Hindawi Publishing Corporation ISRN Toxicology Volume 2013, Article ID 247645, 5 pages http://dx.doi.org/10.1155/2013/247645 Research Article Cross-Reactivity against Naja sumatrana (Black Spitting Cobra) Envenoming from the Haffkine Antivenom in a Mouse Model Gregory Cham,1 Francis Lim,2 Arul Earnest,3 and Ponnampalam Gopalakrishnakone4 1 Emergency Medicine Department, Alexandra Hospital, 378 Alexandra Road, Singapore 159964 2 Singapore Zoological Gardens, 80 Mandai Lake Road, Singapore 729826 3 Centre for Quantitative Medicine, Duke-NUS Graduate Medical School, 8 College Road, Singapore 169857 4 Venom and Toxin Research Programme, Department of Anatomy, Yong Loo Lin School of Medicine, National University of Singapore, 4 Medical Drive, Block MD10, Singapore 117597 Correspondence should be addressed to Gregory Cham; [email protected] Received 8 June 2013; Accepted 11 July 2013 Academic Editors: F. Ducancel, B. K. Mandal, and A. Pessina Copyright © 2013 Gregory Cham et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Naja sumatrana is the dominant cobra species in Malaysia, Singapore, Borneo, and Sumatra, and it does not have specific antivenom. The Haffkine antivenom has been advocated instead. This study aims to determine the efficacy of this antivenom against Naja sumatrana envenoming using a mouse model. Methods.MaleSwissalbinomicewereused.IntravenousLD50 was first determined separately for Naja naja and Naja sumatrana venom. ED50 was determined by preincubating antivenom with each venom at 2.5 LD50 before administering the mixture into the tail vein. Validation was carried out using a challenge test. Each mouse received 111 gofNaja sumatrana venom intramuscularly followed by intraperitoneal administration of dilute Haffkine antivenom. Survival was recorded 24 hours after envenoming. Results.TheLD50 of Naja naja venom was 78.13 g, standard error (SE) 13.3 g. The 50ED of the Haffkine antivenom against Naja naja venom was 45.9 mg, SE 7.5mg. The LD50 and ED50 of Naja sumatrana venom were 55.5 g, SE 12.0 g; and 73.9 mg, SE 12.0 mg, respectively. The intra-peritoneal ED50 against 111 g intramuscular Naja sumatrana venom was 136.95 mg, SE 36.74 mg. Conclusion. The Haffkine polyvalent antivenom exhibited cross-neutralisation against Naja sumatrana venom when used at a higher dose. 1. Introduction (Naja naja), common krait (Bungarus caeruleus), Russell’s viper (Vipera russelli), and saw-scaled viper (Echis carinatus) 1.1. Background and Importance. Naja sumatrana,commonly [7]. However, these species of snakes are not normally known as the black spitting cobra or Equatorial spitting found in Southeast Asia. The antivenom composition of cobra, is the dominant cobra species in Peninsular Malaysia, the Haffkine was chosen for the treatment of black spitting Singapore, Borneo, and Sumatra [1]. It was estimated that the cobra snake bites due to the empirical belief that cobras are highest burden of snake bites exists in South Asia, Southeast originated from the Naja naja. These species are, however, Asia, and sub-Saharan Africa [2].Cobrabitesarecommonin considered to be separate now. It is unknown if the Haffkine Peninsular Malaysia and require significant medical interven- product has any effective paraspecific activity against the Naja tion [3, 4]. However, snake bites are considered uncommon sumatrana venom. There have been no previous papers that in largely urban Singapore [5, 6]. reporttheoutcomesofpatientsgiventhisantivenomforblack Unfortunately specific antivenom therapy against the spitting cobra bites. Naja sumatrana does not exist. The Haffkine antivenom was This study aims to examine if effective cross-neutral- arbitrarily advocated as an antivenom. It is an equine poly- isation exists for Naja sumatrana envenoming when using the valent antivenom raised against the Indian binocellate cobra Haffkine antivenom. 2 ISRN Toxicology 2. Materials and Methods Table 1: Weights of the mice compared between groups. Mean 2.1. Study Design. Using comparable doses of Naja naja or Std. weight Range Naja sumatrana venom, this study analysed the neutralising deviation capabilityoftheHaffkineantivenominamousemodel.This (grams) Group for Naja naja LD was followed by a challenge test by giving a fixed dose of Naja 50 24 29.98 0.99 27.5–32.0 sumatrana venom intramuscularly and subsequent attempt at determination Group for Naja sumatrana rescue with the antivenom. 24 29.42 2.41 25.5–37.5 LD50 determination Group for Naja naja ED 2.2. Materials. Naja naja venom was purchased from Accu- 50 12 28.50 0.95 27.0–30.5 rate Chemical Corporation (New York, USA). Naja suma- determination trana venom was pooled from five local adult Naja sumatrana Group for Naja sumatrana ∘ 12 30.46 1.14 28.0–32.0 cobras housed at the zoo, lyophilised, and stored at −20 C. ED50 determination The antivenom used was a commercial product from the Total 72 29.62 1.72 25.5–37.5 Haffkine Bio-Pharmaceutical Corporation (Mumbai, India). 2.3. Setting and Animal Subjects. Ethics committee approvals fromTanTockSengHospital(TTSH)andSingaporezoo were used. Each was given an intramuscular injection of were first sought. The experiments were carried out in the arbitrarily two times the intravenous LD50 into the left thigh Animal Research Laboratory at TTSH. Only male Swiss muscle from the same batch of reconstituted lyophilised Naja albino mice about five weeks of age were used. The mice were sumatrana venom, in 0.05 mL. After two minutes, this was bred locally at the Laboratory Animals Centre and weighed followed by an intraperitoneal administration of Haffkine between 25.5 and 37.5 grams. antivenom at various concentrations, in two mL. 2.4. Study Protocol. The animals were quarantined for 24 2.5. Measurements. The end point for all the above experi- hours prior to conducting the experiments. Food and water ments was death of the experimental animal. Total number were available ad libitum throughout the study. of deaths was recorded 24 hours after envenoming. 2.4.1. Determining the Lethal Dose (LD50). The LD50 is the dose of venom required to kill 50% of mice within 24 hours. 2.6. Data Analysis. The LD50 and ED50 were calculated Each venom was reconstituted by dissolving lyophilised using the Reed-Muench method [9]. The standard error was venom in normal saline. Further concentrations were ob- calculated according to the method by Pizzi [10]. One-way tained from serial dilutions using additional saline. The lethal ANOVA was used to compare the mean weight of the mice toxicity was determined by injecting 0.2 mL of venom, at for the four groups. Weight was assumed to be normally various concentrations per mouse, into the tail vein [8]. The distributed in the different groups. The Bonferroni method of injections were performed using a 1 mL syringe fitted with comparison was used to adjust for the multiple comparisons. ∘ a 29-gauge needle. The venom was warmed to 37 Cfor30 Level of significance was set at 5%, and data analysis was minutes before injection. Six mice were used for each dilu- carried out using SPSS (Illinois, USA). tion.Themicewereobservedfor24hours. 3. Results 2.4.2. Determining the Effective Dose (ED50). The ED50 is the dose of antivenom required to produce 50% survival of mice In most of the groups compared the mice were similar in within 24 hours when used against intravenous venom at weight. The mean difference in weight for the overall groups = 0.022 2.5 LD50 foreachmouse.Thevenomandantivenomwere of mice was significant ( , by one-way ANOVA). incubated prior to intravenous administration. Lyophilised Pairwise comparisons using the Bonferroni method showed venoms of Naja naja and Naja sumatrana were obtained from significant difference between the weight of mice used for thesamebatchusedforLD50 determination. The Haffkine Naja naja ED50 and that of Naja sumatrana ED50 determina- = 0.028 antivenom used to neutralise both types of cobra venom was tion ( ). However, the actual difference in the means reconstituted from the same batch. Twofold serial dilution was only 1.96 g. Table 1 summarises the weight of the mice using normal saline was made to obtain additional concen- used. trations of antivenom. Venom at 2.5 LD50 and antivenom of The cohort of mice used in the in vivo challenge test was ∘ various concentrations were mixed and incubated at 37 Cfor also significantly smaller than those used in the preincuba- 30 minutes prior to injection. Each mouse was injected with tion experiments. 0.2 mL of venom-antivenom mixture into the tail vein. Four After tail-vein injection of venom, both groups enven- mice were used for each of the dilution groups. The mice were omed with Naja sumatrana and Naja naja venom exhibited observed for 24 hours. weakness and diminished physical activity prior to death. 2.4.3. Haffkine Antivenom versus Naja sumatrana Venom 3.1. LD50 of Cobra Venom. Survival times diminished with Challenge Test. Four groups of four male Swiss albino mice increasing venom doses. Table 2 summarises the survival of ISRN Toxicology 3 Table 2: 24-hour-survival of mice after tail-vein envenoming with Table 4: 24-hour-survival of mice after tail-vein injection of Naja naja venom. Haffkine antivenom incubated with 2.5 LD50 Naja naja venom. Venom (g) Lived Died Mortality % Antivenom dose (mg) Lived Died Mortality % 19.5 6 0 0.00 65.0 4 0 0.00 39.1 6 0 0.00 32.5 0 4 100.00 78.1 3 3 50.00 16.2 0 4 100.00 156.3 0 6 100.00 Table 5: 24-hour-survival of mice after tail-vein injection of Haf- Table 3: 24-hour-survival of mice after tail-vein envenoming with fkine antivenom incubated with 2.5 LD50 Naja sumatrana venom. Naja sumatrana venom. Antivenom dose (mg) Lived Died Mortality % Venom (g) Lived Died Mortality % 104.6 4 0 0.00 19.5 6 0 0.00 52.3 0 4 100.00 39.1 4 2 28.57 26.1 0 4 100.00 78.1 1 5 87.50 156.25 0 6 100.00 Table 6: 24-hour-survival of mice after intra-peritoneal Haffkine antivenom following 111 g intramuscular Naja sumatrana enven- oming.