Table 1.
Details of the antivenom products examined in this study.
Table 2.
Lethal and necrotic toxicity from Eswatini snake venoms in a murine model.
The amount of venom that caused lethality in 50% of mice (LD50) following intravenous injection was determined by Probit analyses, and is reported as μg per mouse, with 95% confidence intervals in parentheses. The minimum amount of venom that caused a lesion approximately 5x5 mm in diameter (MND) following intradermal injection is reported as μg per mouse, with mean lesion area in parentheses. Due to signs of systemic envenoming at higher doses of H. haemachatus venom injection, a smaller lesion area is reported. N.D. = not determined. All experiments used groups of 5 animals.
Fig 1.
ED50 of the three antivenoms against each of the five Eswatini venoms, expressed as μL per mg of venom.
SAIMR Polyvalent (SAVP) shown in teal, Panafrican (ICP) shown in magenta, PANAF Premium (PS&V) shown in blue. ED50 was determined using Probit analysis, data represents the calculated ED50 and error bars represent 95% confidence intervals. The units used (μL per mg of venom) attempts to normalise reporting to the amount of venom used to compensate for differences in number of venom LD50s used, however for clarity, a 3 x LD50 venom dose was used for the following groups; H. haemachatus with PANAF (PS&V) antivenom, N. annulifera with Panafrican (ICP) antivenom, and N. annulifera with PANAF (PS&V) antivenom. All other experiments used 5 x LD50 venom dose.
Table 3.
The preclinical venom-neutralising efficacy (antivenom ED50) of the PANAF (PS&V) and Panafrican (ICP) antivenoms compared to SAIMR Polyvalent in the pre-incubation model of envenoming.
ED50 is defined as the volume of antivenom which protects 50% of mice from the lethal effects of venom. Each experiment used five mice per dose group. The assays utilised a venom challenge dose of 5 x venom LD50s (Table 2), except when it was necessary to reduce the challenge dose 3 x venom LD50s (indicated by red text). In these instances, the maximum volume limit of antivenom that can be injected intravenously was reached without reducing the venom lethality of the 5 x LD50 venom dose, therefore it was necessary to reduce the venom dose to determine an ED50 for the antivenom. Results are reported as ED50 determined by Probit analyses and expressed in (i) volume of antivenom and, in italics, (ii) as μL of antivenom per mg of venom. 95% confidence intervals are reported in parentheses.
Table 4.
The resulting in mean lesion size after intradermal injection with one MND dose of venom preincubated with each of the antivenoms.
The dose of SAIMR Polyvalent in μL was first determined, and the test antivenoms at this fixed volume were then tested to compare lesion size.
Fig 2.
The titre of three antivenoms against five Eswatini venoms determined by end-point titration ELISA.
SAIMR Polyvalent (SAVP) shown in teal, Panafrican (ICP) shown in magenta, PANAF (PS&V) shown in blue, normal horse IgG shown in purple. Panel A: B. arietans. Panel B: D. polylepis. Panel C: H. haemachatus. Panel D: N. annulifera. Panel E: N. mossambica. Data points represent the mean of two replicates and error bars show the standard deviation. The vertical line at the 1 in 62,500 dilution represents the point at which Ig titres of each antivenom were compared.
Fig 3.
Protein profiles of the venom protein components of five Eswatini snake venoms.
A: Venom proteins separated by SDS-PAGE under reducing conditions and stained with Coomassie blue to show all proteinaceous components. B: Immunoblot using normal horse IgG used as primary antibody negative control. C: Immunoblot using SAIMR Polyvalent as primary antibody. D: Immunoblot using Panafrican (ICP) antivenom as primary antibody. E: Immunoblot using PANAF (PS&V) antivenom as primary antibody.
Fig 4.
Plasma clotting activity of Eswatini venoms and their neutralisation by three antivenoms.
Data show the mean area under the curve of nine replicates and error bars represent standard deviation. **** indicates p < 0.0001, ** indicates p < 0.01 and * indicates p < 0.05 by one-way ANOVA. A: Plasma clotting activity of B. arietans, D. polylepis, H. haemachatus, N. annulifera and N. mossambica in comparison to PBS normal clotting control (white bar). B: Effects of antivenoms on neutralising the anticoagulant effects of H. haemachatus venom. C: Effects of antivenoms on neutralising the anticoagulant effects of N. mossambica venom. Normal clotting is shown by ‘PBS control’ bars in white (wells contain no venom), and venom-induced clotting deficiency shown by ‘no antivenom control’ bars in black.
Fig 5.
SVMP activity of Eswatini venoms and their neutralisation by three antivenoms.
*** indicates p < 0.001, **** indicates p < 0.0001 by one-way ANOVA. A: SVMP activity of B. arietans, D. polylepis, H. haemachatus, N. annulifera and N. mossambica in comparison to PBS control. Data show the mean of four replicates and error bars represent standard deviation. Neutralisation of (B) B. arietans and (C) N. annulifera SVMP activity by the three antivenoms SAIMR Polyvalent (SAVP), Panafrican (ICP) and PANAF (PS&V) at different doses, in comparison to a no antivenom control (venom only) showing 100% activity. Data show the mean of four replicates and error bars represent standard deviation.
Fig 6.
PLA2 activity of five venoms from Eswatini and their neutralisation by different antivenoms.
** indicates p < 0.01 by one-way ANOVA. A: PLA2 activity of B. arietans, D. polylepis, H. haemachatus, N. annulifera and N. mossambica. Data show the mean of three replicates and error bars represent standard deviation. Neutralisation of (B) H. haemachatus and (C) N. annulifera PLA2 activity by the three antivenoms SAIMR Polyvalent (SAVP), Panafrican (ICP) and PANAF (PS&V) at different doses, expressed as a percentage of a no antivenom control showing 100% activity. Data show the mean of three replicates and error bars represent standard deviation.