DETERMINATION OF POTENCY AND PARASPECIFIC EFFECTS OF Androctonus crassicauda (OLIVIER, 1807) ANTIVENOM AGAINST Mesobuthus gibbosus (BRULLÉ, 1832) VENOM (SCORPIONES: BUTHIDAE)

Scorpion envenomation remains a real health problem in many countries. In scorpionism cases, it is often recommended that patients be treated with species-specific antivenom. Androctonus crassicauda venom has been used as antigen for antivenom production in Turkey, where this antivenom, called Turkish antivenom, has also been effective in the treatment of envenomation caused by species other than A. crassicauda. The present study aimed at determining the paraspecific effects and potency of the Turkish antivenom against Mesobuthus gibbosus (Brullé, 1832) venom. To assess the venom toxicity and the antivenom efficacy, we determined the Minimum Lethal Dose (MLD) and the Minimum Effective Dose (MED) instead of LD50 and ED50, respectively. Androctonus crassicauda antivenom was capable of neutralizing M. gibbosus venom (20 MLD). This was the first study indicating that A. crassicauda antivenom can be used for the treatment of Mesobuthus gibbosus stings, especially in Aegean Region, Turkey.


INTRODUCTION
Envenomation by arachnids causes significant injuries all over the world.Scorpion sting is the most important type of arachnid envenomation resulting in adult morbidity and pediatric mortality.Scorpionism remains a real health problem in developing countries, especially in tropical and subtropical regions including urban areas (3,5,11,13).
Venom obtained through maceration of telsons of A. crassicauda scorpions has been used as antigen for antivenom.This antivenom, called Turkish antivenom, has also been used against other scorpion species (1,10,17,19).
The present study aimed at determining the paraspecific effects and potency of A. crassicauda antivenom against Mesobuthus gibbosus venom.
Experimental animals: Healthy female Swiss albino mice, weighed 25±1g, were used for determination of the Minimum Lethal Dose (MLD) and Minimum Effective Dose (MED).They were bred at the Animal Facility Center, Faculty of Medicine, Ankara University, Ankara, Turkey.Animals were kept in an experimental room under room temperature (22±2°C) and 60±10% humidity and were fed with commercial rodent pellets ad libitum throughout the experiment.

Venom
Venom was obtained from mature A. crassicauda and M. gibbosus scorpions through electrical stimulation of their telson.The venom was mixed with sterile double distilled water and centrifuged at 15,000rpm for 15min at 4°C.The supernatant was immediately lyophilized and stored at -20°C until use.

Antivenom
Antivenom was prepared from a pool of hyperimmune sera obtained from horses immunized with A. crassicauda venom.

Determination of Minimum Lethal Doses
The Minimum Lethal Dose (MLD) was determined to assess the lethal toxicity of A. crasssicauda and M. gibbosus venoms.Groups of five mice were subcutaneously injected with venom diluted in physiologic saline solution (PSS; 0.9% sodium chloride solution) at doses ranging from 5.0 to 35.0µl/mouse (M.gibbosus venom) and from 2.5 to 15.0µl/mouse (A.crasssicauda venom).The volume of venom injected was kept constant at 500µl/mouse.Control group was injected with PSS only.Following treatment with venom solution, animals were monitored for 48 hours and the number of dead animals was recorded at the end of the experiment.The dose that killed 100 percent animals was considered the MLD.

Serum Neutralization Tests
A solution of A. crassicauda venom (40 MLD) and a solution of M. gibbosus venom (20 MLD), both diluted in PSS to a 2500µl volume, were prepared.Then, each solution was mixed with hyperimmune horse sera and incubated for one hour at 37°C for antigen-antibody reaction.Groups of six mice were subcutaneously injected with the solutions.The injection volume was kept constant at 500µl/mouse.Control groups were injected with PSS only.The number of living mice was recorded after 48h.

Determination of Minimum Effective Doses
Androctonus crassicauda antivenom was prepared at doses ranging from 100µl to 1000µl.The final volume was made up to 5000µl with PSS and the solutions were
To assess the antivenom potency, increasing doses of hyperimmune sera were used (100, 400, 700, 1000µl) while the amounts of A. crassicauda and M. gibbosus venoms were kept constant (40 and 20 MLD, respectively).Changes in the neutralization capacity of the hyperimmune horse sera against A. crassicauda and M. gibbosus venoms were shown in Table 2.The minimum effective dose of the antivenom against both A. crassicauda and M. gibbosus venoms was 400µl.All control mice died.The antivenom dose needed to neutralize M. gibbosus venom was 20 times higher than the MLD of a mouse.The above-mentioned parameters must always be specified for the determination of any venom LD 50 or antivenom potency.As the subcutaneous route provides the highest LD 50 value and is the most frequent route of accidental scorpion envenomation, it should be used to estimate the antivenom potency (7).The venom variability must also be considered during the production of antivenom, which has to be capable of neutralizing the toxic effects of all kinds of venoms from a specific scorpion species (7,16).
Theakston et al. (16) carried out a review of the potency assays used in several countries and noticed that the most acceptable test is the standard murine lethality assay (determination of LD 50 of venom and ED 50 of antivenom).However, considerable variations were found such as the way the test was carried out, the volumes injected, the routes used for injection of venom/antivenom mixtures, the LD 50 value, the mouse strain used and the animals' weight range.
The antivenom activities should be expressed according to national or regional standards and a toxin neutralization unit must be used.

O.
Ozkan et al.DETERMINATION OF POTENCY AND PARASPECIFIC EFFECTS OF Androctonus crassicauda (OLIVIER, 1807) ANTIVENOM AGAINST Mesobutus gibbosus (BRULLÉ, 1832) VENOM (SCORPIONES: BUTHIDAE).J. Venom.Anim.Toxins incl.Trop.Dis., 2007, 13, 2, p. 503 incubated at 37 o C for 60 min.Then, 500µl of each solution was subcutaneously injected into groups of six mice previously injected with 20 MLD of M. gibbosus venom or 40 MLD of A. crassicauda venom.Positive control groups were only injected with 20 MLD of M. gibbosus venom and 40 MLD of A. crassicauda venom diluted in PSS.After administration, animals were monitored for 48 hours and the number of living animals was recorded.The antivenom doses that prevented 100% deaths in the groups were considered the minimum effective doses (MED).
Ozkan et al.DETERMINATION OF POTENCY AND PARASPECIFIC EFFECTS OF Androctonus crassicauda (OLIVIER, 1807) ANTIVENOM AGAINST Mesobutus gibbosus (BRULLÉ, 1832) VENOM (SCORPIONES: BUTHIDAE).J. Venom.Anim.Toxins incl.Trop.Dis., 2007, 13, 2, p. 505 transvaalicus in South Africa; Mesobuthus tamulus and Palamneus swammerdami in (7)rifi et al.(7)reported that the difficulties in standardizing the venom quality and the LD 50 determination are in part related to: the species used; the animals' geographical origin, age and body weight; the season and procedures of venom extraction; the number of specimens milked; the breeding conditions; the venom injection route; and the LD 50 determination method.