Received date: October 25, 2017; Accepted date: November 15, 2017; Published date: November 19, 2017
Citation: Parveen G, Khan MF, Ali H, Ibrahim T, Shah R (2017) Determination of Lethal Dose (LD50) of Venom of four Different Poisonous Snakes found in Pakistan. Biochem Mol Biol J. Vol.3 No.3:18. doi: 10.21767/2471-8084.100046
Pakistan is the highly fertile region for the envenoming and deadly snakes. The envenoming fatality and snake bite cases are increasing day by day not only in Pakistan but also in World. In Pakistan, almost 40,000 snake bite cases and 8000 fatal cases has been identified. This high-risk issue needs to be addressed with an easy accessible and affordable treatment by producing anti snake venom serum. LD50 (50% Lethal Dose) of venom is the prime parameter to determine the toxicity and lethality of venom extracted from the four poisonous snakes present in the Pakistan. The main objective of this study waste produces highly potent and costeffective anti-snake venom serum by the determination of LD50. The venom was extracted from four different species of snakes i.e., Echis carinatus, Vipera russelli, Bungares caeruleus and Naja naja (Cobra) present in Biological Production Division of NIH, Pakistan. The four to five serial dilutions were injected intravenously into the mice tail and observations were recorded to calculate the LD50 of each species by Reed and Munch method in Bacteriology section of Quality Control Laboratory, National Institute of Health, Islamabad. Then 3 to 5 fold LD50 is the neutralization dose of Anti-snake venom serum used for the calculation of ED50 of each batch/lot of anti-snake venom serum(as per WHO). The results of the study shows that LD50 of Naja Naja (Cobra) lies approximate between 6 to 7 μg/dose, Echis carinatus (Saw Scaled Viper) 11 to 12 μg/dose, Vipera russelli (Russel viper) 5 to 6 μg/dose and Bungares caeruleus (Krait) 4 to 5 μg/dose in intravenous injection of dilution.
Venom; LD50; Anti snake venom serum; ED50
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  in Nepal, 1,000 deaths per year  by snake bite, in Sri Lanka, around 33,000 snake bite victims are reported annually . The fatality rate is almost 20% . Snakes are cold-blooded vertebrates and provoke a high number of human deaths due to envenoming characteristic .
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 .
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 .
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 . The LD50 for snake venom was first determined in mice by Meier and Theakston in 1986 . 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 .
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.
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)
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)
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
|Weight of mice (g)||Route of inoculation/dose in ml||No. of Survival after 24Hrs.|
(Saw scaled viper)
|1||32||8||18-20||Intravenous in Tail||0|
|1||20||8||18-20||Intravenous in Tail||0|
|1||16||8||18-20||Intravenous in Tail||0|
|1||16||8||18-20||Intravenous in Tail||0|
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
|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.
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 . 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 . 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 . 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  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 . 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 . 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 . 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 .
Interestingly, the venom of Naja naja (cobra snake) is far more poisonous than viper venoms, in agreement with previously described values in literature. . 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 . 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 . 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 .
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. . 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 .
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 . This high-quality venom [26,27] provokes the production of highest quality of anti-snake venom serum.
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.
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.