Elsevier

Toxicon

Volume 45, Issue 4, 15 March 2005, Pages 489-499
Toxicon

Enzymatic characterization, antigenic cross-reactivity and neutralization of dermonecrotic activity of five Loxosceles spider venoms of medical importance in the Americas

https://doi.org/10.1016/j.toxicon.2004.12.009Get rights and content

Abstract

Loxosceles spiders have a wide distribution in the temperate and tropical regions of the world. Loxoscelism is characterized by necrotic skin ulceration at the bite site and, less commonly, a systemic illness that may be fatal. The purpose of this study was to characterize and compare aspects of the major medically important Loxosceles spider venoms in a standardized manner, particularly considering their neutralization by two Brazilian antivenoms. By SDS-PAGE (12% acrylamide), Loxosceles deserta, Loxosceles gaucho, Loxosceles intermedia, Loxosceles laeta and Loxosceles reclusa venoms had similar electrophoretic profiles, with the major protein bands of 32–35 kDa. All venoms exhibited gelatinolytic, caseinolytic and fibrinogenolytic activities in vitro with a large array of proteases, mainly between 18.1 and 31.8 kDa. Most of these enzymes were metalloproteases as this activity was abolished by 1,10-phenanthroline. Hyaluronidase activity was detected in a protein band of approximately 44 kDa in all venoms. Sphingomyelinase activity was demonstrated in all five venoms. Antigenic cross-reactivity, by Western blotting, was also observed among all venoms studied using commercial equine antivenoms produced in Brazil (Institute Butantan and CPPI). These antivenoms recognized mainly components between 25 and 40 kDa in all venoms with several minor components of >89 kDa. Strong cross-reactivity was also seen among all venoms through the ELISA technique (titre range: 64,000–512,000). All venoms (5 μg doses) induced a similar local reaction when injected intradermally into the flank of rabbits, demonstrating dermonecrosis, hemorrhage, vasoconstriction, edema, and erythema. However, no reaction was observed when each venom was pre-incubated (1 h, 37 °C) with Brazilian commercial sera prior to injection. The antivenoms also abolished the sphingomyelinase activity in vitro, suggesting the venoms of the major medically important Loxosceles spider species have generally similar toxic and enzymatic characteristics. Thus, as Brazilian commercial antivenoms are able to neutralize the dermonecrosis induced by Loxosceles venoms of diverse geographical origin, clinical studies should be undertaken on the potential for a single global Loxosceles antivenom.

Introduction

Loxosceles spiders have a wide distribution from equatorial to subtemperate regions. More than 50 species have been described, found in nearly all environments, including urban habitats. However, only a few species have been implicated in human envenomation (Hogan et al., 2004, Vetter and Bush, 2002). In South America, Loxosceles gaucho (Brazil), Loxosceles intermedia (Brazil) and Loxosceles laeta (Brazil, Peru, Chile) are considered medically important species (Ministry of Health, 1998, Schenone et al., 1989, Martinez-Vargas, 1987). In North America, the species Loxosceles deserta and Loxosceles reclusa are mainly responsible for cases of loxoscelism (Hogan et al., 2004). The diagnosis of Loxosceles envenomation is usually based on the clinical examination findings and patient history, since the spider is seldom identified. For that reason, the real incidence of envenomation is unknown and the misdiagnosis is frequent (Isbister, 2004, Vetter and Bush, 2002). According to the Brazilian Ministry of Health (1998), around 2100 bites occur every year, mainly in Southern and Southeastern states of that country. In the United States around 2400 Loxosceles spider bites are reported (Litovitz et al., 1999).

Loxoscelism is a hemolytic-necrotizing syndrome. Cutaneous loxoscelism (CL) is characterized by a dermonecrotic lesion at the bite site that can take weeks to heal. Rarely, besides the local damage, viscerocutaneous loxoscelism (VCL) can occur, causing disseminated intravascular coagulation (DIC), hemolysis, and acute renal failure. These complications are the main cause of death in VCL (Schenone et al., 1989, Futrell, 1992, Sezerino et al., 1998, Cacy and Mold, 1999, Barbaro and Cardoso, 2003).

Investigation of various species of Loxosceles venoms have demonstrated the presence of various enzymes including proteases, hydrolases, lipases, hyaluronidase, alkaline phosphatase and collagenase. A key venom component, sphingomyelinase D, is responsible for inducing dermonecrosis as well as complement-dependent hemolysis. The mechanism of action is a very complex, multifactorial process involving interactions with the cellular membrane as well as the extracellular matrix, leading to complement, platelet and polymorphic neutrophil (PMN) activation, with cytokine and chemokine release (Futrell, 1992, Patel et al., 1994, Tambourgi et al., 1995, Tambourgi et al., 1998, Gomez et al., 1999a, Málaque et al., 1999, Veiga et al., 2000, Young and Pincus, 2001, Silva et al., 2004).

Despite the fact that loxoscelism is a leading cause of spider bite morbidity in some regions (Hogan et al., 2004, Ministry of Health, 1998), a definitive therapy is not established. Furthermore, the effectiveness of current treatments is still controversial and not fully defined (Hogan et al., 2004). In Brazil, the mainstay of Loxosceles envenomation treatments is antivenom therapy and corticosteroid administration (Barbaro and Cardoso, 2003). Dapsone and corticosteroids are treatments also employed in the United States (Hogan et al., 2004), but the use of these modalities remains controversial.

Studies investigating Loxosceles venom have used diverse approaches and methodologies, making the comparison of results obtained difficult. The aim of this work was to study the five principal medically important Loxosceles spider venoms (L. deserta, L. gaucho, L. intermedia, L. laeta and L. reclusa) in a uniform manner to compare their enzymatic activities and to determine the extent of their antigenic cross-reactivity. The second aim was to evaluate the ability of Brazilian commercial anti-Loxosceles antivenoms to neutralize the dermonecrotic and sphingomyelinase activities of the venoms.

Section snippets

Animals and venoms

Adult New Zealand white rabbits (3–4 kg) were provided by the Butantan Institute animal house. Specimens of adult L. gaucho spiders were collected in the Brazilian state of São Paulo and L. intermedia and L. laeta in the states of Paraná and Santa Catarina, Brazil, respectively. The spiders were kept in quarantine for 1 week without food before venom collection. The venoms were obtained as previously described (Barbaro et al., 1992a). The L. reclusa and L. deserta venoms were also obtained by

Analysis of the venoms by SDS-PAGE

Fig. 1 shows the electrophoretic pattern of the five venoms used. At least 15 components were revealed in all venoms (12% acrylamide). The profile of Loxosceles venoms were very similar with major bands being located around 32 kDa (L. deserta, L. reclusa and L. laeta) and 35 kDa (L. gaucho and L. intermedia venoms). However, many different components were also located around this region demonstrating high variability among the venoms. Bands with less intense staining above 89 kDa were also present

Discussion

Loxoscelism is a necrotizing-hemolytic envenomation syndrome reported in diverse regions of the world (Hogan et al., 2004). Most patients bitten by Loxosceles spiders present with a local inflammatory reaction that could evolve into dermonecrosis. Less commonly, a systemic reaction can occur (Barbaro and Cardoso, 2003). There are several factors that contribute to the severity of loxoscelism. Key factors include the spider species (Barbaro and Cardoso, 2003), gender and ontogenetic stage (Kent

Acknowledgements

This paper was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP grant 02/01003-9) and University of Melbourne collaborative research grant scheme. The authors are indebted to Ms Mary Cameron for excellent technical assistance. We also thank Dr Manying Uip, Associate Professor Margaret J. Morris, Dr Anna R. Young, Ms Jenny M. Steen, Ms Joanne Cook and Ms Matilde de Paula for their help during the execution of this project. We would also like to thank Dr Hernan Gomez,

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