Development and Efficacy of the Antivenom Specific to Severe Envenomations in Morocco and North Africa: Advancements in Scorpion Envenomation Management

Abstract

Scorpion envenomation poses a global public health issue, with an estimated 1,500,000 cases worldwide annually resulting in 2600 deaths. North Africa, particularly Morocco, experiences severe envenomations, mainly attributed to Androctonus mauretanicus and Buthus occitanus in Morocco, and Buthus occitanus and Androctonus australis hector in Algeria and Tunisia, with case numbers often underestimated. Current treatment relies mainly on symptomatic approaches, except in Morocco, where management is limited to symptomatic treatment due to controversies regarding specific treatment. In Morocco, between 30,000 and 50,000 scorpion envenomation cases are reported annually, leading to hundreds of deaths, mainly among children. Controversies among clinicians persist regarding the appropriate course of action, often limiting treatments to symptomatic measures. The absence of a specific antivenom for the venoms of the most lethal scorpions further exacerbates the situation. This study aims to address this gap by developing a monovalent antivenom against the endemic and most dangerous scorpion, Androctonus mauretanicus. The antivenom was produced by immunizing albino rabbits with a mixture of Androctonus mauretanicus venom collected from high-risk areas in Morocco. Immunizations were performed by subcutaneous injections at multiple sites near the lymphatic system, following an immunization schedule. Production control of neutralizing antibody titers was conducted through immunodiffusion. Once a sufficient antibody titer was achieved, blood collection was performed, and the recovered plasma underwent affinity chromatography. The efficacy of purified IgG was evaluated by determining the ED₅₀ in mice, complemented by histological and immunohistochemical studies on its ability to neutralize venom-induced tissue alterations and the neutralization of toxins bound to receptors in the studied organs. The monovalent antivenom demonstrated specificity against Androctonus mauretanicus venom and effective cross-protection against the venom of the scorpions Buthus occitanus and Androctonus australis hector, highly implicated in lethal envenomations in the Maghreb. This study shows that the developed monovalent antivenom exhibits notable efficacy against local scorpions and a surprising ability to neutralize the most lethal envenomations in North Africa. These results pave the way for a new, more specific, and promising therapeutic approach to countering severe scorpion envenomations, especially in Morocco, where specific treatment is lacking.

Keywords: antivenom; cross-reactivity; immunization; neutralization; scorpion venom; toxicity.

Figure 1

SDS-PAGE profile of Am scorpion venom. Lane 1 contains molecular weight markers in kDa; Lane 2 contains Am venom.

Figure 2

Double immunodiffusion illustrating the precipitation arcs resulting from the interaction between the Am venom (in the central well) and the neutralizing antibodies of the rabbit plasma (in the peripheral wells), indicating the production of antibodies. The peripheral wells contain increasing dilutions of rabbit plasma collected on days 21 (a), 35 (b), and 42 (c) after the first immunization.

Figure 3

Double immunodiffusion illustrating the precipitation arcs resulting from the interaction between the Bo (a) and Aah (b) venoms (in the central well) and the neutralizing antibodies of the rabbit plasma (in the peripheral wells), indicating the production of antibodies. The peripheral wells contain increasing dilutions of rabbit plasma collected on day 42 after the first immunization.

Figure 4

Chromatogram of the purification profile of specific antibodies obtained by affinity chromatography.

Figure 5

SDS-PAGE electrophoresis and component percentage of the plasma (A,A’) and purified serum (B,B’).

Figure 6

Western blotting using the produced antivenom against the Am, Bo and Aah venom. Lane 1 contains molecular weight markers in kDa; Line 2 contains Am venom; Lane 3 contains Bo venom; Lane 4 contains Aah venom.

Figure 7

Antivenom neutralization of tissue alterations induced in brain tissue by Am, Bo and Aah scorpion venoms (×10). (A): Image corresponds to the control group. (B–D): Images correspond to the groups treated with the scorpion venoms. (E–G): Images correspond to groups treated with the antivenom 2 h delay. Bv: blood vessel; N: neuron; Vd: vasodilatation; Ed: edema; Lc: loss of cellularity; Hr: hemorrhage.

Figure 8

Antivenom neutralization of tissue alterations induced in cardiac tissue by Am, Bo and Aah scorpion venoms (×10). (A): Image corresponds to the control group. (B–D): Images correspond to the groups treated with the scorpion venoms. (E–G): Images correspond to groups treated with the antivenom 2 h delay Mf: muscle fiber; Dmf: degeneration of myocardium; Cong: congestion.

Figure 9

Antivenom neutralization of toxins fixed to tissue receptors at the level of heart tissue by the effect of the venoms of Am, Bo and Aah. (A): The control group. (B–D): Groups treated with the scorpion venoms. (E–G): Groups treated with the antivenom 2 h delay. Calibration bar = 50 µm. Brown areas are positive for venom and complex venom–antivenom. Detection in nerve cells (red arrow), vascular endothelial cells (black arrow), and inflammatory cells (green arrow).

Figure 10

Antivenom neutralization of toxins fixed to tissue receptors at the level of heart tissue after the effect of the venoms of Am, Bo and Aah. (A): The control group. (B–D): Groups treated with the scorpion venoms. (E–G): Groups treated with the antivenom 2 h delay. Calibration bar = 50 µm. Brown areas are positive for venom and complex venom–antivenom. Detection in cardiac cells (blue arrow), vascular endothelial cells (black arrow), and inflammatory cells (green arrow).