Enzymatic Activity And Brine Shrimp Lethality Of Venom From The Large Brown Spitting Cobra (Naja Ashei) And Its Neutralization By Antivenom Mitchel Otieno Okumu ( [email protected] ) University of Nairobi https://orcid.org/0000-0002-9316-990X James Mucunu Mbaria University of Nairobi College of Agriculture and Veterinary Sciences Joseph Kangangi Gikunju Jomo Kenyatta University of Agriculture and Technology Paul Gichohi Mbuthia University of Nairobi College of Agriculture and Veterinary Sciences Vincent Odongo Madadi University of Nairobi College of Biological and Physical Sciences Francis Okumu Ochola Moi University Research note Keywords: Snake venom phospholipase A2, brine shrimp lethality assay, snake venom toxicity, Naja ashei Posted Date: April 20th, 2020 DOI: https://doi.org/10.21203/rs.3.rs-20006/v1 License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published on July 6th, 2020. See the published version at https://doi.org/10.1186/s13104-020-05167-2. Page 1/13 Abstract Objective There has been little focus on the enzymatic and lethal activities of Naja ashei venom and their neutralization by antivenom. This study aimed to determine the snake venom phospholipase A2/svPLA2 activity and brine shrimp lethality of N. ashei venom and their neutralization by two antivenoms (I and II). The venom of other snakes in East Africa including the puff adder (Bitis arietans), green mamba (Dendroaspis angusticeps), black mamba (Dendroaspis polylepis), Egyptian cobra (Naja haje), red spitting cobra (Naja pallida), and the Eastern forest cobra (Naja subfulva) were used for comparison. Results: N. subfulva venom had the highest svPLA2 activity while D. angusticeps venom had the least activity. N. subfulva venom was the most toxic in the 24-hour brine shrimp lethality assay (BSLA), while N. ashei venom was the most toxic in the 48 and 72-hour assays. N. haje venom was the least toxic in all assays. One ml of antivenom I neutralized 0.075 µg of svPLA2 in N. ashei venom compared to 0.051 µg by antivenom II. Antivenom I was ineffective in neutralizing N .ashei venom-induced lethality but 1 ml of antivenom II neutralized 0.21 mg of N. ashei venom. Introduction More than 5 million snakebites occur every year disproportionately affecting the poorest nations globally [1]. Up to 138, 000 people die from snakebite and 400, 000 are disabled [1]. Cobras are venomous snakes that may be spitting or non-spitting in nature [2]. They are often cultural icons and objects of reverence [3, 4]. African spitting cobras are associated with morbidity and mortality in sub-Saharan Africa [3–11]. Toxicological/pathological effects include blistering, edema, necrosis, respiratory paralysis and ophthalmia [8–13]. Naja ashei is one of the African spitting cobras and is a category 1 snake in Kenya, Ethiopia, Somalia, Uganda, and a category 2 snake in Tanzania [14]. Figure 1. Category 1 snakes are highly venomous, cause many bites, and are associated with high levels of morbidity, disability or mortality [14]. Category 2 snakes are highly venomous, may cause morbidity, mortality, disability or death but lack epidemiological/clinical data to implicate them in snakebite [14]. The skull structure, mitochondrial DNA, venom composition, antiproliferative, and antibacterial properties of N. ashei have previously been reported [3, 7, 8, 15–17]. However, there has been little focus on the enzymatic, and lethal effects of this venom and the capacity of antivenom to neutralize them. This study aimed to evaluate the svPLA2 activity and lethal effects of N. ashei venom and the capacity of commercially available antivenom to neutralize these effects. Materials And Methods Snake venom Venom was extracted from 21 specimens of wild-caught puff adders (B. arietans), green mambas (D. angusticeps), black mambas (D. polylepis), large brown spitting cobras (N. ashei), Egyptian cobras (N. Page 2/13 haje), red spitting cobras (N. pallida), and Eastern forest cobras (N. subfulva) maintained at the Bioken Snake Farm (Kenya). Table S1. Venoms were snap frozen, lyophilized, and stored as powder at -20 °C. They were reconstituted in phosphate-buffered saline (PBS) at the time of use. Animals (brine shrimp) One hundred grams of brine shrimp eggs were commercially sourced from yourshstuff in the Borough of Lebanon, New Jersey, USA (Batch number; X001M8M5IZ). The eggs were hatched at the Department of Public Health, Pharmacology, and Toxicology, University of Nairobi and brine shrimp larvae were used for experiments on venom lethality and its neutralization. Antivenom Antivenoms used in this study are described in Table S2 and were acquired from hospitals in Kenya. Protein content determination of the venoms and antivenoms Lowry’s method was used [18]. Serial dilutions (0.05-2.0 mg/ml) of bovine serum albumin were prepared in triplicate, 0.2 ml of each dilution was pipetted into 10 ml glass tubes, 2 ml of alkaline copper sulphate was added to each tube and incubated at room temperature for 10 minutes. 0.2 ml of Folin-Ciocalteau reagent was added and tubes incubated for 30 minutes. Absorbance was read at 660 nm. Protein content was inferred from the calibration curve [18]. Table S3. SvPLA2 activity of venoms and neutralization of N. ashei venom by antivenom The svPLA2 activity of the venoms was determined by the methods of Haberman and Hardt 1972 and Felix Silva et al [19, 20] with modications. 10 ml of a 1:3 v/v suspension of egg yolk in phosphate- buffered saline (PBS) was added to 90 ml PBS and 300 ml of a 1% w/v agarose solution. 125 µl of 0.1 mM Calcium chloride (CaCl2) was added to the mixture, which was poured into sterile Petri dishes and 6 mm wells were made on the solidied media. Figure S1. 10 µl of venoms (0.5–22.5 µg/ml) were pre- incubated at 37 °C for 60 minutes and pipetted into the wells. These were incubated at 50 °C for 24 hours. A 1:10, v/v solution of Carbol Fuchsin (CF) was used to visualize the halos and Vernier calipers were used for measurement. PBS was used as a negative control. The minimum phospholipase concentration (MPC50) was calculated using regression analysis. All experiments were performed in triplicate. Neutralization of svPLA2 activity The method of Iwanaga and Suzuki 1979 was used with modications. [21]. 10 µl of a 2 × MPC50 dose of N. ashei venom was mixed with 20 µl of antivenom (25–400 µg/ml) in 96-well ELISA plates, incubated at 37 °C for 20 minutes and 200 µl of substrate (1.1% egg yolk suspension in 0.1M PBS + 125 µl 0.2 mM CaCl2) was added to each well, incubated at room temperature and the change in absorbance of the substrate was measured spectrophotometrically from 0 to 30 minutes at 620 nm [21]. All experiments Page 3/13 were performed in triplicate (Figure S2) and the % PLA2 activity was calculated by taking the absorbance of the wells with venom alone as 100%. Table S4. Venom induced brine shrimp lethality The method of Meyer et al 1982 was used [22]. Ten, 48-hour old brine shrimp larvae were transferred from a hatching trough to 5 ml sample vials. Figure S3. Aliquots (5, 50 and 500 µl) of 5 mg/ml stock solutions of the venoms were pipetted into the sample vials and made up to the mark with marine salt solution (38.5% w/v) to a nal concentration of 10, 100, and 1000 µg/ml respectively. PBS was used as control. Surviving larvae were counted after 24, 48, and 72 hours. All experiments were performed in quintuples. Probit analysis was used to determine the median lethal concentration (LC50)[23–25] and the toxicity of the venoms was classied using toxicity indices [26, 27]. Neutralization of brine shrimp induced lethality by antivenom The World Health Organization (WHO) protocol was used with modications [14]. Various doses of antivenoms (25–400 µl of 100 mg/ml stock solutions) were mixed with a 2LC50 dose of N. ashei venom, incubated at 37 °C for 30 minutes, and added to the vials. Figure S3. Surviving larvae were counted after 24 hours. The least amount of antivenom required to prevent death in 50% of brine shrimps was calculated using probit analysis. Results svPLA2 activities of the venoms was dose-dependent. Table 1. N. subfulva venom had the highest activity while D. angusticeps venom had the least activity. Wells containing venom alone recorded the largest decrease in absorbance. Table S4. Regression analysis established that one ml of antivenom I neutralized 0.075 µg of N. ashei venom (Figure S3) while one ml of antivenom II neutralized 0.051 µg of N. ashei venom. (Figure S4). N. subfulva venom was the most toxic in the 24-hour lethality assay, N. ashei venom was the most toxic in the 48 and 72-hour assays, and N. haje venom was the least toxic at all the time intervals. Table 2. N. ashei, N. pallida, and N. subfulva venoms were toxic at all the time intervals while N. haje venom was non-toxic at all the time intervals. B. arietans venom was non-toxic after 24 hours but toxic after 48 and 72 hours. Table 2. Discussion svPLA2 make up to 30% of viperid and elapid venoms [28, 29], and the local effects of venomous snakebite in sub-Saharan Africa have been linked to svPLA2’s [12, 30–33]. Our results suggest that the Eastern forest cobra had the highest svPLA2 activity while mamba venoms had the least activity. This observation seems to corroborate clinical reports on venomous snakebite in Sub-Saharan Africa where Page 4/13 cobra bites are associated with severe muscle and tissue damage, and painful progressive swelling which are generally absent in mamba bites [30, 34–39].