Determination of Lethal Dose (LD50) of Venom of four Different Poisonous Snakes found in Pakistan

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].


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 Received: October 25, 2017; Accepted: November 15, 2017; Published: November 19, 2017 Abstract 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. LD 50 (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 LD 50 . 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 LD 50 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 LD 50 is the neutralization dose of Anti-snake venom serum used for the calculation of ED 50 of each batch/lot of anti-snake venom serum(as per WHO). The results of the study shows that LD 50 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. The assessment of snake venom median lethal doses (LD 50 ) 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 (LD 50 ). The LD 50 value is defined as the amount of a substance (or venom) causing death of 50% injected mice [10]. The LD 50 for snake venom was first determined in mice by Meier and Theakston in 1986 [11]. According to their study the approximate LD 50 for Naja naja snake was determined in mice to be equal to 0.05 μg/g body weight.

Keywords
The potency of anti-snake venom serum is expressed by its action of neutralizing the LD 50 of snake venom. The toxic effect of venom is expressed in term of its LD 50 . This LD 50 also helped in the establishment of the effective titer of the anti-snake venom serum as median effective dose (ED 50 ). 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 LD 50 of venom of different poisonous snakes present in Pakistan and its evaluation in terms of LD 50 . The findings from this investigation will help in the production of highly potent and cost effective anti-snake venom serum in Pakistan.

Procedure
The LD 50 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 LD 50 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.

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 LD 50 were calculated by Reed and Munch   [19]. According to geographical conditions and other factors, venom composition varies widely as demonstrated in values. In another study LD 50 of Krait species is reported to be 0.30 μg/g [14]. Further research and online data of LD 50 of Krait's venom obtained from Australian Venom and Toxin database depicts the LD 50 value 0.169 μg/g which is also inconsistent to our results [15]. Bungarus candidus venom show high lethal activity with an I.V. LD 50 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 LD 50 of crude venom of Bungarus candidus depicts the variation in the lethality of venom with an I.V. LD 50 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 ( 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 LD 50 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 LD 50 values obtained from this study can effectively be utilized to develop potent anti-venom serum in Pakistan. The determination of LD 50 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 LD 50 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 LD 50 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.