Toxicity of crude and detoxified Tityus serrulatus venom in anti-venom-producing sheep

Abstract

Specific anti-venom used to treat scorpion envenomation is usually obtained from horses after hyperimmunization with crude scorpion venom. However, immunized animals often become ill because of the toxic effects of the immunogens used. This study was conducted to evaluate the toxic and immunogenic activities of crude and detoxified Tityus serrulatus (Ts) venom in sheep during the production of anti-scorpionic anti-venom. Sheep were categorized into three groups: G1, control, immunized with buffer only; G2, immunized with crude Ts venom; and G3, immunized with glutaraldehyde-detoxified Ts venom. All animals were subjected to clinical exams and supplementary tests. G2 sheep showed mild clinical changes, but the other groups tolerated the immunization program well. Specific antibodies generated in animals immunized with either Ts crude venom or glutaraldehyde-detoxified Ts venom recognized the crude Ts venom in both assays. To evaluate the lethality neutralization potential of the produced sera, individual serum samples were pre-incubated with Ts crude venom, then subcutaneously injected into mice. Efficient immune protection of 56.3% and 43.8% against Ts crude venom was observed in G2 and G3, respectively. Overall, the results of this study support the use of sheep and glutaraldehyde-detoxified Ts venom for alternative production of specific anti-venom.

Keywords: Tityus serrulatus; anti-venom; glutaraldehyde detoxification; scorpion; sheep.

Fig. 1. Electrocardiogram of sheep inoculated with crude Ts venom. II derivation, 50 mm/sec, with 10 mm/mV sensibility. (A) QR complex (red circle). (B) QS complex (red circle). (C) qR complex (red circle).

Fig. 2. Enzyme-linked immunosorbent assay (ELISA) characterization of the anti-Ts venom sheep antibodies, per group. Reactivity of sheep sera immunized with either PBS (G1, white bars), crude Ts venom (G2, black bars) or glutaraldehyde detoxified Ts venom (G3, grey bars). Serum from each animal was pooled by group and used at different dilutions (1/100; 1/500; 1/1,000; 1/5,000; 1/10,000 and 1/50,000). Plates were coated with 100 µL of Ts crude venom (10 µg/mL). Three independent experiments were conducted and plotted values are the means ± SEM of triplicates (error bars).

Fig. 3. ELISA characterization of the anti-Ts venom sheep antibodies, per animal. Reactivity of individual sheep serum. In G1, animals 1, 2, 3 and 4 were immunized with PBS (A). In G2, animals 5, 6, 7 and 8 were immunized with crude Ts venom (B). In G3, animals 9, 10, 11 and 12 were immunized with glutaraldehyde detoxified Ts venom (C). Serum from each animal was used at different dilutions (1/100; 1/500; 1/1,000; 1/5,000; 1/10,000 and 1/50,000). Plates were coated with 100 µL of Ts crude venom (10 µg/mL). Values are means of duplicates from two independent experiments. Anti-Ts sheep serum that was previously produced by the injection of eight doses of 500 µg of Ts venom and emulsified in Freund's adjuvant was used as a positive control (C+).

Fig. 4. Western blotting characterization of individual sheep sera. A total of 20 µg Ts venom were subjected to 18% sodium dodecyl sulphate polyacrylamide gel electrophoresis and then transferred to nitrocellulose membranes. The membrane was then analysed against each individual serum at 1/1,000 dilution, and revealed with 3,3′-diaminobenzidine/chloronaphthol. P, molecular weight marker; Lanes 1–4, serum from animals immunized with PBS (G1); Lanes 5–8, serum from animals immunized with crude Ts venom (G2); Lanes 9–12, serum from animals immunized with glutaraldehyde-detoxified Ts venom (G3); Lane C+, anti-Ts sheep serum which was previously produced by the injection of eight doses of 500 µg of Ts venom emulsified in Freund's adjuvant.