites in India. Indian J Med Res 138: 38.
2. Nagaraju K, Kannappan N (2015) Survey on pattern of snake bite cases admitted in South Indian Tertiary Care Hospitals. International J Pharma Sci Res 6: 4362.
3. Gutiérrez JM, Warrell DA (2013) The need for full integration of snakebite envenoming within a global strategy to combat the neglected tropical diseases: the way forward. PLoS Negl Trop Dis 7: e2162.
4. Poudyal V, Paudal K (2017) A hospital based study on snake bite poisoning in adults in the western region of Nepal. J Chitwan Med College 6: 33-38.
5. Dayananda K, Reddy PJM (2013) Epidemiological study of snakebite cases admitted in Victoria Hospital, Bangalore. Int J Med Med Sci 46(3): 1304.
6. Shuting L, Jingqiang W (2004) Proteomic characterization of two snake venoms: Naja naja atra and Agkistrodon halys. Biochem J 384: 119-127.
7. Meier J, Theakston R (1986) Approximate LD50 determinations of snake venoms using eight to ten experimental animals. Toxicon 24: 395-401.
8. Warrell D (1995) Clinical toxicology of snakebite in Asia. Handbook of clinical toxicology of animal venoms and poisons. CRC Press, Boca Raton, USA.
9. Amir A, Zahri NAH, (2016). Image classification for snake species using machine learning techniques. International Conference on Computational Intelligence in Information System, Springer.
10. Oukkache N, Jaoudi RE (2014). Evaluation of the lethal potency of scorpion and snake venoms and comparison between intraperitoneal and intravenous injection routes. Toxins 6: 1873-1881.
11. Tohamy AA, Mohamed AF (2014) Biological effects of Naja haje crude venom on the hepatic and renal tissues of mice. J King Saud Uni-Sci26: 205-212.
12. Standardization WECoH (2010) "WHO Guidelines for the Production Control and Regulation of Snake Antivenom Immunoglobulins."
13. Christense P (1979) Production and standardization of antivenin. Snake venoms, Springer: 825-846.
14. Tu A (1991) Handbook of Natural Toxins: Reptile Venoms and Toxins, CRC Press.
15. Thomas S (1999) "LD50." LD50 Scores for various snakes, from https://www.seanthomas.net/oldsite/ld50tot.html.
16. Kankonkar R, Rao S (1972) Efficacy of Haffkine Institute polyvalent antivenin against Indian snake venoms. Indian J Med Res 60: 512-516.
17. Hallermann PU (2016) Global Reptile BioBlitz.
18. Englemann WE, Obst F (1984) Snakes: Biology, behavior and relationship to man. Croom-helm publishing co. london.
19. Mirajkar K, More S (2006) Preliminary studies with a neurotoxin obtained from Bungarus caeruleus venom. J Venom Anim Toxins Incl Trop Dis 12: 78-90.
20. Tan C, Tan N (2015) Toxinology of snake venoms: The Malaysian context. Snake Venoms Eds: 1-37.
21. Oukkache N, Lalaoui M (2012) General characterization of venom from the Moroccan snakes Macrovipera mauritanica and Cerastes cerastes. J Venom Anim Toxins Incl Trop Dis 18(4): 411-420.
22. Riaz Z, Zaman M (2015) Bio-physiological effects of ld50 of crude venom of black pakistaní cobra (Naja Naja karachiensis) in mice. J Anim Plant Sci 25: 1344-1348.
23. Wong KY, Tan CH (2016) Venom and purified toxins of the spectacled cobra (Naja naja) from Pakistan: Insights into toxicity and antivenom neutralization. Am J Trop Med Hyg 94: 1392-1399.
24. Casewell NR, Wagstaff SC (2014) Medically important differences in snake venom composition are dictated by distinct postgenomic mechanisms. Proceedings of the National Academy of Sciences 111: 9205-9210.
25. Omale S, Aguiyi J (2012) Effects of the ethanolic extract of Parinari Curatellifolia on blood clotting factors in rats pretreated with venom of Naja nigricolis. Drug Invention Today 4 (4).
26. Ali SA, Yang DC (2013) Venom proteomic characterization and relative antivenom neutralization of two medically important Pakistani elapid snakes (Bungarus sindanus and Naja naja). J Proteomics 89: 15-23.
27. Ali Z, Begum M (1990) Snake bite: A medical and public health problem in Pakistan." Snakes of medical importance (Asia Pacific region) Singapore National University Singapore: 447-461.
28. Kasturiratne A, Wickremasinghe AR (2008) The global burden of snakebite: A literature analysis and modelling based on regional estimates of envenoming and deaths. PLoS Med 5: e218.
29. Tanaka GD, Maria de Fátima DF (2010) Diversity of Micrurus snake species related to their venom toxic effects and the prospective of antivenom neutralization. PLoS Negl Trop Dis 4: e622.
30. World Health Organization (2010) WHO guidelines for the production, control and regulation of snake antivenom immunoglobulins. Geneva, Switzerland.