Antigenic Cross-Reactivity, Enzymatic Characterization And Neutralization Of Sphingomyelinase And Dermonecrotic Activities Of Medically Important Loxosceles Spider Venoms

^{1,3Barbaro, K.C.; 2Knysak, I.; 2Martins, R.; 3,4,5}Hogan, C. and ³Winkel, K.

1 Lab. Immunopathology, 2 Lab. Arthropods, Butantan Institute, São Paulo, SP Brazil; 3 Australian Venom Research Unit, University of Melbourne, VIC, Australia; 4 Dep. Emerg. Med., Allegheny General Hospital, Pittsburgh, PA, USA and 5 Dep. Emerg. Med., Medical College of Virginia/VCU Hospital, Richmond, VA, USA.

Loxosceles spider venom is a complex mixture of components with toxic and/or enzymatic activities. Bites caused by Loxosceles spider are characterized by necrotic skin ulceration at the bite site and, less commonly, a systemic illness that may be fatal. The aim of this work was to characterize and compare some aspects of the major medically important Loxosceles spider venoms, particularly their neutralization by Brazilian antivenom since this spider has a wide distribution in temperate and tropical regions of the world. By SDS-PAGE, L. deserta, L. gaucho, L. intermedia, L. laeta and L. reclusa venoms showed a similar electrophoretic profiles with the major bands located around 32 kDa (L. deserta and L. laeta venoms) and 35 kDa (L. gaucho, L. intermedia and L. reclusa venoms). The venoms also had several minor components of between 82-220 kDa. All venoms exhibited gelatinolytic, caseinolytic, fibrinogenolytic and hyaluronidase activities in vitro with a large array of caseinolytic and gelatinolytic proteases mainly between 20 - 30 kDa. Hyaluronidase activity was detected in a band around 41 kDa in all venoms. Most of these components are metalloproteinases since rather enzymatic activities were abolished after incubation with 1,10-phenanthroline. Antigenic cross-reactivity was observed among the five venoms studied using commercial antisera produced in Brazil (AAS: AntiArachnidic Serum and PSLAS: Poly Specific Loxosceles Anti Serum). By ELISA there was intense cross-reactivity amongst all venoms (titre range: 64,000-512,000). By Western blotting the sera recognized mainly components of between 25-40 kDa in all venoms. Several minor components >83 kDa were also revealed. All venoms (5 µg) induced a similar local reaction in vivo, characterized by dermonecrosis, hemmorhage/palor and oedema/erythema when injected intradermal (id) injection into the rabbit flank. However no local reaction was observed when each venom was incubated (1h, 37°C) with Brazilian commercial sera prior to i.d. injection. By spectrophotometric method using TNPAL- sphingomyelin as substrate, sphingomyelinase activity was demonstrated in all venoms studied. Both antisera were able to neutralize this activity. These results demonstrate that the venom of the major medically important Loxosceles spider species have generally similar toxic and enzymatic characteristics and that the Brazilian commercial antisera are able to neutralize the effects of these venoms in sphingomyelinase assay as well as a rabbit model of dermonecrotic arachnidism.

Support by: FAPESP 02/01003-9, Universityof Melbourne

Correspondence to: kbarbaro@butantan.gov.br