Direct Correlation between Diffusion of Loxosceles reclusa Venom and Extent of Dermal Inflammation

Abstract

Abstract. Objectives: Envenomation by Loxosceles species (brown recluse) spiders results in large dermal inflammatory lesions. Venom-induced dermal inflammation occurs indirectly via soluble mediators of inflammation. This study aimed to explore whether the anatomic extent of dermonecrotic arachnidism is due to the cascade of soluble proinflammatory mediators elicited by venom deposited at the bite site, or due to diffusion of the venom per se. Methods: Three New Zealand white rabbits received intradermal L. reclusa venom (3-g) injections in the flank. At the time of maximum dermal inflammation (24 hr), paired 4-mm dermal biopsies were obtained in 2-cm intervals extending 0 to 12 cm from the inoculation site. Normal dermal tissue was obtained from the opposite flank to serve as a negative control. One biopsy sample from each interval was homogenized and assayed for myeloperoxidase (MPO) activity and for the presence of venom via an enzyme immunoassay (EIA). The other paired dermal biopsy was sectioned, and examined for the presence of polymorphonuclear neutrophils (PMNs) by microscopy. Lesional areas were measured using digital images imported into imaging software. Results: Mean Ϯ SD lesional diameter 24 hours post inoculation measured 9.18 Ϯ 0.64 cm. Venom was detected in biopsies 0 to 10 cm from the injection site. As expected, the highest venom concentrations were measured at the inoculation site (4.28 Ϯ 3.9 ng/4 mm). In addition, PMNs and MPO were detected up to 8 and 10 cm from the inoculation site, respectively. Neither PMNs nor MPO was detected in tissue absent of venom (kappa = 0.88, p < 0.001). Conclusions: Loxosceles venom diffuses from the envenomation site. The extent of dermal inflammation mirrors the extent of Loxosceles venom diffusion. This observation implies that the venom itself defines the extent and magnitude of tissue injury following Loxosceles envenomation.

A new assay for the detection of Loxosceles species (brown recluse) spider venom

“…21 Our Loxosceles species ELISA assay was specific for Loxosceles species venom when relevant amounts of venom (likely to be recovered from a dermal lesion) were tested. We have observed that venom amounts of less than 40 ng per 100 μL of homogenized tissue are detected from dermal punch biopsy specimens obtained 24 hours after Loxosceles species venom inoculation into rabbit dermis 19,20 and in 1 confirmed case of L arizonica human envenomation. 24 A sensitive and specific Loxosceles species venom assay is clinically needed.…”

“…The approximate amount of venom deposited from a single spider bite is 2,000 ng (personal communication, Charles Kristensen, Spider Pharm Inc, Yarnell, AZ, April 2001), whereas 40 ng is the maximum amount of Loxosceles species venom we recover from 100 μL of homogenized rabbit dermis 24 hours after simulated spider bites. 19,20 Similar amounts of L reclusa venom and bovine serum albumin served as positive and negative controls, respectively. Study and control preparations were coded and placed in Eppendorf tubes of identical appearance before performing the assay to ensure blinding of the investigator performing the ELISA.…”

“…23 The ELISA is capable of detecting less than 0.1 ng of Loxosceles species venom. 19,20 We gathered every domestic North American spider venom commercially available at the time of the investigation. A total of 17 venoms, including 14 spider venoms (Spider Pharm, Yarnell, AZ), 2 scorpion venoms (Spider Pharm), and 1 bee venom (Sigma, St. Louis, MO) were studied (Table).…”

“…19,20 Polyclonal antibodies for the ELISA were raised in rabbits by using unfractionated L reclusa venom. The antibodies were raised and then purified from serum by means of protein A column liquid chromatography, as previously described.…”

“…We developed a polyclonal antibody-based Loxosceles species ELISA as a research tool. 19,20 The assay is capable of detecting less than 0.1 ng of venom. 19 Although the exact amount of venom deposited during a native spider bite is unknown, an estimated amount is 2,000 ng on the basis of observations from commercial spider-milking procedures (personal communication, Charles Kristensen, Spider Pharm Inc, Yarnell, AZ, April 2001).…”